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Huntington's Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD. Previous studies have implicated DNA damage response in HD pathogenesis. Gene transcription and RNA processing has also emerged as molecular mechanisms associated with HD. Here we used multiple approaches to identify HTT interactors in the context of DNA damage stress. Our results indicate that HTT interacts with many proteins involved in the regulation of interconnected DNA repair/remodeling and RNA processing pathways. We present evidence for a role for HTT in double strand break repair mechanism. We demonstrate HTT functional interaction with a major DNA damage response kinase DNA-PKcs and association of both proteins with nuclear speckles. We show that S1181 phosphorylation of HTT is regulated by DSB, and can be carried out (at least in vitro ) by DNA-PK. Furthermore, we show HTT interactions with RNA binding proteins associated with nuclear speckles, including two proteins encoded by genes at HD modifier loci, TCERG1 and MED15, and with chromatin remodeling complex BAF. These interactions of HTT may position it as an important scaffolding intermediary providing integrated regulation of gene expression and RNA processing in the context of DNA repair mechanisms., Competing Interests: Conflicts of Interest: The authors have no relevant financial or non-financial interests to disclose

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a single mutation in the huntingtin gene (HTT). Normal HTT has a CAG trinucleotide repeat at its N-terminal within the range of 36. However, once the CAG repeats exceed 37, the mutant gene (mHTT) will encode mutant HTT protein (mHTT), which results in neurodegeneration in the brain, specifically in the striatum and other brain regions. Since the mutation was discovered, there have been many research efforts to understand the mechanism and develop therapeutic strategies to treat HD. HTT is a large protein with many post-translational modification sites (PTMs) and can be modified by phosphorylation, acetylation, methylation, sumoylation, etc. Some modifications reduced mHTT toxicity both in cell and animal models of HD. We aimed to find the known kinase inhibitors that can modulate the toxicity of mHTT. We performed an in vitro kinase assay using HTT peptides, which bear different PTM sites identified by us previously. A total of 368 kinases were screened. Among those kinases, cyclin-dependent kinases (CDKs) affected the serine phosphorylation on the peptides that contain S1181 and S1201 of HTT. We explored the effect of CDK1 and CDK5 on the phosphorylation of these PTMs of HTT and found that CDK5 modified these two serine sites, while CDK5 knockdown reduced the phosphorylation of S1181 and S1201. Modifying these two serine sites altered the neuronal toxicity induced by mHTT. Roscovitine, a CDK inhibitor, reduced the p-S1181 and p-S1201 and had a protective effect against mHTT toxicity. We further investigated the feasibility of the use of roscovitine in HD mice. We confirmed that roscovitine penetrated the mouse brain by IP injection and inhibited CDK5 activity in the brains of HD mice. It is promising to move this study to in vivo for pre-clinical HD treatment.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues-very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by mutant huntingtin (mHTT). Phosphorylation at serine-421 (pS421) of mHTT has been shown to be neuroprotective in cellular and rodent models. However, the genetic context of these models differs from that of HD patients. Here we employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, we generated isogenic hiPSC lines in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phospho-resistant alanine (S421A). We observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although we observed no effect of S421D on mHTT clearance or axonal transport, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, our analysis revealed modulation of several aspects of mitochondrial form and function. These include mitochondrial surface area, volume, and counts, as well as improved mitochondrial membrane potential and oxidative phosphorylation. Our study validates the protective role of pS421 on mHTT and highlights a facet of the relationship between mHTT and mitochondrial changes in the context of human physiology with potential relevance to the pathogenesis of HD.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, is highly expressed in the brain, but its function in the adult brain remains not well understood. In this study, we identify NLK as an interactor of huntingtin protein (HTT). We report that NLK levels are significantly decreased in HD human brain and HD models. Importantly, overexpression of NLK in the striatum attenuates brain atrophy, preserves striatal DARPP32 levels and reduces mutant HTT (mHTT) aggregation in HD mice. In contrast, genetic reduction of NLK exacerbates brain atrophy and loss of DARPP32 in HD mice. Moreover, we demonstrate that NLK lowers mHTT levels in a kinase activity-dependent manner, while having no significant effect on normal HTT protein levels in mouse striatal cells, human cells and HD mouse models. The NLK-mediated lowering of mHTT is associated with enhanced phosphorylation of mHTT. Phosphorylation defective mutation of serine at amino acid 120 (S120) abolishes the mHTT-lowering effect of NLK, suggesting that S120 phosphorylation is an important step in the NLK-mediated lowering of mHTT. A further mechanistic study suggests that NLK promotes mHTT ubiquitination and degradation via the proteasome pathway. Taken together, our results indicate a protective role of NLK in HD and reveal a new molecular target to reduce mHTT levels., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

The pathogenesis of HD and HDL2, similar progressive neurodegenerative disorders caused by expansion mutations, remains incompletely understood. No systematic quantitative proteomics studies, assessing global changes in HD or HDL2 human brain, were reported. To address this deficit, we used a stable isotope labeling-based approach to quantify the changes in protein abundances in the cortex of 12 HD and 12 control cases and, separately, of 6 HDL2 and 6 control cases. The quality of the tissues was assessed to minimize variability due to post mortem autolysis. We applied a robust median sweep algorithm to quantify protein abundance and performed statistical inference using moderated test statistics. 1211 proteins showed statistically significant fold changes between HD and control tissues; the differences in selected proteins were verified by Western blotting. Differentially abundant proteins were enriched in cellular pathways previously implicated in HD, including Rho-mediated, actin cytoskeleton and integrin signaling, mitochondrial dysfunction, endocytosis, axonal guidance, DNA/RNA processing, and protein transport. The abundance of 717 proteins significantly differed between control and HDL2 brain. Comparative analysis of the disease-associated changes in the HD and HDL2 proteomes revealed that similar pathways were altered, suggesting the commonality of pathogenesis between the two disorders.

Abnormal protein interactions of mutant huntingtin (Htt) triggered by polyglutamine expansion are thought to mediate Huntington's disease (HD) pathogenesis. Here, we explored a functional interaction of Htt with protein arginine methyltransferase 5 (PRMT5), an enzyme mediating symmetrical dimethylation of arginine (sDMA) of key cellular proteins, including histones, and spliceosomal Sm proteins. Gene transcription and RNA splicing are impaired in HD. We demonstrated PRMT5 and Htt interaction and their co-localization in transfected neurons and in HD brain. As a result of this interaction, normal (but to a lesser extend mutant) Htt stimulated PRMT5 activity in vitro. SDMA of histones H2A and H4 was reduced in the presence of mutant Htt in primary cultured neurons and in HD brain, consistent with a demonstrated reduction in R3Me2s occupancy at the transcriptionally repressed promoters in HD brain. SDMA of another PRMT5 substrate, Cajal body marker coilin, was also reduced in the HD mouse model and in human HD brain. Finally, compensation of PRMT5 deficiency by ectopic expression of PRMT5/MEP50 complexes, or by the knock-down of H4R3Me2 demethylase JMJD6, reversed the toxic effects of mutant Htt in primary cortical neurons, suggesting that PRMT5 deficiency may mediate, at least in part, HD pathogenesis. These studies revealed a potential new mechanism for disruption of gene expression and RNA processing in HD, involving a loss of normal function of Htt in facilitation of PRMT5, supporting the idea that epigenetic regulation of gene transcription may be involved in HD and highlighting symmetric dimethylation of arginine as potential new therapeutic target.

Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine repeat within the HD gene product, huntingtin. Huntingtin, a large (347 kDa) protein containing multiple HEAT repeats, acts as a scaffold for protein-protein interactions. Huntingtin-induced toxicity is believed to be mediated by a conformational change in expanded huntingtin, leading to protein misfolding and aggregation, aberrant protein interactions and neuronal cell death. While many non-systematic studies of huntingtin interactions have been reported, they were not designed to identify and quantify the changes in the huntingtin interactome induced by polyglutamine expansion. We used tandem affinity purification and quantitative proteomics to compare and quantify interactions of normal or expanded huntingtin isolated from a striatal cell line. We found that proteins preferentially interacting with expanded huntingtin are enriched for intrinsically disordered proteins, consistent with previously suggested roles of such proteins in neurodegenerative disorders. Our functional analysis indicates that proteins related to energy production, protein trafficking, RNA post-transcriptional modifications and cell death were significantly enriched among preferential interactors of expanded huntingtin. Expanded huntingtin interacted with many mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD. Furthermore, expanded huntingtin interacted with the stress granule-associated proteins Caprin-1 and G3BP and redistributed to RNA stress granules under ER-stress conditions. These data demonstrate that a number of key cellular functions and networks may be disrupted by abnormal interactions of expanded huntingtin and highlight proteins and pathways that may be involved in HD cellular pathogenesis and that may serve as therapeutic targets.

Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.

N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. The mechanisms of polyglutamine neurotoxicity, and cellular responses are not fully understood. We have studied gene expression profiles by short oligo array using an inducible PC12 cell model expressing an N-terminal huntingtin fragment with expanded polyglutamine (Htt-N63-148Q). Mutant huntingtin Htt-N63 induced cell death and increased the mRNA and protein levels of activating transcription factor 3 (ATF3). Mutant Htt-N63 also significantly enhanced ATF3 transcriptional activity by a promoter-based reporter assay. Overexpression of ATF3 protects against mutant Htt-N63 toxicity and knocking down ATF3 expression reduced Htt-N63 toxicity in a stable PC12 cell line. These results indicated that ATF3 plays a critical role in toxicity induced by mutant Htt-N63 and may lead to a useful therapeutic target.

Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.

Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington's disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.

GDF-8 is a negative regulator of skeletal muscle mass. The mechanisms which regulate the biological activity of GDF-8 have not yet been elucidated. Analogous to the TGF-beta system, GDF-8 propeptide binds to and inhibits the activity of GDF-8. In these studies, we define the critical domain of the GDF-8 propeptide necessary for inhibitory activity. Two molecules of GDF-8 propeptide monomer inhibit the biological activity of one molecule of GDF-8 homodimer. Although the propeptide contains N-linked glycosylation when synthesized in mammalian cells, this glycosylation is not necessary for the inhibition of GDF-8. Taking advantage of the bacterial expression system, we express and purify GDF-8 propeptide which retains full inhibitory activity. To define the functional regions of the propeptide, we express a series of truncated GST-propeptide fusion proteins and examined their inhibitory activity. We observe that fusion proteins containing the C-terminal region (amino acid residues 99-266) are very stable, but do not exhibit inhibitory activity; while fusion proteins containing the N-terminal region (amino acid residues 42-115) are labile but contain essential inhibitory activity. The data suggest that the C-terminal region may play a role in the stability of the GDF-8 propeptide and that the inhibitory domain is located in the region between amino acids 42 and 115.

The activities of presenilin 1 (PS1) and 2 modulate the proteolytic processing of amyloid precursor proteins to produce Abeta1-42, and mutations in these proteins are associated with an accelerated rate of Abeta deposition. PS1 and PS2 themselves are subject to a highly-regulated endoproteolytic cleavage to generate stable 27 kDa N-terminal and 17 kDa C-terminal fragments. Here, we examined the relationship between the regulated cleavage of PS1 and the acceleration of Abeta deposition in transgenic mice that co-express Mo/Hu APPswe and varied levels mutant PS1 (A246E variant). The steady-state levels of the N- and C-terminal fragments of mutant PS1 in mice expressing low levels of mRNA were similar to that of mice expressing high levels of mRNA. Only mice expressing high levels of transgene mRNA accumulated uncleaved full-length protein. In mice co-expressing low levels of PS1A246E mRNA with Mo/Hu APPswe the age of appearance of Abeta deposits was similar to that of mice co-expressing expressing Mo/Hu APPswe with very high levels of mutant PS1. Our findings demonstrate that the levels of accumulated human PS1 N- and C-terminal fragments do not increase in proportion to the level of transgene mRNA and that similarly, the magnitude by which mutant PS1 accelerates the deposition of beta-amyloid is not proportional to the level of transgene expression.

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Depression of myostatin activity leads to increased muscle growth and carcass lean yield. In an attempt to down-regulate myostatin, transgenic mice were produced with a ribozyme-based construct or a myostatin pro domain construct. Though the expression of the ribozyme was detected, muscle development was not altered by the ribozyme transgene. However, a dramatic muscling phenotype was observed in transgenic mice carrying the myostatin pro domain gene. Expression of the pro domain transgene at 5% of beta-actin mRNA levels resulted in a 17-30% increase in body weight (P < 0.001). The carcass weight of the transgenic mice showed a 22-44% increase compared with nontransgenic littermates at 9 weeks of age (16.05 +/- 0.67 vs. 11.16 +/- 0.28 g in males; 9.99 +/- 0.38 vs. 8.19 +/- 0.19 g in females, P < 0.001). Extreme muscling was present throughout the whole carcass of transgenic mice as hind and fore limbs and trunk weights, all increased significantly (P < 0.001). Epididymal fat pad weight, an indicator of body fat, was significantly decreased in pro domain transgenic mice (P < 0.001). Analysis of muscle morphology indicated that cross-sectional areas of fast-glycolytic fibers (gastrocnemius) and fast-oxidative glycolytic fibers (tibialis) were larger in pro domain transgenic mice than in their controls (P < 0.01), whereas fiber number (gastrocnemius) was not different (P > 0.05). Thus, the muscular phenotype is attributable to myofiber hypertrophy rather than hyperplasia. The results of this study suggest that the over-expression of myostatin pro domain may provide an alternative to myostatin knockouts as a means of increasing muscle mass in other mammals., (Copyright 2001 Wiley-Liss, Inc.)

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.