Dataset Information

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

ABSTRACT: Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

SUBMITTER: Schulze-Krebs A

PROVIDER: S-EPMC8396294 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

Schulze-Krebs Anja A Canneva Fabio F Stemick Judith J Plank Anne-Christine AC Harrer Julia J Bates Gillian P GP Aeschlimann Daniel D Steffan Joan S JS von Hörsten Stephan S

International journal of molecular sciences 20210818 16

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic r ...[more]

PMID: 34445621

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Project description:Huntington Disease (HD) is a dominantly inherited, relentlessly progressive neurodegenerative disease. Caused by a polyglutamine expansion in the mutant huntingtin protein (mhtt), HD pathogenesis impairs function in the cerebral cortex and in medium spiny neurons of the striatum and involves transcriptional dysregulation of a number of genes. Of these genes, silencing of genes related to mitochondrial function is believed to explain metabolic dysfunction in rodent models of HD. Here we show that transglutaminase 2 (TG2), which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes. TG2 inhibition by RNA knockdown, genetic deletion, or administration of a novel irreversible, peptide-based TG2 inhibitor (ZDON) de-repressed two established regulators of mitochondrial function, PGC-1? and cytochrome c in a cell model of HD. TG2 must localize to non-coding or coding regions of these mitochondrial metabolic genes to silence their transcription. As expected, TG2 inhibition reversed the increased susceptibility of HD mouse cells and human HD myoblasts to the mitochondrial toxin, 3-nitroproprionic acid (3-NP); however, protection mediated by TG2 inhibition was not associated with improved mitochondrial bioenergetics. Indeed, an unbiased array analysis indicated that TG2 inhibition leads to normalization of not only mitochondrial genes but of nearly 40% of genes that are dysregulated in HD mouse striatal neurons, including chaperone and histone genes. Indeed, TG2 interacts directly with Histone 3 in the nucleus. Moreover, TG2 inhibition significantly attenuated photoreceptor degeneration in a Drosophila model of HD and protected mouse HD striatal neurons (YAC128) from NMDA- induced toxicity. Altogether these findings demonstrate that TG2 mediates its deleterious effects in HD by contributing to broad transcriptional dysregulation of genes representing many cellular functions. These studies define a novel HDAC-independent epigenetic strategy for treating neurodegeneration. To evaluate the effect of TG2 inhibition (treatment with ZDON, Boc-DON, CystamineA, and Control) in striatal cell lines from a transgenic model of Huntington disease (Q111) vs. control (Q7). One sample (WT.boc.3, 4068264015_G) failed the QC test and was excluded from further analyses.

Project description:The precise degradation of dysfunctional mitochondria by mitophagy is essential for maintaining neuronal homeostasis. HTT (huntingtin) can interact with numerous other proteins and thereby perform multiple biological functions within the cell. In this study, we investigated the role of HTT during mitophagy and analyzed the impact of the expansion of its polyglutamine (polyQ) tract. HTT is involved in different mitophagy steps, promoting the physical proximity of different protein complexes during the initiation of mitophagy and recruiting mitophagy receptors essential for promoting the interaction between damaged mitochondria and the nascent autophagosome. The presence of the polyQ tract in mutant HTT affects the formation of these protein complexes and determines the negative consequences of mutant HTT on mitophagy, leading to the accumulation of damaged mitochondria and an increase in oxidative stress. These outcomes contribute to general mitochondrial dysfunction and neurodegeneration in Huntington disease.Abbreviations: AMPK: AMP-activated protein kinase; ATG13: autophagy related 13; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene-diamine-tetra-acetic acid; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; FUNDC1: FUN14 domain containing 1; HD: Huntington disease; HRP: horseradish peroxidase; HTT: huntingtin; LC3-II: lipidated form of MAP1LC3/LC3; mtDNA: mitochondrial deoxyribonucleic acid; MTDR: MitoTracker Deep Red; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1, autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; OCR: oxygen consumption rate; OPTN: optineurin; OXPHOS: oxidative phosphorylation; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PINK1: PTEN induced putative kinase 1; PLA: proximity ligation assay; PMSF: phenylmethylsulfonyl fluoride; polyQ: polyglutamine; PtdIns3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; Rot: rotenone; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TMRM: tetramethylrhodamine methyl ester; UB: ubiquitin; ULK1: unc-51 like kinase 1.

Project description:Huntington Disease (HD) is a dominantly inherited, relentlessly progressive neurodegenerative disease. Caused by a polyglutamine expansion in the mutant huntingtin protein (mhtt), HD pathogenesis impairs function in the cerebral cortex and in medium spiny neurons of the striatum and involves transcriptional dysregulation of a number of genes. Of these genes, silencing of genes related to mitochondrial function is believed to explain metabolic dysfunction in rodent models of HD. Here we show that transglutaminase 2 (TG2), which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes. TG2 inhibition by RNA knockdown, genetic deletion, or administration of a novel irreversible, peptide-based TG2 inhibitor (ZDON) de-repressed two established regulators of mitochondrial function, PGC-1α and cytochrome c in a cell model of HD. TG2 must localize to non-coding or coding regions of these mitochondrial metabolic genes to silence their transcription. As expected, TG2 inhibition reversed the increased susceptibility of HD mouse cells and human HD myoblasts to the mitochondrial toxin, 3-nitroproprionic acid (3-NP); however, protection mediated by TG2 inhibition was not associated with improved mitochondrial bioenergetics. Indeed, an unbiased array analysis indicated that TG2 inhibition leads to normalization of not only mitochondrial genes but of nearly 40% of genes that are dysregulated in HD mouse striatal neurons, including chaperone and histone genes. Indeed, TG2 interacts directly with Histone 3 in the nucleus. Moreover, TG2 inhibition significantly attenuated photoreceptor degeneration in a Drosophila model of HD and protected mouse HD striatal neurons (YAC128) from NMDA- induced toxicity. Altogether these findings demonstrate that TG2 mediates its deleterious effects in HD by contributing to broad transcriptional dysregulation of genes representing many cellular functions. These studies define a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Project description:Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. Expanded mutant HTT (mHTT) is toxic and leads to regional atrophy and neuronal cell loss in the brain, which occurs earliest in the striatum. Therapeutic lowering of mHTT in the central nervous system (CNS) has shown promise in preclinical studies, with multiple approaches currently in clinical development for HD. Quantitation of mHTT in the cerebrospinal fluid (CSF) is being used as a clinical pharmacodynamic biomarker of target engagement in the CNS. We have previously shown that the CNS is a major source of mHTT in the CSF. However, little is known about the specific brain regions and cell types that contribute to CSF mHTT. Therefore, a better understanding of the origins of CSF mHTT and whether therapies targeting mHTT in the striatum would be expected to be associated with significant lowering of mHTT in the CSF is needed. Here, we use complementary pharmacological and genetic-based approaches to either restrict expression of mHTT to the striatum or selectively deplete mHTT in the striatum to evaluate the contribution of this brain region to mHTT in the CSF. We show that viral expression of a mHTT fragment restricted to the striatum leads to detectable mHTT in the CSF. We demonstrate that targeted lowering of mHTT selectively in the striatum using an antisense oligonucleotide leads to a significant reduction of mHTT in the CSF of HD mice. Furthermore, using a transgenic mouse model of HD that expresses full length human mHTT and wild type HTT, we show that genetic inactivation of mHTT selectively in the striatum results in a significant reduction of mHTT in the CSF. Taken together, our data supports the conclusion that the striatum contributes sufficiently to the pool of mHTT in the CSF that therapeutic levels of mHTT lowering in the striatum can be detected by this measure in HD mice. This suggests that CSF mHTT may represent a pharmacodynamic biomarker for therapies that lower mHTT in the striatum.

Project description:Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Dataset Information

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

Publications

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

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