Ramasamy Thangavel

Phosphorylation of Tau by Fyn: Implications for Alzheimer's Disease

The abnormal phosphorylation of tau protein on serines and threonines is a hallmark characteristic of the neurofibrillary tangles of Alzheimers disease (AD). The discovery that tau could be phosphorylated on tyrosine and evidence that Aβ signal transduction involved tyrosine phosphorylation led us to question whether tyrosine phosphorylation of tau occurred during the neurodegenerative process. In this study we determined that human tau tyr18 was phosphorylated by the src family tyrosine kinase fyn. By developing both polyclonal and monoclonal probes specific for phospho-tyr18, we found that the phosphorylation of tau at tyr18 occurred at early developmental stages in mouse but was absent in the adult. Our phosphospecific probes also revealed that paired helical filament preparations exhibited phospho-tyr18 reactivity that was sensitive to phosphotyrosine-specific protein phosphatase treatment. Moreover, immunocytochemical studies indicated that tyrosine phosphorylated tau was present in the neurofibrillary tangles in AD brain. However, the staining pattern excluded neuropil threads and dystrophic neurites indicating that tyrosine phosphorylated tau was distributed in AD brain in a manner dissimilar from other abnormally phosphorylated tau. We also found evidence suggesting that differentially phosphorylated tau existed within degenerating neurons. Our data add new support for a role for fyn in the neurodegenerative process.

Expression of markers for both neuronal and glial cells in human amniotic epithelial cells

Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. We attempted to detect various developmental antigens specific to neural cells by immunocytochemical methods. The cultured HAE cells displayed positive immunoreactivity to RC1, vimentin, A2B5, neurofilament proteins, microtubule-associated protein 2 (MAP2) and MAP2 kinase. In addition, the cells also demonstrated immunoreactivity to glial fibrillary acidic protein, CNPase, myelin basic protein and galactocerebroside. The appearance rate of positive cells was more than 50% in cells positive to RC1, A2B5, vimentin or neuronal markers, and 20-30% to glial cell markers. Double staining showed the heterogeneous appearance of oligodendrocyte lineage cells. These data indicate that HAE cells may have the putative multipotentiality of neurons, astrocytes and oligodendrocytes.

The parahippocampal gyrus in Alzheimer's disease. Clinical and preclinical neuroanatomical correlates.

Abstract: The human parahippocampal gyrus forms a large part of the limbic lobe along the ventromedial part of the temporal cortical mantle. It is a variable and complicated cortex in terms of structure, and the latter is aggravated further by interfaces with the anterior insula anteriorly and the cingulate gyrus and occipital lobe posteriorly. Additional complications relate to its lateral border with the temporal cortex and especially the sulcal configurations that define this junction. The rhinal sulcus, which separates parahippocampal and temporal cortices in other species, including the anthropoid apes, is either lacking or rudimentary in the human brain. Thus, defining this junction requires cytoarchitectural examination and precludes the use of mere inspection of sulcal existing patterns. The cortical areas that form the parahippocampal gyrus are vulnerable to pathological changes in Alzheimers disease (AD), and its entorhinal and perirhinal subdivisions are both the most heavily damaged cortical areas and the focus for disease onset. The neurons that acquire neurofibrillary tangles (NFTs) occupy the junction of the isocortical mantle with the limbic cortical mantle, but share, or partially share, a vulnerability phenotype with large neurons in both domains. The differential expression of this phenotype across time creates the false impression of NFT spread in cross‐sectional comparisons of AD brains. The questions of what this phenotype is and why it is expressed first in the perirhinal and entorhinal cortices of the parahippocampal gyrus are the central molecular biological/neuroanatomical questions in understanding the etiology of AD.

Human amniotic epithelial cells are promising transgene carriers for allogeneic cell transplantation into liver

AbstractAs human amniotic epithelial tissue is formed on about the eighth day after fertilization, human amniotic epithelial cells (hAEC) may have multipotency to differentiate into various organs, such as brain, heart, or liver. In this study, we showed evidence of the synthesis and excretion of albumin by hAEC, by immunostaining and enzyme-linked immunoassay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses revealed the expression of albumin mRNA and protein, respectively. In addition, hAEC also demonstrated immunoreactivity to genetic markers of liver lineage, such as human serum albumin and α-fetoprotein. Transplanted hAEC to Scid mouse liver showed positive immunoreactivity to albumin and α-fetoprotein. Genetically modified cells containing the β-galactosidase (LacZ) gene (AxCALacZ) were integrated in liver parenchyma. Human polymorphic gene analysis in Scid mouse liver after the implantation of hAEC showed that these Scid mouse livers obviously contained this human-specific gene until day 7 after the cell transplantation. As hAEC do not cause any acute rejection by allotransplantation, we conclude that hAEC may be useful as a transgene carrier to treat patients with inherited liver diseases.

Evidence for active acetylcholine metabolism in human amniotic epithelial cells: applicable to intracerebral allografting for neurologic disease.

Human amniotic epithelial (HAE) cells have been used for allotransplantation in patients with lysosomal storage disease due to lack of expression of HLA antigens. Previously, we have reported the expression of differentiation markers for both neural stem cells, and neuron and glial cells. In the present study, we investigated the presence of choline acetyltransferase (ChAT) and acetylcholine (ACh) in HAE cells using different experimental approaches. Cultured HAE cells showed strong immunoreactivity against ChAT antibody. ChAT activity in primary cells was 24.9 +/- 8.5 pmol/mg protein/h. Using HPLC with electrochemical detection, ACh was detected in both cell incubation media and cell pellets indicating that these cells synthesize and release ACh in a time-dependent manner. Additional confirmation of this hypothesis was gained from the data obtained from RT-PCR and Western blot analyses which revealed the expression of ChAT mRNA and ChAT protein, respectively, in HAE cells. Results of the present study suggest that HAE cells can possibly be applied for intracerebral allografting to treat neurologic diseases in which cholinergic neurons are damaged.

Glia maturation factor modulates β-amyloid-induced glial activation, inflammatory cytokine/chemokine production and neuronal damage

Glia maturation factor (GMF), discovered and characterized in our laboratory, is a highly conserved protein primarily localized in mammalian central nervous system. Previously we demonstrated that GMF is required in the induced production of proinflammatory cytokines and chemokines in brain cells. We now report that ventricular infusion of human amyloid beta peptide1-42 (Abeta1-42) in mouse brain caused glial activation and large increases in the levels of GMF as well as induction of inflammatory cytokine/chemokine known for launching the neuro inflammatory cascade in Alzheimers disease (AD). To test the hypothesis that GMF is involved in the pathogenesis of AD, we infused Abeta1-42 in the brain of GMF-deficient (GMF-KO) mice, recently prepared in our laboratory. GMF-deficient mice showed reduced glial activation and significantly suppressed proinflammatory cytokine/chemokine production following Abeta infusion compared to wild type (Wt) mice. The decrease in glial activation in the GMF-KO mice is also associated with significant reduction in Abeta induced loss of pre-synaptic marker, synaptophysin, and post-synaptic density protein-95 (PSD 95). We also examined the potential relationship between GMF or lack of it with learning and memory using the T-maze, Y-maze, and water maze, hippocampal-dependent spatial memory tasks. Our results show that memory retention was improved in GMF-KO mice compared to Wt controls following Abeta infusion. Diminution of these Abeta1-42 effects in primary cultures of GMF-KO astrocyte and microglia were reversed by reconstituted expression of GMF. Taken together, our results indicate a novel mediatory role of GMF in the neuro-inflammatory pathway of Abeta and its pro-inflammatory functions.

Dendritic arbors of developing retinal ganglion cells are stabilized by β1-integrins

The architecture of dendritic arbors is a defining characteristic of neurons and is established through a sequential but overlapping series of events involving process outgrowth and branching, stabilization of the global pattern, and synapse formation. To investigate the roles of cadherins and beta1-integrins in maintaining the global architecture of the arbor, we used membrane permeable peptides and transfection with dominant-negative constructs to disrupt adhesion molecule function in intact chick neural retina at a stage when the architecture of the ganglion cell (RGC) arbor is established but synapse formation is just beginning. Inactivation of beta1-integrins induces rapid dendrite retraction, with loss of dynamic terminal filopodia followed by resorption of major branches. Disruption of N-cadherin-beta-catenin interactions has no effect; however, dendrites do retract following perturbation of the juxtamembrane region of N-cadherin, which disrupts N-cadherin-mediated adhesion and initiates a beta1-integrin inactivating signal. Thus, developing RGC dendritic arbors are stabilized by beta1-integrin-dependent processes.

Glia Maturation Factor Induces Interleukin-33 Release from Astrocytes: Implications for Neurodegenerative Diseases

Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Multiple sclerosis (MS) involve activation of glial cells and release of inflammatory mediators leading to death of neurons. Glia maturation factor (GMF) is up-regulated in the central nervous system (CNS) in these neurodegenerative diseases. Interleukin-33 (IL-33) is highly expressed constitutively in the CNS. We have treated mouse astrocytes, mixed culture with glial cells and neurons, and only neurons with GMF and/or IL-33 in vitro. Both GMF and IL-33-induced chemokine (C-C motif) ligand 2 (CCL2) release in a dose and time-dependent manner. We report that GMF induced IL-33 release, and that IL-33 augments GMF-induced tumor necrosis factor-alpha (TNF-α) release from mouse astrocytes. IL-33 induces CCL2, TNF-α and nitric oxide release through phosphorylation of ERK in mouse astrocytes. Incubation of mixed culture containing glial cells and neurons or only neuronal culture with IL-33 reduced the number of neurons positive for microtubule-associated protein 2. In conclusion, IL-33 augments GMF-mediated neuroinflammation and may provide a new drug target for neurodegenerative and autoimmune diseases.

Protection of MPTP-induced neuroinflammation and neurodegeneration by Pycnogenol

Oxidative stress and inflammation play a crucial role in Parkinsons disease (PD) pathogenesis and may represent a target for treatment. Current PD drugs provide only symptomatic relief and have limitations in terms of adverse effects and inability to prevent neurodegeneration. Flavonoids have been suggested to exert human health benefits by its anti-oxidant and anti-inflammatory properties. Therefore, in the present study, using 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP)-induced mouse model of Parkinsonism, we investigated the neuroprotective potential of bioflavonoid compound Pycnogenol® (PYC), an extract of Pinus maritime bark. MPTP injected mice developed significantly severe oxidative stress and impaired motor coordination at day 1 and day 7 postinjection. This was associated with significantly increased inflammatory responses of astrocyte and microglia as assessed by ionized calcium binding adaptor molecule 1 (Iba 1) and glial fibrillary acidic protein (GFAP) immunohistochemistry, and nuclear transcription factor-κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the striata by Western blot. Additionally, there was significant upregulation of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) expression in the striata of MPTP injected mice compared to saline controls. The MPTP-induced neuroinflammation, neurodegeneration and behavioral impairments were markedly repudiated by treatment with PYC. These results suggest that PYC protects dopaminergic neurons from MPTP-induced toxicity in the mouse model of PD. Thus, the present finding of PYC-induced adaptation to oxidative stress and inflammation could suggest a novel avenue for clinical intervention in neurodegenerative diseases including PD.

Enhanced Expression of Glia Maturation Factor Correlates with Glial Activation in the Brain of Triple Transgenic Alzheimer’s Disease Mice

We previously demonstrated that glia maturation factor (GMF), a brain specific protein, isolated, sequenced and cloned in our laboratory, induce expression of proinflammatory cytokines and chemokines in the central nervous system. We also reported that the up-regulation of GMF in astrocytes leads to the destruction of neurons suggesting a novel pathway of GMF-mediated cytotoxicity of brain cells, and implicated its involvement in the pathogenesis of inflammatory neurodegenerative diseases. In the present study, we examined the expressions of GMF in triple-transgenic Alzheimer’s disease (3xTg-AD) mice. Our results show a 13-fold up-regulation of GMF and 8–12-fold up-regulation of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1β, interferon gamma (IFN-γ), and chemokine (C–C motif) ligand 2 (CCL2) and C–X–C motif chemokine 10 (CXCL10/IP-10) mRNA as determined by quantitative real-time RT-PCR in the brain of 3xTg-AD mice as compared to non-transgenic (Non-Tg) mice. In conclusion, the increase in GMF and cytokine/chemokine expression was correlated with reactive glial fibrillary acidic protein positive astrocytes and ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglia in 3xTg-AD mice.