Kelly M Hares

Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro.

Bone marrow-derived mesenchymal stem cells (MSCs) are of therapeutic interest in a variety of neurological diseases. In this study, we wished to determine whether human MSCs secrete factors which protect cultured rodent cortical neurons from death by trophic factor withdrawal or nitric oxide (NO) exposure. Medium conditioned by MSCs attenuated neuronal death under these conditions, a process which was dependent on intact PI(3)kinase/Akt pathway signaling. Trophic withdrawal and NO exposure in cultured cortical neurons led to reduction in Akt signaling pathways, whereas NO administration activated p38 MAPkinase in neuronal cultures. Addition of MSC-conditioned medium significantly activated the PI3kinase/Akt pathway and in neurons exposed to NO, MSC-conditioned medium reduced p38 signaling. We show that MSCs secrete brain-derived neurotrophic factor (BDNF) and addition of anti-BDNF neutralising antibodies to MSC-conditioned medium attenuated its neuroprotective effect. Exposure of neurons to BDNF increased activation of Akt pathways and protected neurons from trophic factor withdrawal. These observations determine the mechanisms of neuroprotection offered by MSC-derived factors and suggest an important role for BDNF in neuronal protection.

Mesenchymal stem cell‐secreted superoxide dismutase promotes cerebellar neuronal survival

J. Neurochem. (2010) 114, 1569–1580.

Purkinje Cell Pathology and Loss in Multiple Sclerosis Cerebellum.

Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition.

Accumulation of cortical hyperphosphorylated neurofilaments as a marker of neurodegeneration in multiple sclerosis

Background: Axonal loss and grey matter neuronal injury are pathological processes that contribute to disease progression in multiple sclerosis (MS). Axon damage has been associated with changes in the phosphorylation state of neurofilaments and the presence of axonal spheroids. Perikaryal accumulation of abnormally phosphorylated neurofilament proteins has been reported in some neurodegenerative diseases. Objectives: The objective of this article is to determine whether abnormally phosphorylated neurofilament accumulates in neuronal perikarya in demyelinated MS cortex. Methods: We used an antibody to hyperphosphorylated neurofilament-H (SMI-34) to assess the level and distribution of this antigen in paraffin sections of cerebral cortex from cases of neuropathologically confirmed MS and controls. We also examined the relationship of neurofilament phosphorylation to cortical demyelination. Results: The number of SMI-34-positive neuronal somata was significantly higher in the MS cortex than the control cortex. As a proportion of the total number of neurons present (i.e. taking account of neuronal loss), the proportion of SMI-34-positive neurons was also significantly higher in the demyelinated and non-demyelinated MS cortex than the control cortex. Conclusions: MS is associated with the widespread accumulation of hyperphosphorylated neurofilament protein in neuronal somata, with the most marked accumulation in regions of cortical demyelination. This aberrant localisation of hyperphosphorylated neurofilament protein may contribute to neuronal dysfunction and degeneration in MS patients.

Mesenchymal Stem Cells Restore Frataxin Expression and Increase Hydrogen Peroxide Scavenging Enzymes in Friedreich Ataxia Fibroblasts

Dramatic advances in recent decades in understanding the genetics of Friedreich ataxia (FRDA)—a GAA triplet expansion causing greatly reduced expression of the mitochondrial protein frataxin—have thus far yielded no therapeutic dividend, since there remain no effective treatments that prevent or even slow the inevitable progressive disability in affected individuals. Clinical interventions that restore frataxin expression are attractive therapeutic approaches, as, in theory, it may be possible to re-establish normal function in frataxin deficient cells if frataxin levels are increased above a specific threshold. With this in mind several drugs and cytokines have been tested for their ability to increase frataxin levels. Cell transplantation strategies may provide an alternative approach to this therapeutic aim, and may also offer more widespread cellular protective roles in FRDA. Here we show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. We demonstrate that normal human mesenchymal stem cells (MSCs) induce both an increase in frataxin gene and protein expression in FRDA fibroblasts via secretion of soluble factors. Finally, we show that exposure to factors produced by human MSCs increases resistance to hydrogen peroxide-mediated toxicity in FRDA fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest, for the first time, that stem cells may increase frataxin levels in FRDA and transplantation of MSCs may offer an effective treatment for these patients.

Reduced axonal motor protein expression in non-lesional grey matter in multiple sclerosis

Background: Multiple sclerosis (MS) is a neurological disease characterised by central nervous system inflammation, demyelination, axonal degeneration and neuronal injury. Preventing neuronal and axon damage is of paramount importance in attempts to prevent disease progression. Intact axonal transport mechanisms are crucial to axonal integrity and evidence suggests these mechanisms are disrupted in MS. Anterograde axonal transport is mediated to a large extent through the kinesin superfamily proteins. Recently, certain kinesin superfamily proteins (KIF5A, KIF1B and KIF21B) were implicated in MS pathology. Objectives: To investigate the expression of KIF5A, KIF21B and KIF1B in MS and control post-mortem grey matter. Methods: Using both quantitative real-time polymerase chain reaction (PCR) and Immunodot-blots assays, we analysed the expression of kinesin superfamily proteins in 27 MS cases and 13 control cases not linked to neurological disease. Results: We have shown significant reductions in KIF5A, KIF21B and KIF1B messenger ribonucleic acid (mRNA) expression and also KIF5A protein expression in MS grey matter, as compared to control grey matter. Conclusion: We have shown significant reductions in mRNA and protein levels of axonal motor proteins in the grey matter of MS cases, which may have important implications for the pathogenesis of neuronal/axonal injury in the disease.

Changes in Expression of the Antioxidant Enzyme SOD3 Occur Upon Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells In Vitro

The discovery that mesenchymal stem cells (MSCs) secrete SOD3 may help explain studies in which MSCs have direct antioxidant activities both in vivo and in vitro. SOD3 is an antioxidant enzyme that dismutes toxic free radicals produced during inflammatory processes. Therefore, MSC production and secretion of active and therapeutically significant levels of SOD3 would further support the use of MSCs as a cellular based antioxidant therapy. The aim of this study was therefore to investigate in vitro if MSC differentiation down the adipogenic, chondrogenic, and osteogenic lineages influences the expression of the antioxidant molecule SOD3. Human bone marrow MSCs and their differentiated progeny were cultured under standard conditions and both the SOD3 gene and protein expression examined. Following adipogenesis, cultures demonstrated that both SOD3 protein and gene expression are significantly increased, and conversely, following chondrogenesis SOD3 protein and gene expression is significantly decreased. Following osteogenesis there were no significant changes in SOD3 protein or gene expression. This in vitro study describes the initial characterization of SOD3 expression and secretion by differentiated MSCs. This should help guide further in vivo work establishing the therapeutic and antioxidative potential of MSC and their differentiated progeny.

Reductions in neuronal peroxisomes in multiple sclerosis grey matter

Background: Peroxisomes are organelles in eukaryotic cells with multiple functions including the detoxification of reactive oxygen species, plasmalogen synthesis and β-oxidation of fatty acids. Recent evidence has implicated peroxisomal dysfunction in models of multiple sclerosis (MS) disease progression. Objectives: Our aims were to determine whether there are changes in peroxisomes in MS grey matter (GM) compared to control GM. Methods: We analysed cases of MS and control GM immunocytochemically to assess peroxisomal membrane protein (PMP70) and neuronal proteins. We examined the expression of ABCD3 (the gene that encodes PMP70) in MS and control GM. Analyses of very long chain fatty acid (VLCFA) levels in GM were performed. Results: PMP70 immunolabelling of neuronal somata was significantly lower in MS GM compared to control. Calibration of ABCD3 gene expression with reference to glyceraldehyde 3-phsophate dehydrogenase (GAPDH) revealed overall decreases in expression in MS compared to controls. Mean PMP70 counts in involved MS GM negatively correlated to disease duration. Elevations in C26:0 (hexacosanoic acid) were found in MS GM. Conclusions: Collectively, these observations provide evidence that there is an overall reduction in peroxisomal gene expression and peroxisomal proteins in GM neurons in MS. Changes in peroxisomal function may contribute to neuronal dysfunction and degeneration in MS.

Axonal motor protein KIF5A and associated cargo deficits in multiple sclerosis lesional and normal-appearing white matter

Understanding the causes of axonal pathology remains a key goal in the pursuit of new therapies to target disease progression in multiple sclerosis (MS). Anterograde axonal transport of many proteins vital for axonal viability is mediated by the motor protein KIF5A, which has been linked to several neurological diseases. This study aimed to investigate the expression of KIF5A protein and its associated cargoes: amyloid precursor protein (APP) and neurofilament (NF) in post mortem MS and control white matter (WM) and to determine if KIF5A expression is influenced by the presence of MS risk single nucleotide polymorphisms (SNPs) identified in the region of the KIF5A gene.

Reductions in kinesin expression are associated with nitric oxide-induced axonal damage

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra‐axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time‐dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO‐exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders.