Increased p75 neurotrophin receptor expression in the canine distemper virus model of multiple sclerosis identifies aldynoglial Schwann cells that emerge in response to axonal damage

Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide regeneration-promoting cells recruitable for therapeutic purposes. There is accumulating evidence that aldynoglial cells with Schwann cell-like growth-promoting properties emerge in the lesioned CNS. However, the characterization of these cells and the signals triggering their in situ generation have remained enigmatic. In the present study, we used the p75 neurotrophin receptor (p75(NTR) ) as a marker for Schwann cells to study gliogenesis in the well-defined canine distemper virus (CDV)-induced demyelination model. White matter lesions of CDV-infected dogs contained bi- to multipolar, p75(NTR) -expressing cells that neither expressed MBP, GFAP, BS-1, or P0 identifying oligodendroglia, astrocytes, microglia, and myelinating Schwann cells nor CDV antigen. Interestingly, p75(NTR) -expression became apparent prior to the onset of demyelination in parallel to the expression of β-amyloid precursor protein (β-APP), nonphosphorylated neurofilament (n-NF), BS-1, and CD3, and peaked in subacute lesions with inflammation. To study the role of infiltrating immune cells during differentiation of Schwann cell-like glia, organotypic slice cultures from the normal olfactory bulb were established. Despite the absence of infiltrating lymphocytes and macrophages, a massive appearance of p75(NTR) -positive Schwann-like cells and BS-1-positive microglia was noticed at 10 days in vitro. It is concluded that axonal damage as an early signal triggers the differentiation of tissue-resident precursor cells into p75(NTR) -expressing aldynoglial Schwann cells that retain an immature pre-myelin state. Further studies have to address the role of microglia during this process and the regenerative potential of aldynoglial cells in CDV infection and other demyelinating diseases.     

Vimentin-positive astrocytes in canine distemper: a target for canine distemper virus especially in chronic demyelinating lesions?

In canine distemper demyelinating leukoencephalitis (DL), caused by canine distemper virus (CDV), astrocytes represent the main virus target. In these cells, glial fibrillary acidic protein (GFAP) is the main intermediate filament, whereas vimentin occurs early in the astrocytic lineage and is replaced gradually by GFAP. To further characterize the role of astrocytic infection in dogs with DL, an animal model for multiple sclerosis, formalin-fixed paraffin-embedded cerebella were investigated immunohistochemically and by immunofluorescence. The expression and morphological alterations of these intermediate filaments were also determined by immunofluorescence studies of CDV-infected canine mixed brain cell cultures. In acute distemper lesions, the astrocytic response was mainly composed of GFAP- and CDV-positive cells. In contrast, vimentin-positive astrocyte-like cells were present in advanced lesions, which represented the main cell type harboring the pathogen, indicating a change in cell tropism and/or susceptibility of glial cells during lesion progression in CDV encephalomyelitis. Canine cell cultures were composed of GFAP-positive astrocytes, vimentin-positive cells and other glial cells. Following infection with the CDV-R252 strain, GFAP-positive astrocytes, especially multinucleated syncytial giant cells, displayed a disrupted cytoskeleton, whereas vimentin-positive cells though more frequently infected did not show any alteration in the filament network. This indicates increased vulnerability of mature GFAP-positive astrocytes compared to immature, vimentin-positive astrocytes. The latter, however, exhibited increased susceptibility to CDV. To conclude, the present findings indicate a change in cell tropism of CDV and/or the occurrence of less differentiated astrocytes representing a permanent source for virus infection and spread in advanced lesions of DL.     

Involvement of brain‐derived neurotrophic factor (BDNF) in the functional elimination of synaptic contacts at polyinnervated neuromuscular synapses during development

We use immunohistochemistry to describe the localization of brain‐derived neurotrophic factor (BDNF) and its receptors trkB and p75^NTR in the neuromuscular synapses of postnatal rats (P6–P7) during the synapse elimination period. The receptor protein p75^NTR is present in the nerve terminal, muscle cell and glial Schwann cell whereas BDNF and trkB proteins can be detected mainly in the pre‐ and postsynaptic elements. Exogenously applied BDNF (10 nM for 3 hr or 50 nM for 1 hr) increases ACh release from singly and dually innervated synapses. This effect may be specific for BDNF because the neurotrophin NT‐4 (2–8 nM) does not modulate release at P6–P7. Blocking the receptors trkB and p75^NTR (with K‐252a and anti‐p75‐192‐IgG, respectively) completely abolishes the potentiating effect of exogenous BDNF. In addition, exogenous BDNF transiently recruits functionally depressed silent terminals, and this effect seems to be mediated by trkB. Calcium ions, the L‐type voltage‐dependent calcium channels and protein kinase C are involved in BDNF‐mediated nerve ending recruitment. Blocking experiments suggest that endogenous BDNF could operate through p75^NTR receptors coupled to potentiate ACh release in all nerve terminals because the anti‐p75‐192‐IgG reduces release. However, blocking the trkB receptor (K‐252a) or neutralizing endogenous BDNF with the trkB‐IgG fusion protein reveals a trkB‐mediated release inhibition on almost mature strong endings in dual junctions. Taken together these results suggest that a BDNF‐induced p75^NTR‐mediated ACh release potentiating mechanism and a BDNF‐induced trkB‐mediated release inhibitory mechanism may contribute to developmental synapse disconnection. © 2009 Wiley‐Liss, Inc.     

Nerve growth factor attenuates proliferation of astrocytes via the p75 neurotrophin receptor

The p75 neurotrophin receptor has been implicated in the regulation of multiple cellular functions that differ depending on the cell context. We have observed that p75^NTR is strongly induced on astrocytes as well as neurons in the hippocampal CA3 region after seizures; however, the function of this receptor on these glial cells has not been defined. We have employed a primary culture system to investigate the effects of neurotrophins on astrocytes. Treatment of hippocampal astrocytes with nerve growth factor (NGF) caused a reduction in cell number, but did not elicit an apoptotic response, in contrast to hippocampal neurons. Instead, activation of p75^NTR by NGF attenuated proliferation induced by mitogens such as EGF or serum. These studies demonstrate the cell type specificity of neurotrophin functions in the brain. © 2009 Wiley‐Liss, Inc.     

Schwann cell p75 neurotrophin receptor modulates small fiber degeneration in diabetic neuropathy

Diabetic neuropathy has an incidence as high as 50% of diabetic patients and is characterized by damage to neurons, Schwann cells and blood vessels within the peripheral nervous system. The low-affinity neurotrophin receptor p75 (p75^NTR), particularly expressed by the Schwann cells in the peripheral nerve, has previously been reported to play a role in developmental myelination and cell survival/death. Increased levels of p75^NTR, in the endoneurium and plasma from diabetic patients and rodent models of disease, have been observed, proposing that this receptor might be involved in the pathogenesis of diabetic neuropathy. Therefore, in this study, we addressed this hypothesis by utilizing a mouse model of selective nerve growth factor receptor (Ngfr) deletion in Schwann cells (SC-p75^NTR-KO). Electron microscopy of sciatic nerves from mice with high fat diet induced obesity demonstrated how loss of Schwann cell–p75^NTR aggravated axonal atrophy and loss of C-fibers. RNA sequencing disclosed several pre-clinical signaling alterations in the diabetic peripheral nerves, dependent on Schwann cell p75^NTR signaling, specially related with lysosome, phagosome, and immune pathways. Morphological and biochemical analyses identified abundant lysosomes and autophagosomes in the C-fiber axoplasm of the diabetic SC-p75^NTR-KO nerves, which together with increased Cathepsin B protein levels corroborates gene upregulation from the phagolysosomal pathways. Altogether, this study demonstrates that Schwann cell p75^NTR deficiency amplifies diabetic neuropathy disease by triggering overactivation of immune-related pathways and increased lysosomal stress.     

The p75 neurotrophin receptor: at the crossroad of neural repair and death

The strong repair and pro-survival functions of neurotrophins at their primary receptors, TrkA, TrkB and TrkC, have made them attractive candidates for treatment of nervous system injury and disease. However, difficulties with the clinical implementation of neurotrophin therapies have prompted the search for treatments that are stable, easier to deliver and allow more precise regulation of neurotrophin actions. Recently, the p75 neurotrophin receptor (p75^NTR) has emerged as a potential target for pharmacological control of neurotrophin activity, supported in part by studies demonstrating 1) regulation of neural plasticity in the mature nervous system, 2) promotion of adult neurogenesis and 3) increased expression in neurons, macrophages, microglia, astrocytes and/or Schwann cells in response to injury and neurodegenerative diseases. Although the receptor has no intrinsic catalytic activity it interacts with and modulates the function of TrkA, TrkB, and TrkC, as well as sortilin and the Nogo receptor. This provides substantial cellular and molecular diversity for regulation of neuron survival, neurogenesis, immune responses and processes that support neural function. Upregulation of the p75^NTR under pathological conditions places the receptor in a key position to control numerous processes necessary for nervous system recovery. Support for this possibility has come from recent studies showing that small, non-peptide p75^NTR ligands can selectively modify pro-survival and repair functions. While a great deal remains to be discovered about the wide ranging functions of the p75^NTR, studies summarized in this review highlight the immense potential for development of novel neuroprotective and neurorestorative therapies.     

Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model of selective Müller cell ablation

Retinal diseases such as macular telangiectasis type 2 (MacTel), age‐related macular degeneration (AMD) and diabetic retinopathy (DR) affect both neurons and blood vessels. Treatments addressing both at the same time might have advantages over more specific approaches, such as vascular endothelial growth factor (VEGF) inhibitors, which are used to treat vascular leak but are suspected to have a neurotoxic effect. Here, we studied the effects of an intravitreal injection of triamcinolone acetonide (TA) in a transgenic model in which patchy Müller cell ablation leads to photoreceptor degeneration, vascular leak, and intraretinal neovascularization. TA was injected 4 days before Müller cell ablation. Changes in photoreceptors, microglia and Müller cells, retinal vasculature, differential expression of p75 neurotrophin receptor (p75^NTR), tumor necrosis factor‐α (TNFα), the precursor and mature forms of neurotrophin 3 (pro‐NT3 and mature NT3) and activation of the p53 and p38 stress‐activated protein kinase (p38/SAPK) signaling pathways were examined. We found that TA prevented photoreceptor degeneration and inhibited activation of microglial and Müller cells. TA attenuated Müller cell loss and inhibited overexpression of p75^NTR, TNFα, pro‐NT, and the activation of p53 and p38/SAPK signaling pathways. TA not only prevented the development of retinal vascular lesions but also inhibited fluorescein leakage from established vascular lesions. TA inhibited overexpression of VEGF in transgenic mice but without affecting its basal level expression in the normal retina. Our data suggest that glucocorticoid treatment may be beneficial for treatment of retinal diseases such as MacTel, AMD, and DR that affect both neurons and the vasculature. GLIA 2014;62:1110–1124     

Gamma-secretase-independent role for cadherin-11 in neurotrophin receptor p75 (p75NTR) mediated glioblastoma cell migration

The p75 neurotrophin receptor (p75^NTR) undergoes γ-secretase-mediated regulated intramembrane proteolysis and is involved in glioblastoma cell migration and invasion. Consistent with previous reports, in this study we show that p75NTR increases U87-MG glioblastoma cell migration, which is reversed by inhibition of γ-secretase activity. However, we show that expression or stabilization of the γ-secretase-generated p75^NTR intracellular domain (ICD) is not sufficient to induce U87-MG glioblastoma cell migration, and that exogenous expression of p75^NTR ICD inhibits p75^NTR-mediated glioblastoma cell (U87-MG and U373-MG) migration. To identify pathways and to determine how p75^NTR mediates glioblastoma migration we utilized a microarray approach to assess differential gene expression profiles between parental U87-MG and cells stably expressing wild-type p75^NTR, a γ-secretase cleavage-resistant chimeric p75^NTR mutant (p75FasTM) and the γ-secretase-generated p75^NTR-ICD, which mimics constitutively cleaved p75^NTR receptor. In our microarray data analysis we identified a subset of genes that were constitutively up-regulated in wild-type p75^NTR cells, which were also repressed in p75^NTR ICD expressing cells. Furthermore, our data revealed among the many differentially expressed genes, cadherin-11 (Cdh-11), matrix metalloproteinase 12 and relaxin/insulin-like family peptide receptor 2 as constitutively up-regulated in wild-type p75^NTR cells, independent of γ-secretase activity. Consistent with a role in glioblastoma migration, we found that U87-p75^NTR cells express higher levels of Cdh-11 protein and that siRNA-mediated knockdown of Cdh-11 resulted in a significant decrease in p75^NTR-mediated glioblastoma cell migration. Therefore, we hypothesize that p75^NTR can impact U87-MG glioblastoma cell migration in a γ-secretase-independent manner through modulation of specific genes, including Cdh-11, and that both γ-secretase-independent and -dependent mechanisms are involved in p75^NTR-mediated U87-MG glioblastoma cell migration.     

Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination. Received: 13 September 1995 / Revised: 26 February 1996 / Accepted: 1 April 1996     

Chronically denervated rat schwann cells respond to GGF in vitro

C-erbB receptor/neuregulin signalling plays a significant role in Schwann cell function. In vivo, Schwann cells up-regulate expression of c-erbB receptors in the first month after injury, but receptor expression is down-regulated with time to levels that are not detectable immunohistochemically. The inability of chronically denervated Schwann cells to respond adequately to signals derived from regenerating axons may be one reason why delayed repair of an injured peripheral nerve frequently fails. We have examined the effects of GGF on denervated Schwann cells in vitro. A modified delayed dissociation technique was used to obtain adult rat Schwann cells from the distal stumps of transected sciatic nerves which had been acutely (7 days) or chronically (2–6 month) denervated. We found that in vitro denervated Schwann cells invariably expressed p75^NTR and c-erbB receptors. There was a progressive decrease in total cell yield and the percentage of cells with Schwann cell phenotype (p75^NTR and/S-100 or/laminin or /GFAP or/c-erbB positive); proliferation rate; migratory potential; and expression of the cell adhesion molecules N-CAM and N-cadherin, with increasing time of denervation. Addition of GGF2 had a significant stimulatory effect upon Schwann cell proliferation and migration, and an increased proportion of Schwann cells expressed N-CAM and N-cadherin, suggesting that these responses were mediated via GGF/c-erbB signalling. Our results support the view that it may be possible to manipulate chronically denervated Schwann cells so that they become more responsive to signals derived from regrowing axons. GLIA 24:290–303, 1998. © 1998 Wiley-Liss, Inc.     

Canine distemper virus does not infect oligodendrocytes in vitro

Dissociated canine brain cell cultures were infected with virulent canine distemper virus (CDV). Double immunofluorescent labelling was done to simultaneously demonstrate viral antigen and specific glial cell markers. Virus containing oligodendrocytes were not found at any stage of the infection. A certain proportion of the infected cells were shown to be astrocytes. It was concluded that CDV has no obvious tropism for oligodendrocytes which could explain the mechanism of demyelination in distemper in vivo.     

Differential expression of HNK-1 and p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK-1 epitope and the neurotrophin receptor p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK-1 and p75(NTR), respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75(NTR) but not of HNK-1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK-1+ cellular debris. The cultivation-induced downregulation of HNK-1 expression in Schwann cells and upregulation of p75(NTR) in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK-1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75(NTR) expression in OECs in situ remain to be elucidated. Interestingly, HNK-1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research.     

Knockout of p75NTR does not alter the viability of striatal neurons following a metabolic or excitotoxic injury

Following metabolic or excitotoxic injury to the striatum, there is de novo expression of the low-affinity p75 neurotrophin receptor (p75^NTR). The novel expression of this pan neurotrophin receptor in rodents occurs within the lesion core and surrounding area, creating a division between viable and nonviable tissue. The present series of experiments sought to elucidate whether the p75^NTR expression seen following metabolic and excitotoxic injury alters neuronal viability within the striatum. Toward this end, we compared the extent of striatal lesion created with quinolinic acid (QA) or 3-nitropropionic acid (3-NP) in p75^NTR null and wild-type mice. Using stereological techniques, we found that the lesion volume and neuronal cell counts between p75^NTR null and wild-type mice were similar 1, 2, and 4 weeks post-QA or -3-NP lesion. The results indicate that the expression of p75^NTR within reactive astrocytes in the mouse striatum is not a key factor in protecting neuronal cell death following metabolic and excitotoxic insults.     

Uptake of parasite‐derived vesicles by astrocytes and microglial phagocytosis of infected erythrocytes may drive neuroinflammation in cerebral malaria

Astrocytes and microglia are activated during cerebral malaria (CM) and contribute to the production and release of several mediators during neuroinflammatory processes. Whether these changes are the consequence of a direct crosstalk between glial cells and the malarial parasite and how these cells participate in the pathogenesis of CM is not yet clear. We therefore examined the interaction of astrocytes and microglia with Plasmodium berghei ANKA‐infected red blood cells using primary cell cultures derived from newborn C57BL/6 mice. We observed a dynamic transfer of vesicles from the parasite to astrocytes within minutes of contact, and the phagocytosis of infected red blood cells by microglia. Differential gene expression studies using the Affymetrix GeneChip^® microarray, and quantitative PCR analyses showed the increase in expression of the set of genes belonging to the immune response network in parasite activated astrocytes and microglia. Interestingly, expression of these genes was also significantly upregulated in brains of mice dying from CM compared with uninfected mice or infected mice that did not develop the neuropathology. Accumulation of parasite‐derived vesicles within astrocytes, and the phagocytosis of infected red blood cells by microglia induced a subsequent increase in interferon gamma inducible protein 10 (IP10) in both the brain and plasma of infected mice at the onset of CM, confirming a role for this molecule in CM pathogenesis. Altogether, these observations point to a possible role for glial cells in the neuropathological processes leading to CM. GLIA 2016 GLIA 2017;65:75–92     

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques

CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-μm-thick serial sections were stained with H & E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection. Received: 4 March 1999 / Revised: 9 June 1999 / Accepted: 28 June 1999     

Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis

The potential interplay of glial cells with T cells during viral induced inflammation was assessed by comparing major histocompatibility complex molecule upregulation and retention on astrocytes and microglia. Transgenic mice expressing green fluorescent protein under control of the astrocyte-specific glial fibrillary acidic protein promoter were infected with a neurotropic coronavirus to facilitate phenotypic characterization of astrocytes and microglia using flow cytometry. Astrocytes in the adult central nervous system up-regulated class I surface expression, albeit delayed compared with microglia. Class II was barely detectable on astrocytes, in contrast to potent up-regulation on microglia. Maximal MHC expression in both glial cell types correlated with IFN-gamma levels and lymphocyte accumulation. Despite a decline of IFN-gamma concomitant to virus clearance, MHC molecule expression on glia was sustained. These data demonstrate distinct regulation of both class I and class II expression by microglia and astrocytes in vivo following viral induced inflammation. Furthermore, prolonged MHC expression subsequent to viral clearance implies a potential for ongoing presentation.