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

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.

Whether CSPG-PTPσ signaling may play a role in a large injury such as stroke has not been investigated. By genetically and pharmacologically restricting the inhibitory properties of sulfated proteoglycans, we investigated the untoward effects of CSPGs on two major neurorepair mechanisms—axonal sprouting and the generation of new neuroblasts as well as their migration after stroke. We also explored the potential molecular pathways through which CSPGs modulate adult NSC biology.

The transmembrane receptor protein tyrosine phosphatase-sigma (PTPσ) has been identified as a major receptor for the inhibitory actions of CSPGs (). To modulate proteoglycan-mediated inhibition over large regions, we have used systemic agents that could block chondroitin sulfate-glycosaminoglycan (CS-GAG) interactions with this receptor in the presence of any evolving lesion without the need to directly impale the CNS parenchyma. Intracellular sigma peptide (ISP), a peptide mimetic of the PTPσ regulatory wedge region with a TAT domain to facilitate membrane and CNS penetration (), was designed for this purpose. ISP has very high specificity for PTPσ (). Importantly, after systemic delivery, ISP rapidly enters the CNS and leads to significant axonal sprouting with restored sensory motor and bladder function after acute contusive cord injury in adult rats () and has also led to the enhanced migration, differentiation, and remyelination by oligodendrocyte precursor cells (OPCs) with functional recovery in mouse models of MS () and SCI ().

In models of stroke (), chondroitinase ABC (ChABC) has been used therapeutically by targeted injection into the spinal cord. While results were encouraging, the effects were limited likely due to minimal spread of the enzyme. To overcome the limitations of native ChABC, several labs have shown successful long-term and/or widespread delivery and efficacy in stroke and SCI models using thermostabilized () and viral-mediated formulations of chondroitinase (), although the potential complications of direct in vivo administration remained.

Ablation studies have suggested that newly born neuroblasts may contribute to functional recovery after stroke, despite the low numbers that can survive as maturing neurons (). Although stroke stimulates this process, the endogenous response is inadequate (). Due to the hostile environment in the damaged brain, many of the newly born neurons approach but cannot invade the stroke peri-infarct region to intermingle with surviving neuropil and they mostly die within 1 week after their birth (). This indicates the need for strategies that can enhance both the survival and migration of newly born neuroblasts. Stroke also induces axonal sprouting in the brain in the form of new ipsilateral local circuits as well as expanded intercortical connections and descending projection reorganizations (). However, sprouting is also insufficient in the lesioned mammalian CNS. One critical repair-limiting factor for both neurons and neural stem cells (NSCs) is the family of potently inhibitory ECM molecules known as chondroitin sulfate proteoglycans (CSPGs) (). Certain CSPGs are upregulated in abundance in glial scars after brain or spinal cord injury (SCI) (). CSPGs in the scar limit regeneration through the lesion but they also severely restrain potential neuroplasticity around and beyond the lesion perimeter (). CSPGs have also been suggested to curtail the access of progenitor cells to remyelinate cord and multiple sclerosis (MS) lesions ().

Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes.

Stroke profoundly alters the lives of affected individuals and is one of the leading causes of death and disability worldwide (). Current treatment strategies are largely neuroprotective and all are limited by narrow time windows (). However, the potential for regeneration/plasticity in the post-stroke CNS is still possible for weeks or even longer, which may provide an extended opportunity for treatment (). Two potential processes for repair are axonal sprouting and neurogenesis (). Understanding how these endogenous mechanisms may be further stimulated to contribute to recovery will help in the development of novel therapeutic interventions.

Since cognitive decline is also a major cause of disability in stroke survivors, we also examined the effect of ISP treatment on cognitive function. The Barnes maze test was used to evaluate learning/memory function, and our data showed that ISP-treated mice used significantly less time as well as fewer error trials to find the escape hole at 4 weeks post-stroke ( Figure 6 F). This demonstrates that systemic ISP treatment is able to improve multiple aspects of functional recovery, including general locomotor function, specific upper limb fine motor control, as well as cognitive function. To examine the potential effective time window of post-stroke ISP treatment, in a separate group of animals, we tested the efficacy of post-stroke treatment with ISP starting at day 7 after stroke. Our data ( Figure 7 ) show that even when started 1 week after stroke, ISP treatment still effectively improved general locomotor function, specific upper limb sensorimotor function, and cognitive function. Note that in the locomotor function test, and especially in the fine motor tape removal test, the day that ISP-treated animals started to show significant improvements after delayed administration was equally shifted by approximately 1 week ( Figure 7 ). However, the extent of functional recovery was only slightly reduced with delayed peptide administration, suggesting that the potential for neural recovery in stroke animals is still possible with a delayed onset of delivery of our regenerative peptide. Notably, for all three behavioral tests, significant improvements in ISP-treated mice in either post-day 1 or post-day 7 treatment paradigms all reached large effect sizes based on Cohen’s d and coefficient r (), especially toward more chronic time points (3–4 weeks post-stroke; statistical details on effect size provided in Tables S3 and S4 ), suggesting that the observed functional improvements due to ISP treatment are also biologically meaningful. Interestingly, when tested in non-stroke mice, ISP treatment in general did not result in significantly increased locomotor function as was observed in treated stroke mice ( Figure S11 A). At 3 weeks, for the total horizontal movement, the naive ISP group did show a slightly decreased range, which returned to the vehicle group level at 4 weeks. In the adhesive and cognitive functional tests ( Figures S11 B–S11D), there were no significant differences between the Veh-treated or ISP-treated naive groups.

∗ 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.

(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).

(C) At day 1 after stroke, before any treatment, the 2 groups of animals have similar infarct sizes and distributions.

Given the positive effects mediated by ISP treatment in multiple neurorepair-related phenomena, we conducted a series of behavioral experiments to examine whether peptide treatment could enhance functional recovery in stroke animals. We tested the efficacy of continuous post-stroke ISP treatment (1 mg/kg/day, subcutaneously [s.c.], starting at 24 h post-stroke) using our proximal middle cerebral artery occlusion (MCAo) model. Two independent cohorts of young adult (10–12 weeks old) C57bl/6J male mice (total of n = 20 each group) were subjected to transient proximal MCAo surgery (45 min) to induce a large stroke in both striatal and cortical tissue ( Figures 6 A and 6B ), mimicking a human “malignant” stroke (). Stroke mice were subjected to T2-weighted MRI scanning to determine the size of the lesion epicenter and were comingled blindly into two equally distributed groups that either received daily vehicle or ISP treatment starting from 24 h post-stroke onset for 4 weeks. Just before the treatment was started, T2-weighted MRI showed that the two groups of mice had no differences in the extent and location of ischemic injury ( Figures 6 B and 6C). Using computer-monitored automated open field analysis, we found that ISP treatment significantly increased locomotor function at 2–4 weeks after stroke in multiple parameters ( Figure 6 D). Since the most common functional deficits following MCAo stroke are motor impairments of the contralateral upper limb, we also examined the effect of post-stroke ISP treatment on performance in a fine forelimb sensory-motor function test, “adhesive tape removal.” The results showed that post-stroke ISP treatment significantly improved the speed ( Figure 6 E) that mice were able to remove the tape from the contralateral affected paw (right front paw in our model) without any obvious effects on the time to remove the tape from the ipsilateral unaffected paw (left front paw in our model [ Figure S10 ], showing the removal time on the unaffected left paw), suggesting that the result of ISP treatment is specifically related to stroke-induced deficits without untoward side effects in sensory and motor function. Importantly, at the 1-week pre-stroke time point and at post-stroke day 3, the control and the ISP-treated groups showed no differences in behavioral tests (post-stroke days 3 and 7 for open field and post-stroke day 7 for tape removal; Figures 6 D and 6E), validating the equal grouping of animals according to stroke lesion size and supporting a neurorestorative mechanism through ISP treatment.

∗ 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.

(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).

(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.

Given that ISP treatment improved axonal sprouting and the migration of new neuroblasts, preserved the integrity of peri-infarct-associated cortical neurons, and decreased overall atrophy of the stroke brain, we further explored the potential mechanism of ISP on promoting neuronal survival and function by examining the transcriptome changes in the peri-infarct cortical regions. We conducted RNA sequencing (RNA-seq) on tissue from motor cortex near the infarct zone on the stroke side from animals that received ISP or scrambled peptide starting at 1 day post-stroke. Cortical tissues were collected at day 14 post-stroke, a time point that has previously revealed transcriptomic differences in either behaviorally spontaneously recovered or non-recovered mice (). RNA-seq showed that there were 217 genes upregulated and 185 genes downregulated within the motor cortex in ISP-treated mice ( Figure 5 A , complete list of differentially expressed genes are provided in Table S2 ). Gene Ontology (GO) term analysis suggests that the pathways that changed include multiple genes upregulated in the negative regulators of apoptotic pathways (Rffl/Ivns1abp/Nr4a2/Bcl2, Figure 5 B) and downregulated genes in the positive regulators of apoptotic pathways (Pcgf2/Bok/Mif/Prr7/Ndufa13/Wnt4/Wfs1, Figure 5 B). Interestingly, several genes related to axon development (Sema4d/Tnfrsf21/Cdh2/Nr4a2/Dixdc1/Spg20/Picalm/Bcl2, Figure 5 B) were also enriched, which is consistent with the enhanced axonal sprouting in ISP-treated mice. Compared to the RNA-seq data comparing mice that showed spontaneous behavioral recovery to the ones that do not recover after stroke (), we found three overlapping genes from our dataset that underwent similar changes in direction and extent of mRNA levels (cited4, Sag, and Tpbg). We examined the changes in mRNA levels by qRT-PCR in vehicle or ISP-treated peri-infarct cortex of the top 5 differentially expressed genes (Igfn1, Penk, Rasgef1c, Dact2, and Grm2), in addition to cited4, Sag, Tpbg, and genes implicated in cell survival and axon development (Nr4a2, also known as Nurr1, Bcl2, and Sema4d). A majority of the target genes were validated by qRT-PCR, with the exception of lowly abundant mRNAs (Sag and Tpbg) and Bcl2 ( Figure 5 C). We tested commercially available antibodies for these genes and found that NR4A2 (NURR1) expression is downregulated in Veh-treated stroke mice in the peri-infarct cortex, but it is preserved in ISP-treated mice ( Figures 5 D–5G). NURR1cells were mainly NeuNneurons ( Figures 5 E and 5F). Interestingly, although Bcl2 expression changes were not validated by qRT-PCR, they were validated by immunostaining. B cell lymphoma 2 (BCL-2) immunostaining was substantially increased in the ISP-treated peri-infarct area and was mainly colocalized with Iba1microglia or infiltrating macrophages ( Figures 5 H–5J). The expression of BCL-2 in microglial cells is consistent with previous characterizations of BCL-2 expression patterns in the adult rodent brain (). We recently reported an effect of PTPσ inhibition on promoting a beneficial M2-like alternative neuroinflammatory response after SCI (). The upregulation of BCL-2 expression in microglia may facilitate this beneficial inflammatory profile post-stroke. The detailed mechanisms of these target genes warrant further investigation.

(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.

(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.

(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.

We also examined chronic atrophy (evaluated at 30 days post-stroke), which normally occurs within the lesioned side of the brain, in mice treated with either scrambled or ISP peptide started at either 1 day or 7 days after stroke. Compared to vehicle-treated stroke mice, ISP treatment started at either 1 day or 7 days post-stroke significantly decreased the extent of atrophy at 30 days after injury ( Figure S9 ). The end of day 1 post-stroke-initiated treatment resulted in a more substantial decrease in brain atrophy compared to post-stroke treatment begun after 1 week. In addition, we noticed more lesion volume variation in day 7-treated mice compared to day 1-treated mice ( Figure S9 ).

Serotonergic fiber sprouting is well known to increase the tone and excitability of the injured CNS (). We, therefore, examined the 5-HT positive nerve terminals near the peri-infarct area at 4 weeks after stroke. Our data demonstrate that, in control animals, 5-HT fibers were present in the vicinity of the peri-infarct zone but were mainly stopped at the edge of the glial scar ( Figure 4 N). However, in ISP treated stroke mice, the intensity of 5-hydroxytryptamine-positive (5-HT) fibers was increased within the peri-infarct zone and, in addition, serotonergic axons penetrated more deeply into the lesion epicenter, using the reactive scar astrocytes as their substrate ( Figures 4 O–4Q). We also examined the immunoreactive density of excitatory synaptic markers (presynaptic marker VGlut2 and postsynaptic marker Homer) at the peri-infarct cortex and found increased immunoreactivity of both VGlut2 and Homer in ISP-treated stroke mice ( Figure S8 ), but no changes in ISP-treated naive mice ( Figure S8 ), suggesting that post-stroke ISP treatment enhances the post-stroke peri-infarct zone synapse density without affecting the stability of synapses in naive non-stroke mice.

To examine whether post-stroke ISP treatment was also able to enhance axonal sprouting, another main mechanism of potential neurorepair after stroke, we examined axonal projections and sprouting from the contralateral motor-sensory cortex. The axonal tracer biotinylated dextran amine (BDA) (10,000 molecular weight [MW]) was injected in the contralateral intact primary motor-sensory cortex at 14 days post-stroke. Brains were harvested at 2 weeks after BDA injection and axonal projections to the peri-infarct area of the opposite hemisphere via the callosum were evaluated. Our results ( Figures 4 C , 4C′, 4F, and 4F′, quantification in 4H) revealed substantially increased axonal projections in the peri-infarct area in the stroke hemisphere originating from the contralateral cortex in ISP-treated mice. Consistent with this, labeled fiber intensity in the corpus callosum was increased by ISP treatment ( Figures 4 D and 4G, quantification in 4I). Interestingly, while this type of BDA tends to preferentially label anterogradely, in ISP-treated animals, we also observed some labeled neuronal cell bodies within the peri-infarct area in the stroke hemisphere but not in vehicle (Veh)-treated stroke mice ( Figures 4 C′ and 4F′), suggesting enhanced survival of peri-stroke neurons that normally project to the contralateral cortex. Naive non-stroke mice receiving BDA injection on one side of the cortex also showed similar retrogradely labeled neurons on the contralateral side in both Veh or ISP-treated mice following the same experimental timeline ( Figure S7 ), suggesting that the preserved peri-stroke neurons in the ISP-treated group were not likely labeled due to the peptide somehow altering/enhancing retrograde uptake of BDA by cross-callosal neurons since they are also present in non-treated and treated naive mice. Focusing on the spinal cord, the recrossed contingent of BDAcorticospinal axon terminals within the C3–C5 segments originating from the non-lesioned cortex was also significantly increased ( Figures 4 J, 4J′, 4K, and 4K′ with quantification in 4L). Injection volumes of BDA in the contralateral cortex were equal in Veh and ISP-treated stroke mice ( Figure 4 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.

(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).

To further identify whether the increased number of DCXcells in the ipsilateral side of the brain as well as those that migrated into the glial scar area were due to increased proliferation of NSCs, in a different cohort, we harvested stroke mice that were subjected to scrambled peptide or ISP treatment after 14 days, a time when NSC cell proliferation peaks in response to stroke (). As before (), NSCs were labeled at 17 days pre-stroke by TAM, and control or ISP peptides were given daily starting at 1 day after stroke. Mice were harvested at 14 days post-stroke and DCX, tdTomato, and Ki67(proliferating cells) along the SVZ were quantified ( Figure S5 ). There were no differences in the total number of Ki67cells along the SVZ at 14 days post-stroke and there were similar numbers of DCXand tdTomatocells within the same regions ( Figure S5 ). This suggests that ISP treatment does not increase the proliferation of NSCs within the SVZ but, rather, the increased total DCXcells in ISP-treated mice at a later time point (day 30, Figure 3 ) may be the result of enhanced survival of DCXneuroblasts that migrate into the infarct area. Indeed, when we compared the day 14 and day 30 stroke brains, we observed further migration of tdTomatoand DCXcells toward the infarct area at day 30 compared to day 14, with increased DCXcells navigating more deeply into the striatum in ISP-treated mice ( Figure S6 , white arrows in B, E, H, and K). In addition, we observed chain-like tdTomato/DCXnewly born neuroblasts migrating from the lateral wall of the SVZ toward the infarct area, which were associated with astrocytes and blood vessels ( Figures S6 M–S6R), consistent with previous reports on the SVZ origin of neuroblasts in the stroke striatum () and the migration pattern of SVZ-derived neuroblasts after stroke ().

Different from the nestin creERT2-R26YFP reporter mice in which 30% of the DCXwere also yellow fluorescent protein positive (YFP) (), our results using the nestin creERT2-tdTomato (Ai9) mice showed that the vast majority of the tdTomato cells that had migrated into the striatum from the SVZ were clearly differentiating as glia, since they were doubly labeled with GFAP ( Figures 3 D and 3H, arrows; single channel images are included in Figure S4 ). Interestingly, the average number of migrated tdTomatocells in the striatum did not differ between control and ISP-treated mice ( Figure 3 J). However, within the SVZ proper and closely adjacent to its lateral wall, many cells that did contain tdTomato were also DCX Figures 3 B and 3F). Thus, primarily, the migration of DCXneuroblasts is being inhibited by CSPG-PTPσ interactions in the vicinity of the scar. The lack of abundant tdTomato expression in migrated neuroblasts deeper into the infarct zone could be due to downregulated activity of the promoter driving the reporter (tdTomato), which could, in turn, downregulate the reporter allele specifically in differentiating/migrating neuroblasts.

To examine whether our in vitro findings translate to a stroke model, we investigated whether post-stroke ISP treatment would enhance SVZ and striatal neuroblast numbers and the migration of neuroblasts into the infarct area. To examine whether ISP treatment could enhance proliferation, migration, differentiation, and survival of NSCs and their progeny, we used an NSC-specific inducible cell labeling system (), using the Ai9 tdTomato reporter. The nestin creERT2-YFP mouse has been previously used by us and others to label newly born cells after stroke () and has successfully labeled multilineage progeny from nestinNSCs post-stroke. However, the Ai9 tdTomato reporter line has not been tested in a stroke model. Two cohorts of mice were used to examine the generation of new neuroblasts and their migration from the SVZ with WT C57BL/6J mice or the nestin creERT2-tdTomato mice. To ensure the specific labeling of SVZ-derived NSCs but not reactive astrocytes, which also upregulate nestin after stroke, we treated the nestin creERT2-tdTomato mice with tamoxifen (TAM) for 5 days starting 22 days before stroke ( Figure 3 ). The waiting period of 17 days after TAM treatment allowed the clearing of TAM from the brain and did not label any reactive astrocytes at 2 days post-stroke in the striatum while it maintained the labeling of SVZ NSCs ( Figure S2 ). Using this mouse line and consistent with our previous observations (), migrations of DCXand tdTomatocells penetrated into the lesioned striatum at 30 days after stroke ( Figure 3 ). There was minimal migration of DCX or tdTomatocells in the striatum on the contralateral side ( Figures 3 A and 3E). Scrambled or ISP peptides were administered starting 1 day post-stroke and then daily for 30 days. At 30 days post-stroke, on the ischemic side of the brain, ISP treatment resulted in clearly increased numbers of DCXcells as well as their enhanced migration toward the stroke penumbra but had no effect on the contralateral side ( Figures 3 B, 3C, 3F, and 3G, quantification in 3I). This lack of a contralateral effect is consistent with the results that 30 days of ISP treatment in non-stroke mice does not change total DCXor tdTomato cells in the SVZ ( Figure S3 ). Interestingly, total DCXcells that had migrated well into the reactive astroglial infarct area also increased with ISP treatment, suggesting enhanced migration deep into the glial scar surrounding the injured area in the brain ( Figures 3 K–3O). Indeed, not only did the total number of DCXcells increase in ISP-treated mice ( Figures 3 K–3O) but also the total area covered by the DCXcells (quantification in Figure 3 P), the furthest distance migrated from the lateral ventricular wall (quantification in Figure 3 Q), and the furthest distance migrated from the medial glial scar border (quantification in Figure 3 R) significantly increased in ISP-treated mice.

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 DCXneuroblasts (arrows) or tdTomatocells 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 DCXneuroblasts deeply into the lesion (B–D, I, and J). ISP treatment enhances DCXneuroblast migration (G, I, L, and N) but did not change total tdTomatocells (F, H, I, and J). ISP treatment enhanced the total number of DCXcells that penetrated into the glial scarred lesion area at 30 days post-stroke (K–O) and the migration of DCXinto the glial scarred lesion area: (P) total DCXcell 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 andp < 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.

The shh signaling pathway is upregulated in multiple cell types in cortical ischemia and influences the outcome of stroke in an animal model.

CSPG-PTPσ binding is known to modulate multiple signaling events in cells including pathways such as AKT and ERK as well as the recently identified ISP-induced production of the CSPG degrading enzyme, matrix metalloproteinase 2 (MMP2) (). To examine the molecular pathways that play a role in mediating ISP-induced migration enhancement, we incubated neurospheres with control media, ChABC (5 mU/mL), ISP containing media (2.5 μM) alone or with inhibitors for the AKT pathway (LY294002, 10 μM), ERK pathway (PD 98059, 10 μM), and the MMP2 inhibitor (OA-Hy, 100 nM). We observed a similar effect of enhanced migration of neuroblasts out of neurospheres with ChABC treatment ( Figures 2 I and 2J), which further supports the role of CSPGs in the inhibition of adult NSC migration. Interestingly, the inhibition of ERK signaling reversed the effect of ISP on NSC migration, while the AKT inhibitor had no effect ( Figures 2 I and 2J). In addition, our results show that MMP2 inhibition is able to reverse the effect of ISP on NSC migration, consistent with our previous findings () that ISP increases MMP2 production in OPCs while expanding this mechanism to a new type of cell, adult NSC-derived neuroblasts ( Figures 2 I and 2J). Importantly, treatment with the specific inhibitors by themselves when ISP peptide was absent did not affect cell exodus from neurospheres ( Figure 2 J), suggesting that ERK inhibitors and MMP2 inhibitors specifically reverse the activation of ERK signaling and MMP2 upregulation caused by ISP treatment. Western blot and qRT-PCR analyses confirmed that ISP treatment in NSCs activates the phosphorylation of ERK ( Figures 2 L and 2O) without affecting p-Akt levels ( Figures 2 K and 2N). ISP treatment also increased the mRNA level of Mmp2 ( Figures 2 M and 2P).

Since neuroblast migration is critical during adult neurogenesis after CNS injury, we examined the movements of SVZ NSC-derived neuroblasts in the absence or presence of additional CSPGs. Individual neurospheres that were similar in size were picked from neurosphere cultures (days in vitro [DIV] 5–6) and plated upon different concentrations of aggrecan and the translocation of cells from the individual neurospheres was quantified by the migration index. CSPG-containing neurospheres treated with ISP showed increased migration on poly-L-lysine-coated culture surfaces ( Figures 2 E and 2F and Video S1 for time lapse of the migration). In the injured brain, because reactive astrocytes produce additional CSPGs within the substrate around the lesion (), we also tested the migration of NSCs in the presence of an extra aggrecan substrate coating ( Figures 2 E and 2F). We observed further decreases in the migration of NSCs with increased aggrecan concentration, suggesting that CSPGs potently inhibit the migration of NSCs but, more interestingly, we observed the increased migration of NSCs with ISP treatment even with increasing aggrecan concentrations, demonstrating that ISP can reverse the inhibitory effects of CSPGs on adult NSC migration. To further validate our results, we also measured the migration of wild-type (WT) or Ptprs conditional KO (cKO) NSCs in the absence or presence of aggrecan ( Figures 2 G and 2H). To avoid potential compensatory effects during the development of the Ptprs KO, we cultured adult NSCs from floxed Ptprs animals and infected the WT cells and the floxed cells with AAV-CMV-Cre one passage before they were harvested for the neurosphere migration assay (deletion of the floxed Ptprs is validated in Figure S1 ). Ptprs KO NSCs demonstrated enhanced migration compared to WT NSCs both in the absence and presence of aggrecan ( Figures 2 G and 2H).

To test the regulatory role of the CSPG receptor, adult neural stem cells were dissociated and differentiated for 5 days in the presence of ISP. Inhibition of PTPσ by ISP led to increased neurite outgrowth in differentiated NSCs in vitro, while the scrambled peptide had no effect ( Figures 2 A and 2B ). Interestingly, genetic knockout (KO) of RPTPσ in adult NSCs showed similar growth-promoting effects ( Figures 2 C and 2D), confirming and extending the previous work () that inhibition of PTPσ signaling enhances neurite outgrowth in adult differentiated NSC-derived neurons in vitro. Also, NSCs produce CSPGs themselves ( Figure 1 ), which explains why the inhibition of PTPσ enhances NSC neurite elongation without an aggrecan substrate. Therefore, both pharmacological and genetic inhibition of the PTPσ receptor leads to increased neurite growth of adult NSCs in vitro.

(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.

(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.

(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.

(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.

(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.

The regulatory role of CSPGs in neuronal migration and axonal growth has been well established (); however, whether this family of extracellular matrix (ECM) molecules also plays a role in constraining the migration of adult neural progenitor cells after stroke is not known. We examined the expression patterns of CSPGs in cultured neurospheres derived from the adult SVZ ( Figures 1 S–1U), as well as in vivo, within neurogenic regions in 3-month-old mice ( Figures 1 P–1R). CSPGs are abundant in neurosphere cultures and in the SVZ where the adult neural stem cells reside (). We detected CSPGs in the conditioned media derived from cultured neurospheres with mass spectrometry, suggesting that they produce and secrete CSPGs in vitro (full list of top proteins included in Table S1 ). Within the SVZ, the CS56 staining is evident at the laterodorsal corner of the lateral ventricle and extending along the length of the ventricular wall adjacent to the striatum. Co-immunostaining for doublecortin (DCX), a neuroblast and immature neuron marker, shows that the newly born neuroblasts in the SVZ are embedded within a CSPG-containing matrix. These results suggest that CSPGs could play a role in the regulation of adult neural stem cells in the SVZ and, importantly, that the NSCs themselves can produce a CSPG-laden matrix ().

To examine whether CSPGs are enriched in the glial scar after ischemic stroke, we stained sections containing the infarct with the CS56 antibody during the acute (2 days), subacute (7 days), and more chronic stages (14 and 30 days) post-stroke. Indeed, Figures 1 A–1L show that CSPGs are enriched near the border of the lesion both in the cortex (white dashed box with higher magnification images in a’–l’) and striatum (pink dashed box with higher magnification images in a’’–il’) compared to levels in the non-stroke brain ( Figures 1 M–1O), with peak upregulation at day 7 but sustained until day 30, especially near the glial scar. The pattern of CS56 staining in the lesion penumbra is consistent with the CSPG accumulation that occurs in humans () and in scar astrocytes described previously () and with the observations of others who have described CSPG upregulation in the stroke penumbra of rodents (). This indicates that CSPGs may play a role in stroke recovery.

(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.

A review of functional heterogeneity among astrocytes and the CS56-specific antibody-mediated detection of a subpopulation of astrocytes in adult brains.

Discussion

Horn et al., 2012 Horn K.E.

Xu B.

Gobert D.

Hamam B.N.

Thompson K.M.

Wu C.L.

Bouchard J.F.

Uetani N.

Racine R.J.

Tremblay M.L.

et al. Receptor protein tyrosine phosphatase sigma regulates synapse structure, function and plasticity. Sclip and Sudhof, 2020 Sclip A.

Sudhof T.C. LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses. Luo et al., 2018 Luo F.

Tran A.P.

Xin L.

Sanapala C.

Lang B.T.

Silver J.

Yang Y. Modulation of proteoglycan receptor PTPsigma enhances MMP-2 activity to promote recovery from multiple sclerosis. In this study, using a sizable CNS injury model (MCAo), we examined the role of the CSPG-PTPσ signaling pathway not only on axonal sprouting but also on injury-stimulated new neuroblast generation and migration, a phenomenon that has been much less explored. We have shown that post-stroke treatment with ISP improves behavioral recovery even in the delayed phase. Moreover, ISP treatment increased the total number of newly generated neuroblasts migrating well into the peri-infarct area. The peptide also enhanced serotonergic, callosal, and corticospinal tract (CST) regeneration/sprouting while increasing the density of excitatory synaptic markers at the peri-infarct zone. There was also significantly decreased cortical and striatal atrophy. Therefore, alleviating CSPG-mediated inhibition of a variety of neurorepair mechanisms is likely to be the molecular and cellular event that is allowing for enhanced recovery after stroke. Also, ISP may promote recovery from stroke by yet other mechanisms in addition to neuroprotection, neuroblast migration, or neuronal sprouting. It is known that members of the LAR family are important regulators of synaptic plasticity and neuronal physiology (), and they are important in the process of remyelination () phenomena that were not evaluated in this study.

Sayed et al., 2020 Sayed M.A.

Eldahshan W.

Abdelbary M.

Pillai B.

Althomali W.

Johnson M.H.

Arbab A.S.

Ergul A.

Fagan S.C. Stroke promotes the development of brain atrophy and delayed cell death in hypertensive rats. + neurons in the vicinity of the lesion penumbra. Multiple potential mechanisms could contribute to the diminished atrophy, including decreased delayed neuronal death, enhanced axonal remodeling, and altered neuroinflammation. Recent evidence has suggested that the chronic modulation of CSPG signaling via chondroitinase ( Bartus et al., 2014 Bartus K.

James N.D.

Didangelos A.

Bosch K.D.

Verhaagen J.

Yáñez-Muñoz R.J.

Rogers J.H.

Schneider B.L.

Muir E.M.

Bradbury E.J. Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury. Didangelos et al., 2014 Didangelos A.

Iberl M.

Vinsland E.

Bartus K.

Bradbury E.J. Regulation of IL-10 by chondroitinase ABC promotes a distinct immune response following spinal cord injury. Dyck et al., 2018 Dyck S.

Kataria H.

Alizadeh A.

Santhosh K.T.

Lang B.

Silver J.

Karimi-Abdolrezaee S. Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPsigma receptors promotes a beneficial inflammatory response following spinal cord injury. L in hematopoietic stem cells ( Zhang et al., 2019 Zhang Y.

Roos M.

Himburg H.

Termini C.M.

Quarmyne M.

Li M.

Zhao L.

Kan J.

Fang T.

Yan X.

et al. PTPsigma inhibitors promote hematopoietic stem cell regeneration. + microglia/macrophage cells near the peri-infarct zone, which agrees with previous reports of CNS BCL-2 expression patterns in adult brains ( Merry et al., 1994 Merry D.E.

Veis D.J.

Hickey W.F.

Korsmeyer S.J. bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS. Sassone et al., 2013 Sassone J.

Maraschi A.

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Ciammola A. Defining the role of the Bcl-2 family proteins in Huntington's disease. Joy and Carmichael, 2021 Joy M.T.

Carmichael S.T. Encouraging an excitable brain state: mechanisms of brain repair in stroke. Delayed secondary neuronal death contributes to the global tissue atrophy that occurs post-stroke (). Interestingly, we observed less atrophy in ISP-treated stroke animals, which was reflected at the cellular level by the survival of callosal neurons as well as NURR1neurons in the vicinity of the lesion penumbra. Multiple potential mechanisms could contribute to the diminished atrophy, including decreased delayed neuronal death, enhanced axonal remodeling, and altered neuroinflammation. Recent evidence has suggested that the chronic modulation of CSPG signaling via chondroitinase () or the administration of our LAR family receptor blocking peptides in models of compressive SCI drives an anti-inflammatory and potently neuroprotective immune response (). It is likely that a similar pro-regenerative, ISP-induced immune phenotype may develop during ischemia brought on by stroke. The reduction in secondary damage also correlates well with the transcriptome profiling of the ISP-treated mice, such as enrichment of genes that negatively regulate apoptotic pathways as well as genes that are involved in axonal development. Interestingly, a recent study showed that a small pharmacological inhibitor of PTPσ also led to the upregulation of an anti-apoptotic gene BCL-Xin hematopoietic stem cells (), suggesting a shared anti-apoptotic mechanism across different cell types. Our data suggest that BCL-2 protein expression is upregulated in Iba1microglia/macrophage cells near the peri-infarct zone, which agrees with previous reports of CNS BCL-2 expression patterns in adult brains () and further supports that ISP may have a modulatory role in neuroinflammation after injury. Interestingly, our data showed that the same 30-day treatment of ISP in non-stroke mice did not affect the synaptic density in the cortex, suggesting that ISP treatment likely does not affect synaptic stability in the non-stroke brain. It is known that stroke injury stimulates a transcriptomic program in cortical neurons that facilitates synaptic reorganizations and plasticity (), and this could explain why the effects of ISPs on synaptic density and sprouting of axons are more evident after injury.

Grade et al., 2013 Grade S.

Weng Y.C.

Snapyan M.

Kriz J.

Malva J.O.

Saghatelyan A. Brain-derived neurotrophic factor promotes vasculature-associated migration of neuronal precursors toward the ischemic striatum. Lee et al., 2006 Lee S.R.

Kim H.Y.

Rogowska J.

Zhao B.Q.

Bhide P.

Parent J.M.

Lo E.H. Involvement of matrix metalloproteinase in neuroblast cell migration from the subventricular zone after stroke. Tran et al., 2018a Tran A.P.

Sundar S.

Yu M.

Lang B.T.

Silver J. Modulation of receptor protein tyrosine phosphatase sigma increases chondroitin sulfate proteoglycan degradation through cathepsin B secretion to enhance axon outgrowth. Luo et al., 2018 Luo F.

Tran A.P.

Xin L.

Sanapala C.

Lang B.T.

Silver J.

Yang Y. Modulation of proteoglycan receptor PTPsigma enhances MMP-2 activity to promote recovery from multiple sclerosis. Tran et al., 2018a Tran A.P.

Sundar S.

Yu M.

Lang B.T.

Silver J. Modulation of receptor protein tyrosine phosphatase sigma increases chondroitin sulfate proteoglycan degradation through cathepsin B secretion to enhance axon outgrowth. Another strategy that migrating cells or extending axons use to invade inhibitory regions is to produce matrix-degrading enzymes. MMPs have been implicated to guide neuroblast migration post-stroke (). We recently reported a downstream pathway in OPCs regulated by PTPσ that involves specific CSPG-degrading enzymes (). Thus, ISP treatment upregulated the release of MMP2 in OPCs, which allows them to digest their way into CSPG-filled plaques, enhancing their remyelination potential in models of MS (). Interestingly, here, we also report the upregulation of Mmp2 RNAs in adult NSCs by ISP treatment, and the stimulatory effect of ISP on NSC migration is reversed by an MMP2 inhibitor. We have also documented that genetic deletion or ISP blockade of PTPσ in adult DRGs leads to the secretion of cathepsin B, which allows them to degrade and cross a strongly inhibitory gradient of CSPG (). Thus, a conserved signaling pathway in a variety of cell types appears to exist that links PTPσ to very particular matrix-digesting mechanisms that we have shown can be amplified experimentally to enhance the ability of cells or axonal growth cones to navigate within an inhibitory environment.

In summary, our data using both pharmacological and genetic PTPσ inhibition confirms and expands the inhibitory role of CSPGs in axonal plasticity and, in addition, demonstrates a critical role of the CSPG-PTPσ signaling cascade in the regulation of adult neural stem cell migration into regions undergoing scar formation and proteoglycan deposition. Such biological reparations may have implications in both normal physiological function and the regenerative response of the brain after injury.