Gene Expression Alterations in the Cerebellum and Granule Neurons of Cstb−/− Mouse Are Associated with Early Synaptic Changes and Inflammation

Abstract

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited neurodegenerative disease, manifesting with myoclonus, seizures and ataxia, caused by mutations in the cystatin B (CSTB) gene. With the aim of understanding the molecular basis of pathogenetic events in EPM1 we characterized gene expression changes in the cerebella of pre-symptomatic postnatal day 7 (P7) and symptomatic P30 cystatin B -deficient (Cstb−/−) mice, a model for the disease, and in cultured Cstb−/− cerebellar granule cells using a pathway-based approach. Differentially expressed genes in P7 cerebella were connected to synaptic function and plasticity, and in cultured cerebellar granule cells, to cell cycle, cytoskeleton, and intracellular transport. In particular, the gene expression data pinpointed alterations in GABAergic pathway. Electrophysiological recordings from Cstb−/− cerebellar Purkinje cells revealed a shift of the balance towards decreased inhibition, yet the amount of inhibitory interneurons was not declined in young animals. Instead, we found diminished number of GABAergic terminals and reduced ligand binding to GABAA receptors in Cstb−/− cerebellum. These results suggest that alterations in GABAergic signaling could result in reduced inhibition in Cstb−/− cerebellum leading to the hyperexcitable phenotype of Cstb−/− mice. At P30, the microarray data revealed a marked upregulation of immune and defense response genes, compatible with the previously reported early glial activation that precedes neuronal degeneration. This further implies the role of early-onset neuroinflammation in the pathogenesis of EPM1.

Brain inflammation is accompanied by peripheral inflammation in Cstb −/− mice, a model for progressive myoclonus epilepsy

“…As the expression and secretion of chemokines have previously been shown to be altered in cerebellar tissue and primary microglia of Cstb −/− mice [8, 23, 24], we focused our further analyses on brain expression of chemokines CXCL1, CXCL10, and CXCL13, which were increased in the sera of mice at P30. Using immunohistochemistry in Cstb −/− and control mice, we did not detect expression of CXCL1 and only low level of CXCL10 at P14 and P30 (data not shown).…”

“…Expression of CXCL13, which binds CXCR4 receptor and regulates B cell migration [32], has been reported to be enhanced in inflammatory CNS diseases, such as multiple sclerosis and encephalitis [37–40]. Our previous gene expression analysis of P30 Cstb −/− cerebellar tissue revealed a striking (29-fold) upregulation of Cxcl13 [24]. On the contrary, in transcriptomics profiling of in vitro-cultured Cstb −/− microglia, a slight downregulation of Cxcl13 was observed [23], suggesting that the CXCL13 upregulation in Cstb −/− microglia might be specific to the brain in vivo.…”

“…Gene expression profiling of cultured Cstb −/− microglia revealed impaired interferon signaling and also showed altered chemokine expression [23]. Finally, a striking upregulation of Cxcl13 in gene expression profiling of postnatal day 30 (P30) Cstb −/− mouse cerebellum was detected [24]. …”

Immunity and Inflammation in Epilepsy

“…1). Glia activation occurs also in genetic models of epilepsy, such as in rats with spike-and-wave discharges mimicking absence seizures (Akin et al 2011), models of tuberous sclerosis (Wong and Crino 2012), and progressive myoclonus epilepsy of Unverricht -Lundborg type 1 (Tegelberg et al 2012;Joensuu et al 2014). Notably, glia activation occurs during epileptogenesis (i.e., the phase that precedes the onset of the disease and accompanies its progression) (Pitkanen and Engel 2014) both in symptomatic and genetic epilepsy models, and is maintained in the chronic epilepsy phase (when the disease is established).…”

Unverricht-Lundborg Disease: A Case Report and Literature Review

“…CSTB is a cysteine protease inhibitor and controls lysosomal cathepsins [25]. Neurodegeneration has a critical role in PME1 pathogenesis and in PME1 patients' lymphoblastoid cell lines increased catepsin activity were showed resulting in increased sensitivity to oxidative stress-induced cell death in the CSTB knock-out mice [25].…”