Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

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.Telethon-Italy and Autonomous Province of Trento (TCP12013 to M.P.); Association Française contre les Myopathies (AFM-22221 to M.P. and M.B.); PRIN-MUR (2017F2A2C5 to M.P.); National Institutes of Health (1R21NS111768-01 to M.P. and U.B.P.); PROGRAM RARE DISEASES CNCCS-Scarl-Pomezia (M.P.); FONDAZIONE AIRC-Italy (24423 to M.P.); Alzheimer Trento Onlus with the legato Baldrachi (M.B.); the Agence Nationale de la Recherche (ANR-15-JPWG-0003-05 JPND CIRCPROT and ANR-18-CE16-0009-01 AXYON to F.S.) and the Spanish Ministry of Science, Innovation and Universities (RTI2018-096322-B-I00 MCIU/AEI/FEDER-UE to J.J.L.

Phosphorylation of mutant huntingtin at serine 116 modulates neuronal toxicity.

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

Generation and expression of N586-82Q constructs with phospho-site alterations.

<p>(A) Schematic representation of the Htt N586 and the sites altered. Italics indicate previously described phosphorylation sites. Detected sites are in bold. (B) Expression of constructs in primary cortical neurons. (C) Quantification of expression of N586 constructs in primary neurons. Fluorescence was quantified using Volocity, and results are expressed as percent of N586-22Q level of expression. (D) Western blot of expression of N586 constructs with phospho-site alterations in primary cortical neurons. The image shown is representative of 4 replicate experiments.</p

Cell toxicity of N586-82Q constructs with alterations of phospho-sites in primary neurons.

<p>Primary cortical neurons were transfected at DIV5. Automated quantification of nuclear condensation was performed 48(A) Toxicity of constructs with alterations of previously identified Htt phosphorylation sites. (B) Toxicity of constructs with alterations of putative Htt phosphorylation sites located between position 1 and 100. (C) Toxicity of constructs with alterations of putative Htt phosphorylation sites located between 100 and 200. (D) Toxicity of constructs with alterations of putative Htt phosphorylation sites located at the 457–464 cluster. (E) Toxicity of constructs with alterations of putative Htt phosphorylation sites located between 200 and 586. Results are expressed as mean ± sem. n = 5 independent experiments per condition. * p<0.05 compared to N586-82Q.</p

Identified phosphorylarion peptides.

<p>Identified phosphorylarion peptides.</p

Isolation of Huntingtin N511 for mass spectrometry.

<p>(A) Representation of the Htt N511 construct used for transfection. (B) HEK293 cells were transfected with the N511 construct for 24 hours. Protein extracts were immunoprecipitated using Flag antibody and treated with phosphatase inhibitors or phosphatase and separated on a gel. Coomassie Blue detection of proteins in gel shows N511 overexpression. The image shown is representative of 3 replicate experiments. (C) Peptide coverage in two mass spectrometry experiments obtained from trypsin digest of Htt N511. Underlined residues indicate detection, blue indicates previously known phosphorylatable residues, red indicates novel detected sites. (D–E) Examples of mass spectra of peptide showing isotopic distribution for the site at serine 116.</p

Time lapse imaging of toxicity of N586 constructs.

<p>Primary cortical neurons were co-transfected at DIV5. Beginning 24 hours after transfection, GFP positive neurons were imaged every 10 mn for 10 hours. (A) Representative images of cells transfected with N586-22Q (top row), N586-82Q (middle row), or N586-82Q S116A (bottom row) at t = 0 (left column), t = 5 h (center column), and t = 10 h (right column). (B) Quantification of cell survival. For each time point cells were given the value of 100 if alive and 0 if dead. Results are expressed as mean ± sem. n = 200 cells analyzed in 5 independent experiments.</p

Immortalized striatal precursor neurons from Huntington’s disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics

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 two weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including 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

Cysteine Proteases Bleomycin Hydrolase and Cathepsin Z Mediate N-terminal Proteolysis and Toxicity of Mutant Huntingtin*

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