Unravelling Endogenous MicroRNA System Dysfunction as a New Pathophysiological Mechanism in Machado-Joseph Disease

Abstract

Machado-Joseph disease (MJD) is a genetic neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). Despite current efforts, MJD's mechanism of pathogenesis remains unclear and no disease-modifying treatment is available. Therefore, in this study, we investigated (1) the role of the 3' UTR of ATXN3, a putative microRNA (miRNA) target, (2) whether miRNA biogenesis and machinery are dysfunctional in MJD, and (3) which specific miRNAs target ATXN3-3' UTR and whether they can alleviate MJD neuropathology in vivo. Our results demonstrate that endogenous miRNAs, by targeting sequences in the 3' UTR, robustly reduce ATXN3 expression and aggregation in vitro and neurodegeneration and neuroinflammation in vivo. Importantly, we found an abnormal MJD-associated downregulation of genes involved in miRNA biogenesis and silencing activity. Finally, we identified three miRNAs-mir-9, mir-181a, and mir-494-that interact with the ATXN3-3' UTR and whose expression is dysregulated in human MJD neurons and in other MJD cell and animal models. Furthermore, overexpression of these miRNAs in mice resulted in reduction of mutATXN3 levels, aggregate counts, and neuronal dysfunction. Altogether, these findings indicate that endogenous miRNAs and the 3' UTR of ATXN3 play a crucial role in MJD pathogenesis and provide a promising opportunity for MJD treatment.

Keywords: 3′; Machado-Joseph disease; SCA3; UTR; gene therapy; lentivirus; mechanism of disease; microRNA; microRNA machinery dysregulation.

Graphical abstract

Figure 1

Inclusion of the 3′ UTR of Human Ataxin-3 Reduces Expression and Aggregation of Mutant Ataxin-3 in HEK293T Cells (A) Schematic representation of the lentiviral constructs used for the evaluation of the role of ataxin-3 3′ UTR in vitro. Human ataxin-3 3′ UTR (1–3,434 bp) was cloned immediately downstream of the mutATXN3 (72Q) coding sequence. The truncated versions of mutATXN3 with and without the 3′ UTR were constructed in order to promote aggregation of mutATXN3 in vitro. (B) Western blot analysis of HEK293T transfected with equimolar amounts of TmutATXN3 and TmutATXN3-3′ UTR constructs showing aggregated (stacking gel) and soluble forms of TmutATXN3 (∼36 kDa). (C and D) Optical densitometry analysis of ATXN3 fractions normalized with actin showing a significant reduction in aggregated and soluble TmutATXN3 protein levels (n = 3). (E and F) Representative images from laser confocal microscopy of HEK293T transfected with equimolar amounts of TmutATXN3 and TmutATXN3-3′ UTR constructs. The analysis was performed by staining mutATXN3 (red), ubiquitin (green), and nuclei (blue). (F) Quantification of mutATXN3 aggregates per cell was normalized by nuclei counts, confirming western blot results (n = 4). (G) Dual-luciferase activity evaluation from HEK293T transfected with a control construct encoding for firefly luciferase and renilla luciferase (FLuc) or a construct encoding for firefly luciferase bound to ATXN3 3′ UTR and renilla luciferase (FLuc-3′ UTR). The quantitative analysis presented as firefly/renilla ratio (FL/RL) relative to control shows a reduction in FLuc-3′ UTR luminescence activity (n = 4). (H) MutATXN3 mRNA stability assay from Neuro2A cells stably expressing mutATXN3 or mutATXN3-3′ UTR after treatment with actinomycin D for 0, 2, and 4 hr. The qPCR analysis for mutATXN3 mRNA levels normalized with endogenous control (18S) and relative to t = 0 showing a faster decay rate for mutATXN3-3′ UTR mRNA (n = 4 for each time point) is shown. The statistical significance was evaluated with paired Student’s t test (C–G) and unpaired Student’s t test (H) (*p < 0.05 and **p < 0.01). The data are expressed as mean ± SEM. The scale bars represent 5 μm.

Figure 2

Genetic and Pharmacological Blockage of miRNA Biogenesis Blocks ATXN3 3′ UTR Regulatory Effects (A) Schematic representation of the lentiviral constructs used for the modulation of endogenous hDrosha and hDicer through H1 mediated expression of shRNAs targeting hDrosha and hDicer. The GFP or lacZ expression cassettes were inserted in order to follow gene expression in both targeting shRNAs and negative shRNAs (predicted not to target any known human or mouse gene). (B) HEK293T co-transfected with TmutATXN3 and either shRNAs against hDrosha and hDicer or neg shRNAs are efficiently co-transfected as can be observed via reporter gene expression of GFP and lacZ. (C) qPCR analysis of endogenous hDrosha and hDicer mRNA levels 48 hr after co-transfection with shRNAs against hDrosha and hDicer compared to control showing efficient gene knockdown (n = 3). (D) qPCR analysis of TmutATXN3 mRNA levels after hDicer and hDrosha knockdown displays a significant increase in TmutATXN3 mRNA levels (n = 3). (E) Western blot evaluation of TmutATXN3 protein levels (n = 6) in the knockdown conditions when compared to control. The TmutATXN3 aggregated and soluble levels were increased after hDrosha/hDicer knockdown. (F) Western blot analysis of TmutATXN3 protein levels in HEK293T cells stably expressing TmutATXN3-3′ UTR. The cells were maintained during 48 hr in complete DMEM as the control condition or in complete DMEM containing kanamycin at a final concentration of 100 nM (n = 7). The TmutATXN3 protein levels were significantly increased in kanamycin treated cells. The qPCR analysis was normalized with endogenous control (ACTB). The statistical significance was evaluated with paired Student’s t test (*p < 0.05 and **p < 0.01). The data are expressed as mean ± SEM.

Figure 3

The 3′ UTR of ataxin-3 Reduces mutATXN3 Inclusions and Associated Neuronal Dysfunction and Neuroinflammation in a Lentiviral Mouse Model of Machado-Joseph Disease (A) Schematic representation of the lentiviral vectors used for the production of lentivirus used in the evaluation of the role of ATXN3 3′ UTR in vivo and schematic representation of mice stereotaxic procedure. Lentiviral particles encoding for mutATXN3 and mutATXN3-3′ UTR were injected bilaterally in the striatum of 5-week-old C57/BL6 mice at the following coordinates (x: +0.6; y: ±1.8; and z: −3.3). (B) qPCR analysis of gDNA transduction rates of both LV-mutATXN3 and LV-mutATXN3-3′ UTR after infection of HEK293T cells (n = 3). (C–E) Immunohistochemical peroxidase staining using anti-Ataxin3 antibody (1H9 ab), 5 weeks post injection. The control mutATXN3 injected animals displayed a large number of mutant ataxin-3 inclusions (C), which were significantly decreased in the mutATXN3-3′ UTR transduced striatum (D), as quantified in (E) (n = 4). (F–H) Immunohistochemical analysis using an anti-DARPP-32 antibody and lesion identification. The mutATXN3-3′ UTR injected hemisphere displayed a marked reduction in DARPP-32 depleted volume as quantified in (H) (n = 4). (I–K) Fluorescent immunohistochemical analysis of IBA-1 immunoreactivity showing a decrease in mutATXN3-3′ UTR injected animals as quantified in (K) (n = 4). qPCR analysis was normalized with endogenous control (Albumin). The statistical significance was evaluated with paired Student’s t test (**p < 0.01 and ***p < 0.001, n = 4). The data are expressed as mean ± SEM. The scale bars represent 40 μm (C and D) and 200 μm (F, G, I, and J).

Figure 4

Transcriptional Dysregulation of miRNAs Targeting ATXN3 3′ UTR and of RISC-Associated Genes (A) Schematic representation of hATXN3 3′ UTR with respective indication of predicted miRNA binding sites for selected miRNAs (mir-9, mir-181a, and mir-494). (B) miRNA qPCR quantification of mir-9, mir-181a, and mir-494 performed in post-mortem human samples from the dentate nuclei of control (CTRL) or MJD patients (n = 6/5) displaying a general downregulation profile for all miRNAs. (C) miRNA qPCR quantification of mir-9, mir-181a, and mir-494 performed in RNA extracted from SH-SY5Y cells stably expressing WtATXN3 (WT) or mutATXN3 (MUT) (n = 6). The Mir-494 was found to be significantly reduced in SH-SY5Y stably expressing mutATXN3. (D) Immunostaining of differentiated cultures of human neurons derived from diseased (MJD) or control (CTRL) patient neurospheres. (E) miRNA qPCR quantification of mir-9, mir-181a, and mir-494 performed in neurons derived from human neurospheres (n = 4/5). (F) miRNA qPCR quantification of mir-9, mir-181a, and mir-494 performed in cerebellar tissue from MJD transgenic mice (TG) or littermate controls (WT) with 8 weeks of age (n = 8/14). (G and H) Expression profiling of genes involved in the biogenesis and function of miRNAs (DGCR8, FMR1, Drosha, Dicer, DDX6, Ago2, and TARBP2) in cerebellum samples from MJD transgenic mice (TG) or littermate controls (WT) with 8 weeks of age (n = 5). A general downregulation profile for most of the evaluated genes was observed. qPCR analysis for miRNA was normalized with endogenous control mir-103-3p (B and C) or snRNAU6 (E and F). qPCR analysis for mRNA was normalized with endogenous control (GAPDH). The statistical significance was evaluated with unpaired Student’s t test (*p < 0.05, **p < 0.01, and ***p < 0.001). The data are expressed as mean ± SEM. The scale bars represent 40 μm.

Figure 5

Human ATXN3 Expression Is Regulated by mir-9, mir-181a, and mir-494 (A) Schematic representation of the lentiviral vector system used for overexpression of candidate miRNAs. The endogenous genomic sequences encoding for pre-miRNA sequences plus approximately 400 bp of flanking sequences were cloned downstream of an H1 promoter. The mir-Neg sequence was used as control. Simultaneously, the reporter gene expression was mediated by PGK controlled GFP expression. (B) Validation of miRNA overexpression. qPCR for mir-9, mir-181a, and mir-494 in transfected HEK293T cells confirming efficient overexpression for each miRNA (n = 4). (C) MutAtxn3 mRNA levels in HEK293T co-transfected with miRNA constructs and mutATXN3-3′ UTR (n = 4). Mir-181a and mir-494 significantly reduced the mutATXN3 mRNA levels. (D) Western blot analysis of HEK293T cells transfected with mutATXN3-3′ UTR and different miRNA constructs. The optical densitometry analysis of mutATXN3 protein levels was normalized with actin. The results are expressed as ratio ataxin-3/actin relative to mir-neg control (n = 3). All miRNAs significantly reduced mutATXN3 protein levels. (E) Validation of direct interaction between miRNAs and ATXN3 3′ UTR using a dual luciferase assay. Luminescence activity from HEK293T cells co-transfected with a dual luciferase construct containing ATXN3 3′ UTR or a control luciferase vector and miRNA/mir-neg encoding constructs is shown. The results are expressed as a ratio of firefly luminescence/Renilla luminescence (FL/RL) relative to control (LucCTRL) (n = 4). All miRNAs specifically reduced Luc-3′ UTR activity when compared to mir-neg. The qPCR analysis for mRNA was normalized with endogenous control (ACTB). The qPCR analysis for miRNA was normalized with endogenous control (snRNAU6). The statistical significance was evaluated with unpaired Student’s t test (B) or one-way ANOVA comparing all miRNAs to mir-neg (C–E) (*p < 0.05 and **p < 0.01). The data are expressed as mean ± SEM.

Figure 6

In Vivo Overexpression of mir-9, mir-181a, and mir-494 Reduce mutATXN3 Levels and Associated Neuropathology (A) Schematic representation of the stereotaxic procedure. The lentiviruses encoding for mutATXN3-3′ UTR were injected bilaterally in the striatum of 5-week-old C57/BL6 mice. Simultaneously, lentiviruses encoding for mir-9, mir-181a, or mir-494 were co-injected with mutATXN3, while mir-Neg was co-injected as control. (B) Lentiviruses encoding for interest miRNAs efficiently mediate in vivo transduction of mouse striatum, as can be seen through reporter expression of GFP. (C) qPCR analysis confirmed miRNA overexpression levels for each miRNA in injected striatum tissue compared to control. (D, F, H, J, and L) Immunohistochemical peroxidase staining using anti-Ataxin3 antibody (1H9 ab), 5 weeks post injection. The control mutATXN3-3′ UTR/LV-mir-Neg injected animals displayed a large number of mutant ataxin-3 inclusions (D), which were significantly decreased after co-injection with any of the study miRNAs (F, H, and J) as quantified in (L) (n = 4–5). (E, G, I, K, and M) Immunohistochemical analysis using an anti-DARPP-32 antibody and lesion identification. The MutATXN3-3′ UTR/LV-mir-Neg injected hemisphere displayed a higher depletion in DARPP-32 volume when compared to the miRNA injected hemispheres (G, I, and K), as quantified in (M) (n = 5). (N) MutATXN3 mRNA levels quantification after miRNA co-injection. LV-mir-9, mir-181a, and mir-494, significantly reduced the levels of mutATXN3 in vivo (n = 4–5). (O) Evaluation of endogenous mouse ataxin-3 mRNA levels in injected mice. The LV-mir-9 and LV-mir-181a significantly reduced mouse ataxin-3 in vivo (n = 4–5). (P) Schematic representation of mouse ATXN3 3′ UTR displaying predicted mir-9, mir-181a, and mir-494 binding sites. The qPCR analysis for mRNA was normalized with endogenous control (18S). The qPCR analysis for miRNA was normalized with endogenous control (snRNAU6). The statistical significance was evaluated with one-way ANOVA comparing all miRNAs to mir-neg (L–O) (*p < 0.05 and **p < 0.01). The data are expressed as mean ± SEM. The scale bars represent 200 μm (B, E, G, I, and K) and 50 μm (D, F, H, and J).