Early treatment with minocycline following stroke in rats improves functional recovery and differentially modifies responses of peri-infarct microglia and astrocytes

BACKGROUND: Altered neuronal connectivity in peri-infarct tissue is an important contributor to both the spontaneous recovery of neurological function that commonly develops after stroke and improvements in recovery that have been induced by experimental treatments in animal models. Microglia and astrocytes are primary determinants of the environment in peri-infarct tissue and hence strongly influence the potential for neuronal plasticity. However, the specific roles of these cells and the timing of critical changes in their function are not well understood. Minocycline can protect against ischemic damage and promote recovery. These effects are usually attributed, at least partially, to the ability of this drug to suppress microglial activation. This study tested the ability of minocycline treatment early after stroke to modify reactive responses in microglia and astrocytes and improve recovery. METHODS: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex of Sprague-Dawley rats. Minocycline was administered for 2 days after stroke induction and the effects on forelimb function assessed up to 28 days. The responses of peri-infarct Iba1-positive cells and astrocytes were evaluated using immunohistochemistry and Western blots. RESULTS: Initial characterization showed that the numbers of Iba1-positive microglia and macrophages decreased in peri-infarct tissue at 24 h then increased markedly over the next few days. Morphological changes characteristic of activation were readily apparent by 3 h and increased by 24 h. Minocycline treatment improved the rate of recovery of motor function as measured by a forelimb placing test but did not alter infarct volume. At 3 days, there were only minor effects on core features of peri-infarct microglial reactivity including the morphological changes and increased density of Iba1-positive cells. The treatment caused a decrease of 57% in the small subpopulation of cells that expressed CD68, a marker of phagocytosis. At 7 days, the expression of glial fibrillary acidic protein and vimentin was markedly increased by minocycline treatment, indicating enhanced reactive astrogliosis. CONCLUSIONS: Early post-stroke treatment with minocycline improved recovery but had little effect on key features of microglial activation. Both the decrease in CD68-positive cells and the increased activation of astrogliosis could influence neuronal plasticity and contribute to the improved recovery.Wai Ping Yew, Natalia D. Djukic, Jaya S. P. Jayaseelan, Frederick R. Walker, Karl A. A. Roos, Timothy K. Chataway, Hakan Muyderman and Neil R. Sim

Alteration in Superoxide Dismutase 1 Causes Oxidative Stress and p38 MAPK Activation Following RVFV Infection

Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNFα following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells

Mitochondrial dysfunction in amyotrophic lateral sclerosis - a valid pharmacological target?

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the selective death of upper and lower motor neurons which ultimately leads to paralysis and ultimately death. Pathological changes in ALS are closely associated with pronounced and progressive changes in mitochondrial morphology, bioenergetics and calcium homeostasis. Converging evidence suggests that impaired mitochondrial function could be pivotal in the rapid neurodegeneration of this condition. In this review, we provide an update of recent advances in understanding mitochondrial biology in the pathogenesis of ALS and highlight the therapeutic value of pharmacologically targeting mitochondrial biology to slow disease progression.H Muyderman and T Che

Differential effects of the cell cycle inhibitor, olomoucine, on functional recovery and on responses of peri-infarct microglia and astrocytes following photothrombotic stroke in rats

Background: Following stroke, changes in neuronal connectivity in tissue surrounding the infarct play an important role in both spontaneous recovery of neurological function and in treatment-induced improvements in function. Microglia and astrocytes influence this process through direct interactions with the neurons and as major determinants of the local tissue environment. Subpopulations of peri-infarct glia proliferate early after stroke providing a possible target to modify recovery. Treatment with cell cycle inhibitors can reduce infarct volume and improve functional recovery. However, it is not known whether these inhibitors can influence neurological function or alter the responses of peri-infarct glia without reducing infarction. The present study aimed to address these issues by testing the effects of the cell cycle inhibitor, olomoucine, on recovery and peri-infarct changes following photothrombotic stroke. Methods: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex in Sprague-Dawley rats. Olomoucine was administered at 1 h and 24 h after stroke induction. Forelimb function was monitored up to 29 days. The effects of olomoucine on glial cell responses in peri-infarct tissue were evaluated using immunohistochemistry and Western blotting. Results: Olomoucine treatment did not significantly affect maximal infarct volume. Recovery of the affected forelimb on a placing test was impaired in olomoucine-treated rats, whereas recovery in a skilled reaching test was substantially improved. Olomoucine treatment produced small changes in aspects of Iba1 immunolabelling and in the number of CD68-positive cells in cerebral cortex but did not selectively modify responses in peri-infarct tissue. The content of the astrocytic protein, vimentin, was reduced by 30% in the region of the lesion in olomoucine-treated ratsWai Ping Yew, Natalia D. Djukic, Jaya S. P. Jayaseelan, Richard J. Woodman, Hakan Muyderman, and Neil R. Sim

The mitochondrial T1095C mutation increases gentamicin-mediated apoptosis

Abstract not availableHakan Muyderman, Neil R. Sims, Masashi Tanaka, Noriyuki Fuku, Ravinarayan Raghupathi, Dominic Thyagaraja

Glutathione monoethyl ester prevents TDP-43 pathology in motor neuronal NSC-34 cells

Abstract not availableTong Chen, Bradley J. Turner, Philip M. Beart, Lucy Sheehan-Hennessy, Chinasom Elekwachi, Hakan Muyderma

Mutant TDP-43 expression triggers TDP-43 pathology and cell autonomous effects on primary astrocytes: implications for non-cell autonomous pathology in ALS

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular dysfunction of astrocytes. Whether non-cell autonomous effects occur in other forms of ALS, such as TAR DNA binding protein 43 (TDP-43)-related disease, remains unclear. Here, we characterised the impact of mutant TDP-43 expression on primary astrocytes derived from transgenic TDP-43(A315T) mice. Mutant TDP-43 astrocytes revealed evidence for TDP-43 pathology, shown by cytoplasmic TDP-43 inclusions and accumulation in insoluble cell fractions which was exacerbated by proteasomal inhibition. l-glutamate uptake, measured using an [3H]D-aspartate assay, was impaired in mutant TDP-43 astrocytes, while ATP accumulation was abnormal, suggesting mutant TDP-43 induced astrocytic dysfunction. Astrocyte activation coupled with spinal and cortical motor neuron loss in transgenic TDP-43(A315T) mice could imply non-cell autonomous effects of astrocytes in vivo. These data demonstrate mutant TDP-43-mediated cell autonomous effects on astrocytes that may contribute to motor neuron pathology in ALS.Samantha K. Barton; Chew L. Lau; Mathew D.F. Chiam; Doris Tomas; Hakan Muyderman; Philip M. Beart; Bradley J. Turne

Selective transfection of microglia in the brain using an antibody-based non-viral vector

There are currently few approaches to transiently manipulate the expression of specific proteins in microglia of the brain. An antibody directed against an extracellular epitope of scavenger receptor class B, type I (SR-BI) was found to be selectively taken up by these cells in the brain. Other antibodies tested were not internalised by microglia. A vector was produced by linking the SR-BI antibody to polyethyleneimine and binding a DNA plasmid encoding green fluorescent protein. Infusions of this vector into the hippocampus produced a widespread transfection of cells, more than 80% of which were immunoreactive for microglial/macrophage markers. Transfection was not detected in cells expressing markers for astrocytes or neurons. Reporter gene expression was most prominent near the infusion site but was seen in tissue up to 4 mm away. DNA bound to polyethyleneimine alone or to a vector containing a different antibody did not produce transfection in the brain. Single injections of the vector containing the SR-BI antibody into the brain also resulted in transfection of microglia, albeit with lower efficiency. Vector modifications to promote lysis of endosomes or entry of DNA into the nucleus did not increase efficiency. The findings clearly demonstrate the capacity of the SR-BI antibody to selectively target brain microglia. This approach offers considerable potential to deliver DNA and other molecules capable of modifying the function of these cells in vivo