Progressive mitochondrial dysfunction in cerebellar synaptosomes of cystatin B-deficient mice.

The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model (Cstb-/-) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-of-function mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic (Cstb-/-) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in (Cstb-/-) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1.

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

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.

A patient with pontocerebellar hypoplasia type 6: Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome.

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.