Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. METHODS: The expression of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. RESULTS: In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1ß-induced (immuno)proteasome gene expression could be attenuated by rapamycin. SIGNIFICANCE: Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy.

Insulin-Degrading Enzyme Efficiently Degrades polyQ Peptides but not Expanded polyQ Huntingtin Fragments.

Background: Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. Objective: A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Methods: Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. Results: We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. Conclusions: This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments.

Ubiquitin-modifying enzymes in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the N-terminus of the HTT gene. The CAG repeat expansion translates into a polyglutamine expansion in the mutant HTT (mHTT) protein, resulting in intracellular aggregation and neurotoxicity. Lowering the mHTT protein by reducing synthesis or improving degradation would delay or prevent the onset of HD, and the ubiquitin-proteasome system (UPS) could be an important pathway to clear the mHTT proteins prior to aggregation. The UPS is not impaired in HD, and proteasomes can degrade mHTT entirely when HTT is targeted for degradation. However, the mHTT protein is differently ubiquitinated when compared to wild-type HTT (wtHTT), suggesting that the polyQ expansion affects interaction with (de) ubiquitinating enzymes and subsequent targeting for degradation. The soluble mHTT protein is associated with several ubiquitin-modifying enzymes, and various ubiquitin-modifying enzymes have been identified that are linked to Huntington's disease, either by improving mHTT turnover or affecting overall homeostasis. Here we describe their potential mechanism of action toward improved mHTT targeting towards the proteostasis machinery.

Reduction in PA28αß activation in HD mouse brain correlates to increased mHTT aggregation in cell models.

Huntington's disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αß disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αß activated proteasomes in various in vitro models showed that PA28αß improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αß in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αß is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

Regulation of Proteasome Activity by (Post-)transcriptional Mechanisms.

Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ensure proper protein homeostasis. The proteasome is responsible for the degradation of the majority of intracellular proteins, which are often targeted for degradation via polyubiquitination. However, the degradation rate of proteins is also affected by the capacity of proteasomes to recognize and degrade these substrate proteins. This capacity is regulated by a variety of proteasome modulations including (1) changes in complex composition, (2) post-translational modifications, and (3) altered transcription of proteasomal subunits and activators. Various diseases are linked to proteasome modulation and altered proteasome function. A better understanding of these modulations may offer new perspectives for therapeutic intervention. Here we present an overview of these three proteasome modulating mechanisms to give better insight into the diversity of proteasomes.

Co-administration of Anti microRNA-124 and -137 Oligonucleotides Prevents Hippocampal Neural Stem Cell Loss Upon Non-convulsive Seizures.

Convulsive seizures promote adult hippocampal neurogenesis (AHN) through a transient activation of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). However, in a significant population of epilepsy patients, non-convulsive seizures (ncSZ) are observed. The response of NSPCs to non-convulsive seizure induction has not been characterized before. We here studied first the short-term effects of controlled seizure induction on NSPCs fate and identity. We induced seizures of controlled intensity by intrahippocampally injecting increasing doses of the chemoconvulsant kainic acid (KA) and analyzed their effect on subdural EEG recordings, hippocampal structure, NSPC proliferation and the number and location of immature neurons shortly after seizure onset. After establishing a KA dose that elicits ncSZ, we then analyzed the effects of ncSZ on NSPC proliferation and NSC identity in the hippocampus. ncSZ specifically triggered neuroblast proliferation, but did not induce proliferation of NSPCs in the SGZ, 3 days post seizure onset. However, ncSZ induced significant changes in NSPC composition in the hippocampus, including the generation of reactive NSCs. Interestingly, intrahippocampal injection of a combination of two anti microRNA oligonucleotides targeting microRNA-124 and -137 normalized neuroblast proliferation and prevented NSC loss in the DG upon ncSZ. Our results show for the first time that ncSZ induce significant changes in neuroblast proliferation and NSC composition. Simultaneous antagonism of both microRNA-124 and -137 rescued seizure-induced alterations in NSPC, supporting their coordinated action in the regulation of NSC fate and proliferation and their potential for future seizure therapies.