Quantitative Changes in the Mitochondrial Proteome of Cerebellar Synaptosomes From Preclinical Cystatin B-Deficient Mice.

Katarin Gorski,Tuula A Nyman,Albert Spoljaric,Kai Kaila,Brendan J Battersby,Anna-Elina Lehesjoki

doi:10.3389/fnmol.2020.570640

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disorder caused by loss-of-function mutations in the cystatin B (CSTB) gene. Progression of the clinical symptoms in EPM1 patients, including stimulus-sensitive myoclonus, tonic-clonic seizures, and ataxia, are well described. However, the cellular dysfunction during the presymptomatic phase that precedes the disease onset is not understood. CSTB deficiency leads to alterations in GABAergic signaling, and causes early neuroinflammation followed by progressive neurodegeneration in brains of a mouse model, manifesting as progressive myoclonus and ataxia. Here, we report the first proteome atlas from cerebellar synaptosomes of presymptomatic Cstb-deficient mice, and propose that early mitochondrial dysfunction is important to the pathogenesis of altered synaptic function in EPM1. A decreased sodium- and chloride dependent GABA transporter 1 (GAT-1) abundance was noted in synaptosomes with CSTB deficiency, but no functional difference was seen between the two genotypes in electrophysiological experiments with pharmacological block of GAT-1. Collectively, our findings provide novel insights into the early onset and pathogenesis of CSTB deficiency, and reveal greater complexity to the molecular pathogenesis of EPM1.

Highlights

Progressive myoclonus epilepsy, EPM1 (Unverricht-Lundborg disease; OMIM 254800) is an autosomal recessive neurodegenerative disorder characterized by disease onset between 6 and 16 years, severe stimulus-sensitive, treatment-resistant and physically disabling myoclonus, tonicclonic seizures and ataxia, with cognition essentially preserved (Koskiniemi et al, 1974; Kälviäinen et al, 2008)
Synaptosome Isolation Synaptosomes were isolated from the cerebella of P14-aged Cstb−/− and wt mice (n = 5/genotype, males and females) according to the protocol described by Nolt et al (2011), with slight modifications
To investigate the basis for altered GABAergic signaling in Cstb−/− mice, we analyzed the proteome of cerebellar synaptosomes from presymptomatic P14 Cstb−/− and wt mice by LC-ESI-MS/MS

Summary

INTRODUCTION

Progressive myoclonus epilepsy, EPM1 (Unverricht-Lundborg disease; OMIM 254800) is an autosomal recessive neurodegenerative disorder characterized by disease onset between 6 and 16 years, severe stimulus-sensitive, treatment-resistant and physically disabling myoclonus, tonicclonic seizures and ataxia, with cognition essentially preserved (Koskiniemi et al, 1974; Kälviäinen et al, 2008). Altered GABAergic signaling was first noted in cerebella of presymptomatic postnatal day (P) 7 aged Cstb−/− mice during GABAergic synapse development (Joensuu et al, 2014) This presented as increased expression of the genes encoding for GABAA receptor (GABAAR) subunits α6 and δ in cerebellar tissue lysates, and as an imbalance between excitatory and inhibitory postsynaptic currents (EPSCs; IPSCs), and a complete absence of synchronous IPSC bursts in cerebellar Purkinje cells. At this early age, the number of interneurons in the cerebellum was not altered. The data show a robust alteration to the mitochondrial proteome and factors important for intracellular transport and cytosolic ribosomal biogenesis

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