Journal: Neural Regeneration Research

Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology

doi: 10.4103/NRR.NRR-D-23-01666

Figure Lengend Snippet: Mutant AR with polyQ expansion reduces MANF expression in N2a cells. (A) A schematic of AR0Q and AR48Q plasmids. AR containing 0Q and 48Q were linked with an N-terminal EGFP tag. Created with Microsoft PowerPoint (2312 Build 16.0.17126.20132). (B) Western blotting analysis showed that the AR0Q and AR48Q plasmids expressed the corresponding proteins in the transfected N2a cells. EGFP and AR antibodies were used to detect both AR0Q and AR48Q. 1C2 antibody preferentially reacts with AR48Q. β-tubulin was used as a loading control. (C) Subcellular fractionation was performed using N2a cells transfected with AR0Q and treated with different concentrations (0, 100 nM, 10 nM, or 1 nM) of R1881. Western blotting analysis showed that without R1881, AR0Q was predominantly localized in the cytoplasm, whereas with R1881, AR0Q was predominantly localized in the nucleus. AR antibody was used to detect AR0Q. Lamin B1 was used as a marker for the nucleus (N), and GAPDH was used as a marker for the cytoplasm (C). (D) Subcellular fractionation was performed using N2a cells transfected with AR0Q or AR48Q and treated with or without R1881. Western blotting analysis showed that in the presence of R1881, both AR0Q and AR48Q translocated into the nucleus. AR antibody was used to detect both AR0Q and AR48Q. Lamin B1 was used as a marker for the nucleus (N), and GAPDH was used as a marker for the cytoplasm (C). (E) Fluorescent microscopy images showing the localization of AR0Q and AR48Q in N2a cells treated with or without R1881. Without R1881, AR0Q and AR48Q were present in the cytoplasm and nucleus, whereas with R1881, AR0Q and AR48Q were predominantly localized in the nucleus. DAPI was used to stain nuclei. Scale bars: 20 μm. (F) Western blotting analysis of MANF expression in N2a cells transfected with AR0Q or AR48Q and treated with R1881. AR antibody was used to detect both AR0Q and AR48Q, and MANF antibody was used to detect MANF. β-Actin was used as a loading control. (G) Quantitative analysis of MANF expression ( n = 7, one-way analysis of variance with Tukey’s post hoc test). MANF expression was significantly lower in AR48Q-expressing cells compared with AR0Q-expressing cells. (H) Real-time polymerase chain reaction was performed to measure Manf mRNA levels in N2a cells transfected with AR0Q or AR48Q and treated with R1881 ( n = 4, one-way analysis of variance with Tukey’s post hoc test). The Manf mRNA level was significantly lower in AR48Q-expressing cells than in AR0Q-expressing cells. (I) MTT assay was performed using N2a cells transfected with AR0Q or AR48Q and treated with R1881 ( n = 4, one-way analysis of variance with Tukey’s post hoc test). The viability of AR48Q-expressing cells was significantly lower than that in AR0Q-expressing cells. * P < 0.05, *** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. AR: Androgen receptor; DAPI: 4′,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; MANF: mesencephalic astrocyte-derived neurotrophic factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; polyQ: polyglutamine.

Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).

Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Control, Fractionation, Marker, Microscopy, Staining, Real-time Polymerase Chain Reaction, MTT Assay, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology

doi: 10.4103/NRR.NRR-D-23-01666

Figure Lengend Snippet: Mutant AR with polyQ expansion reduces MANF expression in the brain of male mice. (A) A schematic of the AAV-AR0Q and AAV-AR48Q constructs. AR containing 0Q and 48Q were linked with an N-terminal FLAG tag and were under the control of miniCMV promoter. (B) Western blotting analysis of N2a cells transfected with AAV-AR0Q or AAV-AR48Q plasmids and treated with or without R1881. AR and FLAG antibodies were used to detect both AR0Q and AR48Q, and 1C2 antibody was used to detect AR48Q. β-Actin was used as a loading control. (C) Double immunofluorescence staining using AR (green, Alexa Fluor® 488) and FLAG (red, Alexa Fluor® 594) antibodies showed the localization of AR0Q and AR48Q in the N2a cells treated with or without R1881. Both AR0Q and AR48Q translocated to the nucleus in the presence of R1881. DAPI was used to stain nuclei. Scale bars: 20 μm. (D) A schematic of the stereotaxic injection performed in the mouse cortex. AAV-AR0Q was injected into one side of the cortex and AAV-AR48Q was injected into the contralateral side. (E) Immunofluorescence staining of AR0Q and AR48Q in the cortex of male and female mice. AR0Q and AR48Q were predominantly localized in the nucleus in male mice, with AR0Q being diffuse and AR48Q showing punctate staining (indicated by white arrows). Scale bars: 20 μm. (F) Western blot of AR and MANF expression in the cortex of male mice that were either uninjected (WT), or injected with AAV-AR0Q or AAV-AR48Q. Vinculin was used as a loading control. (G) Quantitative analysis of MANF expression shown in (F) ( n = 5, one-way analysis of variance with Tukey’s post hoc test). MANF expression was significantly decreased in the AR48Q mice compared with the uninjected and AR0Q mice. (H) Double immunofluorescence staining using AR (red, Alexa Fluor® 594) and MANF (green, Alexa Fluor® 488) antibodies in the cortex of male mice injected with AAV-AR0Q or AAV-AR48Q. Scale bar: 50 μm; magnified image, 10 μm. (I) Quantitative analysis of MANF staining intensity shown in H ( n = 8, two-tailed Student’s t -test). MANF staining intensity was significantly decreased in the AR48Q mice compared with the AR0Q mice. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. AAV: Adeno-associated virus; AR: androgen receptor; DAPI: 4′,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; MANF: mesencephalic astrocyte-derived neurotrophic factor; polyQ: polyglutamine; WT: wild-type.

Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).

Techniques: Mutagenesis, Expressing, Construct, FLAG-tag, Control, Western Blot, Transfection, Double Immunofluorescence Staining, Staining, Injection, Immunofluorescence, Two Tailed Test, Virus, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology

doi: 10.4103/NRR.NRR-D-23-01666

Figure Lengend Snippet: Mutant AR with polyQ expansion causes neuronal damage in the cortex of WT mice. (A) Western blotting analysis of AR, NeuN, PSD95, MAP2 and GFAP expression in the cortex of male WT mice injected with AAV-AR0Q or AAV-AR48Q. Vinculin was used as a loading control. (B) Quantitative analysis of NeuN, PSD95, MAP2 and GFAP expression ( n = 3–4, two-tailed Student’s t -test). NeuN, PSD95 and MAP2 expression was significantly decreased, whereas GFAP expression was significantly increased in the AR48Q mice compared with the AR0Q mice. (C) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and NeuN (red, Alexa Fluor® 594) antibodies. Scale bar: 10 μm. (D) Quantitative analysis of NeuN staining intensity ( n = 7, two-tailed Student’s t -test). NeuN staining intensity was significantly decreased in the AR48Q mice compared with the AR0Q mice. (E) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and MAP2 (red, Alexa Fluor® 594) antibodies. Scale bar: 20 μm. (F) Quantitative analysis of MAP2 staining intensity ( n = 6, two-tailed Student’s t -test). MAP2 staining intensity was significantly decreased in the AR48Q mice. (G) Nissl staining images of cortical slices expressing AR0Q or AR48Q. (H) Quantitative analysis of the number of Nissl-positive neurons per image ( n = 5, two-tailed student’s t -test). The number of neurons was significantly decreased in the AR48Q mice compared with the AR0Q mice. (I) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and GFAP (red, Alexa Fluor® 594) antibodies. Scale bar: 20 μm. (J) Quantitative analysis of GFAP staining intensity ( n = 6, two-tailed Student’s t -test). GFAP staining intensity was significantly increased in the AR48Q mice compared with the AR0Q mice. (K) TUNEL assay images. Scale bar: 20 μm. (L) Quantitative analysis of TUNEL staining intensity ( n = 6, two-tailed Student’s t -test) showed that the staining intensity was significantly increased in the AR48Q mice compared with the AR0Q mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. AR: Androgen receptor; EGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; MANF: mesencephalic astrocyte-derived neurotrophic factor; MAP2: microtubule-associated protein 2; NeuN: neuronal nuclei; PSD95: postsynaptic density protein 95; polyQ: polyglutamine; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; WT: wild-type.

Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).

Techniques: Mutagenesis, Western Blot, Expressing, Injection, Control, Two Tailed Test, Double Immunofluorescence Staining, Staining, TUNEL Assay, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology

doi: 10.4103/NRR.NRR-D-23-01666

Figure Lengend Snippet: MANF overexpression ameliorates neuronal damage caused by mutant AR with polyQ expansion. (A) Western blotting analysis of AR, MANF, NeuN, PSD95, MAP2 and GFAP expression in the cortex of male MANF TG mice injected with AAV-AR0Q or AAV-AR48Q. Vinculin was used as a loading control. (B) Quantitative analysis of NeuN, PSD95, MAP2 and GFAP expression ( n = 3, two-tailed Student’s t -test). NeuN, PSD95 and MAP2 expression was similar between the AR48Q mice and the AR0Q mice. GFAP expression was significantly decreased in the AR48Q mice compared with the AR0Q mice. (C) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and NeuN (red, Alexa Fluor® 594) antibodies. Scale bar: 10 μm. (D) Quantitative analysis of NeuN staining intensity ( n = 6, two-tailed Student’s t -test). NeuN staining intensity was similar between the AR0Q and AR48Q mice. (E) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and MAP2 (red, Alexa Fluor® 594) antibodies. Scale bar: 20 μm. (F) Quantitative analysis of MAP2 staining intensity ( n = 7, two-tailed Student’s t -test). MAP2 staining intensity was similar between the AR0Q and AR48Q mice. (G) Nissl staining images of cortical slices expressing AR0Q or AR48Q. Scale bar: 20 μm. (H) Quantitative analysis of the number of Nissl-positive neurons per image ( n = 5, two-tailed Student’s t -test). The number of neurons was similar between the AR0Q and AR48Q mice. (I) Double immunofluorescence staining using FLAG (green, Alexa Fluor® 488) and GFAP (red, Alexa Fluor® 594) antibodies. Scale bar: 20 μm. (J) Quantitative analysis of GFAP staining intensity ( n = 6, two-tailed Student’s t -test). GFAP staining intensity was similar between the AR0Q and AR48Q mice. ** P < 0.01. Data are presented as mean ± SEM. AAV: Adeno-associated virus; AR: androgen receptor; GFAP: glial fibrillary acidic protein; MANF: mesencephalic astrocyte-derived neurotrophic factor; MAP2: microtubule-associated protein 2; NeuN: neuronal nuclei; ns: no significance; PSD95: postsynaptic density protein 95; polyQ: polyglutamine; TG: transgenic.

Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).

Techniques: Over Expression, Mutagenesis, Western Blot, Expressing, Injection, Control, Two Tailed Test, Double Immunofluorescence Staining, Staining, Virus, Derivative Assay, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology

doi: 10.4103/NRR.NRR-D-23-01666

Figure Lengend Snippet: MANF modulates mutant AR aggregation and neurotoxicity. (A) Western blotting analysis comparing soluble and aggregated forms of AR48Q, NeuN, and GFAP in WT and MANF TG mice injected with AAV-AR48Q. FLAG antibody was used to detect AR48Q. HA antibody was used to detect transgenic MANF protein in MANF TG mice. Vinculin was used as a loading control. (B) Quantitative analysis of the ratio of aggregated to soluble AR48Q, NeuN and GFAP expression ( n = 4, two-tailed Student’s t -test). In MANF TG mice, the ratio of aggregated and soluble AR48Q was significantly lower, NeuN expression was significantly higher and GFAP expression was significantly lower. (C) Schematic of the AAV-Ctrl-gRNA and AAV-Manf-gRNA constructs, and the stereotaxic injection performed in the cortex of germline Cas9 mice. AAV-AR48Q and AAV-Ctrl-gRNA were injected into one side of the cortex, and AAV-AR48Q and AAV-Manf-gRNA were injected into the contralateral side. (D) Western blotting analysis comparing soluble and aggregated forms of AR48Q, NeuN and GFAP in the germline Cas9 mice injected with AAV-AR48Q/AAV-Ctrl-gRNA or AAV-AR48Q/AAV-Manf-gRNA. MANF antibody was used to confirm MANF knockdown. Vinculin was used as a loading control. (E) Quantitative analysis of NeuN, GFAP and MANF expression, and the ratio of aggregated and soluble AR48Q ( n = 3, two-tailed Student’s t -test). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. AAV: Adeno-associated virus; AR: androgen receptor; EGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; gRNA: guide RNA; MANF: mesencephalic astrocyte-derived neurotrophic factor; NeuN: neuronal nuclei; TG: transgenic; WT: wild-type.

Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).

Techniques: Mutagenesis, Western Blot, Injection, Transgenic Assay, Control, Expressing, Two Tailed Test, Construct, Knockdown, Virus, Derivative Assay