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Role of CRMP2 and Pin1 in the pathogenesis of Alzheimer’s disease

Final Report Summary - AD PATHOGENESIS (Role of CRMP2 and Pin1 in the pathogenesis of Alzheimer’s disease)

Alzheimer’s disease (AD) is the most common form of dementia and its prevalence is rising due to the global aging of population. Even though a number of important genes involved in the pathogenesis of AD have been identified, molecular mechanisms triggering progressive cognitive impairment and degeneration of neurons are still largely elusive. Increasing evidence indicates that defects in protein phosphorylation together with conformation changes and folding play an important role in the pathogenesis of AD and formation of its characteristic protein aggregates - neurofibrillary tangles (NFTs) and amyloid plaques. Conformational changes at phosphorylated Ser-Pro or Thr-Pro sites are catalyzed by prolyl isomerase Pin1 - so far the only known phospho-specific prolyl isomerase and its deregulation has been linked to pathogenesis of AD and formation of amyloid plaques and NFTs. NFTs are formed mainly from hyperphosphorylated tau aggregates, and the role of tau in AD has been extensively studied. In contrast, the involvement of other proteins found in NFTs, is so far largely unknown. One of them CRMP2 (collapsin response mediator protein 2), which was found hyperphosphorylated by CDK5 kinase in NFTs, is a tubulin-binding protein regulating axon guidance during development of the nervous system.
We based our project on our recent discovery that specifically one isoform of CRMP2 (CRMP2A), phosphorylated by CDK5 kinase at a novel phosphorylation site (Ser27), is stabilized by isomerase Pin1 in distal axons. Notably, all - CRMP2, CDK5 and Pin1 - are known to be important regulators of neural development and their deregulation in adult neurons is characteristic of AD. This indicates, that the molecular mechanism, we described, could regulate both neural development in embryonic stages and pathogenesis of AD in adult/aged neurons.
In the AD Pathogenesis project we aimed to characterize the mechanisms that lead to formation of hyperphosphorylated CRMP2 inclusions in AD, to test which of the two CRMP2 isoforms is involved in CRMP2 hyperphosphorylation and accumulation in neurons and to analyze how high CDK5 activity (induced by amyloid beta peptide or p25 activator), and low level of Pin1 (alone or as combined kinase and conformational stress) affect phosphorylation, stability or localization of CRMP2 isoforms in vitro and in vivo. The aims were divided into 4 aims:
1.Generation of phospho-specific CRMP2A S27 antibodies
2.Characterization of CRMP2A metabolism in Aβ or p25 treated primary WT and Pin1 KO neurons
3.Regulation of CRMP2A and CRMP2B by CDK5 in Pin1KO mice
4.Regulation of CRMP2A and CRMP2B by CDK5 in Pin1 transgenic mice
To address these questions we developed new phospho-specific CRMP2A-pSer27 antibodies, generated new mouse models of combined kinase and conformational stress (p25Tg/Pin1KO mice) and transgenic mice with combined high kinase (CDK5) and isomerase (Pin1) activities specifically localized in cortical pyramidal neurons (generated by in utero electroporation). Moreover, we generated CRMP2 isoform-specific and full deficiency mouse models (CRMP2A KO mice, CRMP2 KO mice, respectively) to analyze the effect of CRMP2A isoform deficiency in vivo. Using these models, we demonstrated that in development, Pin1 and CDK5 regulate axon growth and guidance in Semaphorin3A gradients by stabilizing phosphorylated CRMP2A specifically in distal axons. Next, we found that CRMP2A is highly phosphorylated at Ser27 during development, but that its phosphorylation is gradually reduced in adult and aged nervous system. In contrast, in pathological conditions in mouse AD model (3xTg AD mice) as well as in AD brains we found an aberrant increase of CRMP2 Ser27 phosphorylation in pyramidal neurons co-localizing with several AD markers.
In order to characterize the mechanisms triggering the formation of CRMP2A neuron inclusions, we analyzed the phenotype of Pin1 deficient mice with high CDK5 kinase activity in neurons. We found that in adult mice Pin1 deficiency reduces levels of Ser27-phoshporylated CRMP2A in soluble fractions, which is in accord to what we described in the developing nervous system, but in contrast to the CRMP2A phospho-Ser27 accumulation, we detected in AD neurons. Importantly, we discovered that Pin1 deficiency also reduces phospho-Ser27 CRMP2A solubility leading to increase of the phospho-Ser27 CRMP2 insoluble fraction in brain lysates. Thus, Pin1 may have a dual role in CRMP2A regulation – stabilizing CDK5-phosphorylated CRMP2A and keeping it in the soluble conformation and changes in Pin1 and CDK5 could at least partially account for the formation of CRMP2 inclusions in AD neurons.
Next we showed that Ser27 together with Ser623 phosphorylation significantly reduce axon branching in cortical pyramidal neurons. Furthermore, we detected CRMP2A Ser27 phosphorylation in dendrites of pyramidal neurons and found that CRMP2A deficiency leads to increased length of dendritic spines in vivo, which has been previously found in several AD mouse models. Moreover, we found similar dendritic spine enlargement also in mice with combined Pin1 deficiency and high CDK5 activity suggesting that reduction and inactivation of CRMP2A, which results from deregulation of Pin1 and CDK5, may lead to their dendritic spine pathology.
Together our data show that pathological states of high CDK5 kinase activity and Pin1 deficiency, linked to AD, reduce CRMP2A solubility and promote formation of CRMP2 inclusions. This, together with increased degradation of soluble CRMP2A, depletes the pool of active CRMP2A with direct effect on axon growth, branching and dendritic spine morphology. Our data thus demonstrate that CDK5-CRMP2A-Pin1 signaling regulates not only neural development, but that it also controls CRMP2 in neuropathological conditions linked to AD. Moreover, as Pin1 deficiency has been previously linked to Tau pathology in AD in vivo, upregulation of Pin1 activity in early stages of AD may prevent, or slow down formation of NFTs by modulating both tau and CRMP2 therefore directly promoting neuron survival.
Aside from starting a new line of research in my laboratory, the CIG grant was key in my effort to establish my group as an independent department. This was successfully achieved in the Institute of Physiology, Prague, Czech Republic where a junior department of Molecular Neurobiology was established in 2017 under my supervision. Currently the department consists of the principal investigator, 5 PhD students, 1 undergraduate student and 1 part time secretary. Further information on current projects of the department could be found at the department’s webpage: http://www.fgu.cas.cz/en/departments/molecular-neurobiology.