Janet E. Holley

Astrocyte characterization in the multiple sclerosis glial scar

Dense astrocytic scarring in chronic multiple sclerosis (MS) plaques produces an inhibitory environment which can impede tissue repair. Animal studies have shown that the antigenic phenotype of the most abundant cell type in the brain, the astrocyte, varies depending on astrocyte type and location. To identify the phenotype of scar astrocytes (SAs) in chronic lesions, markers of reactive astrocytes characterized in animal studies were investigated. To date these are the only established markers. Cerebral subventricular deep white matter from normal control, MS normal appearing white matter and lesions (acute, subacute and chronic) were examined by immunohistochemistry and immunoblotting. The antigenic profile of SAs revealed significant modification of astrocyte protein expression in chronic MS lesions. SAs express nestin, embryonic neural cell adhesion molecule, fibroblast growth factor receptor 4, epidermal growth factor receptor, nerve growth factor and a subpopulation of SAs also express basic fibroblast growth factor. These are in addition to the expected markers glial fibrillary acidic protein, vimentin, and the tenascins C and R. Therefore, an SA antigenic phenotype has now been defined. This knowledge may allow the development of therapeutic strategies that prevent scar formation and promote tissue repair.

Increased blood vessel density and endothelial cell proliferation in multiple sclerosis cerebral white matter.

Multiple sclerosis (MS) is primarily considered an inflammatory demyelinating disease, however the role of vasculature in MS pathogenesis is now receiving much interest. MS lesions often develop along blood vessels and alterations in blood brain barrier structure and function, with associated changes in the basement membrane, are pathological features. Nevertheless, the possibility of angiogenesis occurring in MS has received little attention. In this study we used triple label enzyme immunohistochemistry to investigate blood vessel density and endothelial cell proliferation in MS samples (n=39) compared with control tissue to explore evidence of angiogenesis in MS. The results showed that in all MS samples examined blood vessel density increased compared with controls. The greatest increase was found in subacute lesions where numbers of positively stained vessels increased from 43.9+/-8.5% in controls to 84.2+/-13.3% (P=0.001). Furthermore, using an antibody against endoglin (CD105), a specific marker of proliferating endothelial cells, which are characteristic of angiogenesis, we have shown that vessels containing proliferating endothelial cells were more pronounced in all MS tissue examined (normal-appearing white matter, acute, subacute and chronic lesions, P>or=0.027) compared with control and this was greatest in the MS normal-appearing white matter (68.8+/-19.8% versus 10.58+/-6.4%, P=0.003). These findings suggest that angiogenesis may play a role in lesion progression, failure of repair and scar formation.

Peroxiredoxin V in multiple sclerosis lesions: predominant expression by astrocytes

Oxidative stress is implicated in the pathogenesis of multiple sclerosis (MS). Defence against oxidative damage is mediated by antioxidants. Peroxiredoxin V (PRDX V) is an intracellular anti-oxidant enzyme with peroxynitrite reductase activity. It is increased during inflammation, when free radical production intensifies, and is protective in an animal model of brain injury. However, little is known about PRDX V expression in the human brain. We investigated PRDX V expression in white matter from normal human brain (n = 5) and MS patients (n = 18), using immunohistochemistry and immunoblotting. A global increase in PRDX V was evident in MS normal-appearing white matter (NAWM) but the most striking increase was in astrocytes in MS lesions. PRDX V- positive hypertrophic reactive astrocytes were seen in acute lesions where inflammation was present. Yet surprisingly, in chronic lesions (CL), where inflammation has abated and a glial scar formed, there was strong PRDX V staining of post-reactive, scar astrocytes. Furthermore, immunoblotting analysis of tissue from two MS cases confirmed a substantial increase in PRDX V expression in CL compared with NAWM from the same individual. This might indicate ongoing oxidative stress despite the absence of histologically defined inflammation. Further investigations of this phenomenon will be of interest for therapeutic targeting. Multiple Sclerosis 2007; 13: 955—961. http://msj.sagepub.com

Tenascin C induces a quiescent phenotype in cultured adult human astrocytes

Astrocytic scar formation occurs subsequent to brain and spinal cord injury and impedes repair. The exact mechanisms of scar formation have yet to be elucidated but it is known that astrocytes within the scar have a different antigenic phenotype from normal or reactive astrocytes. Astrocyte cell culture offers a suitable system to identify factors that induce the scar phenotype as well as factors that reverse this process and that may help identify therapeutic strategies to treat astrogliosis. However, when placed in standard culture conditions, astrocytes become activated/reactive and express molecules characteristic of scar tissue in vivo. In the present study, we made use of this phenomenon to identify culture conditions that change the activated phenotype of cultured astrocytes into one characteristic of normal quiescent astrocytes. In particular, we examined the effect of extracellular matrix (ECM) proteins found in the human brain, on the phenotype of human adult astrocytes. Significantly fewer astrocytes expressed scar properties when grown on tenascin‐C (TN‐C) than those cultured on other ECM proteins or poly‐L‐lysine‐coated dishes. TN‐C also significantly reduced the proliferation rate of the astrocytes in vitro. In addition, further manipulation of culture conditions induced partial astrocyte reactivation. Our findings suggest that astrocytes grown on TN‐C revert to a quiescent, nonactivated state that is partially reversible. This raises the possibility that therapeutic strategies aimed at manipulating TN‐C levels during CNS injury may help reduce astrocytic scarring.

CD20+inflammatory T-cells are present in blood and brain of multiple sclerosis patients and can be selectively targeted for apoptotic elimination

BACKGROUND A subset of T-cells expresses the B-cell marker CD20 and in rheumatoid arthritis secretes Interleukin (IL)-17. IL-17 secreting T-cells (Th17) have also been implicated in the inflammatory response in the central nervous system in multiple sclerosis (MS) and may be a potential target for elimination by biologic therapeutics. ScFvRit:sFasL comprises of a rituximab-derived antibody fragment scFvRit genetically fused to human soluble FasL that specifically eliminated T-cells. OBJECTIVE To determine the presence and phenotype of CD20+T-cells in blood and brain of MS patients. Second, to determine whether scFvRit:sFasL can selectively eliminate CD20+T-cells. After CD20-selective binding, scFvRit:sFasL is designed to trigger FasL-mediated activation-induced cell death of T-cells, but not B-cells. METHODS Flow cytometry and immunohistochemistry were used to screen for CD20+inflammatory T-cells in MS blood and brain tissue. ScFvRit:sFasL pro-apoptotic activity was evaluated by Annexin-V/PI staining followed by flow cytometry assessment. RESULTS Peripheral blood (n=11) and chronic but not active lesions of MS patient brains (n=5) contained CD20+inflammatory T-cells. Activated CD20+T-cells were predominantly CD4+and secreted both IL-17 and INF-γ. ScFvRit:sFasL triggered CD20-restricted FasL-mediated activation-induced cell death in peripheral blood CD20+T-cells, but not CD20+B-cells. CONCLUSION CD20+inflammatory T-cells are present in blood and chronic brain lesions of MS patients. ScFvRit:sFasL selectively eliminated CD20+T-cells and may eliminate pathogenic T-cells without B-cell depletion.

Targeting oxidative stress-related diseases: organochalcogen catalysts as redox sensitizers.

Tumor cells proliferate under conditions of oxidative stress. A novel therapeutic approach would be to enhance the cellular effects of the reactive oxygen species formed under these conditions by supplementation with a redox catalyst. This provides a means to target and specifically destroy cancer cells via oxidation of redox-sensitive proteins, such as transcription factors, while leaving cells with a normal redox balance largely unaffected. We have previously reported a preliminary observation on the effects of pro-oxidant catalysts that enhance cancer cell death. This paper presents a detailed in vitro investigation into the mechanism of action of synthetic glutathione peroxidase mimics on a model Sp1 transcription factor peptide. The structure and redox potential of these mimics correlate with their ability to catalyze the oxidation of this zinc-binding motif by H(2)O(2) and these compounds promise therapeutic potential by promoting H(2)O(2)-induced PC12 cell death.

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis

BackgroundEndoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction.MethodsWe assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression.ResultsWe found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines.ConclusionsER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

Calnexin is necessary for T cell transmigration into the central nervous system

In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS.

Manipulation of oxygen and endoplasmic reticulum stress factors as possible interventions for treatment of multiple sclerosis: evidence for and against.

Multiple sclerosis (MS) is normally considered a chronic inflammatory disease of the central nervous system (CNS), where T-cells breaching the blood brain barrier react against proteins of the axonal myelin sheaths, leading to focal plaques and demyelination in the brain and spinal cord. Many current therapies are immunosuppressive in nature and are designed to target the immune system at an early stage of the disease. But there is no cure and MS may evolve into a neurodegenerative disease, where immunomodulatory treatments appear less effective. Neurodegeneration is influenced by oxidative and endoplasmic reticulum (ER) mediated stress which can be induced independently of immune processes. Since 1970, MS patients have been self-managing their long term symptoms using hyperbaric oxygen and reporting improvement in their symptoms, especially bladder control. In contrast, the majority of clinical trial evidence does not support the views of patients. Therefore does oxygen under pressure affect brain tissue by modulating oxidative or ER stress at the cellular level resulting in CNS tissue repair or deterioration? This chapter reviews our understanding and the role of oxidative and ER stress in the context of employing hyperoxia treatments to treat MS and evaluate its effects on neural cells.