Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke

Abstract

In addition to neuroprotective strategies, neuroregenerative processes could provide targets for stroke recovery. However, the upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) impedes innate regenerative efforts. Here, we examine the regulatory role of PTPσ (a major proteoglycan receptor) in dampening post-stroke recovery. Use of a receptor modulatory peptide (ISP) or Ptprs gene deletion leads to increased neurite outgrowth and enhanced NSCs migration upon inhibitory CSPG substrates. Post-stroke ISP treatment results in increased axonal sprouting as well as neuroblast migration deeply into the lesion scar with a transcriptional signature reflective of repair. Lastly, peptide treatment post-stroke (initiated acutely or more chronically at 7 days) results in improved behavioral recovery in both motor and cognitive functions. Therefore, we propose that CSPGs induced by stroke play a predominant role in the regulation of neural repair and that blocking CSPG signaling pathways will lead to enhanced neurorepair and functional recovery in stroke.

Keywords: CP: Neuroscience; CSPGs; PTPσ; axonal regeneration and sprouting; functional recovery; neurogenesis; proximal MCAo; stroke.

Figure 1.. Accumulation of CSPGs near the infarct border at 2–30 days after stroke and NSCs

(A–L) CSPG staining in stroke brains. Higher magnification (a’–l”). Representative images are shown with >3 mice with similar results for each time point. (M–R) CSPGs staining in non-stroke brain within the cortex or SVZ niche (higher magnification inp’-r’). (S–U) CSPGs are present in ex vivo adult SVZ neurospheres. Lower right panel shows CSPGs detected by mass spectrometry in conditioned media from neurosphere cultures (see Table S1 for full list). Scale bar: 50 μm.

Figure 2.. Inhibition of CSPG-PTPσ signaling leads to increased neurite outgrowth and migration of SVZ NSCs

(A and B) Inhibition of PTPσ by ISP shows increased neurite outgrowth compared to controls. (C and D) Primary Ptprs cKO adult NSCs (AAV-Cre infected Ptprs floxed NSCs) also show increased neurite outgrowth compared to WT. Total of more than 50 cells were quantified from 3 tissue culture wells. Representative data shown from at least 3 independent experiments. (E and F) Increased CSPG concentrations lead to decreased migration of adult NSCs grown as neurospheres, and ISP leads to increased migration from SVZ neurospheres. (G and H) PTPσ deletion in adult NSCs also results in enhanced migration under both basal conditions and with additional CSPG coating (aggrecan 1 or 10 μg/mL). #, p < 0.05 or ###, p < 0.001 compared to no aggrecan; **p < 0.01 and ***p < 0.001 compared to control peptide or WT. (I and J) ISP enhances NSCs migration via disinhibition of the ERK pathway and upregulation of MMP2 activity. **p < 0.01 and ***p < 0.001. (K–P) ISP treatment of NSCs led to increased p-ERK levels, while it had no effect on pAkt levels. ISP increases Mmp2 mRNA levels. **p < 0.01. For neurosphere migration assays, each data point represents 1 neurosphere, and data were pooled from 2–3 independent experiments. ANOVA for multiple group analysis and Student’s t test for 2 group analyses.

Figure 3.. ISP treatment increased newly born neuroblast migration deep into the striatum 1 month after stroke

For lineage tracing, Nestin creER-tdTomato mice were used. Stroke mice receiving vehicle (A–D, K, and M) or ISP treatment (E–H, L, and N) starting from day 1 post-stroke for 30 days. Contralateral side showing minimal migration of DCX⁺ neuroblasts (arrows) or tdTomato⁺ cells into the striatum (A, E, I, and J). Stroke strongly enhances the migration of newly born astrocytes (double-positive for tdTomato and GFAP, arrows in D and H; for single channel images, see Figure S4), but disallows the migration of DCX⁺ neuroblasts deeply into the lesion (B–D, I, and J). ISP treatment enhances DCX⁺ neuroblast migration (G, I, L, and N) but did not change total tdTomato⁺ cells (F, H, I, and J). ISP treatment enhanced the total number of DCX⁺ cells that penetrated into the glial scarred lesion area at 30 days post-stroke (K–O) and the migration of DCX⁺ into the glial scarred lesion area: (P)total DCX⁺ cell migrated area (Q) furthest distance migrated from the lateral wall of ventricle and (R) furthest distance migrated from the border of the glia scar in striatum. **p < 0.01 and ***p < 0.001, ANOVA for (I and J) and Student’s t test for (O–R). Each data point represents the average of 1 animal (average for each animal is obtained by quantifying multiple brain sections expanding the stroke infarct volume). Data were combined from 2 independent cohorts of mice.

Figure 4.. Post-stroke ISP treatment enhances axonal sprouting from the contralateral cortex after stroke

(A) Schematic representation of the BDA injection site (contralateral to stroke side) and corticospinal tract (CST). (B–M) ISP enhances contralateral cross-callosal projections to the stroke side (B–D, vehicle (Veh)-treated stroke mice; E–G, ISP-treated stroke mice). Quantification in (H) and (I). ISP-treated mice show more callosal projecting neuronal cell bodies retrogradely labeled by BDA directly adjacent to the lesion. (C and F and higher magnification shown in C′ and F′). ISP also enhances the corticospinal tract (CST) sprouting from the non-stroke cortex to the contralateral side across the midline of the cervical spinal cord (J and K and J′ and K′ showing higher magnification). Quantification for CST cross-midline sprouting in (L). Note that BDA injection volume at the non-stroke cortex is similar in Veh or ISP-treated mice (representative images in B and E and quantification in M). (N and O) ISP treatment enhances the density of 5-HT⁺ axons that are in the peri-infarct area and crossing the glial scar region (quantification shown in P and Q). Scale bar: 100 μm (n = 3 mice for each group; multiple brain and spinal cord sections were analyzed for each mouse, and average was used as a single data point for statistical analysis). **p < 0.01 and ***p < 0.001, Student’s t test.

Figure 5.. RNA-seq from the peri-infarct cortex of ISP treated versus Veh-treated stroke mice shows differentially regulated genes

(a) DEGs that can be clustered into GO pathways (B) such as regulators of apoptotic signaling pathways, axon development pathways, and pathways that are involved in responses to stress. (C) Validation of the top selected gene expression by qRT-PCR (n = 7 for each group). (D–G) Nurr1 (Nr4a2) expression is decreased in peri-infarct cortex in stroke mice (arrows in D) but partially restored in ISP-treated mice (n = 4 for each group). Nurr1 expression is mainly detected in the NeuN⁺ neurons in the peri-infarct zone. (H–J) Bcl2 expression is upregulated in ISP-treated peri-infarct zone enriched in Iba1⁺ cells not GFAP⁺ reactive astrocytes (n = 4 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test for (C) and 2-way ANOVA for (J) and (G). Scale bar=50 μm. See Table S2 for complete list of DEGs.

Figure 6.. Post-stroke ISP treatment leads to enhanced functional recovery in mice

(A) Experimental timeline. (B) Representative MRI images at different coronal levels. (C) At day 1 after stroke, before any treatment, the 2 groups of animals have similar infarct sizes and distributions. Post-stroke ISP treatment leads to enhanced general and fine locomotor functions as well as improved cognitive skills. (D) General locomotor performance was measured by automated open field chambers for 1 h. (E and F) Fine motor function was measured by the adhesive tape removal test (E) and cognitive function was measured by Barnes maze at 4 weeks after stroke (F). *p < 0.05, **p < 0.01, ***p < 0.001, 2-way repeated-measures (RM) ANOVA for (D) and (E) and Student’s t test for (F). Each data point represents an individual mouse, data pooled from 2 independent cohorts.

Figure 7.. Post-stroke ISP treatment starting at post-stroke day 7 also leads to enhanced behavioral functional recovery in mice

(A) Experimental timeline. (B) Representative MRI images at different coronal levels. (C) At day 1 after stroke, before any treatment, the 2 groups of animals have similar infarct sizes and distributions. (D–F) Post-stroke ISP treatment leads to enhanced general locomotor function measured by automated open field chambers for 1 h (D), increased fine motor function measured by the adhesive tape removal test (E), and improved cognitive function measured by Barnes maze at 4 w after stroke (F). *p < 0.05, **p < 0.01. Two-way RM ANOVA for (D) and (E) and Student’s t test for (F). Each data point represents an individual mouse; data from 1 cohort.