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Connectivity Correlate of Molecular Pathology in Neurodegeneration

Periodic Reporting for period 3 - ConCorND (Connectivity Correlate of Molecular Pathology in Neurodegeneration)

Reporting period: 2020-06-01 to 2021-11-30

To this day, mechanisms underlying the major neurodegenerative diseases remain poorly understood. Focus on identifying mechanisms determining selective neuronal vulnerability have shed light on cell autonomous aspects of the pathology, however this extensive effort has not yet materialized into a valid therapy for degenerative diseases of the nervous system. An intriguing but universal observation in patients as well as in preclinical models of these diseases is the early and selective alterations in intrinsic neuronal excitability properties as well as in its neuronal circuitry that is involved in maintaining excitation-inhibition balance of the central nervous system. The reason behind the early alterations in neuronal excitability and whether these alterations directly regulate pathomechanisms associated with degeneration or whether they are complex compensatory responses occurring during the disease course remains to be investigated. Moreover, the precise contribution of altered circuits to the disease remains unelucidated and challenging due to the long and largely silent presymptomatic phase of the disease. The recent generation of genetic tools that allows us to modify the activity of specific neuronal components within a neuronal network, now enables us to perform experiments where we can prematurely modify the activity or alter excitation-inhibition balance and correlate those modifications with onset of disease and pathological hallmarks in preclinical models. Notably, such an approach will be used by us to identify circuit associated signaling pathways, which are prematurely impaired in disease and those will be further targeted via gene therapy to provide neuroprotection.

Neurodegenerative disorders 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, neurodegenerative diseases remain incurable and currently there are no therapeutic options to slow down the progression of these disease or associated symptoms.

In the grant proposal ConCorND, we will investigate early changes in a defined neuronal circuit and examine how those early changes contribute to the later development of the neurodegenerative pathology. To this end, we propose to work on the cerebellar circuit using neurodegenerative model of Spinocerebellar ataxia 1 (SCA1). In SCA1, Purkinje cell selectively degenerate, causing the appearance of ataxia and other fatal symptoms. Herein, we will perform the targeted modification of precisely defined the cerebellar circuit in SCA1 in order to identify and untangle the precise process that drives disease from compensatory mechanisms occurring during the disease course. Secondly, we will specifically label cerebellar synapses and using electron microscopy dissect spatio-temporal kinetics of the morphological changes within synaptic inputs onto degenerating Purkinje cells. For this, we will combine 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 the cerebellum of murine models of SCA1.
Work Period June 2020-November 2021

Using Spinocerebellar ataxia 1 (SCA1) as neurodegenerative disease 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 Purkinje cells (PCs).
To this end until now, we have performed the following experiments
1) Characterization of the timing of the earliest cerebellar circuit deficits during development and identification of molecules involved in those deficits combining confocal imaging and electrophysiology. Notably, in SCA1during development, the flop variant of the GluR2 AMPA receptor is dominant and causes faster desensitization than its flip counterpart, which is dominant in the WT cerebellum. We correlated this to the higher increase in glutamate transporters as well as show that overexpressing the flop variant of GluR2 during a critical time window of development is neuroprotective. Binda F et al., manuscript in preparation.

2) Mass Spectrometry analysis of the PC soma and cerebellar molecular layer identified the impact of dysregulated PCs Ca2+ homeostasis on TrkB signaling. In collaboration with Liebscher lab (LMU Munich) in vivo two photon Ca2+ imaging in PC spines revealed a higher portion of active SCA1 spines compared to WT mice. This Ca2+ overload in mutant PC spines lead to the activation of Calpain-2, a cytosolic protease, which aberrantly cleaved and attenuated TrkB signaling in SCA1. Chronic inhibition of Calpain-2 activity in SCA1 mice sustains TrkB signalling, thereby delaying pathology. Pernaci C et al. submitted.
Carla Pernaci will defend her PhD thesis in March 2022.

3) Combining in vivo two photon imaging, chemogenetics, electrophysiology and proteomics, we identify that within the different cerebellar neuronal population, the molecular layer interneurons (MLI) are significantly more active in SCA1 mice when compared to WT, even though the MLIs do not degenerate. Further, we provide proof of concept that major inhibitory tone from the MLIs to the PCs directly impairs motor coordination. We identify molecular correlates within MLIs that render them hyperactive and validate those in human SCA1 patient-derived interneurons. Pilotto F, Douthwaite C, et al manuscript in preparation.

4) Importantly, we applied our hypothesis associated with a causal role for early circuit dysfunction to yet another neurodegenerative disease i.e. Amyotrophic lateral sclerosis and found critical signaling mechanisms involving ER-mitochondria tether in deaccelerating the disease kinetics. Pilotto F, et al., under revision.
Federica Pilotto will defend her PhD thesis in June 2022.
Establishment of a collaboration with Dr. Sabine Liebscher for in vivo calcium imaging in awake and behaving SCA1 mice. Several important data sets have all been obtained which not only consolidate our chemogenetic circuit modulation data but also provide insights into global network changes as disease progresses
We are currently consolidating this project by examining how sensory and motor inputs are integrated within the cerebellum and if those are impaired during the silent presymptomatic phase of the disease. We are confident to have two publications from these collaborations.
We have also established routine electrophysiology in the lab on cerebellar slices and are now expanding into in vivo recordings and intend to study the association of the cerebellum in cognition and two manuscripts have been generated.
Further, our observation of developmental deficits in cerebellar circuits has opened a new direction of research, where besides neurodegeneration we explore the role of these deficits in neurodevelopmental disorders. Social interaction experiments are ongoing in the lab.
Reconstructed single cerebellar Purkinje Cell