An abnormal CAG repeat expansion occurring in the first exon of the huntingtin gene (HTTex1) is the cause of Huntington’s disease (HD). In the N-terminal region, the huntingtin protein (HTT) contains 3 phosphorylatable residues - threonine 3 (T3) and serines 13 and 16 (S13/S16) - that, upon phosphorylation, strongly modulate aggregation and toxicity of mutant HTTex1 fragments. Here, we present evidence that phosphorylation of T3 prevents the diphosphorylation of S13/S16, while its dephosphorylation increases the chances of S13/S16 diphosphorylation in mutant HTTex1-expressing cells. This single observation has massive potential relevance for the understanding of HTTex1 structure and function and the treatment of HD.

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat located in exon 1 of the HTT gene^[1]. Expression of polyglutamine-expanded exon 1 (HTTex1) is sufficient to cause the disease in animal models^[2][3]. Interestingly, the polyglutamine tract is flanked in its N-terminus by a short amphipathic alpha-helix composed of 17 amino acids (N17)^[4][5] that control nucleo-cytoplasmic shuttling, membrane binding, aggregation and toxicity of mutant HTT^{[6][7][8][9][10][11][12][13][14][15][16][17][18]}.

Mounting evidence suggests that N17 phosphorylation is associated with reduced HTT accumulation and neurotoxicity^{[19][20][21][22][23][24][25]}. Double phosphomimic substitutions at S13 and S16 reverted histopathology^[26]. Single T3, S13 or S16 phosphorylation events are enough to completely block HTT aggregation in cell lines but had cell-, tissue- or developmental stage-dependent effects in Drosophila models of HD^[23]. Mimicking T3 phosphorylation ameliorates polyQ-mediated toxicity in a Drosophila model of HD, but increases the amount of insoluble HTTex1 species^[8]. Recent studies on HTTex1 peptides demonstrated that inducing phosphorylation^[25][27][28], or adding a negative charge (e.g. aspartic acid mutations)^[24] to the T3 residue, leads to a decrease in fibril elongation^[23]. Furthermore, T3 phosphorylation was found to be reduced in samples from HD models and clinical patients, further supporting a key role for T3 in HD pathogenesis^[25].

In this study- we aimed at elucidating a possible relationship between the phosphorylation state of T3, S13 and S16 in the huntingtin exon 1 fragments.

Single phosphorylation events at T3, S13 or S16 are enough to completely abolish HTTex1 aggregation in living mammalian cells, and several phosphatase inhibitors are able to prevent HTTex1 aggregation^[23] (Fig. 1A). In order to know if these inhibitors prevent HTTex1 aggregation by means of its phosphorylation at serines 13 and 16, we tested their effect on serine phosphorylation by means of immunoblotting with an anti-phosphoserine 13/16 antibody (Fig. 1B) (see Suppl. Info. for full names). The immunoblots show two bands corresponding to 97QHTTex1-Venus1 and 97QHTTex1-Venus2 constructs, as we carried out these experiments in a bimolecular fluorescence complementation (BiFC) model of HD (see Methods). Although 97QHTTex1-expressing cells treated with B5, B7, C4, and C5 inhibitors showed a trend towards increased levels of serine phosphorylation, no statistically significant differences were detected (Fig. 1C). However, when the phosphatase inhibitors were applied to T3A phosphoresistant constructs (Fig. 1D), a striking increase was observed in HTTex1 serine phosphorylation, with the exception of the protein phosphatase 1/2A inhibitor (B2). We were not able to detect S13/S16 diphosphorylation in T3D constructs, either in the presence or absence of phosphatase inhibitors (data not shown). Taken together, these results suggest that T3 phosphorylation state controls serine phosphorylation in HTTex1. We cannot rule out the possibility that the structural changes induced by mutations in T3 could facilitate or prevent access to the phosphoserine antibody to its epitope. However, this is still a very relevant observation for the HD field, as N17 phosphorylation has attracted great interest for the putative treatment of HD. Serine phosphorylation has been shown to prevent HTT aggregation and toxicity in cell and animal models of the disease^{[19][21][23][26]}, and current evidence also indicates that T3 could be relevant to HD pathology^{[8][23][24][25][27][28][29]}. Recent work on synthetic peptides bearing the first 19 amino acids of HTTex1 suggests that cross-talk between phosphorylation at T3 and S13 and/or S16 might regulate the alpha-helical conformation of N17 in vitro^[30].

Our results strongly support that there is significant interaction between T3 and S13/S16 residues. Further research should be carried out to elucidate this interesting relationship.

The HTTex1 bimolecular fluorescence complementation system was previously described^[23][31]. Briefly, two constructs for the expression of Venus 1 (1-157 amino acids) and Venus 2 (158-236 amino acids) fused to HTTex1 were transfected into human H4 neuroglioma cells (ATCC HTB-148, LGC Standards, Barcelona, Spain) and treated with the different phosphatase inhibitors or the vehicle (final concentration DMSO 0.1% v/v) for 24 h (Table I) (Enzo Life Sciences, Lausen, Switzerland). Filter trap assays and immunoblotting were carried out as previously described^[23]. Total anti-N17 and anti-phosphoserine 13/16 antibodies were kindly provided by Dr. Ray Truant (McMaster University, ON, Canada)^[20][21]. Data were plotted and analyzed by means of Sigmaplot software (Systat Software, Inc., San Jose, CA, USA). Data are shown as the average of 3 independent experiments and error bars represent the standard deviation. Data were analyzed by means of a one-way ANOVA followed by the Holm-Sidak test of means. Significance was accepted at p<0.01.

FH and TFO were supported by a seed grant from the European Huntington´s Disease Network (EHDN). FH is currently supported by Project LISBOA-01-0145-FEDER-007660 (Cellular Structural and Molecular Microbiology) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT (Ref. IF/00094/2013 and PTDC/MED-NEU/31417/2017). TFO was supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain. JBS was supported by FCT/MCTES (Ref. SFRH/BD/85275/2012 and CBQ/04612/ICL3535).

We thank Dr. Ray Truant (McMaster University, ON, Canada) for providing the antibodies used in this study. We thank Prof. Hilal A. Lashuel (EPFL, Lausanne, Switzerland) for helpful comments.

The authors declare no conflicts of interest.

Not applicable.

No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.