Rashmi Parihar

The malin–laforin complex suppresses the cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin–proteasome system

Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin-proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin-laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins.

The SCN1A gene variants and epileptic encephalopathies

The voltage-gated sodium channels are fundamental units that evoke the action potential in excitable cells such as neurons. These channels are integral membrane proteins typically consisting of one α-subunit, which forms the larger central pore of the channel, and two smaller auxiliary β-subunits, which modulate the channel functions. Genetic alterations in the SCN1A gene coding for the α-subunit of the neuronal voltage-gated sodium ion channel, type 1 (NaV 1.1), is associated with a spectrum of seizure-related disorders in human, ranging from a relatively milder form of febrile seizures to a more severe epileptic condition known as the Dravet syndrome. Among the epilepsy genes, the SCN1A gene perhaps known to have the largest number of disease-associated alleles. Here we present a meta-analysis on the SCN1A gene variants and provide comprehensive information on epilepsy-associated gene variants, their frequency, the predicted effect on the protein, the ethnicity of the affected along with the inheritance pattern and the associated epileptic phenotype. We also summarize our current understanding on the pathophysiology of the SCN1A gene defects, disease mechanism, genetic modifiers and their clinical and diagnostic relevance.

Decreased O-Linked GlcNAcylation Protects from Cytotoxicity Mediated by Huntingtin Exon1 Protein Fragment

Background: Earlier reports indicate that O-GlcNAcylation might be protective in neurodegenerative disorders. Results: Suppressing O-GlcNAcylation modulates autophagy to enhance the viability of neuronal cells expressing cytotoxic mutant huntingtin exon 1 protein (mHtt). Conclusion: O-GlcNAcylation regulates the clearance of mHtt by modulating the fusion of autophagosomes with lysosomes. Significance: This regulatory mechanism emerges as a novel therapeutic strategy for Huntington disease. O-GlcNAcylation is an important post-translational modification of proteins and is known to regulate a number of pathways involved in cellular homeostasis. This involves dynamic and reversible modification of serine/threonine residues of different cellular proteins catalyzed by O-linked N-acetylglucosaminyltransferase and O-linked N-acetylglucosaminidase in an antagonistic manner. We report here that decreasing O-GlcNAcylation enhances the viability of neuronal cells expressing polyglutamine-expanded huntingtin exon 1 protein fragment (mHtt). We further show that O-GlcNAcylation regulates the basal autophagic process and that suppression of O-GlcNAcylation significantly increases autophagic flux by enhancing the fusion of autophagosome with lysosome. This regulation considerably reduces toxic mHtt aggregates in eye imaginal discs and partially restores rhabdomere morphology and vision in a fly model for Huntington disease. This study is significant in unraveling O-GlcNAcylation-dependent regulation of an autophagic process in mediating mHtt toxicity. Therefore, targeting the autophagic process through the suppression of O-GlcNAcylation may prove to be an important therapeutic approach in Huntington disease.

Satellite III non-coding RNAs show distinct and stress-specific patterns of induction

The heat shock response in human cells is associated with the transcription of satellite III repeats (SatIII) located in the 9q12 locus. Upon induction, the SatIII transcripts remain associated with the locus and recruit several transcription and splicing factors to form the nuclear stress bodies (nSBs). The nSBs are thought to modulate epigenetic changes during the heat shock response. We demonstrate here that the nSBs are induced by a variety of stressors and show stress-specific patterns of induction. While the transcription factor HSF1 is required for the induction of SatIII locus by the stressors tested, its specific role in the transcriptional process appears to be stress dependent. Our results suggest the existence of multiple transcriptional loci for the SatIII transcripts and that their activation might depend upon the type of stressors. Thus, induction of SatIII transcripts appears to be a generic response to a variety of stress conditions.

Spatial positions of homopolymeric repeats in the human proteome and their effect on cellular toxicity

Proteins with homopolymeric repeat tracts are very common in the human proteome. Intriguingly, some but not all repeat tracts show length variation in the population and, in a few, the expansion of repeat tract beyond the normal length is associated with neurodegenerative and developmental disorders. In this study we have addressed questions such as why some amino acid residues are favored in longer repeat tracts and why repeat tracts show terminal bias. Using cell biological assays for repeat tracts fused to green fluorescent protein we show here that homopolymeric repeats that are beyond their naturally occurring length in the proteome are cytotoxic in nature. This toxicity is further modulated by the length of the peptide that bears the repeat and the spatial location of the repeat within the peptide. Thus, the cellular toxicity appears to be one of the selective processes that regulate the evolution of homopolymeric repeats in the proteome.

Human satellite-III non-coding RNAs modulate heat shock-induced transcriptional repression

ABSTRACT The heat shock response is a conserved defense mechanism that protects cells from physiological stress, including thermal stress. Besides the activation of heat-shock-protein genes, the heat shock response is also known to bring about global suppression of transcription; however, the mechanism by which this occurs is poorly understood. One of the intriguing aspects of the heat shock response in human cells is the transcription of satellite-III (Sat3) long non-coding RNAs and their association with nuclear stress bodies (nSBs) of unknown function. Besides association with the Sat3 transcript, the nSBs are also known to recruit the transcription factors HSF1 and CREBBP, and several RNA-binding proteins, including the splicing factor SRSF1. We demonstrate here that the recruitment of CREBBP and SRSF1 to nSBs is Sat3-dependent, and that loss of Sat3 transcripts relieves the heat-shock-induced transcriptional repression of a few target genes. Conversely, forced expression of Sat3 transcripts results in the formation of nSBs and transcriptional repression even without a heat shock. Our results thus provide a novel insight into the regulatory role for the Sat3 transcripts in heat-shock-dependent transcriptional repression. Highlighted Article: The satellite-III non-coding RNAs, expressed only during heat shock in human cells, recruit transcription factors to their nuclear foci, contributing to heat-shock-induced transcriptional repression.

Identification and characterization of novel splice variants of the human EPM2A gene mutated in Lafora progressive myoclonus epilepsy.

The EPM2A gene, defective in the fatal neurodegenerative disorder Lafora disease (LD), is known to encode two distinct proteins by differential splicing; a phosphatase active cytoplasmic isoform and a phosphatase inactive nuclear isoform. We report here the identification of three novel EPM2A splice variants with potential to code for five distinct proteins in alternate reading frames. These novel isoforms, when ectopically expressed in cell lines, show distinct subcellular localization, interact with and serve as substrates of malin ubiquitin ligase-the second protein defective in LD. Two phosphatase active isoforms interact to form a heterodimeric complex that is inactive as a phosphatase in vitro, suggesting an antagonistic function for laforin isoforms if expressed endogenously in significant amounts in human tissues. Thus alternative splicing could possibly be one of the mechanisms by which EPM2A may regulate the cellular functions of the proteins it codes for.

Proline repeats, in cis- and trans-positions, confer protection against the toxicity of misfolded proteins in a mammalian cellular model.

A broad range of neurodegenerative disorders result from the cytotoxicity conferred by aberrantly folded mutant proteins. Intriguingly, the cytotoxicity and aggregation property of a few mutant proteins are known to be modulated by the flanking sequences. One of such modulators is the proline repeat tract. Using a mammalian cellular model, we show here that proline repeat tract, both in cis- and in trans-positions, ameliorate the cytotoxicity of wide range of misfolded proteins coded by synthetic constructs. We further show that the proline repeat tract could possibly confer protection against the cytotoxicity of misfolded proteins by altering their conformation at the time of their synthesis. Thus, our study elucidates the mechanism by which the proline repeat tract might ameliorate the toxicity of misfolded proteins, and opens up new therapeutic modalities for disorders caused by cytotoxic misfolded proteins.

Interdependence of laforin and malin proteins for their stability and functions could underlie the molecular basis of locus heterogeneity in Lafora disease

ABSTRACTLafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the EPM2A gene coding for the laforin protein phosphatase or the NHLRC1 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each other’s function. Using cell biological assays we demonstrate here that (i) malin promotes its own degradation via auto-ubiquitination, (ii) laforin prevents the auto-degradation of malin by presenting itself as a substrate and (iii) malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each other’s stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneity observed in LD.

Association of the GRM4 gene variants with juvenile myoclonic epilepsy in an Indian population.

1Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208 016, India 2Department of Biochemistry, G. R. Medical College, Gwalior 474 009, India 3Nizam’s Institute of Medical Sciences, Banjara Hills, Punjagutta, Hyderabad 500 034, India 4Krishna Institute of Medical Science, Secunderabad 500 003, India 5Department of Neurology, Govind Ballabh Pant Hospital, Jawaharlal Nehru Marg, New Delhi 110 002, India