Journal: International Journal of Molecular Sciences

Article Title: Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

doi: 10.3390/ijms19092788

Figure Lengend Snippet: TaqMan quantitative RT-PCR analysis of dopamine-differentiated neural stem cells (NSCs). ( A ) Expression level of genes markers of NSCs and their progeny ( NESTIN , SOX1 , SOX2 PAX6 , β-Tub-III , MBP , Olig-2 , GFAP ) under control media (orange) and DIF1 media derived dopaminergic differentiation (green) and from pluripotent undifferentiated CJ01 iPSC (blue); ( B ) expression level of genes markers of the midbrain dopaminergic lineage ( TH , LMX1A , EN1 , NURR1 , PITX3 , and dopamine decarboxylase ( DDC ) enzyme that convert L-DOPA to dopamine) in NSC differentiated under control media (orange) or DIF1 media (green) and from undifferentiated CJ01 iPSC (blue), confirming the increase in the dopaminergic neuronal differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3. Error bars represent relative expression ± S.D.

Article Snippet: The Q-PCR for the NSCs and dopaminergic cell markers was performed using the following TaqMan gene expression assays (ThermoFisher Scientific): NES (Hs00707120_s1), MBP (Hs00921945_m1), SOX1 (Hs01057642_s1), SOX2 (Hs01053049_s1) and PAX6 (Hs01088112_m1); TH (H200165941_m1), LMX1A (Hs00892663_m1), DDC (Hs01105042_m1), EN1 (Hs00154977_m1), NR4A2 (Hs00428691_m1) and PITX3 (Hs00374504_m1).

Techniques: Quantitative RT-PCR, Expressing, Control, Derivative Assay

Journal: bioRxiv

Article Title: Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype

doi: 10.1101/2023.04.26.538430

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) post-injection. Data 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) post-injection. (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: bioRxiv

Article Title: Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype

doi: 10.1101/2023.04.26.538430

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 were analyzed by two-way ANOVA with Tukey’s multiple comparison test. (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, ±S.E.M. (shaded areas), as indicated. (C) Average amplitudes: DA transient amplitudes are presented in µM ±S.E.M. 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 ±S.E.M. 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: bioRxiv

Article Title: Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype

doi: 10.1101/2023.04.26.538430

Figure Lengend Snippet: Stereological analysis of SNc neurons. Pitx3 IRES-tTA males and female mouse VTA were bilaterally injected with either AAV9-TRE-eGFP or AAV9-TRE-shRit2 and brains were fixed, sectioned and stained with cresyl violet and TH-specific antibodies at the LT timepoint. Total and TH+ cell numbers were counted as described in Methods. LT male and female data were pooled. LT Rit2 KD significantly decreased total Nissl+ ( A. * p=0.02) and TH+ ( B. **p=0.008) cells, but did not significantly affect the %neurons that were TH+ ( C. p=0.64) in SNc. Unpaired, two-tailed Student’s t test, n=7.

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, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype

doi: 10.1101/2023.04.26.538430

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. (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: bioRxiv

Article Title: Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype

doi: 10.1101/2023.04.26.538430

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 (B) Methylphenidate (MPH) treatment: ST shRit2 mice received either saline, 5mg/kg MPH, or 0.5mg/kg DMI, and were retested 15 min post-injection. Left: Raw rotarod results presented as latency to fall during the trial. Right: Paired pre- and post-test rotarod performance indices. 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 ±20mg/kg L-DOPA and were retested 1 hour post-injection. Left: Raw rotarod results. Right: Paired pre- and post-test rotarod performance indices. 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 ±20mg/kg L-DOPA and were retested 1 hour post-injection. Left: Raw rotarod results. Right: Paired pre- and post-test rotarod performance indices. 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