Journal: Nature Communications

Article Title: Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease

doi: 10.1038/s41467-021-25651-y

Figure Lengend Snippet: HdhQ7 and HdhQ111 cells were transfected with small interfering RNA (siRNA) control (siCon) or GSK3 (siGSK3) for 3 days, followed by CHIR99021 (3 µM) treatment for 48 h. a Mitochondrial membrane potential (MMP) was examined by TMRM staining; quantification of TMRM density is shown ( n = 8). b Cell death was measured by lactate dehydrogenase (LDH) release after 16 h serum starvation ( n = 4). Neuro2a cells were transfected with vectors expressing control guide RNA (gRNA) or GSK3β gRNA and Cas9. After confirming GSK3 knockout (Supplementary Fig. ), cells were transfected with Myc-tagged Q23-Htt (Q23) or Q73-Htt (Q73) constructs for 12 h, followed by a 2-day incubation with CHIR99021 (3 μM) or vehicle (Veh). c Representative TMRM images of the indicated groups. Scale bar = 30 μm. d Quantification of TMRM fluorescence density is shown ( n = 3). e Cell death in the indicated groups was measured by LDH release ( n = 9). f Heat maps representing the effects of 12 GSK3 inhibitors on MMP (left) and cell viability following serum withdrawal (right). All molecules were assessed at 8 doses ranging from 0.078 to 10 μM. MMP data are presented as the area under the dose–response curve of TMRM signal increase relative to DMSO-treated cells. Viability data are presented as the integral of the increase in non-apoptotic cells across all doses. AF: compound autofluorescence prevents an accurate assessment. TMRM scale: 0–20, viability scale: 0–1500. The CHIR99021 structure and its chemical analog, CHIR98014, are shown on the right. All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test in a , b , two-way ANOVA with Šídák’s post hoc test in d , and two-way ANOVA with Dunnett’s post hoc test in e . Data are representative of at least three independent experiments. Exact p values are shown in the figures.

Article Snippet: To knock down CAST in neurons differentiated from human iPSCs, lentiviral particles containing control or CAST shRNA (Origene, Rockville, MD, USA) were added into medium (MOI 12:1) with polybrene (4 μg/mL) (MilliporeSigma).

Techniques: Transfection, Small Interfering RNA, Control, Membrane, Staining, Expressing, Knock-Out, Construct, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease

doi: 10.1038/s41467-021-25651-y

Figure Lengend Snippet: a Knock-down efficiency of CAST and calpain I ( CAPN1 ) short hairpin RNA (shRNA) was confirmed by western blotting. b Mitochondrial membrane potential (MMP) was assessed using a TMRM fluorescence probe in control shRNA (shCon) or CAST shRNA (shCAST) expressing cells treated with CHIR99021 (chir) or vehicle (veh) ( n = 4). c Cell viability was assessed by MTT assay in shCon- or shCAST-expressing cells treated with CHIR99021 or vehicle ( n = 6). d MMP was measured by TMRM staining in control shRNA (shCon)- or sh CAPN1 -expressing cells treated with CHIR99021 or vehicle ( n = 4). e Cell viability was measured by MTT assay in shCon or sh CAPN1 cells treated with CHIR99021 or vehicle ( n = 8). Mixed striatal neurons were differentiated from iPS cells of patients with HD and normal subjects. Twenty days after neuronal differentiation, neurons were infected with lentivirus expressing GFP-labeled shCon or shCAST. f Knock-down efficiency of shCAST in neurons derived from HD patient iPS cells was confirmed by western blotting. Two days after infection, neurons derived from iPS cells from normal subjects (Nor) and HD patients (HD) were treated with CHIR99021 at 1 μM for 5 consecutive days. g MMP was assessed using a TMRM fluorescence probe ( n = 4). Cell viability was measured by MTT assay after brain-derived neurotrophic factor (BDNF) withdrawal for 12 h ( n = 4). h Neurons were stained with anti-MAP2 (red)/anti-GAD67 (blue) or anti-β-tubulin (clone Tuj1) (blue)/anti-DARPP-32 (red) to indicate dendritic and axonal morphology, respectively. Scale bar = 30 μm. i Quantification of MAP2 + neuronal dendrite length ( n = 23 cells/group). j Quantification of Tuj1 + neuronal axon length ( n = 28 cell/group). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures.

Article Snippet: To knock down CAST in neurons differentiated from human iPSCs, lentiviral particles containing control or CAST shRNA (Origene, Rockville, MD, USA) were added into medium (MOI 12:1) with polybrene (4 μg/mL) (MilliporeSigma).

Techniques: Knockdown, shRNA, Western Blot, Membrane, Fluorescence, Control, Expressing, MTT Assay, Staining, Infection, Labeling, Derivative Assay

Journal: Nature Communications

Article Title: Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease

doi: 10.1038/s41467-021-25651-y

Figure Lengend Snippet: a HdhQ7 and HdhQ111 cells were treated with DMSO (vehicle) or CHIR99021 (3 μM) for 2 days, with cells stained with anti-Tom20 antibody to assess mitochondrial morphology. Left: representative images. Scale bar = 30 μm. Right: histogram showing the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted ( n = 3). b Total levels of the indicated proteins classified into three pathways (mitochondrial dynamics, biogenesis, and autophagy) were examined and quantified. The mean relative protein level value was presented in a dual-color heat map. ** p = 0.0055, Q111-CHIR99021 versus Q111-Veh. Relative protein abundance scale: 0.5–2.0. c Mitochondria were isolated from the striatum of 12-week-old R6/2 and WT littermates treated with vehicle or CHIR99021 and subjected to western blotting. The relative abundance of mitochondrial Drp1 was quantitated (histogram). d Mitochondrial fractions were isolated from HdhQ7 cells expressing control shRNA (shCon) or CAST shRNA (shCAST) after treatment with 3-nitroproponic acid (3-NP) and/or CHIR99021. Representative immunoblots and quantification of Drp1 relative levels ( n = 4). e Representative images of anti-Tom20 staining (green) in shCon- or shCAST-expressing cells treated with Veh or CHIR99021. Scale bar = 30 μm. f Histogram shows the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted ( n = 5). g Representative images of anti-Tom20 staining (green) in shCon- or calpain I shRNA (sh CAPN1 )-expressing HdhQ111 cells treated with Veh or CHIR99021. Scale bar = 30 μm. h Histogram shows the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted ( n = 6). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures. i Summary graphic depicting the ability of CHIR99021 to stabilize CAST and promote mitoprotection and neuroprotection.

Article Snippet: To knock down CAST in neurons differentiated from human iPSCs, lentiviral particles containing control or CAST shRNA (Origene, Rockville, MD, USA) were added into medium (MOI 12:1) with polybrene (4 μg/mL) (MilliporeSigma).

Techniques: Staining, Quantitative Proteomics, Isolation, Western Blot, Expressing, Control, shRNA

Journal: Journal of Neuroscience

Article Title: Calpains Are Downstream Effectors of bax-Dependent Excitotoxic Apoptosis

doi: 10.1523/jneurosci.2345-11.2012

Figure Lengend Snippet: Figure 3. Calpain inhibition using RNA interference technology and overexpression of the endogenous calpain inhibitor calpastatin attenuateNMDA-inducedexcitotoxicapoptosis.A,NSC34cellsweretransfectedwithshRNAplasmidsagainstthelargesubunitofcalpain I(shRNAI),calpainII(shRNAII),orscrambleshRNA.ReducedexpressionlevelsofcalpainIandIIaftershRNAexpressioncomparedwith scramble were assessed by Western blotting 36 h after transfection. Probing for -actin served as a loading control. Experiments were repeatedthreetimeswithsimilarresults.B,C,Cell-deathanalysisafter100MNMDAtreatmentfor5minassayedover24h(B)and300 MNMDAfor60mintreatmentassayedover4h(C).CorticalneuronsweretransfectedwithshRNAplasmidsasdescribedabove. A plasmid expressing GFP allowed for the identification of transfected neurons. The number of PI-positive cells in trans- fected neurons was quantified (n 125–160 cells per time point quantified). Experiments were performed in triplicate in threeseparatecultures.*p0.05comparedwithNMDA-treated,scrambletransfectedcontrols(ANOVA,posthocTukey’stest). D,E,Corticalneuronsweretransfectedwithaplasmidexpressingtheendogenouscalpaininhibitor,calpastatinfusedwithGFP,or aplasmidexpressingGFPaloneasacontrol.Neuronsweretreatedwith100MNMDAfor5minandassayedover24h(D)and300 MNMDAfor60minandassayedover4h(E).ThenumberofPI-positivecellsintransfectedneuronswasquantified(n142–173 cells per time point quantified). *p 0.05 compared with NMDA-treated controls (ANOVA, post hoc Tukey’s). Experiments were repeated three times with independent culture preparations with similar results.

Article Snippet: For overexpression of calpastatin, cells were transfected with a vector expressing GFP– calpastatin (MG209946; OriGene).

Techniques: Inhibition, Over Expression, Western Blot, Transfection, Control, Plasmid Preparation, Expressing