Genetic ablation of inositol 1,4,5-Trisphosphate receptor type 2 (IP3R2) fails to modify disease progression in a mouse model of Spinocerebellar Ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020, PTDC/NEUNMC/3648/2014 and COMPETE-FEDER (POCI-01-0145-FEDER-016818); fellowships to DCG (2021.08121.BD), DMF (SFRH/BD/147947/2019), JSC (SFRH/BD/140624/2018), ANC (SFRH/BPD/118779/2016), AVF (UMINHO/BIL-CNCG/2022/11), SGG (SFRH/BD/101298/2014), and JFV (2020.05109.BD); FCT Scientific Employment Stimulus (CEEC)—Individual Call position to SDS (CEECIND/00685/2020); grants from the Bial Foundation (037/18) and “the la Caixa” Foundation (LCF/PR/HR21/52410024) to JFO; and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also supported by grants from the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122 and national funds through the Foundation for Science and Technology (FCT)

A comprehensive characterization of cerebellar pathology in an animal model of sca3

Dissertação de mestrado em Ciências da SaúdeO cerebelo é uma estrutura cerebral crucial com um papel definido no funcionamento motor, sendo responsável pela coordenação motora, postura e manutenção do equilíbrio. A disfunção cerebelar manifesta-se frequentemente em movimentos descoordenados e desequilíbrio (ataxia), que afeta negativamente atividades diárias. A ataxia hereditária dominante mais prevalente em todo o mundo é a Ataxia Espinocerebelosa Tipo 3 (SCA3), uma doença neurodegenerativa progressiva, neuropatologicamente caracterizada por perda neuronal em regiões específicas, para a qual ainda não existe um tratamento eficaz. Em modelos de murganho de SCA3 - incluindo o modelo transgénico usado neste estudo - CMVMJD135, a sintomatologia motora é observada antes de ocorrer manifestações neuropatológicas passiveis de ser detetadas. Evidências de disfunção neuronal, particularmente no cerebelo - uma área afetada na SCA3 tem sido recentemente relatadas, juntamente com evidências de anormalidades fisiológicas, morfológicas e moleculares nas células de Purkinje (CP), os neurónios eferentes do cerebelo. No entanto, a contribuição da disfunção cerebelar para a fisiopatologia da SCA3 permanece por elucidar. No CMVMJD135 - um modelo animal bem estabelecido da doença, as CP apresentam defeitos eletrofisiológicos que acompanham os défices motores, verificando-se antes de degeneração cerebelar evidente. Para compreender as alterações cerebelares (e das CP) que contribuem para a patogénese da SCA3, investigámos a arquitetura das CP e dos seus aferentes sinápticos em murganhos CMVMJD135 numa fase pós-sintomática da doença. Este estudo revelou que, murganhos transgénicos apresentam alterações arquitetónicas neuronais e sinápticas nas mesmas regiões onde foram anteriormente relatados distúrbios electrofisiológicos, nomeadamente uma redução acentuada da complexidade dendrítica de CP detetada através da coloração de Golgi-Cox, e um aumento na inervação das Climbing Fibers, observada por meio de imunomarcação para o transportador vesicular de glutamato 2. A análise de microscopia eletrónica de transmissão da ulta-estrutura da sinapse cerebelar revelou um aumento da largura da fenda sináptica nos animais transgénicos. Além disso, estendemos o nosso conhecimento relativamente aos mecanismos envolvidos na disfunção cerebelar através de uma análise proteómica quantitativa do cerebelo, que sugeriu o envolvimento de vias nucleares e sinápticas, incluindo alterações metabólicas e do citoesqueleto, como potencialmente contribuindo para a disfunção cerebelar no modelo CMVMJD135 da SCA3. Este estudo destaca alterações morfológicas e moleculares potencialmente relevantes para a doença que poderão ser usadas como biomarcadores em estudos pré-clínicos, e que poderão constituir novos alvos terapêuticos promissores.The cerebellum is a crucial brain structure with a well-established role in motor functioning, being accountable for motor coordination, posture, and balance maintenance. Cerebellar dysfunction commonly manifests through uncoordinated movement and imbalance (ataxia), which detrimentally impacts daily activities. The most prevalent dominantly inherited ataxia worldwide is Spinocerebellar ataxia type 3 (SCA3), a progressive neurodegenerative disorder neuropathologically characterized by neuronal loss in specific brain regions, for which a disease-modifying treatment is still lacking. In SCA3 mouse models - including the CMVMJD135 transgenic mouse herein used, the motor-related symptomatology is observed before any gross pathology. Accumulating evidence of neuronal dysfunction, particularly at the cerebellum - a key SCA3 affected area-, has revealed physiological, morphological, and molecular abnormalities in the sole output neurons of the cerebellum, the Purkinje Cells (PC). However, how cerebellar (and PC) dysfunction contributes to SCA3 pathophysiology remains poorly understood. In the CMVMJ135 transgenic mouse model – a well-established mouse model of the disease, region dependent PC electrophysiological defects accompany motor impairments before overt cerebellar degeneration. To deepen our understanding of cerebellar (and PC) abnormalities contributing to SCA3 pathogenesis, we investigated PC and synaptic afferents architecture in late-symptomatic CMVMJD135 mice (where the phenotype is fully established). Our work unveiled that fully-symptomatic transgenic mice present neuronal and synaptic architectural changes in the same regions where physiological disturbances were previously reported, namely a marked reduction of dendritic complexity of PC detected through Golgi-Cox staining and a marked increase in Climbing Fiber innervation, observed through immunostaining for the vesicular glutamate transporter 2. Moreover, transmission electron microscopy analysis of the cerebellar synapse ultrastructure unveiled a significant increase of synaptic cleft width in transgenic mice. Furthermore, we extended our knowledge on the mechanisms involved in cerebellar pathology through an unbiased quantitative proteomic analysis of this brain region, which suggested the involvement of nuclear and synaptic-related pathways, including metabolic and cytoskeletal changes, as potentially contributing to cerebellar dysfunction in late-symptomatic SCA3 mice. Overall, this study highlights potential disease-relevant morphological and molecular readouts that can be used as biomarkers in pre-clinical studies and pinpoint molecular perturbations underlying disease, which may provide promising therapeutic targets for future drug development.The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho, and at The Centre for Neuroscience and Cell Biology (CNC). Financial support was provided by grants from the National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020. This work has also been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI – Portuguese Platform of Bioimaging PPBI-POCI-01-0145- FEDER-022122