Journal: Neurochemical Research

Article Title: MicroRNA-124-3p Modulates Alpha-Synuclein Expression Levels in a Paraquat-Induced in vivo Model for Parkinson’s Disease

doi: 10.1007/s11064-024-04130-y

Figure Lengend Snippet: Effects of miR-124-3p in the expression of dopaminergic neuronal markers in the SN and striatum of a rat PQ model in vivo. (a) Design and timeline of the experimental animal procedure. Male Wistar rats were subjected to intranigral injection with 250 nM miR-124-3p, or saline, followed by four i.p injections of saline or PQ (10 mg/kg), separated by one day. Five days after the last PQ injection, the brains were collected for western blot analysis. Expression levels of TH in the (b) SN and (c) striatum, and (d) PITX3 in the SN of adult Wistar rats treated with saline, PQ or 250 nM miR-124-3p + PQ. (e) Representative western blot protein bands of PITX3 (32 kDa) and actin (42 kDa). TH and PITX3 protein levels were normalized against actin. Data are expressed as a percentage of the controls (saline) ± SEM. Protein expression in the control condition (saline) was set to 100%. N = 3 or 4 rats. * p < 0.05 compared to the saline group and ## p < 0.01 compared to the PQ-treated group using the one-way ANOVA followed by Sidak’s multiple comparison test. Abbreviations: i.p, intraperitoneal; TH, tyrosine hydroxylase; PITX3, pituitary homeobox 3; PQ, paraquat; SN, substantia nigra

Article Snippet: Then, 30 µg of total protein was loaded in a 12.5% or 15% polyacrylamide gel, followed by electrophoresis and transfer onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK) for 90 min. Membranes were blocked using 5% milk or 5% bovine serum albumin and then incubated with the following primary antibodies: mouse anti-TH (1:5000; BD Biosciences), rabbit anti-PITX3 (1:1000; Invitrogen), mouse anti-α-synuclein (1:500; BD Biosciences); rabbit anti-pS129-α-synuclein (1:200; Santa Cruz Biotechnology), goat anti-NOX1 (1:200; Santa Cruz Biotechnology), mouse anti-Rac1 (1:750; Millipore), rabbit anti-p47 phox (1:1000; Santa Cruz Biotechnology), mouse anti-actin (1:5000; BD Biosciences) and mouse anti-beta-tubulin (1:5000; BD Biosciences); and the secondary antibodies conjugated to horseradish peroxidase—HRP (chicken anti-goat, goat anti-rabbit or goat anti-mouse; 1:5000; all from Santa Cruz Biotechnology).

Techniques: Expressing, In Vivo, Injection, Saline, Western Blot, Comparison

Journal: NPJ Parkinson's Disease

Article Title: Silencing Parkinson’s risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability

doi: 10.1038/s41531-024-00648-8

Figure Lengend Snippet: Pitx3 IRES-tTA mouse midbrains were bilaterally injected with either AAV9-TRE-eGFP or -shRit2 and mice were assessed either 4–5 weeks (ST) or 25 weeks (LT) postinjection. Data are presented as mean ±S.E.M., and were analyzed by two-way repeat measures ANOVA with Sidak’s multiple comparison test ( a– h ), or two-way ANOVA with Tukey’s multiple comparison test (I-L) . Top: Experimental schematic. Pitx3 IRES-tTA mouse midbrains were bilaterally injected with either AAV9-TRE-eGFP or -shRit2 and mice were assessed either 4-5 weeks (ST) or 25 weeks (LT) postinjection. a – d Accelerating Rotarod: Mice were assessed over 3 consecutive trials as described in Methods. Rit2 silencing in DA neurons abolished motor learning in male ST. a Trial: p = 0.001, F (2, 41) = 8.95; Virus: p = 0.002, F (1, 24) = 11.69; trial x virus: p = 0.03; * p < 0.005; n = 9-11) and LT b . Trial: p = 0.01, F (2, 34) = 5.22; Virus: p = 0.02, F (1, 17) = 7.02; ** p = 0.005, n = 8-11) mice, but had no effect on ST c . Trial: p = 0.002, F (2, 48) = 6.82; Virus: p = 0.72, F (1, 48) = 0.12; n = 11–12) or LT d . Trial: p = 0.002, F (2, 48) = 6.82, Virus: p = 0.72, F (1,48) = 0.12, n = 8-10) female motor learning. e–h Fixed-speed Rotarod: Mice were assessed over the indicated consecutive speeds as described in Methods . e,f. Rit2 silencing did not affect ST males e . Rate: p < 0.0001, F (5,70) = 11.59, Virus: p = 0.48, F (1,14) = 0.53, n = 8), but significantly diminished LT male rotarod performance. f Rate: p < 0.0001, F (5,102) = 46.88, Virus: p < 0.0001, F (1,102) = 98.31, Rate x Virus: p < 0.0001, F (5,102) = 5.34; *** p < 0.001, **** p < 0.0001, n = 8-11) g,h. Rit2 silencing had no effect on female rotarod performance at the ST timepoint g . RPM: p < 0.0001, F (2, 35) = 12.79, Virus: p = 0.22, F (1,15) = 1.637; n = 8) but significantly diminished performance at the LT timepoint h . RPM: p < 0.0001, F (5,108) = 6.29; Virus: p = 0.006, F (1,108) = 12.48; n = 10). i–l Challenge balance beam: Mice were assessed on the challenge balance beam as described in Methods . Mean foot fault numbers ( i,j ) and beam traversal times (seconds) ( k,l ) were analyzed. Rit2 silencing had no effect on ST male foot faults, but increased LT male foot faults (I. Time: p = 0.0007, F (1,32) = 14.23; Virus: p < 0.0001, F (1, 32) = 23.83; Time x virus: p = 0.02, F (1, 32) = 6.01; *** p = 0.004, **** p < 0.0001, n = 8–10) and traversal times k . Time: p = 0.004, F (1,32) = 9.52; Virus: p = 0.01, F (1, 32) = 7.02; Time x virus: p = 0.03, F (1, 32) = 5.17; ** p < 0.01, n = 8–10). Rit2 silencing had no effect on ST female foot faults, but increased LT female foot faults j . Time: p = 0.42, F (1,31) = 0.68; Virus: p = 0.0002, F (1, 31) = 18.59; Time x virus: p = 0.03, F (1, 31) = 5.36; *** p = 0.0003, n = 8-10) and traversal times l . Time: p = 0.10, F (1,31) = 2.85; Virus: p = 0.003, F (1, 31) = 10.64; Time x virus: p = 0.0008, F (1, 31) = 13.95; ** p = 0.005, *** p = 0.0001, n = 8-10).

Article Snippet: Quantitative PCR was performed using the Applied Biosystems® 7500 Real-Time PCR System Machine and software or using the Bio-Rad C1000 Touch Thermal Cycler with CFX96 Real-Time system and software using Taqman® gene expression assays for mouse Rit2 (Mm0172749_mH), TH (Mm00447557_m1), DAT (Mm00438388_m1), DRD2 (Mm00438541_m1), Pitx3 (Mm01194166_g1), Nurr1 (Nr4a2, Mm00443060_m1), Rit1 (Mm00501400_m1), Vps35 (Mm00458167_m1).

Techniques: Injection, Comparison, Virus

Journal: NPJ Parkinson's Disease

Article Title: Silencing Parkinson’s risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability

doi: 10.1038/s41531-024-00648-8

Figure Lengend Snippet: Ex vivo fast-scan cyclic voltammetry: Pitx3 IRES-tTA mouse VTA were bilaterally injected with either AAV9-TRE-eGFP ( n = 6) or AAV9-TRE-shRit2 ( n = 5-7) and electrically evoked DA transients were measured ex vivo in acute dorsal striatum as described in Methods . Values are presented as mean ±S.E.M. and were analyzed by two-way ANOVA with Tukey’s multiple comparison test. Data were acquired from n = 5-8 DS slices prepared from 3-4 independent mice per viral condition. a Representative voltammograms: Voltammograms displaying evoked current over voltage cycles and time, in slices from eGFP- and shRit2-injected mice, recorded in either ACSF or 25nM L-741,626, as indicated. Arrowheads indicate delivery of single, squared wave pulse. b Dopamine transients: Representative evoked DA transients in slices in slices from eGFP- and shRit2-injected mice, recorded in either ACSF or 25nM L-741,626. (shaded areas), as indicated. c Average amplitudes: DA transient amplitudes are presented in µM. Virus: p = 0.03, F (1, 20) = 2.83; Drug: p = 0.02, F (1, 20) = 6.58; * p < 0.05. d Average decay tau, presented in seconds. Virus: p = 0.16, F (1, 23) = 2.08; Drug : p < 0.0001, F (1, 23) = 27.43; ** p < 0.01.

Article Snippet: Quantitative PCR was performed using the Applied Biosystems® 7500 Real-Time PCR System Machine and software or using the Bio-Rad C1000 Touch Thermal Cycler with CFX96 Real-Time system and software using Taqman® gene expression assays for mouse Rit2 (Mm0172749_mH), TH (Mm00447557_m1), DAT (Mm00438388_m1), DRD2 (Mm00438541_m1), Pitx3 (Mm01194166_g1), Nurr1 (Nr4a2, Mm00443060_m1), Rit1 (Mm00501400_m1), Vps35 (Mm00458167_m1).

Techniques: Ex Vivo, Injection, Comparison, Virus

Journal: NPJ Parkinson's Disease

Article Title: Silencing Parkinson’s risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability

doi: 10.1038/s41531-024-00648-8

Figure Lengend Snippet: Quantitative Immunoblotting . Pitx3 IRES-tTA mouse VTA were bilaterally injected with either AAV9-TRE-eGFP or AAV9-TRE-shRit2. Striatal lysates were collected 4-5 weeks (ST) or 5-6mo (LT) post-injection and αSyn and pS129-αSyn levels were measured by quantitative immunoblot, normalized to actin, as described in Methods . Tops: Representative immunoblots showing two independent mouse lysates per virus. Average values ± S.E.M. and presented, and N values indicate the number of independent striata assessed. a–d αSyn levels: shRit2 had no effect on total αSyn levels in males at either ST a . p = 0.46, n = 6) or LT b . p = 0.62, n = 7-9) timepoints. ST shRit2 significantly increased αSyn in females ( c . * p = 0.04, n = 6), but had no effect in LT females as compared to controls ( d . p = 0.50, n = 6). e–h pS129- αSyn levels : pS129-αSyn was significantly increased by shRit2 in ST e. * p = 0.02, n = 6) and LT f . * p = 0.04, n = 7-9) males. pS129-αSyn trended towards a significant increase by shRit2 in ST females g . p = 0.06, n = 6) and was significantly increased in LT females h . * p = 0.04, n = 7-9). Two-tailed, unpaired, Student’s t test.

Article Snippet: Quantitative PCR was performed using the Applied Biosystems® 7500 Real-Time PCR System Machine and software or using the Bio-Rad C1000 Touch Thermal Cycler with CFX96 Real-Time system and software using Taqman® gene expression assays for mouse Rit2 (Mm0172749_mH), TH (Mm00447557_m1), DAT (Mm00438388_m1), DRD2 (Mm00438541_m1), Pitx3 (Mm01194166_g1), Nurr1 (Nr4a2, Mm00443060_m1), Rit1 (Mm00501400_m1), Vps35 (Mm00458167_m1).

Techniques: Western Blot, Injection, Virus, Two Tailed Test

Journal: NPJ Parkinson's Disease

Article Title: Silencing Parkinson’s risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability

doi: 10.1038/s41531-024-00648-8

Figure Lengend Snippet: Accelerating rotarod rescue studies . a Experimental schematic . Pitx3 IRES-tTA male mouse VTA were bilaterally injected with AAV9-TRE-shRit2 and were assessed on the accelerating rotarod at the indicated timepoints as described in Methods . Mice were initially tested for three trials, injected ±the indicated treatment drugs (I.P), and were retested for an additional three trials. Performance indices for each mouse were calculated pre- and post-test, and performance were assessed with a two-tailed, paired Student’s t test. N values indicate the number of independent mice. b Methylphenidate (MPH) treatment: ST shRit2 mice received either saline, 5 mg/kg MPH, or 0.5 mg/kg DMI, and were retested 15 min postinjection. Left: Raw rotarod results presented as average latency to fall during the trial, ±S.E.M. Right: Paired pre- and post-test rotarod performance indices for each independent mouse assessed. shRit2 mice treated with MPH performed significantly better than pre-injection (* p = 0.03), whereas performance was not enhanced by either saline (p = 0.28) or DMI ( p = 0.60), n = 5-6. c L-DOPA treatment on ST shRit2 males: ST shRit2 mice were treated ±20 mg/kg L-DOPA and were retested 1 hour postinjection. Left: Raw rotarod results presented as average latencies ±S.E.M. Right: Paired pre- and post-test rotarod performance indices for each independent mouse. shRit2 mice treated with L-DOPA (* *p = 0.005), but not saline ( p = 0.98) performed significantly better than pre-injection, n = 6–7. d L-DOPA treatment on LT shRit2 males: LT shRit2 mice were treated ±20 mg/kg L-DOPA and were retested 1 hour postinjection. Left: Raw rotarod results presented as average latencies ±S.E.M. Right: Paired pre- and post-test rotarod performance indices for each independent mouse. Neither L-DOPA ( p = 0.48) nor saline ( p = 0.82) treatment significantly improved performance as compared to pre-injection performance, n = 6.

Article Snippet: Quantitative PCR was performed using the Applied Biosystems® 7500 Real-Time PCR System Machine and software or using the Bio-Rad C1000 Touch Thermal Cycler with CFX96 Real-Time system and software using Taqman® gene expression assays for mouse Rit2 (Mm0172749_mH), TH (Mm00447557_m1), DAT (Mm00438388_m1), DRD2 (Mm00438541_m1), Pitx3 (Mm01194166_g1), Nurr1 (Nr4a2, Mm00443060_m1), Rit1 (Mm00501400_m1), Vps35 (Mm00458167_m1).

Techniques: Injection, Two Tailed Test, Saline