Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Jan 18.
Published in final edited form as: Glia. 2021 Apr 3;69(8):1950–1965. doi: 10.1002/glia.24003

CHPG Enhances BDNF and Myelination in Cuprizone-Treated Mice through Astrocytic Metabotropic Glutamate Receptor 5

Kyle S Saitta 1,2, Lauren D Lercher 2, Danielle M Sainato 2, Ashish Patel 2, Yangyang Huang 2, W Geoffrey McAuliffe 2, Cheryl F Dreyfus 2
PMCID: PMC9847144  NIHMSID: NIHMS1862459  PMID: 33811383

Abstract

It is well recognized that astrocytes can produce factors known to affect the myelination process. One such factor, brain-derived neurotrophic factor (BDNF), can enhance the differentiation of oligodendrocyte lineage cells following a demyelinating lesion. Our previous work indicated that enhancing astrocyte-derived BDNF via injection of a general agonist of Group I/II metabotropic glutamate receptors (mGluRs) into the lesion increased myelin proteins in the cuprizone model of demyelination after 4 hours. To determine if this observation has potential therapeutic significance, we now use a more specific mGluR agonist, 2-chloro-5-hydroxyphenylglycine (CHPG), which binds to mGluR5, to examine effects on myelination through the clinically relevant approach of a peripheral injection. In initial studies, intraperitoneal injection of CHPG resulted in an increase in myelin proteins within the lesioned corpus callosum. These effects were blocked when either BDNF or the CHPG receptor, mGluR5, was deleted from glial fibrillary acidic protein (GFAP)+ astrocytes or when the BDNF receptor, tropomyosin receptor kinase B (TrkB), was deleted from proteolipid protein (PLP)+ oligodendrocytes. Moreover, injection of CHPG over 2 weeks not only elevated BDNF and myelin proteins, but also enhanced myelination and reversed behavioral deficits. Interestingly, effects on myelin and myelin proteins were not seen in the control animals, indicating that a lesion is critical in eliciting effects. Taken together, the data suggest that the mGluR agonist CHPG may be a potential therapeutic strategy for treating demyelinating diseases and that it works by enhancing the release of BDNF from astrocytes.

Keywords: CHPG, BDNF, cuprizone, astrocytes, oligodendrocytes, myelin, metabotropic glutamate receptors

Graphical Abstract

graphic file with name nihms-1862459-f0001.jpg

Main Points:

Intraperitoneal injection of CHPG stimulated mGluR5 on astrocytes to release BDNF and impact myelin within a lesion.

BDNF from astrocytes directly affected oligodendrocytes.

Stimulation of oligodendrocytes reversed deficits in myelination and behavior.

Introduction

While the central nervous system (CNS) has an innate ability to remyelinate axons, this process is often impaired in demyelinating diseases such as multiple sclerosis (MS) (Franklin, 2002; Franklin & Ffrench-Constant, 2008; Franklin & Ffrench-Constant, 2017). Cellular changes within the environment of a demyelinating lesion may contribute to the inability of oligodendrocytes to remyelinate. Therefore, one approach to this issue is to manipulate the local cellular environment, rendering it more suitable for regeneration and repair of the myelin sheath.

To approach this issue, our lab has focused on brain-derived neurotrophic factor (BDNF), which enhances the differentiation of oligodendrocytes in culture (Du et al., 2003; Du et al., 2006). In BDNF +/− mice that have decreased levels of BDNF, reductions in myelin proteins are evident during development (VonDran et al., 2010; Xiao et al., 2010) and following cuprizone treatment (VonDran et al., 2011). Intracerebroventricular administration of BDNF or a BDNF mimetic increases myelin proteins after 6 weeks of cuprizone treatment (Fletcher et al., 2018; Nguyen et al., 2019). Furthermore, deletion of the BDNF high-affinity receptor from oligodendrocytes during cuprizone treatment or after removal from cuprizone has detrimental effects on myelin (Huang et al., 2020). Together, the studies suggest that BDNF aids in the repair of a demyelinating lesion.

In determining how to take advantage of these observations, our lab considered possible endogenous sources of BDNF within the lesion site and how to enhance levels of BDNF to increase myelin proteins. We found that the Group I/II metabotropic glutamate receptor (mGluR) agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) induced BDNF release from astrocytes in culture (Jean et al., 2008). To examine effects in vivo, ACPD was injected directly into the lesion of cuprizone-fed mice. Strikingly, not only did ACPD increase BDNF levels in the lesion site, but myelin proteins also increased within 4 hours (Fulmer et al., 2014). Moreover, deletion of astrocyte-derived BDNF inhibited the effects of ACPD, suggesting that astrocytes were the source of this neurotrophin. Astrocytes were found to possess both BDNF and relevant mGluRs, making these cells optimally positioned to enhance myelination after a lesion.

These previous studies, although suggestive of the role of mGluR agonists on myelination, were limited to the role of a general mGluR agonist, ACPD, on myelin proteins. They did not, however, address the possibility that a specific mGluR agonist could affect myelination itself or reverse behavioral deficits in a therapeutically important manner. Moreover, they did not define the direct effects of ACPD on astrocytes mediated through mGluRs.

In the present work, we explored for the first time the potential therapeutic utility of mGluR agonists. First, we evaluated effects of an mGluR agonist administered via intraperitoneal injection. Second, we assessed long-term agonist treatment to determine outcomes on behavior and myelin itself. We chose the small molecule 2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, and investigated its effects on the cuprizone-lesioned corpus callosum. Within 1 day, intraperitoneal injection of CHPG elevated BDNF and myelin proteins in the lesion site. These effects were eliminated after 2-methyl-6-(phenylethynyl)pyridine (MPEP), a specific mGluR5 antagonist, was injected into the lesion site and when mGluR5 was deleted from astrocytes, suggesting in new findings that astrocytes are the direct target for CHPG’s actions. Moreover, when BDNF was deleted from astrocytes or the BDNF high-affinity tropomyosin receptor kinase B (TrkB) receptor was deleted from proteolipid protein (PLP)+ oligodendrocyte lineage cells, the CHPG effect was also reduced, suggesting that astrocyte-derived BDNF may interact directly with oligodendrocytes to increase myelin proteins within the lesion site.

To evaluate effects on myelination itself and the possibility that the drug may reverse behavioral deficits, CHPG was administered for 2 weeks. Intraperitoneal treatment increased numbers of myelinated axons and the thickness of the myelin sheath, as well as reversed behavioral deficits. Interestingly, CHPG did not affect myelin proteins, myelin or behavior in control-fed mice. Collectively, these new studies indicate that administration of an mGluR5 agonist may be a potential therapeutic approach to promote myelination in the diseased brain by directly stimulating the production and release of BDNF from astrocytes to stimulate oligodendrocytes in the demyelinated lesion site.

Materials and Methods

Experimental animals

Mice were managed by the Rutgers Robert Wood Johnson Animal Facility. Animal maintenance, husbandry, housing, transportation, and use were in compliance with the Laboratory Animal Welfare Act (PL 89-544; PL-91-579) and with NIH guidelines (NIH Manual Chapter 4206). Mice were housed in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle and maintained on standard mouse chow with water ad libitum prior to cuprizone treatment.

Wild-type mice and inducible conditional knockout mice in which BDNF or mGluR5 was deleted from glial fibrillary acidic protein (GFAP)+ cells were on a C57BL/6 background. Inducible conditional knockout mice in which TrkB was deleted from PLP+ cells were on a C57/Balbc/129 background.

For experiments in which BDNF was conditionally deleted from GFAP+ cells, hGFAP-CreERT2-BDNFfl/fl-eGFP mice or BDNFfl/fl-eGFP mice lacking cre expression (used as controls) were utilized. hGFAP-CreERT2-BDNFfl/fl-eGFP mice were generated by crossing hGFAP-CreERT2 mice (MMRRC) with BDNFfl/fl mice (The Jackson Laboratory). hGFAP-CreERT2-BDNFfl/fl mice were then mated to another reporter mouse (astrocyte cre reporter; ACR, MMRRC), in which GFAP+ cells express β-gal before and e-GFP after tamoxifen-induced recombination (Casper & McCarthy, 2006). Importantly, these mice have been shown to exhibit no evidence of nonastrocytic recombination in tamoxifen-induced adult mice (Casper et al., 2007). We have previously described the successful deletion of BDNF from GFAP+ cells, reporting a level of 49% recombination (percentage GFAP cells exhibiting recombination/total GFAP+ cells) in these hGFAP-CreERT2-BDNFfl/fl-eGFP mice (Fulmer et al., 2014).

Similarly, hGFAP-CreERT2-mGluR5fl/fl-eGFP mice or mGluR5fl/fl-eGFP mice lacking cre expression (used as controls) were used for experiments in which mGluR5 was conditionally deleted from GFAP+ cells. hGFAP-CreERT2-mGluR5fl/fl-eGFP mice were generated by crossing hGFAP-CreERT2 mice (MMRRC) with mGluR5fl/fl mice (a gift from Anis Contractor and Jian Xu, Northwestern University, Chicago, Illinois). hGFAP-CreERT2-mGluR5fl/fl mice were then mated to the ACR reporter mice (MMRRC).

In a third set of mice, PLP-CreERT2-TrkBfl/fl-Rosa26-lacZ mice or TrkBfl/fl-Rosa26-lacZ mice lacking cre expression (used as controls) were used for experiments in which TrkB was conditionally deleted from PLP+ cells. PLP-CreERT2-TrkBfl/fl-Rosa26-lacZ mice were generated by crossing PLP-CreERT2 mice (a gift from Ueli Suter, ETH Zürich, Zürich, Switzerland), TrkBfl/fl mice (a gift from Luis Parada, University of Texas Southwestern, Dallas, Texas), and Rosa26-lacZ mice (a gift from Michael Matise, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ). These mice exhibited a 67% deletion of TrkB+CC1+/total CC1+ cells compared to controls (Huang et al., 2020).

To induce the deletion of BDNF or mGluR5 from astrocytes, or TrkB from oligodendrocytes, 1 mg tamoxifen (Cat# T5648, Sigma-Aldrich) dissolved in 10% ethanol in sunflower seed oil was injected intraperitoneally twice daily for 5 days at 7-9 weeks of age, corresponding to the week before being placed on cuprizone feed. Both Cre− and Cre+ mice were treated with tamoxifen.

Cuprizone treatment

Demyelination was induced by feeding 8-10-week-old male mice 0.2% cuprizone (Cat# C9012, Sigma-Aldrich) milled into mouse feed (Envigo Teklad, TD.01453) for 4 or 6 weeks. The control feed was identically processed, but without cuprizone supplementation (Envigo Teklad, TD.00217). Cuprizone and control feed were changed every 2 to 3 days and animal cages were changed every 1-2 weeks.

Intraperitoneal and stereotaxic injections

Based on studies in which ACPD was administered intraperitoneally (Jantzie et al., 2010), initial studies with CHPG (Cat# 3695, Tocris Bioscience) focused on a dose response of 10, 20, and 40 mg/kg. CHPG was dissolved in saline and mice were administered 0.02 mL/g body weight. These pilot studies revealed that 40 mg/kg was the lowest dose in which CHPG increased BDNF, MBP, and PLP in cuprizone-fed mice at 24 hours when compared to saline-injected cuprizone-fed, saline-injected control-fed, and CHPG-injected control-fed mice. Therefore, this dose was chosen for our studies in which effects of CHPG are observed at 24 hours. Injections of 40 mg/kg CHPG were given either 24 hours before or 6 and 24 hours before euthanasia. Alternatively, 20 mg/kg CHPG was injected every other day for 2 weeks with euthanasia occurring 24 hours after the last injection. Controls received 0.9% saline injections at similar times.

Stereotaxic injections of MPEP (Cat# 1212, Tocris Bioscience) were made into the corpus callosum of mice at coordinate bregma −1.0 mm and 0.5 mm lateral to the sagittal sinus. A Hamilton syringe was lowered to a depth of 1.875 mm to target the midcaudal corpus callosum over the fimbria-fornix. 2 μL of 150 μM MPEP or 0.9% saline vehicle was infused. This concentration of MPEP has been shown to be effective for mGluR5 inhibition when perfused directly into the brain (Chau et al., 2011). 40 mg/kg CHPG or saline was then injected intraperitoneally one hour following stereotaxic injection based on the reported half-life of MPEP (Anderson et al., 2003; Hodge et al., 2006).

Western blot

The midline of the corpus callosum overlying the fimbria-fornix and rostral hippocampus, from bregma −0.06 to −2.06, was dissected using a mouse brain matrix that allows 1 mm coronal cuts along the rostral-caudal axis. Tissue was immediately frozen at −80°C before being lysed by triturating tissue in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1% CHAPS, 0.5% NP-40, 1% Triton X-100, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 20 μg/mL soybean trypsin inhibitor, 50 mM NaF, 1 mM PMSF, 0.5 μM microcystin, and 1 mM ortho-vanadate. Protein concentrations were determined using a BCA protein assay kit (Cat# 23223 and Cat# 23224, Pierce).

Antibodies for Western blots included a rabbit polyclonal antibody to BDNF (1:200, Cat# sc-546, Santa Cruz Biotechnology, RRID:AB_630940), a mouse monoclonal antibody to myelin basic protein (MBP) (1:400, Cat# MCA184S, Serotec, RRID:AB_322319), and a goat polyclonal antibody to PLP (1:1,000, Cat# sc-18529, Santa Cruz Biotechnology, RRID:AB_2165798). A mouse monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000,000, Cat# H86504M, Meridian Life Science, RRID:AB_151542) was used as a loading control.

For the analysis of BDNF, MBP, PLP, and GAPDH, 10 μg protein was run on 12% Bis-Tris gels (Cat# NP0341BOX, Invitrogen). Protein was then transferred to a PVDF membrane (Cat# IPVH00010, Millipore) and membranes were exposed to HRP-linked anti-rabbit (Cat# NA934, GE Healthcare, RRID:AB_772206), anti-mouse (Cat# NA931, GE Healthcare, RRID:AB_772210), or anti-goat (Cat# 705-035-003, Jackson ImmunoResearch, RRID:AB_2340390) antibodies as appropriate. All Western blots were visualized with a chemiluminescence system (Cat# ORT2655, Cat# ORT2755, Perkin Elmer Western Lighting Plus ECL) and data were analyzed using Quantity One V 4.2.1 software (Bio-Rad, RRID:SCR_014280).

Immunohistochemistry

Brains from mice perfused with 4% paraformaldehyde (PFA) were post-fixed in PFA for 2 hours followed by 30% sucrose/PBS for 48 hours. Brains were embedded in OCT (Cat# 4583, Tissue Tek) and frozen at −80°C before serial sections were obtained using a Leica cryostat. Sections were stained and then evaluated using a Leica fluorescent microscope equipped with a Lumenera Infinity3S-1UR CCD camera. Infinity Analyze software was used for image capture. Within each experiment, controls and treatment groups were treated identically and simultaneously during the fixation and staining procedures.

Serial sections were taken in the coronal plane from the basal forebrain to the caudal hippocampus and images were obtained with a 40× objective lens from the midline of the corpus callosum (a width of 240 μm) overlying the fimbria-fornix and rostral hippocampus, from bregma −0.06 to −2.06. An equal number of matched sections were analyzed for each treatment group. For CC1+ staining, 30 matched 20 μm sections separated by 80 μm were counted per mouse. Double counting was corrected for based on cell body diameter and section thickness allowing for an estimation of cell number (Abercrombie, 1946). For analysis of mGluR5+GFAP+ co-staining, a more limited area was counted, from bregma −1.06 to −2.06, using 17 matched 14 μm sections separated by 56 μm. Cells and cellular profiles were counted using the ImageJ Cell Counter plug-in and analysis was done blinded to treatment groups.

Antibodies for immunohistochemistry included mouse monoclonal antibodies to CC1 (1:800, Cat# OP80, Calbiochem, RRID:AB_2057371) and GFAP (1:500, Cat# sc-33673, Santa Cruz Biotechnology, RRID:AB_627673) and a rabbit polyclonal antibody to mGluR5 (1:500, Cat# AB5675, Millipore, RRID:AB_2295173). The secondary antibodies used were AlexaFluor 488 donkey anti-mouse IgG (Cat# 715-545-151, Jackson ImmunoResearch Labs, RRID:AB_2341099) and Cy3 donkey anti-rabbit IgG (Cat# 711-165-152, Jackson ImmunoResearch Labs, RRID:AB_2307443).

Transmission electron microscopy

Following cardiac perfusion with 4% paraformaldehyde/2.5% glutaraldehyde, the brain was removed and sectioned sagitally in 150 μm sections using a Leica vibratome. Sections of the midline of the corpus callosum were post-fixed in 1% osmium tetroxide, stained with uranyl acetate, dehydrated with graded ethanols, and embedded in Poly/Bed812 resin (Polysciences, Inc.). Thin sections (1 μm) were stained with toluidine blue for orientation and ultrathin sections (0.13 μm) were subsequently collected on copper grids. Images of the axons cut in cross section were obtained using a transmission electron microscope (Philips CM12) at 5000× magnification. For each mouse, images were taken in the corpus callosum immediately caudal to its junction with the fornix. To count percentage of myelinated fibers, the entire field (14.8 μm × 9.7 μm) of 10 images was analyzed per mouse for myelinated axons and unmyelinated axons and the data are presented as percent of the total number. ImageJ software (RRID:SCR_003070) was used to calculate the axonal diameter, myelin thickness, and g-ratios; 150 axons per mouse were randomly selected for evaluation using the ImageJ G Ratio plug-in. The g-ratio represents the ratio of the diameter of the axon to the diameter of the axon plus myelin. To assess changes associated with the demyelination and remyelination processes specifically within myelinated fibers, axons with g-ratios >0.95 (unmyelinated axons) were not analyzed (Mason et al., 2001; Zhou et al., 2012). Analysis was done blinded to treatment groups.

Balance beam test

A round plexiglass rod, 1.3 cm in diameter and 70 cm in length, was placed 17 cm above soft bedding and used to test motor coordination. This test took place over 2 consecutive days: 1 day of training and 1 day of testing. A black box was placed at the end of the beam as the finish point. Nesting materials from home cages were placed in the black box to attract the mouse to the finish point. On training days, each mouse crossed the beam 2 times. On the test day, times to cross the beam and the number of paw slips were recorded with a video camera. Video analysis was done blinded to treatment groups. 12 mice per treatment group were used for analysis.

Wire hang test

A round metal wire, 2 mm in diameter and 43 cm in length, was used to test grip strength. The wire was attached to two poles at a height of 43 cm above a cage with soft bedding to break the fall. Mice began the test with the forelimbs attached to the wire and the time to fall was recorded with a video camera. Video analysis was done blinded to treatment groups. 8 mice per treatment group were used for analysis.

Data analysis

For each experiment, a control- and cuprizone-fed mouse injected with saline or CHPG were compared. In some cases, cuprizone-fed mice injected with saline or CHPG were also injected with MPEP or vehicle and compared with one another. In other cases, tamoxifen-injected Cre+ or Cre− mice were fed cuprizone and then injected with saline or CHPG. Each experiment was repeated ≥ 3 times and the data are presented as ± SEM.

Statistical analysis was performed with GraphPad PRISM 8 software (GraphPad Prism, RRID:SCR_002798). Statistical differences were determined using ANOVA followed by Tukey’s multiple comparisons test or paired t-test as appropriate.

For analysis of g-ratio vs. axon diameter, linear regression analysis was used to compare differences in slopes between cuprizone-fed mice ± CHPG using GraphPad PRISM 8 software. 150 axons per mouse were randomly selected for analysis using the ImageJ G Ratio plug-in. Conditions were considered significant at p < 0.05.

Results

CHPG increased BDNF and myelin proteins in the demyelinated corpus callosum after 24 hours

In previous work, injection of the mGluR Group I/II agonist ACPD directly into the lesioned corpus callosum increased BDNF and myelin proteins (Fulmer et al., 2014). To determine if a more selective mGluR agonist can mimic the effects of ACPD even when administered through the more therapeutically relevant route of a peripheral injection, mice were fed cuprizone for 4 weeks before receiving intraperitoneal injections of the Group I mGluR agonist CHPG (40 mg/kg) 24 hours and 6 hours prior to dissection. Tissue from the midline of the corpus callosum was then collected and processed for Western blot. As was shown with ACPD, cuprizone treatment resulted in significant decreases in BDNF, MBP, and PLP protein levels in saline vehicle-injected mice. Although CHPG did not fully recover the deficits in these proteins at 24 hours when compared to control-fed mice, CHPG significantly increased BDNF, MBP, and PLP in mice when its treatment was compared to cuprizone-fed control animals (Fig. 1). Interestingly, CHPG had no effect in these control-fed mice. This result was similar to that observed in previous studies where it was found that cuprizone-fed, but not control-fed mice, express elevated astrocytic mGluR5 and GFAP (Fulmer et al., 2014), priming the astrocytes to respond to CHPG and making them a target to impact myelin.

Figure 1. Cuprizone-lesioned mice exhibited increases in BDNF and myelin proteins 24 hours after intraperitoneal injection of CHPG.

Figure 1.

Open in a new tab

Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle 6 and 24 hours prior to dissection. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected control-fed (Ctrl) mice, analyzed by ANOVA followed by Tukey’s multiple comparisons test. *Significantly different from saline-injected and CHPG-injected control-fed mice, *p<0.05, #Significantly different from saline-injected cuprizone-fed mice, #p<0.05. Each experiment consisted of one mouse per treatment group and each experiment was repeated four times. Data are presented as mean ± SEM.

To determine if changes in myelin proteins were due to an increase in myelin proteins per cell or due to an increase in numbers of myelinating cells, immunofluorescent staining for CC1+ oligodendrocytes was performed. Cuprizone treatment caused a decrease in CC1+ cell numbers in saline-injected mice, but CHPG had no effect on these cells in either control-fed or cuprizone-fed mice (Fig. 2). This observation suggests that CHPG increased myelin proteins per cell when assessed after 24 hours.

Figure 2. CHPG did not affect CC1+ oligodendrocyte cell numbers at 24 hours, suggesting that CHPG increased myelin proteins per cell.

Figure 2.

Open in a new tab

CC1 immunofluorescent staining in the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle 6 and 24 hours prior to perfusion. Data presented as percent saline-injected control-fed (Ctrl) mice, analyzed by ANOVA followed by Tukey’s multiple comparisons test. *Significantly different from saline-injected and CHPG-injected control-fed mice, *p<0.05. Each experiment consisted of one mouse per treatment group and each experiment was repeated three times. Data are presented as mean ± SEM. Scale bar, 20 μm.

CHPG required astrocyte-derived BDNF and TrkB on oligodendrocytes to elicit its effects

In previous work, we found that ACPD elevated BDNF when injected into the corpus callosum and that this was due to the release of BDNF into the lesion site (Fulmer et al., 2014). The increase in BDNF was associated with an increase in myelin proteins. Moreover, the source of BDNF appeared to be the astrocytes as ACPD’s effects on myelin proteins were blocked when astrocyte-derived BDNF was deleted. Now, to determine if this was true when CHPG was injected intraperitoneally, we utilized tamoxifen-inducible conditional knockout mice similar to those used in our early work. These mice exhibit a level of 49% recombination (percentage GFAP cells exhibiting recombination/total GFAP+ cells) (Fulmer et al., 2014). After 4 weeks on cuprizone, CHPG was injected intraperitoneally 24 hours and 6 hours prior to dissection and the corpus callosum was examined for changes in myelin proteins. Mice deficient in astrocytic BDNF were unable to respond to CHPG (Fig. 3A).

Figure 3. CHPG-elicited increases in BDNF and myelin proteins were blocked when BDNF was deleted from astrocytes or when TrkB was deleted from oligodendrocytes.

Figure 3.

Open in a new tab

A) Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of hGFAP-CreERT2-BDNFfl/fl-eGFP mice (Cre+) or BDNFfl/fl-eGFP mice lacking cre expression (Cre−) subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle 6 and 24 hours prior to dissection. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected cuprizone-fed mice. *Significantly different, *p<0.05, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Each experiment consisted of one mouse per treatment group and each experiment was repeated four times. Data are presented as mean ± SEM. B) Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of PLP-CreERT2-TrkBfl/fl-eGFP mice (Cre+) or TrkBfl/fl-eGFP mice lacking cre expression (Cre−) subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle 6 and 24 hours prior to dissection. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected cuprizone-fed mice. *Significantly different, *p<0.05, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Each experiment consisted of one mouse per treatment group and each experiment was repeated four times. Data are presented as mean ± SEM.

Oligodendrocytes in the corpus callosum (VonDran et al., 2011; Huang et al., 2020) as well as other brain regions (Du et al., 2003; Du et al., 2006; VonDran et al., 2010; Xiao et al., 2010), express TrkB receptors. These receptors can affect the development of oligodendrocytes (Wong et al., 2013). Moreover, deletion of TrkB from PLP+ oligodendrocyte lineage cells using PLP-CreERT2-TrKBfl/fl-lacZ mice impacts the response of these cells to cuprizone-elicited demyelination (Huang et al., 2020). These mice exhibit a 67% decrease in TrkB+CC1+ cells relative to total CC1+ cells upon tamoxifen treatment (Huang et al., 2020). Therefore, we for the first time made use of these mice to determine if CHPG-elicited effects on myelin proteins were due to direct actions on oligodendrocyte lineage cells. As noted in Fig 3B, the deletion of TrkB from PLP+ cells resulted in a total inability of CHPG to elevate myelin proteins. Interestingly, the increases in BDNF were also blocked, suggesting that oligodendrocytes may also contribute BDNF to CHPG’s actions. Future studies are necessary to address this intriguing possibility.

CHPG required astrocytic mGluR5 to elicit its effects

Previous work indicated that ACPD increased BDNF and myelin proteins in the lesion site, but did not determine if its effects were mediated through actions of a specific mGluR on a specific cell type. Two approaches were taken to address this problem. First, since CHPG is typically associated with mGluR5 activation (Doherty et al., 1997) (but see Kammermeier et al., 2012), a single stereotaxic injection of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) was made directly into the cuprizone-lesioned corpus callosum at a concentration of 150 μM one hour before a single intraperitoneal injection of CHPG. While CHPG again increased BDNF, MBP, and PLP in the absence of MPEP, mice that received MPEP prior to CHPG had protein levels that were no different than in mice that received MPEP prior to saline (Fig. 4A), indicating a complete blockage of CHPG’s effects. These data suggest not only that mGluR5 was important for CHPG to elicit its effects, but also that CHPG was acting within the lesion site even after peripheral administration.

Figure 4. Pharmacological inhibition of mGluR5 within the corpus callosum or genetic deletion of mGluR5 from astrocytes blocked the actions of peripherally administered CHPG.

Figure 4.

Open in a new tab

A) Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle 1 hour following stereotaxic injection of the mGluR5 antagonist MPEP (150 μM) or saline. Mice were dissected 24 hours after CHPG injection. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected cuprizone-fed mice. *Significantly different, *p<0.05, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Each experiment consisted of one mouse per treatment group and each experiment was repeated five times. Data are presented as mean ± SEM. B) mGluR5+GFAP+ immunofluorescent staining in the corpus callosum of hGFAP-CreERT2-mGluR5fl/fl-eGFP mice (Cre+) or mGluR5fl/fl-eGFP mice lacking cre expression (Cre−) subjected to a 4-week cuprizone lesion. Data presented as percent mGluR5+GFAP+ cellular profiles per total GFAP+ cellular profiles relative to Cre− mice. *Significantly different from Cre− cuprizone control, *p<0.05, analyzed by paired t-test. Each experiment consisted of one mouse per treatment group and each experiment was repeated three times. Data are presented as mean ± SEM. Scale bar, 20 μm. C) Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of hGFAP-CreERT2-mGluR5fl/fl-eGFP mice (Cre+) or mGluR5fl/fl-eGFP mice lacking cre expression (Cre−) subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (40 mg/kg) or 0.9% saline (Sal) vehicle. Mice were dissected 24 hours after CHPG injection. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected cuprizone-fed mice. *Significantly different, *p<0.05, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Each experiment consisted of one mouse per treatment group and each experiment was repeated five times. Data are presented as mean ± SEM.

Second, while in previous studies we found that astrocytes in the lesion contain both mGluR5 and BDNF, we did not test whether mGluRs on astrocytes were necessary to elicit changes in astrocyte function (Fulmer et al., 2014). A formal possibility is that CHPG does not work directly on astrocyte, but rather on other cells in the lesion site. To test this possibility, we made use of inducible conditional knockout mice that delete mGluR5 from GFAP+ cells following tamoxifen administration, similar to those used previously to examine astrocytes during developmental epilepsy (Umpierre et al., 2019). To determine the successful deletion of mGluR5 from astrocytes, tamoxifen was administered to mice prior to 4 weeks of cuprizone treatment. Immunofluorescent staining for mGluR5 and GFAP in Cre− and Cre+ mice revealed a 51% decrease in mGluR5+GFAP+ cellular profiles relative to total GFAP+ cellular profiles in Cre+ mice (Fig. 4B).

Using these mice, we determined the role of mGluR5 on astrocytes following administration of CHPG. Tamoxifen was injected to Cre− and Cre+ mice that were then fed cuprizone for 4 weeks before receiving a single intraperitoneal injection of CHPG or saline. Control Cre− mice were able to reverse deficits in myelin proteins in response to CHPG. However, this effect was fully prevented in mice in which astrocytic mGluR5 was deleted, indicated by the protein levels in CHPG-injected Cre+ mice that were no different than saline-injected Cre+ mice (Fig. 4C). These results suggest for the first time that CHPG required mGluR5 on astrocytes to exert its effects. Moreover, to the best of our knowledge, this is the first evidence that an mGluR5 agonist has direct actions on astrocytes in vivo to affect release of BDNF and the subsequent increase in myelin proteins following a demyelinating lesion.

CHPG enhanced myelination and behavior in cuprizone-fed mice when injected every other day for 2 weeks

To determine if CHPG’s effects could be maintained over time, and therefore have therapeutic potential, injections of CHPG (20 mg/kg) were started after 4 weeks of cuprizone treatment and continued to be administered every other day for an additional 2 weeks while remaining on cuprizone feed. Control mice received saline injections. Elevations in BDNF and myelin proteins were maintained over this time period (Fig. 5A), suggesting that multiple doses of CHPG can prolong its effects over time. CHPG totally reversed PLP deficits in cuprizone-fed mice, which were no longer significantly different from control-fed animals. In addition, the drug partially reversed decreases in BDNF and MBP at this time point. Again, no CHPG effects were observed in control-fed mice, suggesting that CHPG was only effective after the development of a lesion. To determine if CHPG impacted numbers of oligodendrocytes at this time point, numbers of CC1+ cells were counted. Interestingly, in contrast to effects seen after 24 hour treatment with CHPG, now the drug significantly increased numbers of oligodendrocytes (Fig. 5B).

Figure 5. After 2 weeks, BDNF and myelin proteins remained elevated after multiple intraperitoneal injections of CHPG with increased numbers of CC1+ oligodendrocytes.

Figure 5.

Open in a new tab

A) Western blots demonstrated BDNF, MBP, and PLP protein levels in the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (20 mg/kg) or 0.9% saline (Sal) vehicle every other day for an additional 2 weeks while remaining on cuprizone feed. Graph represents densitometric analysis of Western blots normalized to GAPDH and presented as percent saline-injected control-fed (Ctrl) mice, analyzed by ANOVA followed by Tukey’s multiple comparisons test. *Significantly different from saline-injected and CHPG-injected control-fed mice, *p<0.05, #Significantly different from saline-injected cuprizone-fed mice, #p<0.05. Each experiment consisted of two control mice ± CHPG and two or four cuprizone mice ± CHPG. Each experiment was repeated eight times. Data are presented as mean ± SEM. B) CC1 immunofluorescent staining in the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (20 mg/kg) or 0.9% saline (Sal) vehicle every other day for an additional 2 weeks while remaining on cuprizone feed. Data presented as percent saline-injected cuprizone-fed (Cupz) mice, analyzed by paired t-test. *Significantly different from saline-injected cuprizone-fed mice, *p<0.05. Each experiment consisted of one mouse per treatment group and each experiment was repeated six times. Data are presented as mean ± SEM. Scale bar, 20 μm.

Although previous studies in our lab have suggested that mGluR agonists can increase myelin proteins within a lesion, we have never determined if they can affect myelination itself. To address the potential of mGluR agonists to effect myelin structure, transmission electron microscopy of the corpus callosum was performed. These studies revealed that CHPG-elicited elevations in myelin proteins and oligodendrocytes corresponded to improvements in myelin (Fig. 6A). Although CHPG did not affect axon diameter in either control-fed or cuprizone-fed mice (Fig. 6B), it did affect the percent of myelinated axons (Fig. 6C). Small but significant decreases in the average g-ratio, indicative of increased myelin thickness, were also noted after cuprizone feed (Fig. 6D). Furthermore, linear regression analysis of g-ratio against axon diameter indicated that CHPG treatment resulted in a significant increase in slope compared to vehicle-treated animals and that it acted preferentially on smaller diameter axons (Fig. 6E). Of importance, CHPG-elicited myelin improvements after cuprizone were not significantly different from control-fed mice, suggesting complete recovery of myelin. Moreover, as was the case with myelin protein levels, CHPG had no effect on myelin structure in control-fed mice, indicating that a lesion must be evident to see effects of CHPG.

Figure 6. Multiple intraperitoneal injections of CHPG over 2 weeks increased myelin thickness and the percentage of myelinated fibers.

Figure 6.

Open in a new tab

A) Transmission electron microscopy (5000× magnification) of the corpus callosum of mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (20 mg/kg) or 0.9% saline (Sal) vehicle every other day for an additional 2 weeks while remaining on cuprizone feed. Scale bar, 2 μm. Graphs represent the B) mean axon diameter, C) mean percentage of myelinated fibers, or D) mean g-ratio among treatment groups. Data are presented as percent saline-injected control-fed (Ctrl) mice, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Analysis was done blinded to treatment groups. *Significantly different from saline-injected and CHPG-injected control-fed mice, *p<0.05, #Significantly different from saline-injected cuprizone-fed mice, #p<0.05. Each experiment consisted of one mouse per treatment group and each experiment was repeated three times. Data are presented as mean ± SEM. E) Scatter-plot of g-ratio against axon diameter of cuprizone-fed mice ± CHPG. Linear regression analysis revealed a significant difference in slopes between saline-injected and CHPG-injected mice at p<0.05.

To determine if improvements in myelination were associated with a functional consequence, behavioral tests were performed. Cuprizone treatment has been found to impair motor and muscle coordination with a temporal relationship between motor dysfunction and CNS demyelination (Franco-Pons et al., 2007). In addition to the corpus callosum, other brain regions affected by cuprizone have also been identified including the cerebellum (Groebe et al., 2009; Skripuletz et al., 2010) and several gray matter areas (Goldberg et al., 2015), all of which may contribute to the behavioral deficits observed in this model. Here, we made use of the balance beam test to measure balance and motor coordination plus the wire hang test as an indicator of grip strength and muscle coordination. Cuprizone-fed mice performed worse on these tests compared to their control-fed counterparts, as they had more paw slips (Fig. 7A), required more time to cross the balance beam (Fig. 7B), and exhibited less grip strength (Fig. 7C). Excitingly, CHPG completely reversed all of these endpoints in cuprizone-fed mice compared to control-fed mice, suggesting that CHPG attenuated both the myelination and behavioral deficits associated with cuprizone treatment.

Figure 7. Multiple intraperitoneal injections of CHPG over 2 weeks increased motor coordination and grip strength as analyzed by the balance beam test and wire hang test.

Figure 7.

Open in a new tab

A-B) Balance beam assay and C) wire hang test were performed in mice subjected to a 4-week cuprizone (Cupz) lesion and injected intraperitoneally with CHPG (20 mg/kg) or 0.9% saline (Sal) vehicle every other day for an additional 2 weeks while remaining on cuprizone feed. Data are presented as percent saline-injected control-fed (Ctrl) mice, analyzed by ANOVA followed by Tukey’s multiple comparisons test. Analysis was done blinded to treatment groups. *Significantly different from saline-injected and CHPG-injected control-fed mice, *p<0.05, #Significantly different from saline-injected cuprizone-fed mice, #p<0.05 Each experiment consisted of one mouse per treatment group and each experiment was repeated twelve times for the balance beam assay and eight times for the wire hang test. Data are presented as mean ± SEM.

Overall, our studies newly demonstrated that CHPG increased BDNF and myelin proteins. Actions of CHPG were elicited through the mediation of astrocytic mGluR5, astrocyte-derived BDNF, and oligodendroglial TrkB. The effects of CHPG on myelin proteins were evident when injected every other day for 2 weeks. Moreover, CHPG treatment resulted in enhancements in myelin and reversal of behavioral deficits associated with cuprizone treatment. The data suggest that stimulation of astrocytic mGluR5 may be a therapeutic approach to demyelination worth pursuing.

Discussion

In previous studies we found that activation of Group I/II mGluRs by injecting the agonist ACPD directly into the cuprizone-lesioned corpus callosum elevated levels of astrocyte-derived BDNF, leading to increases in myelin proteins (Fulmer et al., 2014). Here we further defined cellular events leading to this observation and investigated whether this finding may have therapeutic significance. In particular, a specific mGluR5 agonist, CHPG, was injected intraperitoneally and its immediate actions as well as those noted after 2 weeks of treatment were assessed. CHPG increased BDNF, MBP, and PLP just 24 hours after administration and these proteins remained elevated when CHPG was injected over a 2-week time frame. Elevations in BDNF and myelin proteins were at least in part due to astrocyte-derived BDNF. Moreover, we report for the first time that astrocyte-associated mGluR5 and oligodendroglial TrkB likewise mediated these responses. Also novel to this work is that multiple intraperitoneal injections of an mGluR agonist enhanced myelination itself and reversed behavioral deficits associated with cuprizone treatment. Collectively, these data suggest that CHPG, a small molecule able to cross the blood-brain barrier, can enhance myelination and reverse behavioral deficits when it is injected intraperitoneally. To the best of our knowledge, this is the first evidence that the stimulation of mGluR5 can affect myelination and reverse behavioral deficits.

Interestingly, we were able to see changes in myelin proteins and BDNF as early as 24 hours after application of CHPG. The relatively quick response to CHPG prompts the question of the cellular and molecular events underlying these increases. We suggest that this observation is consistent with previous studies that demonstrate the rapid release of BDNF from astrocytes in response to metabotropic glutamate receptor stimulation in culture (Jean et al., 2008). The increase in BDNF release from astrocytes is dependent on intracellular calcium stores and the Phospholipase C pathway and is in agreement with experiments that show that specific agonists of Group I mGluRs induce transient increases in intracellular calcium levels within astrocytes of hippocampal slices (Pasti et al., 1997; Shelton & McCarthy, 1999; Nett et al., 2002; Zur Nieden & Deitmer, 2006; Copeland et al., 2017; Umpierre et al., 2019). Other studies from our lab indicate increases in BDNF as early as 4 hours after injection of ACPD into the lesion site (Fulmer et al., 2014).

Moreover, the rapid increases in myelin proteins in response to BDNF are reminiscent of work indicating that myelin proteins may be increased in a quick translationally-dependent manner (Coelho et al., 2009; Laursen et al., 2011; Michel et al., 2015; Ishii et al., 2019). For example, when stimulated with neurotrophin-3, cultured oligodendrocytes increase 35S-methionine incorporation in total oligodendrocyte protein in just 15 minutes (Coelho et al., 2009). This response is suggested to occur through a posttranscriptional mechanism that is dependent on the ERK and PI3K/mTOR pathways (Coelho et al., 2009). Likewise, studies in vivo suggest that ERK2 is important in a posttranscriptional elevation of MBP following a lesion (Michel et al., 2015) and we found, as noted above, increases in myelin protein as early as 4 hours after ACPD. Future studies will explore the possibility that these molecular mechanisms are operative in our observations as well.

In defining the cellular response to CHPG to enhance myelination, we examined effects of the drug after 2 weeks of treatment. At this point, CHPG changes were associated with increased numbers of mature CC1+ cells. We do not yet know whether CHPG was affecting proliferation, differentiation, or survival of oligodendrocyte lineage cells, but this is clearly a topic for future investigation. Similarly, we do not yet know if CHPG can reverse demyelination, a question that can be asked once cuprizone is removed after several weeks.

In the current work, we showed that CHPG does in fact elicit its actions in the brain even when administered peripherally, as evident by the local injection of the mGluR5 antagonist, MPEP, which blocked the effects of intraperitoneally injected CHPG. Moreover, an interesting aspect of our work was that the state of the lesion environment may determine when CHPG is most effective. Because we showed that CHPG’s actions were eliminated when mGluR5 was deleted from astrocytes, we suggest that CHPG required astrocytes to first become reactive and express mGluR5 before being able to elicit its actions. This is apparent by the fact that CHPG was only effective after a lesion but not in control-fed mice when astrocytes have little to no expression of mGluR5 (Fulmer et al., 2014).

Our studies for the first time indicate further that CHPG required the presence of TrkB receptors on PLP+ cells to elicit its effects, suggesting that its ability to increase BDNF was enhanced by the direct stimulation of oligodendrocyte lineage cells by the neurotrophin. In light of these findings, is important to remember that BDNF is not only synthesized by astrocytes, but is also synthesized and released from oligodendrocytes (Dai et al., 2003; Bagayogo & Dreyfus, 2009). This raises the possibility that astrocyte-derived BDNF may influence oligodendrocyte-derived BDNF or other aspects of oligodendrocyte function and contribute in these ways to the effects of CHPG.

Our work is consistent with culture work that supports the possibility that mGluR stimulation elicits positive astrocyte actions. In these studies, treatment with the Group I/Group II ACPD leads to the release of neurotransmitters, including glutamate (Miller et al., 1995; Nakahara et al., 1997; Biber et al., 1999) as well as BDNF (Jean et al., 2008). The stimulation of astrocytes by other mGluR agonists also enhances growth factor release leading to positive effects. Thus, stimulation of Group II mGluRs enhances BDNF (Durand et al., 2017), glial-derived neurotrophic factor (GDNF) (Battaglia et al., 2015), and TGF-β release (Bruno et al., 1998; Caraci et al., 2011) and enhances glutamate uptake (Yao et al., 2005).

Similarly, other studies indicate that activation of Group I mGluRs may also be directly beneficial to glial cells other than astrocytes that are found within the lesion site. For example, the Group I agonist 3,5-dihydroxyphenylglycine (DHPG) reduces oxidative stress and prevents OPC death in culture (Deng et al., 2004; Luyt et al., 2006) and releases BDNF from cultured oligodendrocytes (Bagayogo & Dreyfus, 2009). Agonists of Group I mGluRs on microglia reverse activation of the microglia (Byrnes et al., 2009). Moreover, the application of CHPG itself to the BV2 microglial cell line attenuates cell death, oxidative stress, inflammation, and proliferation, yet enhances BDNF expression under toxic conditions (Qiu et al., 2015; Ye et al., 2017; Huang et al., 2018). These studies that should be pursued in the future suggest that mGluR5 stimulation may influence the lesion environment to manipulate synergy between multiple cells and enhance actions elicited through the astrocyte population.

Interestingly, expression of glial mGluR5, as well as other mGluRs, may be a disease-specific target with importance to human tissue. Studies have shown that mGluRs are upregulated primarily on astrocytes and microglia in human MS lesions compared to control tissue (Geurts et al., 2003; Geurts et al., 2005; Newcombe et al., 2008). However, mGluR5 on astrocytes may have special significance. In the case of astrocytes, upregulated mGluR2/3 as well as 5 is reported on virtually all reactive astrocytes within lesion sites, while mGluR1, 4 and 8 are associated only with a subpopulation of those cells (Geurts et al., 2003; Geurts et al., 2005; Newcombe et al., 2008). On the other hand, microglia and young oligodendrocytes do not upregulate mGluR5 in human samples, although microglia do express mGluR1, 2/3 and 8 within active MS lesions (Geurts et al., 2003; Geurts et al., 2005; Newcombe et al., 2008) and cells morphologically resembling young oligodendrocytes have been reported to exhibit mGluR2/3 (Newcombe et al., 2008).

Furthermore, the role of BDNF in demyelinating diseases like MS may also have human relevance. For example, evidence suggests that levels of BDNF are decreased in patients with MS (Sarchielli et al., 2002; Azoulay et al., 2005; Caggiula et al., 2005; Azoulay et al., 2008; Mehrpour et al., 2015; Vacaras et al., 2017). In addition, some therapeutic agents used for MS elevate levels of BDNF in patients and this typically corresponds to a decrease in the severity of the disease (Azoulay et al., 2005; Mehrpour et al., 2015; Vacaras et al., 2017). Thus, enhancing BDNF levels in patients with these types of diseases may be beneficial.

In summary, we demonstrated that the small molecule mGluR agonist CHPG effectively reversed characteristics associated with a cuprizone-induced demyelinating lesion, including changes in myelination and behavior. Furthermore, we showed for the first time that CHPG’s effects were dependent on astrocytic mGluR5, astrocyte-derived BDNF, and oligodendroglial TrkB. We suggest that astrocytic mGluR5 may therefore be a novel therapeutic target for treating demyelinating diseases and other neurodegenerative diseases known to upregulate astrocytic mGluR5 by taking advantage of the disease environment to promote regeneration and repair.

Acknowledgments:

This work was supported by NIH RO1 NS036647, NIH T32 ES007148, NIH F31 NS098642; The National Multiple Sclerosis Society RG 4257B4/1; and the Rutgers School of Graduate Studies Acceleration and Completion Fellowship. We thank Dr. Anis Contractor and Dr. Jian Xu (Northwestern University, Chicago, Illinois) for providing the mGluR5fl/fl mice.

Footnotes

Conflict of Interest: The authors declare no competing financial interests.

References

  1. Abercrombie M (1946). Estimation of nuclear population from microtome sections. Anat Rec, 94, 239–247. doi: 10.1002/ar.1090940210 [DOI] [PubMed] [Google Scholar]
  2. Anderson JJ, Bradbury MJ, Giracello DR, Chapman DF, Holtz G, Roppe J, King C, Cosford ND & Varney MA (2003). In vivo receptor occupancy of mGlu5 receptor antagonists using the novel radioligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine). Eur J Pharmacol, 473(1), 35–40. doi: 10.1016/s0014-2999(03)01935-6 [DOI] [PubMed] [Google Scholar]
  3. Azoulay D, Urshansky N & Karni A (2008). Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection. J Neuroimmunol, 195(1-2), 186–193. doi: 10.1016/j.jneuroim.2008.01.010 [DOI] [PubMed] [Google Scholar]
  4. Azoulay D, Vachapova V, Shihman B, Miler A & Karni A (2005). Lower brain-derived neurotrophic factor in serum of relapsing remitting MS: reversal by glatiramer acetate. J Neuroimmunol, 167(1-2), 215–218. doi: 10.1016/j.jneuroim.2005.07.001 [DOI] [PubMed] [Google Scholar]
  5. Bagayogo IP & Dreyfus CF (2009). Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway. ASN Neuro, 1(1). doi: 10.1042/an20090006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Battaglia G, Riozzi B, Bucci D, Di Menna L, Molinaro G, Pallottino S, Nicoletti F & Bruno V (2015). Activation of mGlu3 metabotropic glutamate receptors enhances GDNF and GLT-1 formation in the spinal cord and rescues motor neurons in the SOD-1 mouse model of amyotrophic lateral sclerosis. Neurobiol Dis, 74, 126–136. doi: 10.1016/j.nbd.2014.11.012 [DOI] [PubMed] [Google Scholar]
  7. Biber K, Laurie DJ, Berthele A, Sommer B, Tolle TR, Gebicke-Harter PJ, van Calker D & Boddeke HW (1999). Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia. J Neurochem, 72(4), 1671–1680. [DOI] [PubMed] [Google Scholar]
  8. Bruno V, Battaglia G, Casabona G, Copani A, Caciagli F & Nicoletti F (1998). Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J Neurosci, 18(23), 9594–9600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Byrnes KR, Stoica B, Loane DJ, Riccio A, Davis MI & Faden AI (2009). Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity. Glia, 57(5), 550–560. doi: 10.1002/glia.20783 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caggiula M, Batocchi AP, Frisullo G, Angelucci F, Patanella AK, Sancricca C, Nociti V, Tonali PA & Mirabella M (2005). Neurotrophic factors and clinical recovery in relapsing-remitting multiple sclerosis. Scand J Immunol, 62(2), 176–182. doi: 10.1111/j.1365-3083.2005.01649.x [DOI] [PubMed] [Google Scholar]
  11. Caraci F, Molinaro G, Battaglia G, Giuffrida ML, Riozzi B, Traficante A, Bruno V, Cannella M, Merlo S, Wang X, Heinz BA, Nisenbaum ES, Britton TC, Drago F, Sortino MA, Copani A & Nicoletti F (2011). Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective. Mol Pharmacol, 79(3), 618–626. doi: 10.1124/mol.110.067488 [DOI] [PubMed] [Google Scholar]
  12. Casper KB & McCarthy KD (2006). GFAP-positive progenitor cells produce neurons and oligodendrocytes throughout the CNS. Mol Cell Neurosci, 31(4), 676–684. doi: 10.1016/j.mcn.2005.12.006 [DOI] [PubMed] [Google Scholar]
  13. Casper KB, Jones K & McCarthy KD (2007). Characterization of astrocyte-specific conditional knockouts. Genesis, 45(5), 292–299. doi: 10.1002/dvg.20287 [DOI] [PubMed] [Google Scholar]
  14. Chau P, Söderpalm B & Ericson M (2011). The mGluR5 antagonist MPEP elevates accumbal dopamine and glycine levels; interaction with strychnine-sensitive glycine receptors. Addict Biol, 16(4), 591–599. doi: 10.1111/j.1369-1600.2011.00343.x [DOI] [PubMed] [Google Scholar]
  15. Coelho RP, Yuelling LM, Fuss B & Sato-Bigbee C (2009). Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes. Glia, 57(16), 1754–1764. doi: 10.1002/glia.20888 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Copeland CS, Wall TM, Sims RE, Neale SA, Nisenbaum E, Parri HR & Salt TE (2017). Astrocytes modulate thalamic sensory processing via mGlu2 receptor activation. Neuropharmacology, 121, 100–110. doi: 10.1016/j.neuropharm.2017.04.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dai X, Lercher LD, Clinton PM, Du Y, Livingston DL, Vieira C, Yang L, Shen MM & Dreyfus CF (2003). The trophic role of oligodendrocytes in the basal forebrain. J Neurosci, 23(13), 5846–5853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Deng W, Wang H, Rosenberg PA, Volpe JJ & Jensen FE (2004). Role of metabotropic glutamate receptors in oligodendrocyte excitotoxicity and oxidative stress. Proc Natl Acad Sci U S A, 101(20), 7751–7756. doi: 10.1073/pnas.0307850101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Doherty AJ, Palmer MJ, Henley JM, Collingridge GL & Jane DE (1997). (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but no mGlu1, receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology, 36(2), 265–267. doi: 10.1016/s0028-3908(97)00001-4 [DOI] [PubMed] [Google Scholar]
  20. Du Y, Lercher LD, Zhou R & Dreyfus CF (2006). Mitogen-activated protein kinase pathway mediates effects of brain-derived neurotrophic factor on differentiation of basal forebrain oligodendrocytes. J Neurosci Res, 84(8), 1692–1702. doi: 10.1002/jnr.21080 [DOI] [PubMed] [Google Scholar]
  21. Du Y, Fischer TZ, Lee LN, Lercher LD & Dreyfus CF (2003). Regionally specific effects of BDNF on oligodendrocytes. Dev Neurosci, 25(2-4), 116–126. doi: 10.1159/000072261 [DOI] [PubMed] [Google Scholar]
  22. Durand D, Carniglia L, Turati J, Ramirez D, Saba J, Caruso C & Lasaga M (2017). Amyloid-beta neurotoxicity and clearance are both regulated by glial group II metabotropic glutamate receptors. Neuropharmacology, 123, 274–286. doi: 10.1016/j.neuropharm.2017.05.008 [DOI] [PubMed] [Google Scholar]
  23. Fletcher JL, Wood RJ, Nguyen J, Norman EML, Jun CMK, Prawdiuk AR, Biemond M, Nguyen HTH, Northfield SE, Hughes RA, Gonsalvez DG, Xiao J & Murray SS (2018). Targeting TrkB with a Brain-Derived Neurotrophic Factor Mimetic Promotes Myelin Repair in the Brain. J Neurosci, 38(32), 7088–7099. doi: 10.1523/jneurosci.0487-18.2018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Franco-Pons N, Torrente M, Colomina MT & Vilella E (2007). Behavioral deficits in the cuprizone-induced murine model of demyelination/remyelination. Toxicol Lett, 169(3), 205–213. doi: 10.1016/j.toxlet.2007.01.010 [DOI] [PubMed] [Google Scholar]
  25. Franklin RJ (2002). Why does remyelination fail in multiple sclerosis? Nat Rev Neurosci, 3(9), 705–714. doi: 10.1038/nrn917 [DOI] [PubMed] [Google Scholar]
  26. Franklin RJ & Ffrench-Constant C (2008). Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci, 9(11), 839–855. doi: 10.1038/nrn2480 [DOI] [PubMed] [Google Scholar]
  27. Franklin RJM & Ffrench-Constant C (2017). Regenerating CNS myelin - from mechanisms to experimental medicines. Nat Rev Neurosci, 18(12), 753–769. doi: 10.1038/nrn.2017.136 [DOI] [PubMed] [Google Scholar]
  28. Fulmer CG, VonDran MW, Stillman AA, Huang Y, Hempstead BL & Dreyfus CF (2014). Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination. J Neurosci, 34(24), 8186–8196. doi: 10.1523/jneurosci.4267-13.2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Geurts JJ, Wolswijk G, Bo L, van der Valk P, Polman CH, Troost D & Aronica E (2003). Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis. Brain, 126(Pt 8), 1755–1766. doi: 10.1093/brain/awg179 [DOI] [PubMed] [Google Scholar]
  30. Geurts JJ, Wolswijk G, Bo L, Redeker S, Ramkema M, Troost D & Aronica E (2005). Expression patterns of Group III metabotropic glutamate receptors mGluR4 and mGluR8 in multiple sclerosis lesions. J Neuroimmunol, 158(1-2), 182–190. doi: 10.1016/j.jneuroim.2004.08.012 [DOI] [PubMed] [Google Scholar]
  31. Goldberg J, Clarner T, Beyer C & Kipp M (2015). Anatomical Distribution of Cuprizone-Induced Lesions in C57BL6 Mice. J Mol Neurosci, 57(2), 166–175. doi: 10.1007/s12031-015-0595-5 [DOI] [PubMed] [Google Scholar]
  32. Groebe A, Clarner T, Baumgartner W, Dang J, Beyer C & Kipp M (2009). Cuprizone treatment induces distinct demyelination, astrocytosis, and microglia cell invasion or proliferation in the mouse cerebellum. Cerebellum, 8(3), 163–174. doi: 10.1007/s12311-009-0099-3 [DOI] [PubMed] [Google Scholar]
  33. Hodge CW, Miles MF, Sharko AC, Stevenson RA, Hillmann JR, Lepoutre V, Besheer J & Schroeder JP (2006). The mGluR5 antagonist MPEP selectively inhibits the onset and maintenance of ethanol self-administration in C57BL/6J mice. Psychopharmacology (Berl), 183(4), 429–438. doi: 10.1007/s00213-005-0217-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Huang Y, Song YJ, Isaac M, Miretzky S, Patel A, Geoffrey McAuliffe W & Dreyfus CF (2020). Tropomyosin Receptor Kinase B Expressed in Oligodendrocyte Lineage Cells Functions to Promote Myelin Following a Demyelinating Lesion. ASN Neuro, 12, 1759091420957464. doi: 10.1177/1759091420957464 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Huang YY, Zhang Q, Zhang JN, Zhang YN, Gu L, Yang HM, Xia N, Wang XM & Zhang H (2018). Triptolide up-regulates metabotropic glutamate receptor 5 to inhibit microglia activation in the lipopolysaccharide-induced model of Parkinson's disease. Brain Behav Immun, 71, 93–107. doi: 10.1016/j.bbi.2018.04.006 [DOI] [PubMed] [Google Scholar]
  36. Ishii A, Furusho M, Macklin W & Bansal R (2019). Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental myelination and in adulthood. Glia, 67(7), 1277–1295. doi: 10.1002/glia.23602 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jantzie LL, Talos DM, Selip DB, An L, Jackson MC, Folkerth RD, Deng W & Jensen FE (2010). Developmental regulation of group I metabotropic glutamate receptors in the premature brain and their protective role in a rodent model of periventricular leukomalacia. Neuron Glia Biol, 6(4), 277–288. doi: 10.1017/s1740925x11000111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jean YY, Lercher LD & Dreyfus CF (2008). Glutamate elicits release of BDNF from basal forebrain astrocytes in a process dependent on metabotropic receptors and the PLC pathway. Neuron Glia Biol, 4(1), 35–42. doi: 10.1017/s1740925x09000052 [DOI] [PubMed] [Google Scholar]
  39. Kammermeier PJ (2012). The orthosteric agonist 2-chloro-5-hydroxyphenylglycine activates mGluR5 and mGluR1 with similar efficacy and potency. BMC Pharmacol, 12, 6. doi: 10.1186/1471-2210-12-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Laursen LS, Chan CW & Ffrench-Constant C (2011). Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K. J Cell Biol, 192(5), 797–811. doi: 10.1083/jcb.201007014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Luyt K, Varadi A, Durant CF & Molnar E (2006). Oligodendroglial metabotropic glutamate receptors are developmentally regulated and involved in the prevention of apoptosis. J Neurochem, 99(2), 641–656. doi: 10.1111/j.1471-4159.2006.04103.x [DOI] [PubMed] [Google Scholar]
  42. Mason JL, Langaman C, Morell P, Suzuki K & Matsushima GK (2001). Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre. Neuropathol Appl Neurobiol, 27(1), 50–58. [DOI] [PubMed] [Google Scholar]
  43. Mehrpour M, Akhoundi FH, Delgosha M, Keyvani H, Motamed MR, Sheibani B & Meysamie A (2015). Increased Serum Brain-derived Neurotrophic Factor in Multiple Sclerosis Patients on Interferon-beta and Its Impact on Functional Abilities. Neurologist, 20(4), 57–60. doi: 10.1097/nrl.0000000000000053 [DOI] [PubMed] [Google Scholar]
  44. Michel K, Zhao T, Karl M, Lewis K & Fyffe-Maricich SL (2015). Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain. J Neurosci, 35(20), 7850–7865. doi: 10.1523/jneurosci.4380-14.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Miller S, Romano C & Cotman CW (1995). Growth factor upregulation of a phosphoinositide-coupled metabotropic glutamate receptor in cortical astrocytes. J Neurosci, 15(9), 6103–6109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nakahara K, Okada M & Nakanishi S (1997). The metabotropic glutamate receptor mGluR5 induces calcium oscillations in cultured astrocytes via protein kinase C phosphorylation. J Neurochem, 69(4), 1467–1475. [DOI] [PubMed] [Google Scholar]
  47. Nett WJ, Oloff SH & McCarthy KD (2002). Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. J Neurophysiol, 87(1), 528–537. doi: 10.1152/jn.00268.2001 [DOI] [PubMed] [Google Scholar]
  48. Newcombe J, Uddin A, Dove R, Patel B, Turski L, Nishizawa Y & Smith T (2008). Glutamate receptor expression in multiple sclerosis lesions. Brain Pathol, 18(1), 52–61. doi: 10.1111/j.1750-3639.2007.00101.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nguyen HTH, Wood RJ, Prawdiuk AR, Furness SGB, Xiao J, Murray SS & Fletcher JL (2019). TrkB Agonist LM22A-4 Increases Oligodendroglial Populations During Myelin Repair in the Corpus Callosum. Front Mol Neurosci, 12, 205. doi: 10.3389/fnmol.2019.00205 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Pasti L, Volterra A, Pozzan T & Carmignoto G (1997). Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. J Neurosci, 17(20), 7817–7830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Qiu JL, Zhu WL, Lu YJ, Bai ZF, Liu ZG, Zhao P, Sun C, Zhang YB, Li H & Liu W (2015). The selective mGluR5 agonist CHPG attenuates SO2-induced oxidative stress and inflammation through TSG-6/NF-kappaB pathway in BV2 microglial cells. Neurochem Int, 85-86, 46–52. doi: 10.1016/j.neuint.2015.04.007 [DOI] [PubMed] [Google Scholar]
  52. Sarchielli P, Greco L, Stipa A, Floridi A & Gallai V (2002). Brain-derived neurotrophic factor in patients with multiple sclerosis. J Neuroimmunol, 132(1-2), 180–188. doi: 10.1016/s0165-5728(02)00319-3 [DOI] [PubMed] [Google Scholar]
  53. Shelton MK & McCarthy KD (1999). Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ. Glia, 26(1), 1–11. [DOI] [PubMed] [Google Scholar]
  54. Skripuletz T, Bussmann JH, Gudi V, Koutsoudaki PN, Pul R, Moharregh-Khiabani D, Lindner M & Stangel M (2010). Cerebellar cortical demyelination in the murine cuprizone model. Brain Pathol, 20(2), 301–312. doi: 10.1111/j.1750-3639.2009.00271.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Umpierre AD, West PJ, White JA & Wilcox KS (2019). Conditional Knock-out of mGluR5 from Astrocytes during Epilepsy Development Impairs High-Frequency Glutamate Uptake. J Neurosci, 39(4), 727–742. doi: 10.1523/jneurosci.1148-18.2018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vacaras V, Major ZZ & Buzoianu AD (2017). Brain-derived neurotrophic factor levels under chronic natalizumab treatment in multiple sclerosis. A preliminary report. Neurol Neurochir Pol, 51(3), 221–226. doi: 10.1016/j.pjnns.2017.03.002 [DOI] [PubMed] [Google Scholar]
  57. VonDran MW, Clinton-Luke P, Honeywell JZ & Dreyfus CF (2010). BDNF+/− mice exhibit deficits in oligodendrocyte lineage cells of the basal forebrain. Glia, 58(7), 848–856. doi: 10.1002/glia.20969 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. VonDran MW, Singh H, Honeywell JZ & Dreyfus CF (2011). Levels of BDNF impact oligodendrocyte lineage cells following a cuprizone lesion. J Neurosci, 31(40), 14182–14190. doi: 10.1523/jneurosci.6595-10.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wong AW, Xiao J, Kemper D, Kilpatrick TJ & Murray SS (2013). Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation. J Neurosci, 33(11), 4947–4957. doi: 10.1523/jneurosci.3990-12.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xiao J, Wong AW, Willingham MM, van den Buuse M, Kilpatrick TJ & Murray SS (2010). Brain-derived neurotrophic factor promotes central nervous system myelination via a direct effect upon oligodendrocytes. Neurosignals, 18(3), 186–202. doi: 10.1159/000323170 [DOI] [PubMed] [Google Scholar]
  61. Yao HH, Ding JH, Zhou F, Wang F, Hu LF, Sun T & Hu G (2005). Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes. J Neurochem, 92(4), 948–961. doi: 10.1111/j.1471-4159.2004.02937.x [DOI] [PubMed] [Google Scholar]
  62. Ye X et al. (2017). Activated mGluR5 protects BV2 cells against OGD/R induced cytotoxicity by modulating BDNF-TrkB pathway. Neurosci Lett, 654, 70–79. doi: 10.1016/j.neulet.2017.06.029 [DOI] [PubMed] [Google Scholar]
  63. Zhou YX, Pannu R, Le TQ & Armstrong RC (2012). Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination. Neurobiol Dis, 45(1), 196–205. doi: 10.1016/j.nbd.2011.08.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zur Nieden R & Deitmer JW (2006). The role of metabotropic glutamate receptors for the generation of calcium oscillations in rat hippocampal astrocytes in situ. Cereb Cortex, 16(5), 676–687. doi: 10.1093/cercor/bhj013 [DOI] [PubMed] [Google Scholar]

RESOURCES