Final Report Summary - MOLPARK (Molecular mechanisms of neuronal restoration: novel approaches for Parkinson's Disease)
Executive summary:
MOLPARK has been exceptionally successful. We have achieved all deliverables and all but 13 of our 82 milestones and the work has led to 36 publications in leading journals. The main highlights of the MOLPARK program are as follows. A role for histone H2AX in stem cell self-renewal has been established, and we have shown in vivo that stem cell numbers in the adult brain can be manipulated by affecting this regulatory pathway. We have developed a new technology to identify proteins enriched in nascent chromatin during replication of stem cells and embryonic fibroblasts. Stem cells display a unique recruitment of complexes involved in high replication fidelity, epigenetic re-establishment and chromatin remodelling during replication. These results provide evidence for a selective enrichment of specific protein modules in newly synthesized DNA of stem cells that contribute to the preservation of genomic integrity and the epigenome during replication, features that define the unique chromatin organization which underlie stemness. Several tools to investigate the role of Bex1 in stem cell proliferation and differentiation have been developed and conditions have been developed for the use of Bex1 reagents to influence neural stem cell pools.
project Context and Objectives:
summary description of project context and objectives
the goal of MOLPARK was to make major advances in our understanding of the basic cellular and molecular mechanisms underlying the generation and survival of nigrostriatal dopaminergic neurons and the establishment and maintenance of their connections in order to provide a platform for radically new, much needed therapeutic approaches for Parkinson's disease.
to achieve this goal, our plan was to undertake an integrated programme of research focused on understanding the basic cellular and molecular mechanisms that control the generation and survival of dopaminergic nigrostriatal neurons and the establishment and maintenance of their connections with the aim of discovering novel therapeutic approaches for Parkinson's Disease based on replenishing and protecting dopaminergic nigrostriatal neurons and restorating their processes and connections.
main objectives
1. To define the mechanisms important for stem cell self-renewal, differentiation and integration into the midbrain and attempt to exploit these mechanisms to induce or boost existing self-repair processes with the aim of replenishing neurons in Parkinson's disease.
2. To define the mechanisms important for sustaining the survival of dopaminergic nigrostriatal neurons in health and disease with the aim of developing new, effective growth factor-based therapies aimed at protecting neurons in Parkinson's disease.
3. To define the mechanisms that promote the growth of dopaminergic nigrostriatal axons and dendrites with the aim of identifying potential therapeutic strategies based on restoring neural processes in Parkinson's disease.
4. To define the mechanisms that promote and sustain the synaptic connections of dopaminergic nigrostriatal neurons with the aim of restoring connections in Parkinson's disease.
specific aims underpinning these objectives
1. To define the mechanisms important for stem cell self-renewal, differentiation and integration in the midbrain.
here our plan was to focus mostly on the roles of GABA and a small adaptor-like protein called Bex1. Specifically, we planned to investigate the control of stem cell proliferation by GABA by pharmacologically manipulating GABAA receptor function. We planned to investigate the role of Bex1 in regulating stem cell self-renewal and differentiation by manipulating Bex1 expression in cultured cells and in transgenic mice. We wished also to screen for small molecules capable of regulating stem cell self-renewal and screen for small molecules capable of stabilizing fluorescently tagged Bex1. We planned to further investigate validated hits by investigating their actions in stem cell proliferation, survival and differentiation in in vitro assays and subsequently in in vivo paradigms of neural stem cell renewal.
2. To define novel mechanisms for sustaining the survival of DA nigrostriatal neurons in health and disease.
here our plan was to focus on two recently discovered, structurally related neurotrophic factors, CDNF and MANF, and also carry out studies on Ret and DJ-1. CDNF had been shown to protect dopaminergic neurons in a rat 6-OHDA model of Parkinson's disease. We planned to test the efficacy of CDNF in additional models of Parkinson's disease, and test MANF in several Parkinson's disease models. We also planned to generate CDNF and MANF knockout mice and study the dopaminergic nigrostriatal neuron phenotype of these mice. We wished also to identify receptors for these neurotrophic factors. We also planned using several genetic models in mice and Drosophila to further understanding of the role of the Ret receptor tyrosine kinase and DJ-1 (a regulator of PKB/Akt phosphorylation that is associated with autosomal recessive early-onset Parkinson's disease). We also planned to complement these in vivo genetic studies of Ret and DJ-1 with in vitro gain- or loss-of- function mutations of these proteins in cell lines.
3. To define the mechanisms that promote the growth of dopaminergic nigrostriatal axons and dendrites
here our plan was to study the dendritic morphology and axon projections of dopaminergic nigrostriatal neurons in several genetic mouse models, including those with null and conditional mutations in the following genes: Ret, DJ-1, CDNF, MANF, Dvl1 and Wnts. We also planned to study the effects of GDNF, CDNF, MANF and selected members of the TNF superfamily (based on studies of TNF superfamily receptors in dopaminergic neurons) in vitro. We also wished to investigate the role of intrinsic factors in axonal and dendritic growth, including the Runx and Cux transcription factors and the extracellular calcium sensing receptor.
4. To define the mechanisms that promote and sustain the synaptic connections of dopaminergic nigrostriatal neurons.
here our plan was to characterize the effect of dopamine depletion on synapses of the nigrostriatal system. We wished also to examine the effect of selected neurotrophic factors (GDNF, CDNF and MANF) in the maintenance and distribution of synapses in the nigrostriatal system, and planned to test the protective effects of neurotrophic factors on synapses in lesion and transgenic models for Parkinson's disease. We also planned to examine the effect of neurotrophic factors on the release of dopamine in the nigrostriatal system. Finally, we planned to screen for small molecules to restore synaptic connectivity.
project Results:
WP1: Replenishing neurons
1.1 Mechanisms of stem cell proliferation by GABA and histone modification
this task has progressed well. We have shown that the MRN complex and ATM associate with activated H2AX are necessary for ligand induced changes in self-renewal of ES cells. Unexpectedly MDC1 and 53BP1 were shown to be dispensable. During the period of activity, two publications have appeared showing that during DNA damage, H2AX can be phosphorylated on Ser 139 and that this phosphorylation is paralleled by a de-phosphorylation of a newly discovered phospho site Tyr142. De-phosphorylation of Tyr 142 is necessary for recruitment of DNA repair proteins and failure of its dephosphorylation results instead in activation of a death pathway. Most interestingly, we have found that ligand activation of this pathway controlling stem cell self-renewal phosphorylation of both Ser139 and Tyr142 are increased. We found that Ser139 phosphorylation takes place in specific foci of the chromatin while Tyr142 occurs throughout the genome. Based on these findings we have initiated the generation of point mutant mice with amino acid substitutions in these phospho sites to examine its role in adult brain stem cell self-renewal. Analysis of these strains by following all newborn neurons using BrdU has revealed that neither of these phosphosites are critical for the role of H2AX on stem cell proliferation in vivo.
1.2 Screen for small molecules regulating stem cell self-renewal
after extensive evaluation of different strategies and assays we (Ernfors) have decided on an open cell based assay. These assays have now been established allowing for screening of self-renewal of embryonic stem cells, human foreskin fibroblasts, human embryonic neural stem cells and adult mouse neural stem cells. We have obtained the NIH diversity set II library and initiated screens in the above cells. Compounds that increase cell number in embryonic stem cells, human embryonic stem cells and adult mouse neural stem cells but not in fibroblasts will be analysed in greater detail. At present we have approximately 50 promising compounds after screening embryonic stem cells and human embryonic neural stem cells. These have been counter screened on fibroblasts, glioma cells and neural stem cells. We have furthermore, conducted cell cycle profile analysis and analyzed BrdU incorporation for all these compounds. Based on these experiments we have selected one compound reducing and one increasing cell numbers. Dose response, effects on cell numbers over time, efficacy in different lines of ESCs and much more detailed experiments addressing effects and function has been conducted and concluded, and are presently being compiled into two manuscripts which should be submitted.
efforts were made (Actar) in setting up a voltage sensor probe (VSP) assay for studying GABAA in HEK293 cells and, later, stem cells. Difficulties in expressing the different subunits of the GABAA receptor in the correct stoichiometry in a relevant cell type and, also, questions about the ion channel being a suitable drug target for selectivity reasons made us decide on not to continue with the project and, thus, reallocate the work in WP1 and also, spend more time in WP4, where more effort was desirable. As for WP1, a new possible treatment for glioblastoma by using psychopharmaca was investigated. Glioblastoma is a highly aggressive malignant brain tumor with poor treatment options. A novel approach is the use of conventional anti-cancer treatments in combination with other therapies, such as psychopharmaca (Nordenberg et al., Int J Oncol. 2010, Oct;37(4):1043-51, In vitro novel combinations of psychotropics and anti-cancer modalities in U87 human glioblastoma cells.) Sertindole is an antipsychotic drug with a balanced inhibiting effect on dopamine D2, serotonin 5HT2 and a1-adrenergic receptors as well as ion channels, important for cell proliferation. Glioma stem cells were exposed to Sertindole and viability was measured after 48 hours. A dose response cytotoxic effect was seen in the glioma cells, which were more sensitive to the drug than differentiated cells. When investigating the cells morphologically, it was discovered that Sertindole causes volume changes in the treated glioma cells. In vivo xenograft experiments were performed in zebra fish and mouse, showing that Sertindole was cytotoxic to the glioma in the brain of zebrafish, with no negative effect on the viability of the fish. The mouse study could not be completed due to the severity of the tumours, which killed the mice.
1.3 Mechanisms of enhancing the endogenous stem cell pool size and self-repair by GABAA receptor signalling interference
despite having the potential for massive self-renewal, neural stem cells (NSCs) undergo limited cell division in vivo and it is unclear how stem cell pool size and cellular output in the adult brain is regulated. In this task the objective was to examine if H2AX signaling mechanisms operate in the adult brain and restrict self-renewal of endogenous stem cells. We find that the recruitment of the PI3 kinase related kinase (PIKK) signaling pathway and histone H2AX phosphorylation following GABAA receptor activation limits subventricular zone (SVZ) proliferation. Consequently, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions by pharmacological modulation or by genetically targeting H2AX activation. Unexpectedly, we can conclude from these studies that changes in self-renewal have long-lasting consequences on stem cell numbers, niche size and neuronal output. These results establish a mechanism that continuously limits self-renewal and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC self-renewal may contribute to the limited self-repair capacity of the damaged brain. This task is completed.
1.4 Molecular Mechanism by which Bex1 regulates stem cell self-renewal
Bex1 has been identified as an interactor of the intracellular domain of p75NTR, the low affinity neurotrophin receptor (Vilar, et al., EMBO Journal, 2006). It is a member of a family of 5 proteins in mice and rats (Bex1, 2, 3, 4, and 6) of which Bex1, 2, 4 and 6 show high sequence homology at the protein level. Vilar, et al., showed that overexpression of Bex1 inhibited neuronal differentiation in PC12 cells as well as in neural progenitor cells isolated from the rat postnatal subventricular zone (SVZ). Starting from these results we (Ibanez) decided to further investigate the role of Bex1 in neuronal progenitor cells in vivo and to elucidate the molecular mechanism of Bex1 action in these cells.
1.4.1 To elucidate cellular and molecular mechanisms of Bex1 function in mitotically active cells, with emphasis on neural stem cells and neuronal precursors
to be able to investigate the cellular and molecular mechanisms of Bex1function in mitotically active cells, two tools have been generated: I) a neurosphere culture system has been established in the lab; II) a retroviral expression system has been set up.
in order to modify Bex1 levels specifically in mitotically active cells, retroviral vectors derived from Moloney murine leukemia virus have been generated. The cDNAs of wildtype as well as of mutant Bex1 fused to GFP (Bex1?NLS) have been cloned into the viral backbone under the control of the CMV promoter to achieve a strong overexpression. To knockdown Bex1 in these cells, the shRNA sequence used by Vilar et al. has been modified to fit the mouse sequence and has been cloned into the retroviral backbone under the control of the U6 promoter.
A lack of effect of Bex1 overexpression on proliferation of neuronal precursor cells was further confirmed by BrdU incorporation experiments: We investigated BrdU incorporation after 1 and 2 days of growth factor withdrawal (to induce differentiation). In both conditions Bex1 did not influence the proliferation rate of precursor cells.
1.4.2 To elucidate the physiological relevance of Bex1 function for neuronal stem cell renewal and differentiation in vivo
we had planned to investigate the role of Bex1 on stem cell renewal and differentiation in the SVZ of young postnatal mice. Throughout life, the SVZ continues to produce neuronal progenitor cells that subsequently migrate via the RMS into the olfactory bulb. These precursors start to differentiate during their migration, and eventually integrate into the mature neuronal network of the olfactory bulb. Since the numbers of progenitors generated in this brain area is extremely high, and since Bex1 is expressed within the olfactory bulb (Koo et al., The Journal of Comparative Neurology, 2005), this is an ideal model system to study Bex1 function on proliferation, differentiation and survival.
1.4.3 To investigate the possibility of enhancing the size of endogenous stem cell pools by modulating Bex1 stability
in preliminary experiments a system has been established in the lab that will allow us to determine the formation of neurospheres from single cells. By plating virus-infected cells at clonal density we can assess the rate of sphere formation from modified cells as well as from control cells. In addition we will measure the size of the generated spheres and thus indirectly measure proliferative capacity of individual cells.
publications:
adameyko I, Lallemend F, Aquino JB, Pereira JA, Topilko P, Muller T, Fritz N, Beljajeva A, Mochii M, Liste I, Usoskin D, Suter U, Birchmeier C, Ernfors P. Cell. 2009. Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin. Cell, 139(2):366-79.
aquino JB, Lallemend F, Marmigere F, Adameyko II, Golemis EA, Ernfors P. 2009. The retinoic acid inducible Cas-family signaling protein Nedd9 regulates neural crest cell migration by modulating adhesion and actin dynamics. Neuroscience. 15;162(4):1106-19.
staaf S, Oerther S, Lucas G, Mattsson JP, Ernfors P. 2009. Differential regulation of TRP channels in a rat model of neuropathic pain. Pain 144(1-2):187-199.
usoskin D, Zilberter M, Linnarsson S, Hjerling-Leffler J, Uhlén P, Harkany T, Ernfors P. 2010 En masse in vitro functional profiling of the axonal mechanosensitivity of sensory neurons. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16336-41.
ernfors, P. 2010. Cellular origin and developmental mechanisms during the formation of skin melanocytes. Exp Cell Res. 1;316(8):1397-407.
staaf S, Franck MC, Marmigere F, Mattsson JP, Ernfors P. 2010. Dynamic expression of the TRPM subgroup of ion channels in developing mouse sensory neurons. Gene Expr Patterns. 10(1):65-74.
fernando R.N. Eleuteri B., Abdelhady1 S., Nussenzweig, A., Andang M., and Patrik Ernfors (2011). Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc. Natl. Acad. Sci. USA. 108(14):5837-5842.
bachy, I., Ernfors, P (2011). The transcription factor Cux-2 marks the specification of an A-delta sublineage of TrkA sensory neurons. Dev Biol. 1;360(1):77-86.
franck, M., Stenqvist, A.m Li, L, Hao, J, Usoskin, D., Wiesenfeld-Hallin Z., Xu, X, and Ernfors, P (2010) Essential role of Ret for defining non-peptidergic nociceptor phenotypes and functions of the adult mouse. Eur. J. Neurosci. 33(8):1385-1400
abdo, H, Li, L., Lallemend, F., Bachy, I., Xu, X-J., Rice, F., Ernfors, P. (2011). Dependence on the transcription factor Shox2 for specification of sensory neurons conveying discriminative touch. Eur. J. Neurosci. 34(10):1529-1541.
adameyko I., Lallemend, F., Furlan, A., Zinin, N., Srinivas Kitambi, S., Blanchart, A., Favaro, R., Nicolis, S., Lübke, M., Muller, T., Birchmeier, C., Suter, U., Takahashi, Y. and Ernfors. P (2011). Sox2 and MITF cross regulatory interactions establish progenitor and melanocyte lineages in the cranial neural crest. Development 139(2):397-410.
kitambi SS, Nilsson ES, Sekyrova P, Ibarra C, Nyah Tekeoh G, Andang M, Ernfors P, Uhlen P. (2012). Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart. BMC Physiol. 26;12(1):3.
lallemend, F., Adameyko I., Furlan, A., and Ernfors. P (2012). Positional differences of axon growth rates between sensory neurons encoded by Runx3. EMBO J. 2012 Aug 17.
aranda, S., Rutishauser, D. and Ernfors, P (2012). Distinct protein interaction networks associated with newly replicated DNA in embryonic stem cells. Submitted.
WP2: Protecting neurons
more than 20 neurotrophic factors (NTFs) can protect dopamine (DA) neurons in animals models of Parkinson's disease (PD) (Bespalov and Saarma, 2007; Lindholm and Saarma, 2010). However, only GDNF, NRTN that are also tested on phase 2 clinical trials for PD (Gill et al., 2003; Andressoo and Saarma, 2008) and two recently discovered NTFs, cerebral dopamine neurotrophic factor, CDNF (Lindholm et al., 2007) and mesencephalic astrocyte-derived neurotrophic factor, MANF (Petrova et al; Molec. Neurosci., 2003; Voutilainen et al, 2009) can also repair substantia nigra (SN) DA neurons. A major focus of this WP was to thoroughly assess the therapeutic potential of CDNF and MANF in animal models of PD, generate knockout mice to understand aspects of the physiology and of these NTFs that could be clinically relevant, and identify the CDNF receptor as thus could represent a potential drug target for PD.
2.1 Neuroprotective effects of CDNF and MANF in mouse MPTP and rat 6-OHDA PD models
we (Saarma) discovered a novel evolutionarily conserved neurotrophic factor, CDNF and found that in rats, CDNF protects and repairs DA neurons in the 6-hydroxydopamine (6-OHDA)-induced unilateral rat model of PD (Lindholm et al., 2007). We then found that intrastriatally injected MANF (Voutilainen et al., 2009) protects nigrostriatal DA nerves from 6-OHDA induced degeneration in rats. Most importantly, MANF also restored the function of nigrostriatal DA system when administered three weeks after 6-OHDA in the striatum (Voutilainen et al., 2009). Modest therapeutic effects of GDNF and NRTN are partially caused by their poor diffusion in the brain. We therefore compared MANF and GDNF diffusion in rat brain and found that MANF is distributed throughout the striatum more readily than GDNF. We also found that the mechanism of MANF action may differ from that of GDNF and CDNF since intrastriatally injected 125I-MANF was transported to the frontal cortex whereas 125I-GDNF and 125I-CDNF were transported to SN. Taken together these results suggested that in addition to CDNF also MANF has significant therapeutic potential for the treatment of PD (Voutilainen et al., 2009). We then decided to carefully compare the therapeutic effects of CDNF with MANF and GDNF using a severe 6-OHDA lesion model in rats. Two weeks after 6-OHDA lesion GDNF, CDNF and MANF were continuously infused into rat striatum with Alzet minipumps at three different concentrations: 1.5 νg, 3.0 νg and 4.5 νg in 24-h during two weeks. The results of this study clearly demonstrated that CDNF at all three concentrations tested was able to regenerate the axons of DA neurons. Moreover at 1.5 νg/24-h and 3.0 νg /24-h CDNF also protected DA neuron cell bodies (Voutilainen et al., 2011). Importantly, MANF tested at the same protein concentrations and GDNF tested at optimal 3.0 νg/24-h concentration showed no statistically significant neurorestorative effect in this severe PD model (Voutilainen et al., 2011). The volume of distribution for MANF in striatum was larger than that of CDNF and significantly larger than that of GDNF. We also treated naïve rats with CDNF and MANF and found no behavioral signs of toxicity. This potentially very important result indicates that CDNF and MANF may have very small side effects when used in PD therapy. These results imply that CDNF is significantly more potent than MANF or GDNF for neurorestorative therapy of PD. Therefore in future therapeutic trials we concentrated on CDNF.
2.2 Neurorestorative effects of CDNF on MitoPark model of Parkinson's disease
we initially planned to use a new mouse model of PD that has been developed in the laboratory of Nilsson at Karolinska Institute. This model is based on conditional knockout mice (MitoPark mice) with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. At that time none of the genetic animal models of PD showed significant neurodegeneration and therefore MitoPark mice looked as an attractive alternative. Two important findings led us to the change of our plans. Firstly, Olson's lab in Stockholm found that GDNF can protect DA neurons in MitoPark model and therefore this model would not give us additional important information on CDNF efficacy compared to GDNF. Then the Kirik lab demonstrated that viral delivery of a-synuclein (α-syn) into rodent SN will induce very efficient neurodegeneration in nigral DA system (Ulusoy et al. Exp. Neurol., 2008). Interestingly, the Björklund lab in Lund found that viral vector-mediated delivery of GDNF was inefficient in preventing wild-type a-synuclein-induced loss of DA neurons and terminals. In addition, GDNF overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson's disease (Decressac et al., Brain, 2011).
2.3 Identification of receptors for CDNF and MANF
CDNF and MANF receptors and mechanisms of action are unknown. CDNF and MANF show neurorestorative effects in rodent (Lindholm et al. 2007; Voutilainen et al., 2009) and monkey models of PD when they are injected into brain parenchyma. However, in vitro, when we have applied CDNF or MANF into the culture medium of DA, sympathetic, sensory or cortical neurons that die due to absence of NTFs, we have not seen any survival promoting effect (Lindholm et al, 2007; Hellmann et al., 2011). This is totally different from the action of all known NTFs. Interestingly, also injection of CDNF and MANF into the brain of naïve rats and mice without pathological lesions cause no obvious changes in the brain (Voutilainen et al. 2011; Airavaara et al., 2009; 2011), whereas for example GDNF injection activates RET receptor, stimulating excessive neurite outgrowth and axonal sprouting. In collaboration with Dr. Krieglstein, Freiburg, we have recently found that CDNF and MANF can rescue DA neurons and the MN9D dopamine cell line in culture, but only after cells have been lesioned with staurosporine or 6-OHDA or ER stress triggered by thapsigargin (Zhou et al. in preparation). In addition, we have also shown that CDNF and MANF by the virtue of their N-terminal saposin-like domain can bind oxidized phospholipids (Lindholm and Saarma., 2010).
these exciting results point to the following possible modes of action of CDNF-MANF:
1) receptors of CDNF and MANF are lesion-induced;
2) Cellular lesion induces the release of additional subunit or cofactor (e.g. oxidized lipid) that activates MANF and CDNF.
2.4 Generation of CDNF and MANF knockout mice
to understand the physiological roles of these new neurotrophic factors in vivo, we generated conventional MANF- and CDNF-deficient mice. The severe phenotype in DmMANF mutant flies (Palgi et al., 2009) suggested that the phenotype of MANF-and CDNF mice might also be severe. Therefore in parallel we created conditional gene-deficient (cKO) mice where MANF or CDNF genes can be deleted in a time- and tissue specific manner. Our idea was that MANF and CDNF KO mice may be useful genetic models for PD.
drosophila mutants lacking MANF have dramatically reduced DA levels, partial loss of DA neurons and complete lack of DA neurites. Thus fly MANF is the lege artis target-field NTF for fly DA neurons. We hypothesized that in mice CDNF and MANF represent target-derived NTFs for midbrain DA neurons and wanted to test this hypothesis on the respective KO and cKO mice.
2.5 In vivo survival of SN neurons in genetic mouse models
dysfunctions in the signalling activity of neurotrophic factors represent one of the pathogenic events that trigger neurodegeneration in the brain. Ret, the receptor for glial cell line-derived neurotrophic factor (GDNF) is specifically required for long-term target innervations and cell survival of a significant fraction of substantia nigra pars compacta (SNpc) DA neurons. DJ-1/PARK7 deletion in mice, on the other hand, resulted in changes in dopamine signaling, but the effects on target innervation and cell survival were very subtle. To investigate the combined effects of Ret and DJ-1 deletions, Klein has generated double mutant mice lacking expression of Ret in midbrain dopaminergic neurons and DJ-1 in all cells of the body (DAT-Cre:Retlx/lx;DJ1 null mice, in short Ret;DJ-1 mutant mice). At 24 month of age Ret;DJ-1 mutant mice suffer from enhanced loss (40%) of dopaminergic neurons compared to Ret single mutant (25%) and DJ-1 single mutant mice (less than 5%, not significant). These results indicate that DJ-1 is required for survival of midbrain dopaminergic neurons which suffer from partial trophic deprivation. In contrast, Ret;DJ-1 mutant mice show no additional loss of target innervation (40% loss in Ret;DJ-1 mutant and in Ret single mutant; no loss in DJ-1 single mutant mice). These findings also suggest that DJ-1 plays no or a minor role in maintaining target innervation (Aron et al., 2010a; 2010b).
2.6 In vivo survival of cells in genetic Drosophila models
klein had proposed to study the cooperation of dRet and DJ-1 in Drosophila. Mutant flies overexpressing constitutive active forms of Ret (MEN2A and MEN2B) in the eye, display a rough eye phenotype due to excessive proliferation, increased cell size, patterning defects and apoptosis. Analysis of sections of adult eyes revealed that the remaining ommatidia were increased in size. The stereotyped regular organization of ommatidia seen in control eyes is absent in dRetMEN2B-expressing eyes, which display random ommatidial rotations. Combined overexpression of RetMEN2B and haploinsufficiency for either DJ-1A or DJ-1B, or complete loss of DJ-1A rescued the rough eye phenotype of GMR-dRetMEN2B flies. Conversely, overexpression of both Ret and DJ-1 led to a more severe phenotype than the ones induced by active dRet alone. Thus, both DJ-1A/B interact genetically with overactive dRet in controlling cell size and differentiation in the developing fly retina.
2.7 In vitro survival of transfected cells carrying gain- or loss-of-function mutations
the biochemical interaction between Ret and DJ-1 in cells derived from knockout mice turned out to be very difficult. Much of the published work on DJ-1 as a modulator of PI3K/Akt signaling could not be reproduced by Klein. Hence, these studies were stopped and more emphasis was put on the fly and mouse work. We have, however, extended our studies towards the interaction of Ret and Pink1/Parkin. Mutations in PINK1 and Parkin are associated with autosomal recessive Parkinson's disease. Pink1 and park mutant Drosophila display phenotypes related to impaired mitochondrial function, including degeneration of indirect flight muscles and the respective Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking.
publications:
airaksinen, M.S. and Saarma, M. (2002). GDNF family neurotrophic factors: receptor mechanisms, biological functions and therapeutic utility. Nature Rev. Neurosci. 3: 383-394.
airavaara M, Shen H, Kuo CC, Peränen J, Saarma M, Hoffer B, Wang Y. (2009) Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J Comp Neurol. 515 (1): 116-124.
airavaara M, Harvey BK, Voutilainen MK, Shen H, Chou J, Lindholm P, Lindahl M, Tuominen RK, Saarma M, Wang Y, Hoffer B. CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice. (2011). Cell Transplantation, in press.
andressoo, J.-O. and Saarma, M. (2008) Signalling mechanisms underlying development and maintenance of dopamine neurons. Curr Opin Neurobiol., 18, 297-306.
aron L, Klein P, Pham TT, Kramer ER, Wurst W, Klein R. (2010a). Pro-survival role for Parkinsons associated gene DJ-1 revealed in trophically impaired dopaminergic neurons. PLoS Biology 8(4): e1000349. doi:10.1371/journal.pbio.1000349
aron, L. and Klein, R. (2010b). Repairing the Parkinsonian brain with neurotrophic factors. Review. Trends in Neurosciences, 2011 Feb;34(2):88-100. Epub 2010 Dec 7.
bespalov, M., Saarma, M. (2007) GDNF receptor complex is an emerging drug target. Trends in Pharmacological Sciences 28 (2), 68-74.
griciuc, A., Aron, L., Roux, M.J. Klein, R. Giangrande, A., and Ueffing M. (2010). Genetic inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila. PLoS Genetics, Aug 26;6(8). Pii: e1001075.
hellman M, Peranen J, Saarma M, and Permi P. (2010) H-1, C-13 and N-15 resonance assignments of the human mesencephalic astrocyte-derived neurotrophic factor. Biomelecular NMR Assignments. 4 (2): 215-217.
hellman M, Arumae U, Yu L-y, Lindholm P, Peranen J, Saarma M. and Permi P.. Neurotrophic factor MANF has a unique mechanism to rescue apoptotic neurons. (2011) J Biol Chem, 286(4):2675-80.(equal contribution).
lindholm, P., Voutilainen, M .H., Laurén, J., Peranen, J., Leppanen, V-M., Andressoo, J.-O. Lindahl, M., Janhunen, S., Kalkkinen, N., Timmusk, T., Tuominen, R. K. and Saarma, M. (2007) Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature, 448, 73-77.
lindholm, P., Peranen, J., Andressoo, J.O. Kalkkinen, N., Kokaia, Z., Lindvall, O., Timmusk, T. and Saarma M. (2008) MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain. Mol Cell Neurosci., 39(3), 356-371.
lindholm P, Saarma M.(2010) Novel CDNF/MANF family of neurotrophic factors. Dev Neurobiol. 70(5): 360-371.
palgi M, Lindström R, Peranen J, Piepponen TP, Saarma M, Heino TI. (2009) Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons. Proc Natl Acad Sci U S A. 106 (7): 2429-2434.
parkash, V., Lindholm, P., Peranen, J., Kalkkinen, N., Oksanen, E., Saarma, M., Leppanen, V-M. and Goldman, A. (2009) The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng.Des. Sel., 22(4): 233-241.
peng, C., Aron, L., Klein, R., Li, M., Wurst, W., Prakash, N., and Le, W. (2011). Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons. J. Neuroscience, Sep 7;31(36):12802-12815.
voutilainen, M.H. Back, S., Porsti, E., Toppinen, L., Lindgren, L., Lindholm, P., Peranen, J., Saarma, M. and Tuominen, R. K. (2009) Neurotrophic factor MANF is neurorestorative in rat model of Parkinson's disease. J. Neurosci., 29(30): 9651-9659. (Saarma, M Corresponding author.)
voutilainen MH, Bäck S, Peranen J, Lindholm P, Raasmaja A, Mannisto PT, Saarma M, Tuominen RK. Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease. (2011) Exp Neurol. 228(1): 99-108.
WP3: Restoring neural processes
we had proposed to study in various experimental paradigms the axonal projections of midbrain DA neurons to the striatum, the size and complexity of the dendritic arbors in these neurons in the intact substantia nigra and assess the growth of neurites from these neurons in dissociated cultures.
3.1 Maintenance and restoration of axonal projections in genetic mouse models
klein investigated the potential synergism between Ret and DJ-1 in maintenance of axonal projections. Axonal projections of midbrain DA neurons to the striatum of DA-specific Ret;DJ-1 double mutant mice (DAT-Cre:Retlx/lx;DJ1 null mice) were quantified by anti-TH immunohistochemistry in sections passing through the striatum compared to age-matched control mice. It was found that aging DA-specific Ret;DJ-1 double mutant mice did not have accelerated loss of SN axons compared to mice that only lack Ret signalling, indicating that DJ-1is not required to maintain target innervation (Aron et al., 2010).
3.2 Lesion models
see WP2.3 for studies with CDNF and MANF.
3.3 The role of intrinsic factors in axonal and dendritic growth
ernfors addressed the role of Cux2 in axon and dendritic growth during development and first characterised its expression in the sensory lineage where it is abundantly expressed. Cux2 was found to be expressed in subpopulations of the three main neuronal sublineages as defined by the expression of neurotrophic factor receptors TrkA, TrkB and TrkC. In particular, it marked a subpopulation of nociceptive TrkA+ neurons that is complete in numbers already at E10.5 and arised during the first, Ngn2-dependent, wave of. These early TrkA+/Cux2+ neurons persist after birth and at this time represent a specific subtype of A-delta nociceptors as seen by expression of TrkA and NF200 but absence of TrpV1. Analysis of Cux2 null mutant (Cux2-/-) mice and gain of function experiments in chicken show that Cux2 is sufficient but not required for specification of Trk+ neuronal subpopulations. Interestingly, Cux2-/- mice are hypersensitive to mechanical, but not to heat or cold, stimuli supporting a requirement in the process of specification of A-delta sublineage of TrkA+ sensory neurons. Cux null mutant mice did not display any deficits of axon and dendritic growth. Hence, our results show that Cux2 is expressed and participates in development of a previously not identified early population of myelinated TrkA+ nociceptors but appears not to constitute an intrinsic determinant regulating axon growth.
3.4 Elaboration, maintenance and restoration of DA neuron dendritic arbors
3.4.1 Influence of CDNF and MANF on dendritic size
saarma has carefully studied the effects of CDNF and MANF on the axonal growth. We have documented the effects of CDNF after single injection of the protein in earlier work (Lindolm et al., 2007) and in continuous infusion of CDNF more recently (Voutilainen et al 2011). Saarma has also shown that MANF either after single injection (Voutilainenn et al., 2009) or after continuous infusion can stimulate the growth and regeneration of the dopaminergic axons. We have not yet studied the effects of CDNF and MANF on the dendritic size mostly because we got several unexpected results which required all the available manpower. However, studies on the effects of CDNF and MANF on dendritic size will be performed soon.
3.4.2 Influence of GDNF and Ret signalling on dendritic size
klein and Davies had planned to study the influence of GDNF on midbrain DA neuron dendritic arbors in organotypic slice cultures from adult and aging wild-type and mutant mice. These studies turned out to be difficult due to the poor survival of DA neurons in these cultures. The work was therefore put on hold to provide more manpower to other projects such as described in WP2.
3.4.3 Role of DJ-1 in dendritic size
this was not done due to the same problem as described in 3.4.2.
3.4.4 Role of Wnt signalling in dendritic size
to begin to examine the contribution of Wnt signalling to the development of dendritic arborisation in medium spiny neurons in the striatum, we examined the Dvl1 mutant mouse. Dvl1 is a scaffold protein required for Wnt signalling. The Dvl1 mutant exhibits defects in dendritic development in cultured hippocampal neurons. We also examined the Wnt7a; Dvl1 double mutant mice for possible defects in dendritic development. Neither single Dvl1 mutant nor the double mutant exhibits detectable defects in the arborisation of medium spiny neurons based on Golgi staining of brain slices. These results suggest that Dvl1 and Wnt7a are not required for the proper dendritic development in the striatum. Further studies are required to examine the contribution of other Wnt factors expressed in the striatum.
3.4.5 The roles of the TNF/TNFR Superfamily in the developing ventral midbrain
classically, members of the tumour necrosis factor/tumour necrosis factor receptor (TNF/TNFR) superfamily have been implicated in the regulation of immune cell proliferation, survival and homeostasis. Recent published and unpublished findings from the Davies laboratory have demonstrated clear roles for several TNF/TNFR proteins in the regulation of peripheral neuronal development. While concerted research efforts over the past decade have successfully contributed to how mDA neurons are generated and the regulation of their phenotype, there is a current paucity of data on the molecular signals that regulate midbrain dopaminergic axonal specification, initiation and elongation during embryonic development. Understanding the cellular and molecular basis of how midbrain dopaminergic neurons project axons and form connections is of critical importance to the success of current therapeutic strategies for the successful treatment of Parkinson's disease.
to investigate if members of the TNF/TNFR superfamily are expressed in the ventral midbrain and target tissues (striatum), a comprehensive, developmental-based and MIQE-compliant RT-qPCR screen was conducted to assay for TNF/TNFR mRNA expression throughout development. Our screen identified several interesting expression patterns for ligands APRIL, TWEAK, BAFF, TWE-PRIL and receptors BCMA, BAFFR, TACI and Fn14 throughout the course of nigrostriatal pathway development. To date, no functions have been ascribed to these proteins during midbrain development. Further immunohistochemical validation of our qPCR data revealed the expression of APRIL and BCMA protein in developing mDA neurons of the ventral midbrain at E12, at a time when midbrain dopaminergic neurons have initiated axonogenesis and rostral axon extension towards their target fields. Further in vitro functional assays conducted using acutely dissociated cultures of embryonic midbrain dopaminergic neurons identified a novel APRIL-BCMA signalling loop that specifically modulates mDA axon elongation. This study is the first to highlight a function for APRIL-BCMA signalling in the developing nervous system, in selectively promoting the growth of mDA neuron axons during a defined window of embryonic development at E12 and E13, but not at E11 and E14. Furthermore, Davies made the first observation that the hybrid mRNA encoding TWE-PRIL is expressed outside of the immune system.
the axon-growth promoting effects of APRIL occur independently of neuronal survival, as evidenced by converging data demonstrating that APRIL neither promotes nor inhibits midbrain dopaminergic neuronal survival in VM cultures established from E12 wild type embryos and APRIL promotes axon outgrowth from mDA neurons in VM cultures established from E12 bax-/- embryos. Utilising function-blocking antibodies to BCMA and TACI, our data strongly suggests that APRIL promotes axonal elongation from mDA neurons by signalling through BCMA and not TACI. Furthermore, APRIL enhances axonal growth in a heparin-sulphate proteoglycan (HSPG)-dependent manner. APRIL promoted mDA axonal growth requires the activity of the canonical NF-kB pathway, as evidenced by the failure of APRIL to promote axon outgrowth from mDA neurons in VM cultures established from E12 p65-/- embryos. Interestingly, GDNF-promoted axon elongation growth is also compromised in these cultures, suggesting that both APRIL and GDNF may enhance axon elongation from mDA neurons through a common signalling pathway. To investigate the physiological relevance of these observations, we conducted studies on midbrain dopaminergic neurons using VM cultures established from E12 april-/- embryos. Consistent with in vitro observations of the effects of recombinant APRIL on mDA axonal growth, genetic ablation of APRIL impairs axonal growth in E12 midbrain dopaminergic neurons. This finding suggests that APRIL may exert its effects via an autocrine or paracrine signalling loop in vitro. However, this does not preclude a role for target-derived APRIL signalling as Davies has shown that APRIL and TWE-PRIL mRNAs are both robustly expressed in the developing striatum. Collectively, these findings indicate that APRIL is an axon growth-promoting factor for embryonic mDA neurons.
publications:
aron L, Klein P, Pham TT, Kramer ER, Wurst W, Klein R. (2010). Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons. PLoS Biology 8(4): e1000349. doi:10.1371/journal.pbio.1000349
dudanova, I., Gatto, G., and Klein, R. (2010). GDNF acts as a chemoattractant to support ephrinA-induced repulsion of limb motor axons. Current Biology 20, Issue 23, 7 December 2010, Pages 2150-2156.
dudanova, I., Kao, T.-J. Herrmann, J.E. Zheng, B., Kania, A., Klein, R. (2012). Genetic evidence for a contribution of EphA:ephrinA reverse signaling to motor axon guidance. J. Neuroscience 32(15):5209 -5215.
wang, L., Klein, R., Zheng, B., and Marquardt, T. (2011). Anatomical Coupling of Sensory and Motor Nerve Trajectory via Axon Tracking. Neuron 71(2):263-77.
WP4: Restoring connections
main achievements:
1)We have established an acute and organotypic brain culture system to examine the contribution of signaling molecules and dopamine depletion in the nigro-striatal system.
2)We demonstrated that several components of the Wnt signaling pathway are present in the adult striatum.
3)We have demonstrated that the Wnt antagonist Dkk1 induces synaptic disassembly in adult neurons.
4)We demonstrated that neutralizing antibodies against Dkk1 restores synaptic degeneration induced by Amyloid- β. These results indicate a key role for Dkk1 in the loss of synapses in neurodegenerative diseases. These results were published (Purro et al, J. Neuroscience, 2012)
5)We have generated a transgenic mouse model that inducibly expresses Dkk1 in the hippocampus and striatum.
6)We showed that expression of Dkk1 in the adult hippocampus results in the loss of synapses and in hippocampal mediated behavior (Lopes, Galli and Salinas, manuscript to be submitted in January 2013)
7)We showed that Wnt signaling is crucial for synaptic maintenance in the adult striatum.
8)We found that defects in synaptic maintenance due to impaired Wnt signaling affects normal striatal-dependent behavior, such as motor coordination and locomotion. (Galli and Salinas, manuscript to be submitted in March 2013)
9)Our preliminary results suggest that long-term defects in synaptic maintenance lead to dopaminergic cell death in the substantia nigra (SN).
4.1. Examining the effect of dopamine depletion on synapses of the nigrostriatal system.
salinas has established an ex-vivo system to examine the contribution of signaling molecules to the formation and maintenance of synapses in the nigro-striatal system. Firstly, we set up acute (4-6 hours) and long-term organotypic (up to 15 day in culture) brain slices cultures that contain the components of the striato-nigral pathway (i.e. striatum and SN). To obtain a significant number of intact connections between the substantia nigra to the striatum in our slices, we made sagittal sections from P15 mouse brains. In these cultures we were able to examine excitatory, inhibitory and dopaminergic synapses by immunofluorescence microscopy. We found that Wnt proteins such as Wnt7a, Wnt3a and Wnt5a distinctly affect the number of synapses in the striatum of acute brain slices. Conversely, we found that Wnt blockade with the Wnt antagonist Dickkopf-1 (Dkk1) significantly decreases the number of synapses in the striatum. These results demonstrate that Wnt signaling is required for the formation of different types of synapses in the striatum.
4.2. To examine the effect of neurotrophic factors in the formation and maintenance of synapses in the dopaminergic system of the basal ganglia
salinas examined the expression of core components of the Wnt signalling pathway in the postnatal striatum at the peak period of synapse formation (P15) and in the adult mice (3 months old), using RT-PCR. We found that several Wnts such as Wnt3, Wnt5a and Wnt7b and the receptors Frizzled (Fz) 3-9 are highly expressed in the adult striatum. Wnt7a, 9b, 10a and 11 and the co-receptors LRP5 and 6 are also expressed. In addition, the secreted Wnt antagonists, secreted Frizzled related proteins (Sfrp 1-3) and Dickkopf-3 (Dkk3) are highly expressed in the striatum. In contrast, Dkk2 and Dkk4 are weakly expressed whereas Dkk1 is not expressed under normal physiological conditions.
to elucidate the in vivo role of Wnt signalling in the maintenance of mature synapses, we have generated an inducible transgenic mouse line that expresses Dkk1 under the control of the tetracycline system. Mice expressing Dkk1 under the regulation of the Tet-O system were crossed with a transgenic line expressing CaMKII rtTA to obtain specific expression of Dkk1 in the hippocampus, striatum and part of the cortex upon induction with Doxycycline (Dox) (Mansuy et al., 1998). RT-PCR analyses of brains of double transgenic mice (TetO-Dkk1 with CaMKII rtTA, hereafter named Dkk1-TG) fed with Dox for 3-28 days revealed a significant increase in Dkk1 mRNA levels in the hippocampus and striatum when compared to Dkk1-TG mice not fed with Dox or to single transgenic mice (Galli et al, 2011).
in adult mice, blockade of Wnt signalling by Dkk1 decreases the number of excitatory and inhibitory synapses, as well as the number of dopamine receptors 1 (D1R) and 2 (D2R) puncta. Blockade of Wnt signalling decreases the number of docked and total vesicles containing the neurotransmitter vGlut1. However, no changes were observed in the number of dendritic spines and dopamine content in the striatum. Blockade of Wnt signalling does not induce cell death, which reinforces the role of Wnts directly in the maintenance of striatal synapses (Galli et al, 2011 and Galli et al, 2012). This defect in synaptic maintenance caused by blockade of Wnt signalling is associated with behavioural deficits such as defects in motor coordination and locomotion induced by psycho-stimulants (amphetamine-induced locomotion).
4.3. To test the synaptic protective function of neurotrophic factors during dopamine depletion in the nigrostriatal system
our preliminary data suggests that long-term but not short-term defects in the maintenance of synapses in the striatum, affects the viability of the dopamine neurons in the SN. We have evaluated the number of TH positive cells in the SN after induction of Dkk1 expression during 1 month and 8 months. We found that the number of TH positive cells is unchanged after 1-month induction. However, we observed a reduction in the number of TH positive cells in the SN after 8 months of Dkk1 expression. These results highlight the importance of synaptic maintenance in cell viability, and shed new light into the potential role for dysfunction of Wnt signaling in neurodegeneration in the striato-nigral system.
4.5. To screen for small molecules that will restore synaptic connectivity
our studies (Salinas) in cultured neurons, brain slices and in transgenic mice have demonstrated that blockade of Wnt signalling with the secreted Wnt antagonist Dkk1 induces synaptic disassembly in the adult brain. Moreover, we found that Dkk1 is downstream of Amyloid- β during synaptic degeneration. These results demonstrate that Dkk1 is a potential therapeutic target for the treatment of neurodegenerative diseases where synapses are compromised. We (Salinas) therefore started a collaborative project (with Actar) to screen for small compounds that block the binding of Dkk1 to its receptor. HEK293 cells expressing a SuperTOPflash construct were transferred from Salinas to Actar. The generation of the cells is described in detail in (Nathans et al., Cell, Vol. 116, 883-895, March 19, 2004, Vascular Development in the Retina and Inner Ear: Control by Norrin and Frizzled-4,a High-Affinity Ligand-Receptor Pair). The SuperTop Flash reporter drives firefly luciferase by 7 LEF/TCF consensus binding sites, to which β -catenin is recruited upon active canonical Wnt-signalling in the cell, thus a measure of intracellular Wnt-activity. A functional Wnt-activity assay was developed by Actar in which small molecules from Actar's screening library of approximately 30.000 compounds were tested. A subgroup of 1000 compounds from the library was selected due to chemical properties such as size, diversity and likelihood to penetrate CNS. From these, 700 compounds were screened in the luciferase reporter cell assay at one concentration. Compounds, which showed a DKK-1 selective pattern of Wnt-signalling, were object to further selection due to druggability and potency in the cell assay and picked for more extensive testing, i.e. dose response in the cell assay and DKK-1 selectivity in binding assays. A DKK-1 binding assay was developed for testing of small molecules in the Corning Epic System, which is a label-free screening platform based on optical biosensor technology. Also, a LRP6/DKK-1 interaction assay was developed, aiming to detect small molecules that interfere with the interaction between DKK-1 and LRP6, one part of a receptor complex to which Wnts and DKK-1 may bind. Confirmation of hits from cell assay by binding assays was not manageable within the project time frame. Confirmed compounds will be examined for their efficacy in cultured neurons and brain slices for their ability to block Dkk1 function and therefore protect synapses.
in addition to our work in the striatum, Salinas has also examined synaptic degeneration in the hippocampus:
1) The Wnt antagonist Dkk1 induces synaptic disassembly in adult neurons. Salinas has previously demonstrated that Wnt signalling plays a role in the formation, growth and function of synapses in the central nervous system. However, Wnts and their signalling components are not only expressed during synaptogenesis but also in the adult. We decided to investigate whether Wnts are also required in the maintenance of synapses in mature neurons. As hippocampal neurons express several Wnts, we decided to use the Wnt antagonist Dkk1 to test the role of Wnts in synaptic maintenance. We found that Dkk1 induces the rapid disassembly of synapses in mature neurons in cultures (Purro et al, J. Neuroscience 2012).
2) Neutralizing antibodies against Dkk1 restores synaptic degeneration induced by Amyloid-β.
several studies suggest a potential link between Dkk1 expression and neurodegenerative diseases such as Alzheimer's disease (AD). Although Dkk1 levels are almost undetected in the adult normal brain, they are elevated in AD patients. Moreover, Dkk1 is also elevated in animal models of AD that over-express mutant Amyloid-β (Aβ). We therefore tested the effect of Amyloid-β on Dkk1 and found that indeed Aβ significantly increases the levels of Dkk1 in brain slices at the time when synapses are lost. Importantly we found that neutralizing antibodies against Dkk1 completely block the effect of Aβ on synapses (Purro et al, J. Neuroscience 2012). These findings highlight the importance of Dkk1 in synaptic degeneration and more importantly they identify Dkk1 as a possible target for the treatment of AD. This work has been highly cited in the media (commentaries in news websites, interview by BBC World Service), highlighted by UCL Media and funding organizations and reviewed twice by Faculty of 1000.
3) We (Salinas) have generated a transgenic mouse model that inducible expresses Dkk1 in the hippocampus and striatum. To begin to elucidate the in vivo role of Wnt signalling at hippocampal synapses, we have used the Dkk1-TG mice. Induction of Dkk1 expression in adult mice for a period of 1-2 weeks does not affect cell viability (based on TUNEL assay and Caspase-3 staining), nor affect the progenitor cell niche (Doublecortin staining) (Lopes et al, 2012). Thus, deficiency in Wnt signalling does not affect cell viability or stem cell number.
4) Expression of Dkk1 in the adult hippocampus results in the loss of synapses and in hippocampal mediated behavior (Lopes, Galli, Cacucci and Salinas, manuscript to be submitted in January 2013). Given the role of Wnt-signalling in excitatory synapses (Ciani et al., 2011) and its newly described role in synaptic maintenance (Purro et al., 2012) we next examined the possible defects in hippocampal dendritic spines in our Wnt signalling deficient animal model. Golgi staining revealed that 2 weeks of continuous expression of Dkk1 in the hippocampus lead to almost 20% reduction in spine size and about 10% decrease in the spine number of CA1 cells. In contrast, no changes were observed in CA3 cells.
consistent with the loss of spines, we found a significant decrease in the number of excitatory synapses (colocalization of pre- and postsynaptic markers) but inhibitory synapses are unaffected. These results suggest that blockade of Wnt signaling with Dkk1 in the adult hippocampus specifically induce the disassembly of excitatory synapses (Lopes et al, 2012).
overall conclusion: Our studies (Salinas) in the hippocampus and striatum demonstrate the Wnt signaling is required for the maintenance of synapses in the adult brain and that pathogenic molecules such as Amyloid-β triggers the expression of the Wnt antagonist Dkk1 to induce synapse loss. Our studies in the striatum strongly suggest that Wnts or activation of the Wnt pathway could protect synapses against synaptic degeneration.
publications:
ciani, L., Boyle, K. A., Dickins, E., Sahores, M., Anane, D., Lopes, D. M., Gibb, A. J. and Salinas, P. C. (2011) Wnt7a signaling promotes dendritic spine growth and synaptic strength through CaMKII, Proc Natl Acad Sci U S A 108(26): 10732-7.
mansuy, I. M., Winder, D. G., Moallem, T. M., Osman, M., Mayford, M., Hawkins, R. D. and Kandel, E. R. (1998) 'Inducible and reversible gene expression with the rtTA system for the study of memory', Neuron 21(2): 257-65.
purro S. A., Dickins E., Salinas P.C. (2012) The Wnt antagonist Dkk1 mediates the synaptic disassembly activity of Aβ: a potential role for Wnt synaptic dysfunction in Alzheimer's Diseases. J. Neuroscience. 32: 3492-3498.
galli S, Millar SE, Salinas P.C. (2011) Nanosymposium: Wnt signalling controls synaptic maintenance in the striatum. Society for Neuroscience Annual Meeting, 12-16 November, Washington Convention Center, Washington DC, USA. (Talk)
galli S, Lopes D, Millar S, Salinas P.C. (2012) Wnt signalling controls synaptic maintenance in the striatum: potential implications in Parkinson's Disease. Society for Neuroscience Annual Meeting. 13-17 October. New Orleans Convention Center. New Orleans, USA.
lopes D.M. Galli S., Millar S., Cacucci F. and Salinas P.C. (2012) Synaptic maintenance mediated by Wnt signalling modulates learning and memory. Society for Neuroscience Annual Meeting. 13-17 October. New Orleans Convention Center. New Orleans, USA.
potential Impact:
the potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
parkinson's disease is one of the most common neurodegenerative disorders with a prevalence of approximately 1% in the population over the age of 65. Patients suffer from impaired motor function characterised by rhythmic tremor, inability to initiate and complete routine movement, muscle rigidity, postural instability and paucity of facial expression. These symptoms are caused by the degeneration and loss of the dopaminergic neurons of the substantia nigra pars compacta that project to the striatum and play a key role in controlling the extrapyramidal motor system.
the mainstay of Parkinson's disease treatment, drugs that restore dopamine levels, is of limited long-term benefit as it does not prevent disease progression. Moreover, because other midbrain dopaminergic systems play key roles in emotional balance and reward (mesolimbic pathway) and motivation, attention, planning and social behaviour (mesocortical dopaminergic pathway), these drugs can cause imbalances in these systems with serious adverse effects. Unfortunately, there has been at best only limited success with a variety of other therapeutic approaches, including cell transplantation, growth factor therapy and selective stimulation by surgically implanted electrodes. New, much needed, therapeutic approaches for this devastating disease will only come from a better understanding of the generation, development and maintenance of dopaminergic neurons and their connections.
the research carried out by MOLPARK has made very significant advances in our understanding of the basic mechanisms that control neuron generation from stem cells, and molecules that sustain the survival of dopaminergic neurons and promote the growth and maintenance of their processes and connections. These major advances in our understanding are providing important new platforms for the development of novel therapies for Parkinsons disease and potentially other neurodegenerative diseases. In addition to the major advances in understanding cellular and molecular aspects of dopaminergic neuron development and function, important translational aspects of our research include the following:
(i). The establishment of screens for small molecule drug leads for regulating stem cell self-renewal.
(ii). We have demonstrated the efficacy of the novel neurotrophic factors CDNF and MANF in protecting midbrain dopaminergic neurons in rodent models of Parkinson's disease and in substantially countering the adverse consequences of impaired dopaminergic neuron function in these models. Importantly, treatment of naïve rats with CDNF and MANF revealed no behavioral signs of toxicity, implying that CDNF and MANF may have few side effects when used in Parkinson's disease therapy. As a result of this work, clinical trials are planned to assess the efficacy of CDNF in Parkinson's disease.
(iii). In addition to potential important translational outcomes of our research for the or Parkinson's disease therapy, our work on stem cell proliferation and differentiation has led to assessments of a promising novel therapeutic approach for glioblastoma, a highly aggressive malignant brain tumor with poor treatment options. This novel approach is based on the use of the antipsychotic drug Sertindole. We have shown that Sertindole exerts a cytotoxic effect on glioma stem cells in culture and is cytotoxic in vivo to the glioma cells grafted into the brain of zebrafish with no negative effect on the viability of the fish.
in summary, our work has not only made a very significant contribution to basic knowledge but has led to preclinical translational research that underpins the development of novel therapeutic approaches for Parkinson's disease and glioma. As such, the work of MOLARK may ultimately help alleviate suffering in the significant proportion of the population and lead to an important source of revenue for the European pharmaceutical industry should preclinical studies come to fruition with the development of therapies that can be taken through the clinic successfully.
dissemination activities
in the time period 2009-2012, the Molpark consortium published 36 original and review articles in peer-reviewed journals. Many publications appeared in the top cell and molecular biology and neuroscience journals such as Cell, Proc. Natl. Acad. Sci USA, EMBO Journal, Journal of Neuroscience, Development, Cell Reports, Neuron, PLoS Biology, PLoS Genetics, Current Biology, Journal of Biological Chemistry.
Project website: http://www.molpark.co.uk
MOLPARK has been exceptionally successful. We have achieved all deliverables and all but 13 of our 82 milestones and the work has led to 36 publications in leading journals. The main highlights of the MOLPARK program are as follows. A role for histone H2AX in stem cell self-renewal has been established, and we have shown in vivo that stem cell numbers in the adult brain can be manipulated by affecting this regulatory pathway. We have developed a new technology to identify proteins enriched in nascent chromatin during replication of stem cells and embryonic fibroblasts. Stem cells display a unique recruitment of complexes involved in high replication fidelity, epigenetic re-establishment and chromatin remodelling during replication. These results provide evidence for a selective enrichment of specific protein modules in newly synthesized DNA of stem cells that contribute to the preservation of genomic integrity and the epigenome during replication, features that define the unique chromatin organization which underlie stemness. Several tools to investigate the role of Bex1 in stem cell proliferation and differentiation have been developed and conditions have been developed for the use of Bex1 reagents to influence neural stem cell pools.
project Context and Objectives:
summary description of project context and objectives
the goal of MOLPARK was to make major advances in our understanding of the basic cellular and molecular mechanisms underlying the generation and survival of nigrostriatal dopaminergic neurons and the establishment and maintenance of their connections in order to provide a platform for radically new, much needed therapeutic approaches for Parkinson's disease.
to achieve this goal, our plan was to undertake an integrated programme of research focused on understanding the basic cellular and molecular mechanisms that control the generation and survival of dopaminergic nigrostriatal neurons and the establishment and maintenance of their connections with the aim of discovering novel therapeutic approaches for Parkinson's Disease based on replenishing and protecting dopaminergic nigrostriatal neurons and restorating their processes and connections.
main objectives
1. To define the mechanisms important for stem cell self-renewal, differentiation and integration into the midbrain and attempt to exploit these mechanisms to induce or boost existing self-repair processes with the aim of replenishing neurons in Parkinson's disease.
2. To define the mechanisms important for sustaining the survival of dopaminergic nigrostriatal neurons in health and disease with the aim of developing new, effective growth factor-based therapies aimed at protecting neurons in Parkinson's disease.
3. To define the mechanisms that promote the growth of dopaminergic nigrostriatal axons and dendrites with the aim of identifying potential therapeutic strategies based on restoring neural processes in Parkinson's disease.
4. To define the mechanisms that promote and sustain the synaptic connections of dopaminergic nigrostriatal neurons with the aim of restoring connections in Parkinson's disease.
specific aims underpinning these objectives
1. To define the mechanisms important for stem cell self-renewal, differentiation and integration in the midbrain.
here our plan was to focus mostly on the roles of GABA and a small adaptor-like protein called Bex1. Specifically, we planned to investigate the control of stem cell proliferation by GABA by pharmacologically manipulating GABAA receptor function. We planned to investigate the role of Bex1 in regulating stem cell self-renewal and differentiation by manipulating Bex1 expression in cultured cells and in transgenic mice. We wished also to screen for small molecules capable of regulating stem cell self-renewal and screen for small molecules capable of stabilizing fluorescently tagged Bex1. We planned to further investigate validated hits by investigating their actions in stem cell proliferation, survival and differentiation in in vitro assays and subsequently in in vivo paradigms of neural stem cell renewal.
2. To define novel mechanisms for sustaining the survival of DA nigrostriatal neurons in health and disease.
here our plan was to focus on two recently discovered, structurally related neurotrophic factors, CDNF and MANF, and also carry out studies on Ret and DJ-1. CDNF had been shown to protect dopaminergic neurons in a rat 6-OHDA model of Parkinson's disease. We planned to test the efficacy of CDNF in additional models of Parkinson's disease, and test MANF in several Parkinson's disease models. We also planned to generate CDNF and MANF knockout mice and study the dopaminergic nigrostriatal neuron phenotype of these mice. We wished also to identify receptors for these neurotrophic factors. We also planned using several genetic models in mice and Drosophila to further understanding of the role of the Ret receptor tyrosine kinase and DJ-1 (a regulator of PKB/Akt phosphorylation that is associated with autosomal recessive early-onset Parkinson's disease). We also planned to complement these in vivo genetic studies of Ret and DJ-1 with in vitro gain- or loss-of- function mutations of these proteins in cell lines.
3. To define the mechanisms that promote the growth of dopaminergic nigrostriatal axons and dendrites
here our plan was to study the dendritic morphology and axon projections of dopaminergic nigrostriatal neurons in several genetic mouse models, including those with null and conditional mutations in the following genes: Ret, DJ-1, CDNF, MANF, Dvl1 and Wnts. We also planned to study the effects of GDNF, CDNF, MANF and selected members of the TNF superfamily (based on studies of TNF superfamily receptors in dopaminergic neurons) in vitro. We also wished to investigate the role of intrinsic factors in axonal and dendritic growth, including the Runx and Cux transcription factors and the extracellular calcium sensing receptor.
4. To define the mechanisms that promote and sustain the synaptic connections of dopaminergic nigrostriatal neurons.
here our plan was to characterize the effect of dopamine depletion on synapses of the nigrostriatal system. We wished also to examine the effect of selected neurotrophic factors (GDNF, CDNF and MANF) in the maintenance and distribution of synapses in the nigrostriatal system, and planned to test the protective effects of neurotrophic factors on synapses in lesion and transgenic models for Parkinson's disease. We also planned to examine the effect of neurotrophic factors on the release of dopamine in the nigrostriatal system. Finally, we planned to screen for small molecules to restore synaptic connectivity.
project Results:
WP1: Replenishing neurons
1.1 Mechanisms of stem cell proliferation by GABA and histone modification
this task has progressed well. We have shown that the MRN complex and ATM associate with activated H2AX are necessary for ligand induced changes in self-renewal of ES cells. Unexpectedly MDC1 and 53BP1 were shown to be dispensable. During the period of activity, two publications have appeared showing that during DNA damage, H2AX can be phosphorylated on Ser 139 and that this phosphorylation is paralleled by a de-phosphorylation of a newly discovered phospho site Tyr142. De-phosphorylation of Tyr 142 is necessary for recruitment of DNA repair proteins and failure of its dephosphorylation results instead in activation of a death pathway. Most interestingly, we have found that ligand activation of this pathway controlling stem cell self-renewal phosphorylation of both Ser139 and Tyr142 are increased. We found that Ser139 phosphorylation takes place in specific foci of the chromatin while Tyr142 occurs throughout the genome. Based on these findings we have initiated the generation of point mutant mice with amino acid substitutions in these phospho sites to examine its role in adult brain stem cell self-renewal. Analysis of these strains by following all newborn neurons using BrdU has revealed that neither of these phosphosites are critical for the role of H2AX on stem cell proliferation in vivo.
1.2 Screen for small molecules regulating stem cell self-renewal
after extensive evaluation of different strategies and assays we (Ernfors) have decided on an open cell based assay. These assays have now been established allowing for screening of self-renewal of embryonic stem cells, human foreskin fibroblasts, human embryonic neural stem cells and adult mouse neural stem cells. We have obtained the NIH diversity set II library and initiated screens in the above cells. Compounds that increase cell number in embryonic stem cells, human embryonic stem cells and adult mouse neural stem cells but not in fibroblasts will be analysed in greater detail. At present we have approximately 50 promising compounds after screening embryonic stem cells and human embryonic neural stem cells. These have been counter screened on fibroblasts, glioma cells and neural stem cells. We have furthermore, conducted cell cycle profile analysis and analyzed BrdU incorporation for all these compounds. Based on these experiments we have selected one compound reducing and one increasing cell numbers. Dose response, effects on cell numbers over time, efficacy in different lines of ESCs and much more detailed experiments addressing effects and function has been conducted and concluded, and are presently being compiled into two manuscripts which should be submitted.
efforts were made (Actar) in setting up a voltage sensor probe (VSP) assay for studying GABAA in HEK293 cells and, later, stem cells. Difficulties in expressing the different subunits of the GABAA receptor in the correct stoichiometry in a relevant cell type and, also, questions about the ion channel being a suitable drug target for selectivity reasons made us decide on not to continue with the project and, thus, reallocate the work in WP1 and also, spend more time in WP4, where more effort was desirable. As for WP1, a new possible treatment for glioblastoma by using psychopharmaca was investigated. Glioblastoma is a highly aggressive malignant brain tumor with poor treatment options. A novel approach is the use of conventional anti-cancer treatments in combination with other therapies, such as psychopharmaca (Nordenberg et al., Int J Oncol. 2010, Oct;37(4):1043-51, In vitro novel combinations of psychotropics and anti-cancer modalities in U87 human glioblastoma cells.) Sertindole is an antipsychotic drug with a balanced inhibiting effect on dopamine D2, serotonin 5HT2 and a1-adrenergic receptors as well as ion channels, important for cell proliferation. Glioma stem cells were exposed to Sertindole and viability was measured after 48 hours. A dose response cytotoxic effect was seen in the glioma cells, which were more sensitive to the drug than differentiated cells. When investigating the cells morphologically, it was discovered that Sertindole causes volume changes in the treated glioma cells. In vivo xenograft experiments were performed in zebra fish and mouse, showing that Sertindole was cytotoxic to the glioma in the brain of zebrafish, with no negative effect on the viability of the fish. The mouse study could not be completed due to the severity of the tumours, which killed the mice.
1.3 Mechanisms of enhancing the endogenous stem cell pool size and self-repair by GABAA receptor signalling interference
despite having the potential for massive self-renewal, neural stem cells (NSCs) undergo limited cell division in vivo and it is unclear how stem cell pool size and cellular output in the adult brain is regulated. In this task the objective was to examine if H2AX signaling mechanisms operate in the adult brain and restrict self-renewal of endogenous stem cells. We find that the recruitment of the PI3 kinase related kinase (PIKK) signaling pathway and histone H2AX phosphorylation following GABAA receptor activation limits subventricular zone (SVZ) proliferation. Consequently, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions by pharmacological modulation or by genetically targeting H2AX activation. Unexpectedly, we can conclude from these studies that changes in self-renewal have long-lasting consequences on stem cell numbers, niche size and neuronal output. These results establish a mechanism that continuously limits self-renewal and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC self-renewal may contribute to the limited self-repair capacity of the damaged brain. This task is completed.
1.4 Molecular Mechanism by which Bex1 regulates stem cell self-renewal
Bex1 has been identified as an interactor of the intracellular domain of p75NTR, the low affinity neurotrophin receptor (Vilar, et al., EMBO Journal, 2006). It is a member of a family of 5 proteins in mice and rats (Bex1, 2, 3, 4, and 6) of which Bex1, 2, 4 and 6 show high sequence homology at the protein level. Vilar, et al., showed that overexpression of Bex1 inhibited neuronal differentiation in PC12 cells as well as in neural progenitor cells isolated from the rat postnatal subventricular zone (SVZ). Starting from these results we (Ibanez) decided to further investigate the role of Bex1 in neuronal progenitor cells in vivo and to elucidate the molecular mechanism of Bex1 action in these cells.
1.4.1 To elucidate cellular and molecular mechanisms of Bex1 function in mitotically active cells, with emphasis on neural stem cells and neuronal precursors
to be able to investigate the cellular and molecular mechanisms of Bex1function in mitotically active cells, two tools have been generated: I) a neurosphere culture system has been established in the lab; II) a retroviral expression system has been set up.
in order to modify Bex1 levels specifically in mitotically active cells, retroviral vectors derived from Moloney murine leukemia virus have been generated. The cDNAs of wildtype as well as of mutant Bex1 fused to GFP (Bex1?NLS) have been cloned into the viral backbone under the control of the CMV promoter to achieve a strong overexpression. To knockdown Bex1 in these cells, the shRNA sequence used by Vilar et al. has been modified to fit the mouse sequence and has been cloned into the retroviral backbone under the control of the U6 promoter.
A lack of effect of Bex1 overexpression on proliferation of neuronal precursor cells was further confirmed by BrdU incorporation experiments: We investigated BrdU incorporation after 1 and 2 days of growth factor withdrawal (to induce differentiation). In both conditions Bex1 did not influence the proliferation rate of precursor cells.
1.4.2 To elucidate the physiological relevance of Bex1 function for neuronal stem cell renewal and differentiation in vivo
we had planned to investigate the role of Bex1 on stem cell renewal and differentiation in the SVZ of young postnatal mice. Throughout life, the SVZ continues to produce neuronal progenitor cells that subsequently migrate via the RMS into the olfactory bulb. These precursors start to differentiate during their migration, and eventually integrate into the mature neuronal network of the olfactory bulb. Since the numbers of progenitors generated in this brain area is extremely high, and since Bex1 is expressed within the olfactory bulb (Koo et al., The Journal of Comparative Neurology, 2005), this is an ideal model system to study Bex1 function on proliferation, differentiation and survival.
1.4.3 To investigate the possibility of enhancing the size of endogenous stem cell pools by modulating Bex1 stability
in preliminary experiments a system has been established in the lab that will allow us to determine the formation of neurospheres from single cells. By plating virus-infected cells at clonal density we can assess the rate of sphere formation from modified cells as well as from control cells. In addition we will measure the size of the generated spheres and thus indirectly measure proliferative capacity of individual cells.
publications:
adameyko I, Lallemend F, Aquino JB, Pereira JA, Topilko P, Muller T, Fritz N, Beljajeva A, Mochii M, Liste I, Usoskin D, Suter U, Birchmeier C, Ernfors P. Cell. 2009. Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin. Cell, 139(2):366-79.
aquino JB, Lallemend F, Marmigere F, Adameyko II, Golemis EA, Ernfors P. 2009. The retinoic acid inducible Cas-family signaling protein Nedd9 regulates neural crest cell migration by modulating adhesion and actin dynamics. Neuroscience. 15;162(4):1106-19.
staaf S, Oerther S, Lucas G, Mattsson JP, Ernfors P. 2009. Differential regulation of TRP channels in a rat model of neuropathic pain. Pain 144(1-2):187-199.
usoskin D, Zilberter M, Linnarsson S, Hjerling-Leffler J, Uhlén P, Harkany T, Ernfors P. 2010 En masse in vitro functional profiling of the axonal mechanosensitivity of sensory neurons. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16336-41.
ernfors, P. 2010. Cellular origin and developmental mechanisms during the formation of skin melanocytes. Exp Cell Res. 1;316(8):1397-407.
staaf S, Franck MC, Marmigere F, Mattsson JP, Ernfors P. 2010. Dynamic expression of the TRPM subgroup of ion channels in developing mouse sensory neurons. Gene Expr Patterns. 10(1):65-74.
fernando R.N. Eleuteri B., Abdelhady1 S., Nussenzweig, A., Andang M., and Patrik Ernfors (2011). Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc. Natl. Acad. Sci. USA. 108(14):5837-5842.
bachy, I., Ernfors, P (2011). The transcription factor Cux-2 marks the specification of an A-delta sublineage of TrkA sensory neurons. Dev Biol. 1;360(1):77-86.
franck, M., Stenqvist, A.m Li, L, Hao, J, Usoskin, D., Wiesenfeld-Hallin Z., Xu, X, and Ernfors, P (2010) Essential role of Ret for defining non-peptidergic nociceptor phenotypes and functions of the adult mouse. Eur. J. Neurosci. 33(8):1385-1400
abdo, H, Li, L., Lallemend, F., Bachy, I., Xu, X-J., Rice, F., Ernfors, P. (2011). Dependence on the transcription factor Shox2 for specification of sensory neurons conveying discriminative touch. Eur. J. Neurosci. 34(10):1529-1541.
adameyko I., Lallemend, F., Furlan, A., Zinin, N., Srinivas Kitambi, S., Blanchart, A., Favaro, R., Nicolis, S., Lübke, M., Muller, T., Birchmeier, C., Suter, U., Takahashi, Y. and Ernfors. P (2011). Sox2 and MITF cross regulatory interactions establish progenitor and melanocyte lineages in the cranial neural crest. Development 139(2):397-410.
kitambi SS, Nilsson ES, Sekyrova P, Ibarra C, Nyah Tekeoh G, Andang M, Ernfors P, Uhlen P. (2012). Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart. BMC Physiol. 26;12(1):3.
lallemend, F., Adameyko I., Furlan, A., and Ernfors. P (2012). Positional differences of axon growth rates between sensory neurons encoded by Runx3. EMBO J. 2012 Aug 17.
aranda, S., Rutishauser, D. and Ernfors, P (2012). Distinct protein interaction networks associated with newly replicated DNA in embryonic stem cells. Submitted.
WP2: Protecting neurons
more than 20 neurotrophic factors (NTFs) can protect dopamine (DA) neurons in animals models of Parkinson's disease (PD) (Bespalov and Saarma, 2007; Lindholm and Saarma, 2010). However, only GDNF, NRTN that are also tested on phase 2 clinical trials for PD (Gill et al., 2003; Andressoo and Saarma, 2008) and two recently discovered NTFs, cerebral dopamine neurotrophic factor, CDNF (Lindholm et al., 2007) and mesencephalic astrocyte-derived neurotrophic factor, MANF (Petrova et al; Molec. Neurosci., 2003; Voutilainen et al, 2009) can also repair substantia nigra (SN) DA neurons. A major focus of this WP was to thoroughly assess the therapeutic potential of CDNF and MANF in animal models of PD, generate knockout mice to understand aspects of the physiology and of these NTFs that could be clinically relevant, and identify the CDNF receptor as thus could represent a potential drug target for PD.
2.1 Neuroprotective effects of CDNF and MANF in mouse MPTP and rat 6-OHDA PD models
we (Saarma) discovered a novel evolutionarily conserved neurotrophic factor, CDNF and found that in rats, CDNF protects and repairs DA neurons in the 6-hydroxydopamine (6-OHDA)-induced unilateral rat model of PD (Lindholm et al., 2007). We then found that intrastriatally injected MANF (Voutilainen et al., 2009) protects nigrostriatal DA nerves from 6-OHDA induced degeneration in rats. Most importantly, MANF also restored the function of nigrostriatal DA system when administered three weeks after 6-OHDA in the striatum (Voutilainen et al., 2009). Modest therapeutic effects of GDNF and NRTN are partially caused by their poor diffusion in the brain. We therefore compared MANF and GDNF diffusion in rat brain and found that MANF is distributed throughout the striatum more readily than GDNF. We also found that the mechanism of MANF action may differ from that of GDNF and CDNF since intrastriatally injected 125I-MANF was transported to the frontal cortex whereas 125I-GDNF and 125I-CDNF were transported to SN. Taken together these results suggested that in addition to CDNF also MANF has significant therapeutic potential for the treatment of PD (Voutilainen et al., 2009). We then decided to carefully compare the therapeutic effects of CDNF with MANF and GDNF using a severe 6-OHDA lesion model in rats. Two weeks after 6-OHDA lesion GDNF, CDNF and MANF were continuously infused into rat striatum with Alzet minipumps at three different concentrations: 1.5 νg, 3.0 νg and 4.5 νg in 24-h during two weeks. The results of this study clearly demonstrated that CDNF at all three concentrations tested was able to regenerate the axons of DA neurons. Moreover at 1.5 νg/24-h and 3.0 νg /24-h CDNF also protected DA neuron cell bodies (Voutilainen et al., 2011). Importantly, MANF tested at the same protein concentrations and GDNF tested at optimal 3.0 νg/24-h concentration showed no statistically significant neurorestorative effect in this severe PD model (Voutilainen et al., 2011). The volume of distribution for MANF in striatum was larger than that of CDNF and significantly larger than that of GDNF. We also treated naïve rats with CDNF and MANF and found no behavioral signs of toxicity. This potentially very important result indicates that CDNF and MANF may have very small side effects when used in PD therapy. These results imply that CDNF is significantly more potent than MANF or GDNF for neurorestorative therapy of PD. Therefore in future therapeutic trials we concentrated on CDNF.
2.2 Neurorestorative effects of CDNF on MitoPark model of Parkinson's disease
we initially planned to use a new mouse model of PD that has been developed in the laboratory of Nilsson at Karolinska Institute. This model is based on conditional knockout mice (MitoPark mice) with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. At that time none of the genetic animal models of PD showed significant neurodegeneration and therefore MitoPark mice looked as an attractive alternative. Two important findings led us to the change of our plans. Firstly, Olson's lab in Stockholm found that GDNF can protect DA neurons in MitoPark model and therefore this model would not give us additional important information on CDNF efficacy compared to GDNF. Then the Kirik lab demonstrated that viral delivery of a-synuclein (α-syn) into rodent SN will induce very efficient neurodegeneration in nigral DA system (Ulusoy et al. Exp. Neurol., 2008). Interestingly, the Björklund lab in Lund found that viral vector-mediated delivery of GDNF was inefficient in preventing wild-type a-synuclein-induced loss of DA neurons and terminals. In addition, GDNF overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson's disease (Decressac et al., Brain, 2011).
2.3 Identification of receptors for CDNF and MANF
CDNF and MANF receptors and mechanisms of action are unknown. CDNF and MANF show neurorestorative effects in rodent (Lindholm et al. 2007; Voutilainen et al., 2009) and monkey models of PD when they are injected into brain parenchyma. However, in vitro, when we have applied CDNF or MANF into the culture medium of DA, sympathetic, sensory or cortical neurons that die due to absence of NTFs, we have not seen any survival promoting effect (Lindholm et al, 2007; Hellmann et al., 2011). This is totally different from the action of all known NTFs. Interestingly, also injection of CDNF and MANF into the brain of naïve rats and mice without pathological lesions cause no obvious changes in the brain (Voutilainen et al. 2011; Airavaara et al., 2009; 2011), whereas for example GDNF injection activates RET receptor, stimulating excessive neurite outgrowth and axonal sprouting. In collaboration with Dr. Krieglstein, Freiburg, we have recently found that CDNF and MANF can rescue DA neurons and the MN9D dopamine cell line in culture, but only after cells have been lesioned with staurosporine or 6-OHDA or ER stress triggered by thapsigargin (Zhou et al. in preparation). In addition, we have also shown that CDNF and MANF by the virtue of their N-terminal saposin-like domain can bind oxidized phospholipids (Lindholm and Saarma., 2010).
these exciting results point to the following possible modes of action of CDNF-MANF:
1) receptors of CDNF and MANF are lesion-induced;
2) Cellular lesion induces the release of additional subunit or cofactor (e.g. oxidized lipid) that activates MANF and CDNF.
2.4 Generation of CDNF and MANF knockout mice
to understand the physiological roles of these new neurotrophic factors in vivo, we generated conventional MANF- and CDNF-deficient mice. The severe phenotype in DmMANF mutant flies (Palgi et al., 2009) suggested that the phenotype of MANF-and CDNF mice might also be severe. Therefore in parallel we created conditional gene-deficient (cKO) mice where MANF or CDNF genes can be deleted in a time- and tissue specific manner. Our idea was that MANF and CDNF KO mice may be useful genetic models for PD.
drosophila mutants lacking MANF have dramatically reduced DA levels, partial loss of DA neurons and complete lack of DA neurites. Thus fly MANF is the lege artis target-field NTF for fly DA neurons. We hypothesized that in mice CDNF and MANF represent target-derived NTFs for midbrain DA neurons and wanted to test this hypothesis on the respective KO and cKO mice.
2.5 In vivo survival of SN neurons in genetic mouse models
dysfunctions in the signalling activity of neurotrophic factors represent one of the pathogenic events that trigger neurodegeneration in the brain. Ret, the receptor for glial cell line-derived neurotrophic factor (GDNF) is specifically required for long-term target innervations and cell survival of a significant fraction of substantia nigra pars compacta (SNpc) DA neurons. DJ-1/PARK7 deletion in mice, on the other hand, resulted in changes in dopamine signaling, but the effects on target innervation and cell survival were very subtle. To investigate the combined effects of Ret and DJ-1 deletions, Klein has generated double mutant mice lacking expression of Ret in midbrain dopaminergic neurons and DJ-1 in all cells of the body (DAT-Cre:Retlx/lx;DJ1 null mice, in short Ret;DJ-1 mutant mice). At 24 month of age Ret;DJ-1 mutant mice suffer from enhanced loss (40%) of dopaminergic neurons compared to Ret single mutant (25%) and DJ-1 single mutant mice (less than 5%, not significant). These results indicate that DJ-1 is required for survival of midbrain dopaminergic neurons which suffer from partial trophic deprivation. In contrast, Ret;DJ-1 mutant mice show no additional loss of target innervation (40% loss in Ret;DJ-1 mutant and in Ret single mutant; no loss in DJ-1 single mutant mice). These findings also suggest that DJ-1 plays no or a minor role in maintaining target innervation (Aron et al., 2010a; 2010b).
2.6 In vivo survival of cells in genetic Drosophila models
klein had proposed to study the cooperation of dRet and DJ-1 in Drosophila. Mutant flies overexpressing constitutive active forms of Ret (MEN2A and MEN2B) in the eye, display a rough eye phenotype due to excessive proliferation, increased cell size, patterning defects and apoptosis. Analysis of sections of adult eyes revealed that the remaining ommatidia were increased in size. The stereotyped regular organization of ommatidia seen in control eyes is absent in dRetMEN2B-expressing eyes, which display random ommatidial rotations. Combined overexpression of RetMEN2B and haploinsufficiency for either DJ-1A or DJ-1B, or complete loss of DJ-1A rescued the rough eye phenotype of GMR-dRetMEN2B flies. Conversely, overexpression of both Ret and DJ-1 led to a more severe phenotype than the ones induced by active dRet alone. Thus, both DJ-1A/B interact genetically with overactive dRet in controlling cell size and differentiation in the developing fly retina.
2.7 In vitro survival of transfected cells carrying gain- or loss-of-function mutations
the biochemical interaction between Ret and DJ-1 in cells derived from knockout mice turned out to be very difficult. Much of the published work on DJ-1 as a modulator of PI3K/Akt signaling could not be reproduced by Klein. Hence, these studies were stopped and more emphasis was put on the fly and mouse work. We have, however, extended our studies towards the interaction of Ret and Pink1/Parkin. Mutations in PINK1 and Parkin are associated with autosomal recessive Parkinson's disease. Pink1 and park mutant Drosophila display phenotypes related to impaired mitochondrial function, including degeneration of indirect flight muscles and the respective Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking.
publications:
airaksinen, M.S. and Saarma, M. (2002). GDNF family neurotrophic factors: receptor mechanisms, biological functions and therapeutic utility. Nature Rev. Neurosci. 3: 383-394.
airavaara M, Shen H, Kuo CC, Peränen J, Saarma M, Hoffer B, Wang Y. (2009) Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J Comp Neurol. 515 (1): 116-124.
airavaara M, Harvey BK, Voutilainen MK, Shen H, Chou J, Lindholm P, Lindahl M, Tuominen RK, Saarma M, Wang Y, Hoffer B. CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice. (2011). Cell Transplantation, in press.
andressoo, J.-O. and Saarma, M. (2008) Signalling mechanisms underlying development and maintenance of dopamine neurons. Curr Opin Neurobiol., 18, 297-306.
aron L, Klein P, Pham TT, Kramer ER, Wurst W, Klein R. (2010a). Pro-survival role for Parkinsons associated gene DJ-1 revealed in trophically impaired dopaminergic neurons. PLoS Biology 8(4): e1000349. doi:10.1371/journal.pbio.1000349
aron, L. and Klein, R. (2010b). Repairing the Parkinsonian brain with neurotrophic factors. Review. Trends in Neurosciences, 2011 Feb;34(2):88-100. Epub 2010 Dec 7.
bespalov, M., Saarma, M. (2007) GDNF receptor complex is an emerging drug target. Trends in Pharmacological Sciences 28 (2), 68-74.
griciuc, A., Aron, L., Roux, M.J. Klein, R. Giangrande, A., and Ueffing M. (2010). Genetic inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila. PLoS Genetics, Aug 26;6(8). Pii: e1001075.
hellman M, Peranen J, Saarma M, and Permi P. (2010) H-1, C-13 and N-15 resonance assignments of the human mesencephalic astrocyte-derived neurotrophic factor. Biomelecular NMR Assignments. 4 (2): 215-217.
hellman M, Arumae U, Yu L-y, Lindholm P, Peranen J, Saarma M. and Permi P.. Neurotrophic factor MANF has a unique mechanism to rescue apoptotic neurons. (2011) J Biol Chem, 286(4):2675-80.(equal contribution).
lindholm, P., Voutilainen, M .H., Laurén, J., Peranen, J., Leppanen, V-M., Andressoo, J.-O. Lindahl, M., Janhunen, S., Kalkkinen, N., Timmusk, T., Tuominen, R. K. and Saarma, M. (2007) Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature, 448, 73-77.
lindholm, P., Peranen, J., Andressoo, J.O. Kalkkinen, N., Kokaia, Z., Lindvall, O., Timmusk, T. and Saarma M. (2008) MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain. Mol Cell Neurosci., 39(3), 356-371.
lindholm P, Saarma M.(2010) Novel CDNF/MANF family of neurotrophic factors. Dev Neurobiol. 70(5): 360-371.
palgi M, Lindström R, Peranen J, Piepponen TP, Saarma M, Heino TI. (2009) Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons. Proc Natl Acad Sci U S A. 106 (7): 2429-2434.
parkash, V., Lindholm, P., Peranen, J., Kalkkinen, N., Oksanen, E., Saarma, M., Leppanen, V-M. and Goldman, A. (2009) The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng.Des. Sel., 22(4): 233-241.
peng, C., Aron, L., Klein, R., Li, M., Wurst, W., Prakash, N., and Le, W. (2011). Pitx3 is a critical mediator of GDNF-induced BDNF expression in nigrostriatal dopaminergic neurons. J. Neuroscience, Sep 7;31(36):12802-12815.
voutilainen, M.H. Back, S., Porsti, E., Toppinen, L., Lindgren, L., Lindholm, P., Peranen, J., Saarma, M. and Tuominen, R. K. (2009) Neurotrophic factor MANF is neurorestorative in rat model of Parkinson's disease. J. Neurosci., 29(30): 9651-9659. (Saarma, M Corresponding author.)
voutilainen MH, Bäck S, Peranen J, Lindholm P, Raasmaja A, Mannisto PT, Saarma M, Tuominen RK. Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease. (2011) Exp Neurol. 228(1): 99-108.
WP3: Restoring neural processes
we had proposed to study in various experimental paradigms the axonal projections of midbrain DA neurons to the striatum, the size and complexity of the dendritic arbors in these neurons in the intact substantia nigra and assess the growth of neurites from these neurons in dissociated cultures.
3.1 Maintenance and restoration of axonal projections in genetic mouse models
klein investigated the potential synergism between Ret and DJ-1 in maintenance of axonal projections. Axonal projections of midbrain DA neurons to the striatum of DA-specific Ret;DJ-1 double mutant mice (DAT-Cre:Retlx/lx;DJ1 null mice) were quantified by anti-TH immunohistochemistry in sections passing through the striatum compared to age-matched control mice. It was found that aging DA-specific Ret;DJ-1 double mutant mice did not have accelerated loss of SN axons compared to mice that only lack Ret signalling, indicating that DJ-1is not required to maintain target innervation (Aron et al., 2010).
3.2 Lesion models
see WP2.3 for studies with CDNF and MANF.
3.3 The role of intrinsic factors in axonal and dendritic growth
ernfors addressed the role of Cux2 in axon and dendritic growth during development and first characterised its expression in the sensory lineage where it is abundantly expressed. Cux2 was found to be expressed in subpopulations of the three main neuronal sublineages as defined by the expression of neurotrophic factor receptors TrkA, TrkB and TrkC. In particular, it marked a subpopulation of nociceptive TrkA+ neurons that is complete in numbers already at E10.5 and arised during the first, Ngn2-dependent, wave of. These early TrkA+/Cux2+ neurons persist after birth and at this time represent a specific subtype of A-delta nociceptors as seen by expression of TrkA and NF200 but absence of TrpV1. Analysis of Cux2 null mutant (Cux2-/-) mice and gain of function experiments in chicken show that Cux2 is sufficient but not required for specification of Trk+ neuronal subpopulations. Interestingly, Cux2-/- mice are hypersensitive to mechanical, but not to heat or cold, stimuli supporting a requirement in the process of specification of A-delta sublineage of TrkA+ sensory neurons. Cux null mutant mice did not display any deficits of axon and dendritic growth. Hence, our results show that Cux2 is expressed and participates in development of a previously not identified early population of myelinated TrkA+ nociceptors but appears not to constitute an intrinsic determinant regulating axon growth.
3.4 Elaboration, maintenance and restoration of DA neuron dendritic arbors
3.4.1 Influence of CDNF and MANF on dendritic size
saarma has carefully studied the effects of CDNF and MANF on the axonal growth. We have documented the effects of CDNF after single injection of the protein in earlier work (Lindolm et al., 2007) and in continuous infusion of CDNF more recently (Voutilainen et al 2011). Saarma has also shown that MANF either after single injection (Voutilainenn et al., 2009) or after continuous infusion can stimulate the growth and regeneration of the dopaminergic axons. We have not yet studied the effects of CDNF and MANF on the dendritic size mostly because we got several unexpected results which required all the available manpower. However, studies on the effects of CDNF and MANF on dendritic size will be performed soon.
3.4.2 Influence of GDNF and Ret signalling on dendritic size
klein and Davies had planned to study the influence of GDNF on midbrain DA neuron dendritic arbors in organotypic slice cultures from adult and aging wild-type and mutant mice. These studies turned out to be difficult due to the poor survival of DA neurons in these cultures. The work was therefore put on hold to provide more manpower to other projects such as described in WP2.
3.4.3 Role of DJ-1 in dendritic size
this was not done due to the same problem as described in 3.4.2.
3.4.4 Role of Wnt signalling in dendritic size
to begin to examine the contribution of Wnt signalling to the development of dendritic arborisation in medium spiny neurons in the striatum, we examined the Dvl1 mutant mouse. Dvl1 is a scaffold protein required for Wnt signalling. The Dvl1 mutant exhibits defects in dendritic development in cultured hippocampal neurons. We also examined the Wnt7a; Dvl1 double mutant mice for possible defects in dendritic development. Neither single Dvl1 mutant nor the double mutant exhibits detectable defects in the arborisation of medium spiny neurons based on Golgi staining of brain slices. These results suggest that Dvl1 and Wnt7a are not required for the proper dendritic development in the striatum. Further studies are required to examine the contribution of other Wnt factors expressed in the striatum.
3.4.5 The roles of the TNF/TNFR Superfamily in the developing ventral midbrain
classically, members of the tumour necrosis factor/tumour necrosis factor receptor (TNF/TNFR) superfamily have been implicated in the regulation of immune cell proliferation, survival and homeostasis. Recent published and unpublished findings from the Davies laboratory have demonstrated clear roles for several TNF/TNFR proteins in the regulation of peripheral neuronal development. While concerted research efforts over the past decade have successfully contributed to how mDA neurons are generated and the regulation of their phenotype, there is a current paucity of data on the molecular signals that regulate midbrain dopaminergic axonal specification, initiation and elongation during embryonic development. Understanding the cellular and molecular basis of how midbrain dopaminergic neurons project axons and form connections is of critical importance to the success of current therapeutic strategies for the successful treatment of Parkinson's disease.
to investigate if members of the TNF/TNFR superfamily are expressed in the ventral midbrain and target tissues (striatum), a comprehensive, developmental-based and MIQE-compliant RT-qPCR screen was conducted to assay for TNF/TNFR mRNA expression throughout development. Our screen identified several interesting expression patterns for ligands APRIL, TWEAK, BAFF, TWE-PRIL and receptors BCMA, BAFFR, TACI and Fn14 throughout the course of nigrostriatal pathway development. To date, no functions have been ascribed to these proteins during midbrain development. Further immunohistochemical validation of our qPCR data revealed the expression of APRIL and BCMA protein in developing mDA neurons of the ventral midbrain at E12, at a time when midbrain dopaminergic neurons have initiated axonogenesis and rostral axon extension towards their target fields. Further in vitro functional assays conducted using acutely dissociated cultures of embryonic midbrain dopaminergic neurons identified a novel APRIL-BCMA signalling loop that specifically modulates mDA axon elongation. This study is the first to highlight a function for APRIL-BCMA signalling in the developing nervous system, in selectively promoting the growth of mDA neuron axons during a defined window of embryonic development at E12 and E13, but not at E11 and E14. Furthermore, Davies made the first observation that the hybrid mRNA encoding TWE-PRIL is expressed outside of the immune system.
the axon-growth promoting effects of APRIL occur independently of neuronal survival, as evidenced by converging data demonstrating that APRIL neither promotes nor inhibits midbrain dopaminergic neuronal survival in VM cultures established from E12 wild type embryos and APRIL promotes axon outgrowth from mDA neurons in VM cultures established from E12 bax-/- embryos. Utilising function-blocking antibodies to BCMA and TACI, our data strongly suggests that APRIL promotes axonal elongation from mDA neurons by signalling through BCMA and not TACI. Furthermore, APRIL enhances axonal growth in a heparin-sulphate proteoglycan (HSPG)-dependent manner. APRIL promoted mDA axonal growth requires the activity of the canonical NF-kB pathway, as evidenced by the failure of APRIL to promote axon outgrowth from mDA neurons in VM cultures established from E12 p65-/- embryos. Interestingly, GDNF-promoted axon elongation growth is also compromised in these cultures, suggesting that both APRIL and GDNF may enhance axon elongation from mDA neurons through a common signalling pathway. To investigate the physiological relevance of these observations, we conducted studies on midbrain dopaminergic neurons using VM cultures established from E12 april-/- embryos. Consistent with in vitro observations of the effects of recombinant APRIL on mDA axonal growth, genetic ablation of APRIL impairs axonal growth in E12 midbrain dopaminergic neurons. This finding suggests that APRIL may exert its effects via an autocrine or paracrine signalling loop in vitro. However, this does not preclude a role for target-derived APRIL signalling as Davies has shown that APRIL and TWE-PRIL mRNAs are both robustly expressed in the developing striatum. Collectively, these findings indicate that APRIL is an axon growth-promoting factor for embryonic mDA neurons.
publications:
aron L, Klein P, Pham TT, Kramer ER, Wurst W, Klein R. (2010). Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons. PLoS Biology 8(4): e1000349. doi:10.1371/journal.pbio.1000349
dudanova, I., Gatto, G., and Klein, R. (2010). GDNF acts as a chemoattractant to support ephrinA-induced repulsion of limb motor axons. Current Biology 20, Issue 23, 7 December 2010, Pages 2150-2156.
dudanova, I., Kao, T.-J. Herrmann, J.E. Zheng, B., Kania, A., Klein, R. (2012). Genetic evidence for a contribution of EphA:ephrinA reverse signaling to motor axon guidance. J. Neuroscience 32(15):5209 -5215.
wang, L., Klein, R., Zheng, B., and Marquardt, T. (2011). Anatomical Coupling of Sensory and Motor Nerve Trajectory via Axon Tracking. Neuron 71(2):263-77.
WP4: Restoring connections
main achievements:
1)We have established an acute and organotypic brain culture system to examine the contribution of signaling molecules and dopamine depletion in the nigro-striatal system.
2)We demonstrated that several components of the Wnt signaling pathway are present in the adult striatum.
3)We have demonstrated that the Wnt antagonist Dkk1 induces synaptic disassembly in adult neurons.
4)We demonstrated that neutralizing antibodies against Dkk1 restores synaptic degeneration induced by Amyloid- β. These results indicate a key role for Dkk1 in the loss of synapses in neurodegenerative diseases. These results were published (Purro et al, J. Neuroscience, 2012)
5)We have generated a transgenic mouse model that inducibly expresses Dkk1 in the hippocampus and striatum.
6)We showed that expression of Dkk1 in the adult hippocampus results in the loss of synapses and in hippocampal mediated behavior (Lopes, Galli and Salinas, manuscript to be submitted in January 2013)
7)We showed that Wnt signaling is crucial for synaptic maintenance in the adult striatum.
8)We found that defects in synaptic maintenance due to impaired Wnt signaling affects normal striatal-dependent behavior, such as motor coordination and locomotion. (Galli and Salinas, manuscript to be submitted in March 2013)
9)Our preliminary results suggest that long-term defects in synaptic maintenance lead to dopaminergic cell death in the substantia nigra (SN).
4.1. Examining the effect of dopamine depletion on synapses of the nigrostriatal system.
salinas has established an ex-vivo system to examine the contribution of signaling molecules to the formation and maintenance of synapses in the nigro-striatal system. Firstly, we set up acute (4-6 hours) and long-term organotypic (up to 15 day in culture) brain slices cultures that contain the components of the striato-nigral pathway (i.e. striatum and SN). To obtain a significant number of intact connections between the substantia nigra to the striatum in our slices, we made sagittal sections from P15 mouse brains. In these cultures we were able to examine excitatory, inhibitory and dopaminergic synapses by immunofluorescence microscopy. We found that Wnt proteins such as Wnt7a, Wnt3a and Wnt5a distinctly affect the number of synapses in the striatum of acute brain slices. Conversely, we found that Wnt blockade with the Wnt antagonist Dickkopf-1 (Dkk1) significantly decreases the number of synapses in the striatum. These results demonstrate that Wnt signaling is required for the formation of different types of synapses in the striatum.
4.2. To examine the effect of neurotrophic factors in the formation and maintenance of synapses in the dopaminergic system of the basal ganglia
salinas examined the expression of core components of the Wnt signalling pathway in the postnatal striatum at the peak period of synapse formation (P15) and in the adult mice (3 months old), using RT-PCR. We found that several Wnts such as Wnt3, Wnt5a and Wnt7b and the receptors Frizzled (Fz) 3-9 are highly expressed in the adult striatum. Wnt7a, 9b, 10a and 11 and the co-receptors LRP5 and 6 are also expressed. In addition, the secreted Wnt antagonists, secreted Frizzled related proteins (Sfrp 1-3) and Dickkopf-3 (Dkk3) are highly expressed in the striatum. In contrast, Dkk2 and Dkk4 are weakly expressed whereas Dkk1 is not expressed under normal physiological conditions.
to elucidate the in vivo role of Wnt signalling in the maintenance of mature synapses, we have generated an inducible transgenic mouse line that expresses Dkk1 under the control of the tetracycline system. Mice expressing Dkk1 under the regulation of the Tet-O system were crossed with a transgenic line expressing CaMKII rtTA to obtain specific expression of Dkk1 in the hippocampus, striatum and part of the cortex upon induction with Doxycycline (Dox) (Mansuy et al., 1998). RT-PCR analyses of brains of double transgenic mice (TetO-Dkk1 with CaMKII rtTA, hereafter named Dkk1-TG) fed with Dox for 3-28 days revealed a significant increase in Dkk1 mRNA levels in the hippocampus and striatum when compared to Dkk1-TG mice not fed with Dox or to single transgenic mice (Galli et al, 2011).
in adult mice, blockade of Wnt signalling by Dkk1 decreases the number of excitatory and inhibitory synapses, as well as the number of dopamine receptors 1 (D1R) and 2 (D2R) puncta. Blockade of Wnt signalling decreases the number of docked and total vesicles containing the neurotransmitter vGlut1. However, no changes were observed in the number of dendritic spines and dopamine content in the striatum. Blockade of Wnt signalling does not induce cell death, which reinforces the role of Wnts directly in the maintenance of striatal synapses (Galli et al, 2011 and Galli et al, 2012). This defect in synaptic maintenance caused by blockade of Wnt signalling is associated with behavioural deficits such as defects in motor coordination and locomotion induced by psycho-stimulants (amphetamine-induced locomotion).
4.3. To test the synaptic protective function of neurotrophic factors during dopamine depletion in the nigrostriatal system
our preliminary data suggests that long-term but not short-term defects in the maintenance of synapses in the striatum, affects the viability of the dopamine neurons in the SN. We have evaluated the number of TH positive cells in the SN after induction of Dkk1 expression during 1 month and 8 months. We found that the number of TH positive cells is unchanged after 1-month induction. However, we observed a reduction in the number of TH positive cells in the SN after 8 months of Dkk1 expression. These results highlight the importance of synaptic maintenance in cell viability, and shed new light into the potential role for dysfunction of Wnt signaling in neurodegeneration in the striato-nigral system.
4.5. To screen for small molecules that will restore synaptic connectivity
our studies (Salinas) in cultured neurons, brain slices and in transgenic mice have demonstrated that blockade of Wnt signalling with the secreted Wnt antagonist Dkk1 induces synaptic disassembly in the adult brain. Moreover, we found that Dkk1 is downstream of Amyloid- β during synaptic degeneration. These results demonstrate that Dkk1 is a potential therapeutic target for the treatment of neurodegenerative diseases where synapses are compromised. We (Salinas) therefore started a collaborative project (with Actar) to screen for small compounds that block the binding of Dkk1 to its receptor. HEK293 cells expressing a SuperTOPflash construct were transferred from Salinas to Actar. The generation of the cells is described in detail in (Nathans et al., Cell, Vol. 116, 883-895, March 19, 2004, Vascular Development in the Retina and Inner Ear: Control by Norrin and Frizzled-4,a High-Affinity Ligand-Receptor Pair). The SuperTop Flash reporter drives firefly luciferase by 7 LEF/TCF consensus binding sites, to which β -catenin is recruited upon active canonical Wnt-signalling in the cell, thus a measure of intracellular Wnt-activity. A functional Wnt-activity assay was developed by Actar in which small molecules from Actar's screening library of approximately 30.000 compounds were tested. A subgroup of 1000 compounds from the library was selected due to chemical properties such as size, diversity and likelihood to penetrate CNS. From these, 700 compounds were screened in the luciferase reporter cell assay at one concentration. Compounds, which showed a DKK-1 selective pattern of Wnt-signalling, were object to further selection due to druggability and potency in the cell assay and picked for more extensive testing, i.e. dose response in the cell assay and DKK-1 selectivity in binding assays. A DKK-1 binding assay was developed for testing of small molecules in the Corning Epic System, which is a label-free screening platform based on optical biosensor technology. Also, a LRP6/DKK-1 interaction assay was developed, aiming to detect small molecules that interfere with the interaction between DKK-1 and LRP6, one part of a receptor complex to which Wnts and DKK-1 may bind. Confirmation of hits from cell assay by binding assays was not manageable within the project time frame. Confirmed compounds will be examined for their efficacy in cultured neurons and brain slices for their ability to block Dkk1 function and therefore protect synapses.
in addition to our work in the striatum, Salinas has also examined synaptic degeneration in the hippocampus:
1) The Wnt antagonist Dkk1 induces synaptic disassembly in adult neurons. Salinas has previously demonstrated that Wnt signalling plays a role in the formation, growth and function of synapses in the central nervous system. However, Wnts and their signalling components are not only expressed during synaptogenesis but also in the adult. We decided to investigate whether Wnts are also required in the maintenance of synapses in mature neurons. As hippocampal neurons express several Wnts, we decided to use the Wnt antagonist Dkk1 to test the role of Wnts in synaptic maintenance. We found that Dkk1 induces the rapid disassembly of synapses in mature neurons in cultures (Purro et al, J. Neuroscience 2012).
2) Neutralizing antibodies against Dkk1 restores synaptic degeneration induced by Amyloid-β.
several studies suggest a potential link between Dkk1 expression and neurodegenerative diseases such as Alzheimer's disease (AD). Although Dkk1 levels are almost undetected in the adult normal brain, they are elevated in AD patients. Moreover, Dkk1 is also elevated in animal models of AD that over-express mutant Amyloid-β (Aβ). We therefore tested the effect of Amyloid-β on Dkk1 and found that indeed Aβ significantly increases the levels of Dkk1 in brain slices at the time when synapses are lost. Importantly we found that neutralizing antibodies against Dkk1 completely block the effect of Aβ on synapses (Purro et al, J. Neuroscience 2012). These findings highlight the importance of Dkk1 in synaptic degeneration and more importantly they identify Dkk1 as a possible target for the treatment of AD. This work has been highly cited in the media (commentaries in news websites, interview by BBC World Service), highlighted by UCL Media and funding organizations and reviewed twice by Faculty of 1000.
3) We (Salinas) have generated a transgenic mouse model that inducible expresses Dkk1 in the hippocampus and striatum. To begin to elucidate the in vivo role of Wnt signalling at hippocampal synapses, we have used the Dkk1-TG mice. Induction of Dkk1 expression in adult mice for a period of 1-2 weeks does not affect cell viability (based on TUNEL assay and Caspase-3 staining), nor affect the progenitor cell niche (Doublecortin staining) (Lopes et al, 2012). Thus, deficiency in Wnt signalling does not affect cell viability or stem cell number.
4) Expression of Dkk1 in the adult hippocampus results in the loss of synapses and in hippocampal mediated behavior (Lopes, Galli, Cacucci and Salinas, manuscript to be submitted in January 2013). Given the role of Wnt-signalling in excitatory synapses (Ciani et al., 2011) and its newly described role in synaptic maintenance (Purro et al., 2012) we next examined the possible defects in hippocampal dendritic spines in our Wnt signalling deficient animal model. Golgi staining revealed that 2 weeks of continuous expression of Dkk1 in the hippocampus lead to almost 20% reduction in spine size and about 10% decrease in the spine number of CA1 cells. In contrast, no changes were observed in CA3 cells.
consistent with the loss of spines, we found a significant decrease in the number of excitatory synapses (colocalization of pre- and postsynaptic markers) but inhibitory synapses are unaffected. These results suggest that blockade of Wnt signaling with Dkk1 in the adult hippocampus specifically induce the disassembly of excitatory synapses (Lopes et al, 2012).
overall conclusion: Our studies (Salinas) in the hippocampus and striatum demonstrate the Wnt signaling is required for the maintenance of synapses in the adult brain and that pathogenic molecules such as Amyloid-β triggers the expression of the Wnt antagonist Dkk1 to induce synapse loss. Our studies in the striatum strongly suggest that Wnts or activation of the Wnt pathway could protect synapses against synaptic degeneration.
publications:
ciani, L., Boyle, K. A., Dickins, E., Sahores, M., Anane, D., Lopes, D. M., Gibb, A. J. and Salinas, P. C. (2011) Wnt7a signaling promotes dendritic spine growth and synaptic strength through CaMKII, Proc Natl Acad Sci U S A 108(26): 10732-7.
mansuy, I. M., Winder, D. G., Moallem, T. M., Osman, M., Mayford, M., Hawkins, R. D. and Kandel, E. R. (1998) 'Inducible and reversible gene expression with the rtTA system for the study of memory', Neuron 21(2): 257-65.
purro S. A., Dickins E., Salinas P.C. (2012) The Wnt antagonist Dkk1 mediates the synaptic disassembly activity of Aβ: a potential role for Wnt synaptic dysfunction in Alzheimer's Diseases. J. Neuroscience. 32: 3492-3498.
galli S, Millar SE, Salinas P.C. (2011) Nanosymposium: Wnt signalling controls synaptic maintenance in the striatum. Society for Neuroscience Annual Meeting, 12-16 November, Washington Convention Center, Washington DC, USA. (Talk)
galli S, Lopes D, Millar S, Salinas P.C. (2012) Wnt signalling controls synaptic maintenance in the striatum: potential implications in Parkinson's Disease. Society for Neuroscience Annual Meeting. 13-17 October. New Orleans Convention Center. New Orleans, USA.
lopes D.M. Galli S., Millar S., Cacucci F. and Salinas P.C. (2012) Synaptic maintenance mediated by Wnt signalling modulates learning and memory. Society for Neuroscience Annual Meeting. 13-17 October. New Orleans Convention Center. New Orleans, USA.
potential Impact:
the potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
parkinson's disease is one of the most common neurodegenerative disorders with a prevalence of approximately 1% in the population over the age of 65. Patients suffer from impaired motor function characterised by rhythmic tremor, inability to initiate and complete routine movement, muscle rigidity, postural instability and paucity of facial expression. These symptoms are caused by the degeneration and loss of the dopaminergic neurons of the substantia nigra pars compacta that project to the striatum and play a key role in controlling the extrapyramidal motor system.
the mainstay of Parkinson's disease treatment, drugs that restore dopamine levels, is of limited long-term benefit as it does not prevent disease progression. Moreover, because other midbrain dopaminergic systems play key roles in emotional balance and reward (mesolimbic pathway) and motivation, attention, planning and social behaviour (mesocortical dopaminergic pathway), these drugs can cause imbalances in these systems with serious adverse effects. Unfortunately, there has been at best only limited success with a variety of other therapeutic approaches, including cell transplantation, growth factor therapy and selective stimulation by surgically implanted electrodes. New, much needed, therapeutic approaches for this devastating disease will only come from a better understanding of the generation, development and maintenance of dopaminergic neurons and their connections.
the research carried out by MOLPARK has made very significant advances in our understanding of the basic mechanisms that control neuron generation from stem cells, and molecules that sustain the survival of dopaminergic neurons and promote the growth and maintenance of their processes and connections. These major advances in our understanding are providing important new platforms for the development of novel therapies for Parkinsons disease and potentially other neurodegenerative diseases. In addition to the major advances in understanding cellular and molecular aspects of dopaminergic neuron development and function, important translational aspects of our research include the following:
(i). The establishment of screens for small molecule drug leads for regulating stem cell self-renewal.
(ii). We have demonstrated the efficacy of the novel neurotrophic factors CDNF and MANF in protecting midbrain dopaminergic neurons in rodent models of Parkinson's disease and in substantially countering the adverse consequences of impaired dopaminergic neuron function in these models. Importantly, treatment of naïve rats with CDNF and MANF revealed no behavioral signs of toxicity, implying that CDNF and MANF may have few side effects when used in Parkinson's disease therapy. As a result of this work, clinical trials are planned to assess the efficacy of CDNF in Parkinson's disease.
(iii). In addition to potential important translational outcomes of our research for the or Parkinson's disease therapy, our work on stem cell proliferation and differentiation has led to assessments of a promising novel therapeutic approach for glioblastoma, a highly aggressive malignant brain tumor with poor treatment options. This novel approach is based on the use of the antipsychotic drug Sertindole. We have shown that Sertindole exerts a cytotoxic effect on glioma stem cells in culture and is cytotoxic in vivo to the glioma cells grafted into the brain of zebrafish with no negative effect on the viability of the fish.
in summary, our work has not only made a very significant contribution to basic knowledge but has led to preclinical translational research that underpins the development of novel therapeutic approaches for Parkinson's disease and glioma. As such, the work of MOLARK may ultimately help alleviate suffering in the significant proportion of the population and lead to an important source of revenue for the European pharmaceutical industry should preclinical studies come to fruition with the development of therapies that can be taken through the clinic successfully.
dissemination activities
in the time period 2009-2012, the Molpark consortium published 36 original and review articles in peer-reviewed journals. Many publications appeared in the top cell and molecular biology and neuroscience journals such as Cell, Proc. Natl. Acad. Sci USA, EMBO Journal, Journal of Neuroscience, Development, Cell Reports, Neuron, PLoS Biology, PLoS Genetics, Current Biology, Journal of Biological Chemistry.
Project website: http://www.molpark.co.uk
