Markus Reindl

Glial cell death induced by overexpression of α-synuclein

α‐Synuclein is present in intracellular protein aggregates that are hallmarks of common neurodegenerative disorders including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. α‐Synuclein is localized in neurons and presynaptic terminals. Under pathological conditions, however, it is also found in glia. The role of α‐synuclein in glial cells and its relevance to the molecular pathology of neurodegenerative diseases is presently unclear. To investigate the consequence of α‐synuclein overexpression in glia, we transfected U373 astrocytoma cells with vectors encoding wild‐type human α‐synuclein or C‐terminally truncated synuclein fused to red fluorescent protein. α‐synuclein immunocytochemistry of transfected astroglial cells revealed diffuse cytoplasmic labeling associated with discrete inclusions both within cell bodies and processes. Susceptibility to oxidative stress was increased in astroglial cells overexpressing α‐synuclein, particularly in the presence of cytoplasmic inclusions. Furthermore, overexpression of α‐synuclein induced apoptotic death of astroglial cells as shown by TUNEL staining. Our in vitro model is the first to replicate salient features of the glial pathology associated with α‐synucleinopathies. It provides a simple testbed to further explore the cascade of events that leads to apoptotic glial cell death in some of these disorders; it may also be useful to assess the effects of therapeutic interventions including antioxidative and antiapoptotic strategies. J. Neurosci. Res. 65:432–438, 2001.

Serum and cerebrospinal fluid antibodies to Nogo-A in patients with multiple sclerosis and acute neurological disorders

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

Increased intrathecal production of apolipoprotein D in multiple sclerosis.

Apolipoprotein D (apoD) is a small glycoprotein responsible for the local transport of small hydrophobic ligands. Within the nervous system, apoD may be an acute phase protein that is upregulated in a variety of neuropathological conditions and is involved in the removal of lipids during nerve cell degeneration and provision of lipids during the regenerative phase. In this study, we measured cerebrospinal fluid (CSF) and serum apoD levels in patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré Syndrome (GBS), infectious inflammatory neurological diseases (IND) and non-inflammatory neurological diseases (NND). We found that mean CSF apoD levels are significantly increased in patients with CIDP/GBS reflecting an acute blood-nerve barrier leakage. In contrast, MS is characterized by an increased intrathecal apoD release as measured by the apoD index. Thus, the results of our study provide the first evidence of an increased intrathecal production of apoD in MS. Moreover, we demonstrate that mean apoD indices are highest in MS patients at the time of their first clinical exacerbation. CSF apoD levels and apoD indices correlate with MS disease duration but not with disability or age. Finally, we found that corticosteroid treatment resulted in significantly elevated CSF apoD levels.

Tumor necrosis factor-α-induced cell death in U373 cells overexpressing α-synuclein

Intracellular α‐synuclein inclusion formation in glial cells is frequently seen in Parkinsons disease and multiple system atrophy. Microglial activation in these neurodegenerative disorders suggests that neuroinflammatory responses might interact with α‐synuclein and contribute to the pathogenesis of these disorders. To study the role of tumor necrosis factor‐α (TNF‐α), an important proinflammatory cytokine produced by microglia, on cells overexpressing α‐synuclein we have used the astrocytoma cell line U373 engineered to express C‐terminally truncated α‐synuclein as a fusion protein with red or green fluorescent proteins. We demonstrate that α‐synuclein overexpression augmented TNF‐α‐induced apoptotic cell death in U373 cells by induction of caspase activation. Furthermore, TNF‐α exposure was associated with significant cytoskeletal changes characterized by altered inclusion composition with loss of cytoskeletal proteins and elevation of high‐molecular‐weight α‐synuclein species. We conclude that α‐synuclein overexpression significantly increases the vulnerability of U373 cells to apoptosis through TNF‐α‐mediated pathways.

A critical comparison of frequently used methods for the analysis of tumor necrosis factor-α expression by human immune cells

A variety of methods have been developed for the measurement of tumor necrosis factor (TNF)-alpha synthesis by immune cells. Here we have compared the results of the most common used methods, including in vitro stimulation of whole blood or peripheral blood mononuclear cell (PBMC) cultures with phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) and RT-PCR analysis of TNF-alpha transcription in unstimulated PBMC. When we used EDTA treated blood samples we observed a significant correlation between the PHA and LPS stimulated TNF-alpha responses in whole blood or PBMC cultures. In contrast, TNF-alpha concentrations obtained from PHA and LPS stimulated whole blood cultures from citrate-treated blood did not show a correlation. We also found that the PHA stimulated TNF-alpha response was significantly higher in PBMC than in whole blood cultures, whereas the highest LPS stimulated TNF-alpha response was observed in citrate-treated blood. Moreover, the TNF-alpha response in both, citrate and EDTA treated whole blood cultures was significantly higher after LPS than after PHA stimulation. In contrast, in PBMC cultures the PHA stimulated TNF-alpha response was significantly higher than the LPS stimulated response. The results of RT-PCR analysis revealed a significant correlation with the PHA stimulated TNF-alpha response, both in whole blood assays and in PBMC cultures. In addition our results demonstrate that these different methods can only be compared when the influence of external factors such as the immediate processing of blood samples or the use of an appropriate anticoagulant and stimulant is considered.

Glial cell line-derived neurotrophic factor enhances survival of GM-CSF dependent rat GMIR1-microglial cells.

Microglial activation and proliferation occur in nearly all forms of brain injury. The aim of this study was to investigate the influence of glial cell-line derived neurotrophic factor (GDNF) on proliferation and/or survival in a GMIR1 rat microglial cell line, which proliferates in response to granulocyte-macrophage-colony stimulating factor (GM-CSF). Endogenous GDNF and its receptor, GFRalpha-1, were detected in GMIR1 cells by ELISA and immunohistochemistry/Western blot, respectively. Recombinant GDNF strongly enhanced GMIR1 cell numbers and BrdU-incorporation, an effect inhibited by GDNF blocking antibodies. Inhibition of cAMP/cGMP dependent protein kinase enhanced the GDNF-induced GMIR1 cell number. The results suggest that GDNF has synergistic survival promoting effects on microglia potentially via autocrine mechanisms.

Antibody response to myelin oligodendrocyte glycoprotein and myelin basic protein depend on familial background and are partially associated with human leukocyte antigen alleles in multiplex families and sporadic multiple sclerosis

We investigated the association of the antibody response to myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) with human leukocyte antigen (HLA) class II alleles in 41 patients with sporadic multiple sclerosis (MS) and 12 multiplex MS families. We found significantly increased antibody response to MOG and MBP in MS patients without any difference to asymptomatic relatives. HLA DRB1*04 was associated with IgM reactivity to MOG in MS patients, and DRB1*15 and DRB5 with anti-MOG IgA among asymptomatic relatives. We conclude that antibody responses to MOG and MBP depend on familial background. Moreover, the humoral immune reactivity against MOG is partially under control of certain HLA class II alleles.

GDNF and TGF-β1 promote cell survival in serum-free cultures of primary rat microglia

Recent evidence indicates that glial cell line-derived neurotrophic factor (GDNF) may influence microglial survival, proliferation, and activation, but this has not yet been tested on isolated primary microglia. We compared the effects of individual and combined application of 10xa0ng/ml GDNF and 1xa0ng/ml transforming growth factor-β1 (TGF-β1) on total cell number, 5-bromo-2-deoxyuridine (BrdU) incorporation, DNA nick-end labelling (TUNEL staining), and nitrite and lactate dehydrogenase (LDH) secretion in serum-free cultures of primary rat microglia. GDNF as well as TGF-β1 enhanced the total number of lectin-positive cells and decreased the number of TUNEL-positive nuclei, while no effect on proliferation was observed. Both factors suppressed the secretion of nitrite during the first 4xa0days of culturing, and GDNF but not TGF-β1 reduced the secretion of LDH in 2-week-old cultures. These findings suggest that GDNF and TGF-β1 support survival of primary microglia in vitro.

Anti-MOG antibody responses in multiple sclerosis(1): Reliable testsystems provide reliable results

4 3 1 Phenotypic and Functional Properties of Transformed ~/~ T Ceil Lines and Clones from MS CSF and Blood R.A. Port. M.S. Freedman, Ottawa GeneraIHospital, Canada 4 3 4 Anti-MeG Antibody Responses in Multiple Sclerosis(I): Refiable Testsystems Provide Refiable Results M. geindl, R. Egg, E Deisenhamm er, University oflnnsbruek, Austria, C. Linington, Max-Planck-lnstitut For Psyehiatry, Martinsried, Germany, T. Berger, University of Ing~bruck, Austria