Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) Diagram of the PC12-HttQ23 and Htt-Q74 proteins and experimental design. ( b ) Cells expressing either Htt-Q23 or Htt-Q74 were cultivated at 37 °C ( c ) in the presence of tetracycline (Dox) for 3 days to induce expression, exposed to heat shock 1 h at 42 °C, allowed to recover at 37 °C for 7 h (HS) and protein extracts immunoblotted with the indicated antibodies. HSF1 bands were quantitated using Quantity One image software (BioRad) and values normalized using GAPDH as loading control and referenced to control at 37 °C ( c ) in the absence of −Dox. ( c ) qRT-PCR analysis of the Hsp70 and Hsp25 genes in Htt-Q74-expressing cells as in B. HS (+Dox) group was compared with HS (−Dox) group. Error bars represent means±s.e.m., n =4. Unpaired t -test, *** P <0.001. ( d ) Analysis of Hsp70 promoter occupancy by HSF1. Error bars represent means±s.e.m., n =3. Unpaired t -test, ** P <0.01. ( e ) Mouse-derived striatal STHdh Q7 and STHdh Q111 cells were cultured at 33 °C ( c ), heat shocked at 42 °C 1 h with recovery at 33 °C for 7 h and immunoblotted with the indicated antibodies. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdh Q7 . ( f ) Striatal samples from Wild type (WT) C57BL/6 or KIQ175 mice at 2, 6 and 12 months analysed by immunoblotting ( n =4). ( g ) Dorsal striatal sections from WT and KIQ175 mice ( n =3) at 6 months assayed by immunohistochemistry (IHC) for mHtt aggregation (1C2), HSF1 and HSF1-S303 phosphorylation using DAPI staining as control. Scale bar: 10 μm. ( h ) Gastrocnemius muscle extracts from WT and KI175 mice ( n =4) of the indicated age, immunoblotted for HSF1, Hsp70 and GAPDH. ( i ) Protein extracts from HD patient striatum and controls immunoblotted with indicated antibodies. ( j ) HSF1 phosphoproteomic analysis under non-pathogenic (−Dox) and pathogenic (+Dox) conditions in hsf1 −/− MEF inducible cell line expressing GFP-Htt-Q74 expressing HSF1. See for uncropped immunoblots. HSF1 represented by the Regulatory domain, DBD; DNA binding domain; LZ1-3 and LZ4; leucine zipper domains.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Quantitative RT-PCR, Derivative Assay, Cell Culture, Western Blot, Immunohistochemistry, Staining, Binding Assay

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a , b ) PC12 cells expressing Htt-Q74 for 1, 2 or 3 days followed by Heat Shock (HS) and recovery as indicated. Control and HS in the absence of Dox correspond to cells incubated during 3 days at 37 °C. ( c ) Htt-Q74 cells Dox-induced or not for 3 days and exposed to 2 μM 17-AAG and/or MG132 (5 μM) for 6 h and extracts immunoblotted with the indicated antibodies. ( d ) Diagram of the effects of 17-AAG and MG132 treatment and Htt-Q74 expression in HSF1. ( e ) Htt-Q74 cells transfected with human influenza haemagglutinin-ubiquitin (HA-Ub) plasmid Dox induced or not and treated with 5 μM MG132 treatment for 6 h. Whole-cell extract and HA immunoprecipitated (IP:HA) and HSF1 immunoprecipitated samples (IP:HSF1) were immunoblotted as indicated. ( f ) Transcript levels for indicated E3 ligases evaluated by qRT-PCR from striatum of WT and KIQ175 mice at 6 months. Error bars represent±s.e.m., ( n =3). Unpaired t -test, * P <0.05. ( g ) Human striatum samples from HD patients and controls and ( h ) Mouse striatum from 12 months old WT and KIQ175 mice were immunoblotted for HSF1 and Fbxw7. ( i ) Fbxw7 siRNA in STHdh Q7 and STHdh Q111 cells using scrambled RNA (Scr) as control. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control at 37 °C in the non-pathogenic STHdh Q7 . ( j ) hsf1 −/− MEFs transfected with WT HSF1 or S303A mutant and Fbxw7-FLAG; samples immunoprecipitated with anti-FLAG and HSF1 detected. ( k ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with WT HSF1 or HSF1-S303A HS and 1 h recovery at 37 °C for 7 h. HSF1 was quantified using Quantity One image software and normalized using GAPDH as loading control and referenced to control (−Dox) expressing WT HSF1. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Software, Mutagenesis, Western Blot

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) Mammalian CK2 holoenzyme subunit composition and function. ( b ) Htt-Q74 cells treated with CK2 kinase inhibitors TID43 or ( c ) Emodin 24 h before Htt-Q74 induction with Dox and heat shocked at 42 °C for 1 h followed by recovery at 37 °C for 7 h and extracts analysed by immunoblotting for Hsp70 and GAPDH. ( d ) Htt-Q74 cells treated with 5 μM TID43 and immunoblotted for HSF1 and P-HSF1-S303. ( e ) Fluorescent images for GFP-Htt-Q74 analysed microscopically in cells treated with DMSO or 1 μM TID43 as described in B. Scale bar: 200 μm. ( f ) Quantification of cells containing GFP-Htt-Q74 aggregates from E expressed as percentage of total number of cells evaluated. Error bars represent±s.e.m., ( n =500 cells). Unpaired t -test ** P <0.05. ( g ) hsf1 −/− MEFs expressing Dox-inducible Htt-Q74-GFP transfected with pcDNA or WT HSF1 and incubated with 1 μM TID43 24 h before Htt-Q74-GFP induction followed by heat shock at 42 °C during 1 and 7 h recovery at 37 °C. Cell viability expressed as % of viable cells under control conditions at 37 °C. Error bars represent±s.e.m., ( n =3). Unpaired t -test n.s., no significant, * P <0.05, ** P <0.01). ( h ) Htt-Q74 cells were transfected with siRNA against CK2β regulatory subunit or ( i ) CK2α and/or CK2α′ catalytic subunits using scrambled siRNA (Scr) as control 24 h before Htt-Q74 induction during 2 days followed by heat shock at 42 °C 1 h and recovery at 37 °C, 7 h. HSF1 was quantitated as in (F2H). All immunoblots shown for each panel contain the samples from the same membrane and were cropped to show only relevant data. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Western Blot, Expressing, Transfection, Incubation

Journal: Nature Communications

Article Title: Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

doi: 10.1038/ncomms14405

Figure Lengend Snippet: ( a ) CK2α, CK2α′ and CK2β protein levels in Htt-Q74 expressing cells under control ( c ) or heat shock conditions at 42 °C for 1 h (HS). CK2 subunit abundance was quantified using Quantity One image software normalized using GAPDH as loading control. CK2α′ ratio is shown and referenced to control (−Dox) cells. ( b ) CK2α, CK2α′ and CK2β striatal mRNA levels from WT and KIQ175 mice at 6 months of age. The value given for the amount of mRNA in the control group (WT) was set as 1. Error bars represent mean±s.e.m., ( n =4 animals). Values for the KIQ175 group were compared to the WT group. Statistical significance was measured by two-tailed unpaired t -test * P <0.05. ( c ) Protein levels for CK2α, CK2α′ and CK2β in the striatum and ( d ) gastrocnemius muscle of WT and KIQ175 mice at 6 months of age ( n =4). ( e ) Coronal section of the striatum of WT and KIQ175 at 6 months of age, showing co-localization of CK2α′ (red) with Ctip2 (green) and Fox1p (magenta) labelled MSNs in merged image. Scale bar: 10 μm. ( f ) CK2α, CK2α′ and CK2β qRT-PCR analysis and ( g ) protein levels in the striatum of HD patients and sex-age matched controls from 3 biospecimen banks . The value given for the amount of mRNA in the control group (C) was set as 1 for each gene. Error bars represent mean±s.e.m., ( n =7). One-tailed unpaired t -test * P <0.05, ** P <0.05, NS, no significant. Values for the Huntington's disease (HD) group were compared to the control (C) group. CK2α′ bands from immunoblots were quantified using Quantity One image software (BioRad) and the protein values were normalized using GAPDH as loading control and referenced to the corresponding age-sex-matched control patient. See for uncropped immunoblots.

Article Snippet: HttQ74 cells were transfected with HA-Ub vector (Addgene #18712) by using Lipofectamine LTX Reagent (Invitrogen) following protocol instructions (Invitrogen protocols 25-0946 W). hsf1 −/− MEF-GFP-Htt-Q74 cells were transfected with pcDNA WT HSF1 or the HSF1 S303A mutant plasmid using Lipofectamine LTX reagent. hsf1 −/− MEFs were electroporated with FBXW7-FLAG tag and WT HSF1 or S303A plasmids using the SE Cell Line Nucleofector.

Techniques: Expressing, Software, Two Tailed Test, Quantitative RT-PCR, One-tailed Test, Western Blot

Journal: eLife

Article Title: Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein

doi: 10.7554/eLife.29123

Figure Lengend Snippet: ( A ) Structure of NCT-504. ( B ) NCT-504 treatment reduces Htt(exon1)-Q103 in PC12 cells. Cells with stable expression of ecdysone-inducible GFP-Htt(exon1)-Q103 (green), induced for 24 hr, and treated with DMSO (top panels) or 23 μM NCT-504 (bottom). Cells stained with DAPI (blue). Scale Bar = 50 μm. ( C ) Concentration-response curve of NCT-504 inhibition of cellular accumulation of GFP-Htt(exon1)-Q103 in PC12 cells. ( D ) NCT-504 inhibition of PIP4K γ binding to an immobilized proprietary active site ligand (DiscoverX KINOME scan https://www.discoverx.com/services/drug-discovery-development-services/kinase-profiling/kinomescan ). ( E ) NCT-504 exhibits dose-dependent inhibition of phosphorylation of PI4P by full length isolated PIP4Kγ. ( F ) The intrinsic ATPase specific activity of full length isolated PIP4Kγ in the absence of PI5P substrate as a function of NCT-504 concentration is the same in the presence (blue) or in the absence (purple) of NCT-504.

Article Snippet: GFP-Htt-exon1-Q23 (Plasmid #40261)1 and GFP-Htt-exon1-Q74 (Plasmid #40262)1 were purchased from Addgene (Cambridge, MA) ( ).

Techniques: Expressing, Staining, Concentration Assay, Inhibition, Binding Assay, Drug discovery, Phospho-proteomics, Isolation, Activity Assay

Journal: eLife

Article Title: Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein

doi: 10.7554/eLife.29123

Figure Lengend Snippet: HEK293T cells were either transfected with GFP-HTT(exon1)-Q23 or GFP-HTT(exon1)-Q74. Two hours after transfection, cells were either treated with DMSO or NCT-504 (2 μM). After 48 hr of treatment, cells were fixed and quantification of the cells transfected with GFP-HTT(exon1)-Q74 for the percentage of cells with aggregates is shown below (results from three independent experiments. Statistical significance was analyzed using paired one tailed student T test ***p<0.005. Bar = 50 μm.

Article Snippet: GFP-Htt-exon1-Q23 (Plasmid #40261)1 and GFP-Htt-exon1-Q74 (Plasmid #40262)1 were purchased from Addgene (Cambridge, MA) ( ).

Techniques: Transfection, One-tailed Test

Journal: eLife

Article Title: Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein

doi: 10.7554/eLife.29123

Figure Lengend Snippet: ( A ) Experimental design of PIP4Kγ inhibitor treatment in neurons. Mouse primary cortical neurons transduced with Htt(exon1)-Q72 lentivirus and treated with PIP4Kγ inhibitor NCT-504 for 48 hr. ( B ) Assessment of baseline toxicity of NCT-504 to find non-toxic concentrations. The indicated concentrations of NCT-504 were applied to neurons for 48 hr and baseline toxicity assessed by in-cell western analysis using neurofilament antibody. To normalize cell number Draq5 and sapphire 700 staining (D + S) was performed. Veh represents vehicle (DMSO) treatment.

Article Snippet: GFP-Htt-exon1-Q23 (Plasmid #40261)1 and GFP-Htt-exon1-Q74 (Plasmid #40262)1 were purchased from Addgene (Cambridge, MA) ( ).

Techniques: Transduction, In-Cell ELISA, Staining

Journal: eLife

Article Title: Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein

doi: 10.7554/eLife.29123

Figure Lengend Snippet: ( A ) NCT-504 reduces the levels of Htt(exon1)-Q72 in primary cortical mouse neurons as measured by western blot analysis using the monoclonal antibody 5TF-1C2, which recognizes polyQ. β-tubulin was used as a loading control. ( B ) Effect of knockdown of PIP4K2C on mHtt aggregates in mouse primary cortical neurons, as measured by two-dimensional resolution of high molecular weight species using AGERA.

Article Snippet: GFP-Htt-exon1-Q23 (Plasmid #40261)1 and GFP-Htt-exon1-Q74 (Plasmid #40262)1 were purchased from Addgene (Cambridge, MA) ( ).

Techniques: Western Blot, Control, Knockdown, High Molecular Weight

Journal: eLife

Article Title: Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein

doi: 10.7554/eLife.29123

Figure Lengend Snippet: ( A ) Effect of knockdown on PIP4Kγ protein assessed by western of N2a cell lysates. This is a Neuro-2a neuroblastoma cell line purchased from the ATCC (RRID: CVCL_0470 ) ( B ) Effect of knockdown of PIP4K2C on formation of mHtt aggregates as measured by two-dimensional resolution of high molecular weight species using AGERA. Htt(exon1) polyglutamine expansion lengths are indicated.

Article Snippet: GFP-Htt-exon1-Q23 (Plasmid #40261)1 and GFP-Htt-exon1-Q74 (Plasmid #40262)1 were purchased from Addgene (Cambridge, MA) ( ).

Techniques: Knockdown, Western Blot, High Molecular Weight

Journal: Free Radical Biology & Medicine

Article Title: Mitochondrial superoxide generation induces a parkinsonian phenotype in zebrafish and huntingtin aggregation in human cells

doi: 10.1016/j.freeradbiomed.2018.10.446

Figure Lengend Snippet: Effects of MitoPQ on huntingtin proteostasis and cell viability in a HD cell model. U2OS cells transfected with GFP-Htt ex1 Q23 or GFP-Htt ex1 Q74 were treated for 24 h with 30 µM MitoPQ, 1 µM rotenone, or solvent (control). (A) Huntingtin expression (EGFP) and nucleus staining (Hoechst 34580). Representative images show cells with Htt in diffuse form (Q23 and Q74) and Htt aggregates (observed only with Q74). (B) Quantification of live cells presenting ( i ) only diffuse huntingtin or ( ii ) containing aggregates; and ( iii ) cell death. n = 6 independent experiments, * P < 0.05 vs. Q23; # P < 0.05 vs. Q74.

Article Snippet: For transfection with EGFP-Htt ex1 Q23 or EGFP-Htt ex1 Q74 (40261 and 40262, Addgene), cells were treated with Opti-MEM, containing 0.5 μg plasmid DNA, 0.5 μL Lipofectamine LTX and 0.5 μL Plus reagent (Invitrogen) for 45 min (37 °C, 5% CO 2 ).

Techniques: Transfection, Solvent, Control, Expressing, Staining

Journal: Molecular Therapy

Article Title: Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant

doi: 10.1016/j.ymthe.2025.10.005

Figure Lengend Snippet: MPZL2 is a frequent cause of mild-to-moderate hearing loss in humans (A) Pedigrees of probands from the Yonsei University Hearing Loss (YUHL) cohort whose hearing loss was caused by variants in MPZL2 . Black symbols represent probands or affected family members, and identified MPZL2 variants are indicated beneath the symbols. Probands from YUHL 941, 957, 965, 1108, and 1178 underwent targeted panel sequencing, whereas the rest underwent whole-exome sequencing. (B) Pure-tone audiograms showing the average hearing thresholds of patients carrying the MPZL2 variants that cause hearing loss. The bold blue line represents the average thresholds obtained from all patients carrying MPZL2 variants, and the bold red line indicates the average thresholds of patients with a homozygous MPZL2 p.Q74∗ variant. Transparent blue lines represent individual patient thresholds. (C) Age-related decline in hearing thresholds in patients carrying MPZL2 variants. Dots indicate individual values, and bold lines indicate the regression line according to age. Only statistically significant regression results (250, 500, and 1,000 Hz) are presented. (D) Domain structure of the MPZL2 protein. The five MPZL2 variants identified in YUHL patients are shown. (E) Overview of the haplotype analysis, which was performed using data from 504 East Asians in the 1KG Project. The gray-colored haplotype represents the haplotype enriched in control samples, whereas the blue-colored haplotype is enriched in cases carrying the MPZL2 p.Q74∗ variant. The two red-colored single-nucleotide polymorphisms indicated, including MPZL2 p.Q74∗, are in strong linkage disequilibrium. The p values were calculated to statistically determine the case-enriched haplotype using a chi-squared test between the case and control groups.

Article Snippet: Mpzl2 p.Q74∗ KI mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Sequencing, Variant Assay, Control

Journal: Molecular Therapy

Article Title: Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant

doi: 10.1016/j.ymthe.2025.10.005

Figure Lengend Snippet: Generation and characterization of Mpzl2 p.Q74∗ knockin mice (A) Schematic illustration of introducing the homologous MPZL2 c.220C>T mutation into the mouse Mpzl2 locus. (B) Representative immunofluorescence images showing MPZL2 protein expression in the organ of Corti from wild-type and Mpzl2 Q74∗ homozygous mice. Scale bars, 20 μm. DC, Deiter’s cell; IBC, inner border cell; IHC, inner hair cell; IPC, inner pillar cell; IPhC, inner phalangeal cell; OHC, outer hair cell; OPC, outer pillar cell. (C) Relative expression levels of endogenous Mpzl2 mRNA in cochleae across the three genotypes. Real-time PCR primer binding sites within the Mpzl2 coding sequence (CDS) are illustrated as forward and reverse. Data are presented as mean ± standard deviation (SD) ( Mpzl2 +/+ , n = 5; Mpzl2 Q74∗/+ , n = 5; Mpzl2 Q74∗/Q74∗ , n = 4). One-way ANOVA test with Dunnett’s multiple comparison corrections was conducted to determine statistical significance; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (D and E) Auditory brainstem response (ABR) (D) and distortion product otoacoustic emission (DPOAE) (E) thresholds at 4, 12, and 24 weeks of age across the three genotypes (4 weeks: Mpzl2 +/+ , n = 8; Mpzl2 Q74∗/+ , n = 8; Mpzl2 Q74∗/Q74∗ , n = 16; 12 weeks: Mpzl2 +/+ , n = 8; Mpzl2 Q74∗/+ , n = 7; Mpzl2 Q74∗/Q74∗ , n = 9; 24 weeks: Mpzl2 +/+ , n = 4; Mpzl2 Q74∗/+ , n = 7; Mpzl2 Q74∗/Q74∗ , n = 10). Data are presented as mean ± SD. For tone ABR and DPOAE thresholds, statistical significance was determined via two-way ANOVA with Tukey’s multiple comparison corrections. For click ABR thresholds, statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison corrections; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (F) Representative whole-mount immunofluorescence images of the organ of Corti at DC or pillar cell (PC) level stained with SOX2 in 24-week-old Mpzl2 p.Q74∗ heterozygous and homozygous mice. Scale bars, 20 μm. (G) Quantification of surviving DC, OPC, IPC, and IPhC/IBC in 24-week-old Mpzl2 p.Q74∗ heterozygous and homozygous mice. Data are presented as mean ± SD. Two-way ANOVA with Bonferroni’s multiple comparison corrections was conducted to determine statistical significance. ns, not significant; ∗∗∗∗ p < 0.0001. (H) Cross-sectional images of the organ of Corti in Mpzl2 p.Q74∗ heterozygous and homozygous mice at 24 weeks of age. Supporting cells were labeled with SOX2, and the basement membrane was stained with type IV collagen. Scale bars, 20 μm. White arrows indicate OPCs, and white triangles indicate DCs.

Article Snippet: Mpzl2 p.Q74∗ KI mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Knock-In, Mutagenesis, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing, Standard Deviation, Comparison, Staining, Labeling, Membrane

Journal: Molecular Therapy

Article Title: Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant

doi: 10.1016/j.ymthe.2025.10.005

Figure Lengend Snippet: AAV-based gene supplementation of exogenous human MPZL2 under control of the CAG promoter induces ototoxicity (A) Schematic diagram of AAV-DJ-CAG-human MPZL2 ( hMPZL2 ) or AAV-PHP.eB-CAG- hMPZL2 used for gene supplementation therapy in Mpzl2 Q74∗/Q74∗ mice. (B) Timeline of the in vivo experiments. AAV was injected into P1–P3 Mpzl2 Q74∗/Q74∗ pups through the round window membrane. After 4 weeks, audiologic tests (ABR and DPOAE) and histologic evaluations were conducted. (C) ABR and DPOAE thresholds of Mpzl2 +/+ ( n = 4), Mpzl2 Q74∗/Q74∗ ( n = 10), Mpzl2 Q74∗/Q74∗ + AAV-DJ-CAG- hMPZL2 (low dose, n = 12; high dose, n = 11), and Mpzl2 Q74∗/Q74∗ + AAV-PHP.eB-CAG- hMPZL2 ( n = 19) mice at 4 weeks of age. Data are presented as mean ± SD. Statistical significance was determined via two-way ANOVA with Tukey’s multiple comparison corrections for tone ABR and DPOAE thresholds and via one-way ANOVA with Tukey’s multiple comparison corrections for click ABR thresholds; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (D) Representative whole-mount images of Mpzl2 Q74∗/Q74∗ and Mpzl2 Q74∗/Q74∗ + AAV-DJ-CAG- hMPZL2 (low and high doses) mice at P28. The top three images represent the hair cell layers, whereas the bottom three images display supporting cell layers across the three groups. Hair cells were labeled with phalloidin, and supporting cells were labeled with SOX2. IHCr, inner hair cell region; OHCr, outer hair cell region; scale bars, 20 μm. (E and F) Quantification of surviving hair (E) and supporting (F) cells across the three groups at P28. Data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparison corrections was conducted to determine statistical significance. ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Mpzl2 p.Q74∗ KI mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Control, In Vivo, Injection, Membrane, Comparison, Labeling

Journal: Molecular Therapy

Article Title: Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant

doi: 10.1016/j.ymthe.2025.10.005

Figure Lengend Snippet: AAV-based human MPZL2 supplementation under control of the EF1α promoter rescues long-term auditory function in Mpzl2 Q74∗/Q74∗ mice (A) Schematic diagram of AAV-DJ-EF1α- hMPZL2 or AAV-PHP.eB-EF1α- hMPZL2 . (B and C) ABR (B) and DPOAE (C) thresholds of Mpzl2 +/+ , Mpzl2 Q74∗/Q74∗ , Mpzl2 Q74∗/Q74∗ + AAV-DJ-EF1α- hMPZL2 , and Mpzl2 Q74∗/Q74∗ + AAV-PHP.eB-EF1α- hMPZL2 mice at 4, 12, and 24 weeks of age (4 weeks: Mpzl2 +/+ , n = 4; Mpzl2 Q74∗/Q74∗ , n = 6; AAV-DJ-EF1α- hMPZL2 , n = 17; AAV-PHP.eB-EF1α- hMPZL2 , n = 4; 12 weeks: Mpzl2 +/+ , n = 4; Mpzl2 Q74∗/Q74∗ , n = 6; AAV-DJ-EF1α- hMPZL2 , n = 7; 24 weeks: Mpzl2 +/+ , n = 3; Mpzl2 Q74∗/Q74∗ , n = 6; AAV-DJ-EF1α- hMPZL2 , n = 5). Data are presented as mean ± SD. Statistical significance was determined via two-way ANOVA with Tukey’s multiple comparison corrections for tone ABR and DPOAE thresholds and via one-way ANOVA with Tukey’s multiple comparison corrections for click ABR thresholds; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (D) Representative whole-mount images of supporting cells at DC and PC layers from the basal cochlear turn of Mpzl2 Q74∗/+ , Mpzl2 Q74∗/Q74∗ , and Mpzl2 Q74∗/Q74∗ + AAV-DJ-EF1α- hMPZL2 mice at 24 weeks of age. Scale bars, 20 μm. (E) Quantification of surviving DCs and OPCs across the three groups at 24 weeks of age. Data are presented as mean ± SD. Two-way ANOVA with Bonferroni’s multiple comparison corrections was performed to determine statistical significance; ns, not significant; ∗∗∗∗ p < 0.0001. (F) Representative cross-sectional images showing the arrangement of supporting cells and MPZL2 expression in the organ of Corti at the basal cochlear turn from Mpzl2 Q74∗/+ , Mpzl2 Q74∗/Q74∗ , and Mpzl2 Q74∗/Q74∗ + DJ-EF1α- hMPZL2 mice at 24 weeks of age. Scale bars, 20 μm. HeC, Hensen’s cell. (G) A gel image showing the specificity of primers for codon-optimized human MPZL2 delivered by AAV-DJ. cDNAs from the cochleae of non-injected Mpzl2 +/+ , as well as non-injected, DJ-CAG- hMPZL2 -injected, and DJ-EF1α- hMPZL2 -injected Mpzl2 Q74∗/Q74∗ mice, were used for amplification (left). Real-time PCR results presenting the relative expression levels of human MPZL2 mRNA in the cochleae of non-injected, DJ-EF1α- hMPZL2 -injected, and DJ-CAG- hMPZL2 -injected Mpzl2 Q74∗/Q74∗ mice ( n = 3, biological replicates) at P28 (right). Data are presented as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison corrections was conducted to determine statistical significance; ∗∗∗ p < 0.001. (H) Western blotting results showing the relative expression levels of human MPZL2 protein (normalized to β-actin expression) in the cochleae of non-injected, DJ-EF1α- hMPZL2 -injected, and DJ-CAG- hMPZL2 -injected Mpzl2 Q74∗/Q74∗ mice ( n = 3, biological replicates) at P28. Data are presented as mean ± SD. An independent t test was conducted to determine statistical significance; ∗∗ p < 0.01. (I) Time-dependent changes in the expression levels of human MPZL2 protein in the cochleae of DJ-EF1α- hMPZL2 -injected Mpzl2 Q74∗/Q74∗ mice at 4 and 24 weeks of age.

Article Snippet: Mpzl2 p.Q74∗ KI mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Control, Comparison, Expressing, Injection, Amplification, Real-time Polymerase Chain Reaction, Western Blot