Inhibition of PTPRS function does not affect the survival or regeneration of dopaminergic neurons but alters synaptic function in the nigrostriatal pathway

Abstract

Parkinson's Disease (PD) is characterized by midbrain dopaminergic (mDA) neuron degeneration in the ventral midbrain, contributing to debilitating motor symptoms. This study investigated whether Protein Tyrosine Phosphatase Receptor Sigma (PTPRS), a known inhibitor of axonal growth through chondroitin sulfate proteoglycan (CSPG) interaction, plays a role in mDA neuron survival, function, and regeneration in PD. Our data show that inhibition of PTPRS using intracellular sigma peptide (ISP) enhances human mDA neuron neurite outgrowth in vitro, suggesting an inhibitory role of this receptor in the differentiation of human embryonic stem cell (hESC)-derived mDA neurons in vitro. However, genetic deletion and pharmacological inhibition of PTPRS does not affect mDA neuron survival, axon density, or motor behavior in 6-OHDA unilateral partially lesioned mice. Similarly, mDA-specific deletion of Ptprs does not affect the basal behavioral measurement in cKO mice, including general locomotion and motor learning. Interestingly, however, Ptprs deletion led to a reduced response in the behavior sensitization to repeated methamphetamine (METH) exposure in cKO mice, suggesting a dampened response in synaptic function in cKO mice under these conditions. Ex vivo voltammetry recording in the striatum indeed shows altered parameters of dopamine (DA) release upon stimulation. These findings indicate that the inhibition of PTPRS function in human mDA neuron progenitors might be a promising strategy for enhancing neurite outgrowth or incorporation in host tissues in transplantation therapies. Finally, our data support that inhibition of PTPRS function in mDA neurons in adult mice in vivo could inhibit METH induced plasticity and behavioral sensitization.

Keywords: Dopaminergic neurons; PTPRS; Parkinson's disease; Synapses.

Fig. 1.

Pharmaceutical inhibition of PTPRS leads to increased TH+ neurites. (A-B) Plots made from publicly available scRNAseq data showing Ptprs expression in (A) mouse (Single nuclei dataset, n.d.) and (B) human (Kamath et al., 2022) mDA neurons. (C) Dual Smad inhibition differentiation protocol and experimental timeline for hESC-derived mDA neurons. (D-E) Representative images of human mDA neuron culture stained for TH, βIII-tubulin, and DAPI following 3-day treatment of (D) scrambled peptide or (E) intracellular sigma peptide (ISP). Quantification of (F) TH+ neurite length, (G) TH+ cell number, (H) βIII-Tubulin+ neurite length, and (I) βIII-Tubulin+ cell number. n = 3 for all groups. Mean ± SEM, *p < 0.05. Student’s t-test was used for statistical analysis. *, p < 0.05. Each data point represents a biological replicate, displaying the average values from technical replicates in that experiment. Scale bar as indicated.

Fig. 2.

Dat^IRES-Cre-Ptprs^flox/fl mouse line effectively recombines floxed alleles in dopaminergic neurons within the substantia nigra. (A) Model for targeting Ptprs gene deletion in mDA neurons. (B) Representative image showing tdTomato labeling colocalization with TH+ cells in the nigrostriatal pathway. (C) Dissected bulk tissue from cerebellum (Cer), substantia nigra (SN), hippocampus (HC), cortex (CTX), and stratum (STR) were collected from Dat^IRES-Cre-Ptprs^wt/wt (WT) and Dat^IRES-Cre-Ptprs^fl/fl (cKO) mice with the blue arrow pointing to the Ptprs recombined allele. (D-E) Increased mDA neuron specificity was achieved with Laser Capture Microdissection (LCM) performed on tdTomato+ cells as seen (D) before and (E) after LCM collection. Blue triangles represent cells targeted for LCM and green dashed circles represent LCM cut site. (F) Electrophoresis gel for the tdTomato+/− SNc LCM single mDA neurons from WT and cKO mice with the blue arrow pointing at the Ptprs recombined allele. Scale bar as indicated.

Fig. 3.

Dat^IRES-Cre-Ptprs^flox/flox mice show no changes in TH+ cell number. Representative immunohistochemistry images of TH DAB staining in the substantia nigra (SN) and ventral tegmental area (VTA) for Dat^IRES-Cre-Ptprs^wt/wt (WT) and Dat^IRES-Cre-Ptprs^fl/fl (cKO) mice at (A) 5–6 months old and (C) 14–22 months old. Unbiased stereological quantification of TH+ cells in the SN and VTA at (B) 5–6 months old and (D) 14–22 months old. Each data point represents the total number of cells per region. Representative immunohistochemistry images of TH DAB staining in the striatum for WT and cKO mice at (E) 5–6 months old and (H) 14–22 months old. Quantification of (F, H) striatum area and (G,J) % TH+ fiber density in the striatum at (F,G) 5–6 month old and (I,J) 14–22 month old mice. Each data point represents the average of a single animal (>3 brain sections per animal). n = 6–8 for each group and the sex of each animal is represented by open circles (females) and open triangles (males). Two-way ANOVA with Tukey’s post hoc pairwise analysis or student’s t-test was used for statistical analysis. Mean ± SEM. ns = not significant. Scale bar as indicated.

Fig. 4.

DatIRES-Cre-Ptprsflox/flox mice have no changes in protein levels of mDA neuron markers. Probing western blot for TH and β-Actin protein levels in the (A) substantia nigra and (E) striatum and DAT and β-Actin protein levels in the (C) substantia nigra and (G) striatum. Regions were collected via tissue punches. Integrated density quantification normalized to β-Actin for TH in the (B) SN and (F) STR and DAT in the (D) SN and (H) STR regions. n = 4 per group with 2 males and females each. The sex of each animal is represented by open circles (females) and open triangles (males). Student’s t-test was used for statistical analysis. Mean ± SEM. ns = not significant.

Fig. 5.

Dat^IRES-Cre-Ptprs^flox/flox mice show no changes in behavioral tests. (A) Behavior timeline for Dat^IRES-Cre-Ptprs^wt/wt (WT) and Dat^IRES-Cre-Ptprs^fl/fl (cKO) mice at 4–6 months old. General locomotion for 23 h at 1 h intervals showing the (B) total distance, (C) average speed, and (D) vertical activity count. Total 23 h locomotion data showing ^® total distance, (F) average speed, and (G) vertical activity count. The 1st hour of the 23 h locomotion test quantification for (H) total distance, (I) average speed, and (J) vertical activity count. Motor coordination was assessed via (K-L) pole test showing (K) descending time and (L) time to turn and (M) rotarod showing latency to fall. Anxiety-like behavior was assessed via the open field test showing (N) % distance in outer zone and (O) % time in outer zone and the elevated plus maze showing (P) % distance in open arms and (Q) % time in open arms. n = 14–39 per group. The sex of each animal is represented by open circles (females) and open triangles (males). Two-way ANOVA with Tukey’s post hoc pairwise analysis or student’s t-test was used for statistical analysis. Mean ± SEM. ns = not significant.

Fig. 6.

Deletion of Ptprs shows no change in the protection or recovery of the nigrostriatal pathway from the 6-OHDA unilateral injection model. (A) Mouse model (left) used for Dat^IRES-Cre-Ptprs^wt/wt (WT) and Dat^IRES-Cre-Ptprs^fl/fl (cKO) in the experimental timeline (right) for the 6-OHDA unilateral partial lesion model. (B) Representative images of the SN showing the contralateral and ipsilateral sides for WT and cKO mice 31 days after 6-OHDA injection. (C) Unbiased stereological quantification of TH+ cells in the contralateral and ipsilateral SN and VTA. (D) Representative images of the striatum with TH DAB staining on the contralateral and ipsilateral sides 31 days after the 6-OHDA injection. Quantification of (E) TH+ fiber area for contralateral and ipsilateral sides and (F) % TH+ fibers remaining. (G) Schematic showing the rotation test methodology. (H) Methamphetamine-induced rotation test showing the number of net rotations 30 days following 6-OHDA injection. n = 5–6 per group. Two-way ANOVA with Tukey’s post hoc pairwise analysis, or student’s t-test was used for statistical analysis. Mean ± SEM. ****p < 0.0001, ns = not significant.

Fig. 7.

Pharmacological inhibition of PTPRS does not improve recovery of the nigrostriatal pathway from the 6-OHDA unilateral injection model.

Fig. 8.

Dat^IRES-Cre-Ptprs^flox/flox mice have altered synaptic functions. (A) Methamphetamine (METH)-induced locomotion test showing total distance in the first 30 min following saline or METH injection. Measured total distance in 10 min intervals for the 1 h test following METH injection on (B) METH day 1, (C) METH day 5, (D) challenge day 7, and ^® challenge day 30. N = 10–11 per group. Two-way ANOVA repeated measures (RM) with Tukey’s post hoc pairwise analysis was used for statistical analysis of METH-induced locomotion data. Representative false color plot and cyclic voltammogram plot for DA in Dat^IRES-Cre-Ptprs^wt/wt (WT) and Dat^IRES-Cre-Ptprs^fl/fl (cKO) mice with (F) no stimulation and (N) KCl stimulation. Before KCl stimulation (G) concentration, (H) duration, (I) frequency, (J) number of events, (K) concentration relative frequency with 0.02uM bins, (L) duration relative frequency with 1 s bins, and (M) frequency relative frequency with 0.02 Hz bins. After KCl stimulation (O) concentration, (P) duration, (Q) frequency, ^® number of events, (S) concentration relative frequency with 0.02 μm bins, (T) duration relative frequency with 1 s bins, and (U) frequency relative frequency with 0.02 Hz bins. N = 3 per group. Student’s t-test was used for statistical analysis. Mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.