Periodic Reporting for period 4 - ConCorND (Connectivity Correlate of Molecular Pathology in Neurodegeneration)
Periodo di rendicontazione: 2021-12-01 al 2022-11-30
NDs represent a major increasing burden to our aging society. Inherited and sporadic forms of neurodegenerative disorders arise mid to late in life by selectively affecting specific neuronal populations within defined regions of the central nervous system. It is well accepted that as the life span of our population increases so does the risk to develop neurodegenerative diseases such as dementia. It is estimated by 2050 neurodegenerative disorders will over take cancer to become leading cause of death in the developed world. Despite the acknowledgement of this issue, NDs remain incurable and currently there are no therapeutic options to slow down the progression of these disease.
In ConCorND, we investigated early changes in defined neuronal circuit and examined how those early changes contribute to the development of the pathology. To this end, we examined cerebellar circuit using mouse model of Spinocerebellar ataxia 1 (SCA1). In SCA1, Purkinje cells (PCs) degenerate, causing ataxia and other fatal symptoms. We performed the targeted modification of precisely defined cerebellar circuit in SCA1, and identified that altered inhibiton drives disease pathology. Secondly, we will dissected spatio-temporal kinetics of the morphological changes within synaptic inputs onto PCs. We combined conditional mouse models with pharmacogenetics, followed by transcriptome, proteome, connectome mapping and behavior to unravel disease-associated processes unfolding at neuronal and circuit level in SCA1.
Using Spinocerebellar ataxia 1 (SCA1) as ND model, our goal is to dissect how early alterations in cerebellar circuits at a structural, molecular and functional level influence and drive the pathological responses in vulnerable PCs.
Aim 1
We identified alteration of the inhibitory component of cerebellar circuitry that led to an increased inhibitory tone onto PCs in Sca1. Based on these observations, we collaborated with Liebscher group at MLU, Munich and combining in vivo two-photon imaging in behaving Sca1 mice together with structural and molecular layer interneuron (MLIN)-specific proteomics, we show that MLIN within the cerebellar network are differentially altered. The earliest deficits are observed within sensory-motor integration as well as brain state-dependent activity changes in the mutant SCA1 cerebellum. Chemogenetic tools were used to modulate MLIN activity, which confirmed that mimicking disease-associated activity changes in MLIN are sufficient to induce SCA1-patholgoy in wildtype mice. MLIN-selective proteomics after chemogenetic modulation revealed a disease-linked molecular signature, accounting for changes in MLIN functionality in SCA1. These results indicate that at a circuit level, molecular changes within MLIN govern cerebellar network dysfunction and promote PC degeneration in SCA1. The study is under review
Aim 2
Cerebellar climbing fibers (CFs) are the main excitatory inputs onto PCs. In SCA1, CFs maturation is impaired that could lead to altered functional and signaling responses within PCs. Using confocal imaging and 3D reconstruction of CFs onto PCs, revealed an increased number of synaptic contacts at early stages of development in SCA1 cerebellar slices, compared to WT. This alteration is accompanied by modification of excitatory transmission at CF to PC synapses. Ex vivo electrophysiological recordings in acute cerebellar slices revealed a pronounced response to CF electrical stimulation on PCs in SCA1 compared to WT. Moreover, we related the observed deficit to higher levels of the flop variant of the GluR2 AMPA receptor, which is known to desensitize faster than GluR2 flip counterpart, These findings indicate developmental deficts in SCA1 circuit, leading to a lasting pathological trace on the adult SCA1 cerebellum. The manuscript is in preparation
Aim 3
To characterize the molecular fingerprints of PCs at developmental stages, we performed mass spectrometry on PCs of SCA1 and WT and identified altered TrkB expression. We found that while BDNF expression is high, that of TrkB is reduced. In vivo two-photon imaging, revealed higher Ca2+ amplitude and frequency in mutant PC dendrites as from presymptomatic stage with correspondingly elevated expression of Ca2+ protease calpain-2, leading to the abnormal cleavage of TrkB. Pharmacological inhibition of calpain restored TrkB expression and alleviated pathological hallmarks. Conversely, PC-specific expression of TrkB delayed disease hallmarks and improved behavioral outcome. Moreover, TrkB overexpression normalized the PC pacemaker activity and restored the expression of Ca2+ buffers. Our findings show that both hyperactivity of PC dendrites and mutant ataxin-1 impair TrkB receptor-mediated signaling, leading to the propagation of the SCA1 pathology. This study is in submission
Novel Findings:
We validated our circuit-associated concept in Amyotrophic lateral sclerosis (ALS). We pharmacologically reduced excitatory cholinergic transmission on motor neurons, leading to the induction of ER stress. Proteomic analysis revealed GRP75 as being altered. We provided evidence that the transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function during the initial stage of ALS. An abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived motor neurons led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium uptake impairments. Sustaining high GRP75 expression in spinal neurons specifically prevented ER stress, normalized mitochondrial function and ameliorated ALS-behavioral phenotype. The manuscript is accepted
Establishment of labeling and mass spectrometry analysis of defined inhibitory interneurons
Establishement of human SCA1 patient-derived GABAergic neurons
3D EM and reconstruction of three different cerebellar synapses on PCs