Modulation of nuclear REST by alternative splicing: a potential therapeutic target for Huntington's disease

Abstract

Huntington's disease (HD) is caused by a genetically mutated huntingtin (mHtt) protein with expanded polyQ stretch, which impairs cytosolic sequestration of the repressor element-1 silencing transcription factor (REST), resulting in excessive nuclear REST and subsequent repression of neuronal genes. We recently demonstrated that REST undergoes extensive, context-dependent alternative splicing, of which exon-3 skipping (∆E₃ )-a common event in human and nonhuman primates-causes loss of a motif critical for REST nuclear targeting. This study aimed to determine whether ∆E₃ can be targeted to reduce nuclear REST and rescue neuronal gene expression in mouse striatal-derived, mHtt-expressing STHdh^Q111/Q111 cells-a well-established cellular model of HD. We designed two morpholino antisense oligos (ASOs) targeting the splice sites of Rest E₃ and examined their effects on ∆E₃ , nuclear Rest accumulation and Rest-controlled gene expression in STHdh^Q111/Q111 cells. We found that (1) the ASOs treatment significantly induced ∆E₃ , reduced nuclear Rest, and rescued transcription and/or mis-splicing of specific neuronal genes (e.g. Syn1 and Stmn2) in STHdh^Q111/Q111 cells; and (2) the ASOs-induced transcriptional regulation was dependent on ∆E₃ induction and mimicked by siRNA-mediated knock-down of Rest expression. Our findings demonstrate modulation of nuclear REST by ∆E₃ and its potential as a new therapeutic target for HD and provide new insights into environmental regulation of genome function and pathogenesis of HD. As ∆E₃ is modulated by cellular signalling and linked to various types of cancer, we anticipate that ∆E₃ contributes to environmentally tuned REST function and may have a broad range of clinical implications.

Keywords: Huntington's disease; PPARγ; REST/NRSF; Stmn2; Syn-1; alternative splicing; antisense oligos; gene therapy; nuclear translocation.

Figure 1

Induction of Rest ∆E₃ by specific ASOs in STHdh^Q7/Q7 and STHdh^Q111/Q111 cells. (A) Schematic structure of Rest gene and targeted splicing sites of the two ASOs (I₂E₃ and E₃I₃). Primers used for polymerase chain reaction (PCR) detection of ∆E₃ are shown by arrows. (B) PCR verification of ASOs‐induced ∆E₃. Cells treated with the ASOs (I₂E₃ and/or E₃I₃, 3 μM of each) or a control oligo were harvested for RNA isolation at 48 hrs post‐treatment. Nested PCR was performed with E₁F₁/E₄R₃ and E₂F₂/E₄R₁ (or E₁F₁/E₄R₁) as the primer set for the 1st‐ and 2nd‐round amplification, respectively. Amplicons were sequence verified, and ratio of the variants with/‐out ∆E₃ for each lane was analysed by GeneTools software (Syngene, Cambridge, UK). (C) Relative expression of the E₂/E₄ (∆E₃) mRNA assayed by qRT‐PCR with E_2/4F₁/R₁. The E₂/E₄ mRNA levels were expressed as folds over the control using Gapdh as the reference. Data are shown as Mean ± S.E.M. anova: F = 165.54, P < 0.0001 for STHdh^Q7/Q7; F = 32.76, P = 0.0006 for STHdh^Q111/Q111. **P < 0.01, ***P < 0.001 compared with I₂E₃ group; ^# P < 0.05 compared with I₂E₃ + E₃I₃ group.

Figure 2

Immunofluorescence analysis of STHdh^Q7/Q7 and STHdh^Q111/Q111 cells with or without ASOs treatment. ICC was performed on with P3 and P6 cells using the antibody sc‐25398 (A) and ab21635 (B) against N‐ and C‐terminal of REST, respectively. Note that the two ASOs were combined for the treatment. Percentage of nuclear REST was analysed by ImageJ, and values of 100 cells were averaged for each group and shown as Mean ± S.E.M. ***P < 0.001 compared with the control STHdh^Q111/Q111 group by Student's t‐test.

Figure 3

Comparison of REST‐controlled gene transcription between STHdh^Q7/Q7 and STHdh^Q111/Q111 cells with or without ASOs and siRNA treatment. (A) Transcriptional regulation of Bdnf, Syn1 and Stmn2 by ASOs and siRNAs. qRT‐PCR‐assayed mRNA levels of the genes were expressed as fold over the control STHdh^Q111/Q111 and shown as Mean±SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with STHdh^Q111/Q111 treated with a control oligo or non‐specific siRNA;. ^# P < 0.05 compared with the E₃I₃ group. (B) Syn1 mis‐splicing of in STHdh^Q7/Q7 and STHdh^Q111/Q111 cells with/‐out ASOs treatment. (C) Rescue of Syn1 mis‐splicing by Rest siRNAs. Note that amplicons shown for (B) and (C) were products of qRT‐PCR with Syn1‐E₈F₁/E₉R_1, Rest‐E₃F₁/E₄R₁ and Gapdh‐F₃/R₃. Ratio of the two Syn1 variants with/‐out I₈ was assessed by GeneTools software (Syngene) and averaged for three occasions. (D) Comparison of transcriptome between STHdh^Q7/Q7 and STHdh^Q111/Q111 with/‐out ASOs treatment. Three RNA samples (Q111‐Control, Q111‐ASOs and Q7‐Control) were transcriptionally profiled, and hierarchical clustering analysis of expression values was carried out using the genes generated by anova comparisons between the samples.

Figure 4

Comparison of Stmn2 (A) and Bdnf (B) protein expression between STHdh^Q7/Q7 and STHdh^Q111/Q111 cells with or without ASOs treatment. Western blotting and ELISA were performed to assay protein expression of Stmn2 and Bdnf, respectively. A representative Western blot for Stmn2 with Gapdh as a loading control was shown as an inset. Data are shown as Mean ± S.E.M. (n = 3). **P < 0.01, ***P < 0.001 compared with STHdh^Q111/Q111 control by Student's t‐test.

Figure 5

Illustration of ∆E₃ as an endogenous, manipulable modulator of REST function. The splicing event ∆E₃, which is common in human and non‐human primates, results in REST∆ protein isoform(s) missing ZF‐5 critical for nuclear targeting and therefore modulates nuclear REST levels and genome function. Note that (1) ∆E₃ is modulated by PPARγ in a cell‐dependent manner, presumably through a cis‐element in E3; and (2) inclusion of E₅ as the last exon, which is mutually exclusive to E₄, was only observed in human but not rodents and non‐human primates 14.