Early loss of cerebellar Purkinje cells in human and a transgenic mouse model of Alzheimer's disease.

BACKGROUND: The cerebellum's involvement in AD has been under-appreciated by historically labeling as a normal control in AD research. METHODS: We determined the involvement of the cerebellum in AD progression. Postmortem human and APPswe/PSEN1dE9 mice cerebellums were used to assess the cerebellar Purkinje cells (PC) by immunohistochemistry. The locomotor and spatial cognitive functions were assessed in 4- to 5-month-old APPswe/PSEN1dE9 mice. Aß plaque and APP processing were determined in APPswe/PSEN1dE9 mice at different age groups by immunohistochemistry and Western blot. RESULTS: We observed loss of cerebellar PC in mild cognitive impairment and AD patients compared with cognitively normal controls. A strong trend towards PC loss was found in AD mice as early as 5 months. Impairment of balance beam and rotorod performance, but no spatial learning and memory dysfunction was observed in AD mice at 4-5 months. Aß plaque in the cerebral cortex was evidenced in AD mice at 2 months and dramatically increased at 6 months. Less and smaller Aß plaques were observed in the cerebellum than in the cerebrum of AD mice. Similar intracellular APP staining was observed in the cerebellum and cerebrum of AD mice at 2 to 10 months. Similar expression of full-length APP and C-terminal fragments were indicated in the cerebrum and cerebellum of AD mice during aging. DISCUSSION: Our study in post-mortem human brains and transgenic AD mice provided neuropathological and functional evidence that cerebellar dysfunction may occur at the early stage of AD and likely independent of Aß plaque.

Modulation of astrocyte phenotype in response to T-cell interaction.

We determined that T-cell astrocyte interaction modulates interleukin-10 (IL-10) production from both cell types. The impact of IL-10 on astrocytes was compared to IL-10 generated from T-cell-astrocyte interactions in vitro. We demonstrated that T-cells directly interact with astrocytes to upregulate gene expression and secretion of IL-10, confirmed by elevated STAT3p/STAT3 expression in astrocytes. IL-10 increased astrocytes proliferation. In addition, IL-10 treatment and CD4+ co-culture shifts primary astrocytes toward a more energetic phenotype. These findings indicate that direct interaction of CD4+ T-cells with astrocytes, activated the IL-10 anti-inflammatory pathway, altering astrocyte phenotype, metabolism, and proliferation.

Endothelin-1 Mediated Decrease in Mitochondrial Gene Expression and Bioenergetics Contribute to Neurodegeneration of Retinal Ganglion Cells.

Endothelin-1 (ET-1) is a vasoactive peptide that is elevated in aqueous humor as well as circulation of primary open angle glaucoma (POAG) patients. ET-1 has been shown to promote degeneration of optic nerve axons and apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms are still largely unknown. In this study, RNA-seq analysis was used to assess changes in ET-1 mediated gene expression in primary RGCs, which revealed that 23 out of 156 differentially expressed genes (DEGs) had known or predicted mitochondrial function, of which oxidative phosphorylation emerged as the top-most enriched pathway. ET-1 treatment significantly decreased protein expression of key mitochondrial genes including cytochrome C oxidase copper chaperone (COX17) and ATP Synthase, H+ transporting, Mitochondrial Fo Complex (ATP5H) in primary RGCs and in vivo following intravitreal ET-1 injection in rats. A Seahorse ATP rate assay revealed a significant decrease in the rate of mitochondrial ATP production following ET-1 treatment. IOP elevation in Brown Norway rats showed a trend towards decreased expression of ATP5H. Our results demonstrate that ET-1 produced a decrease in expression of vital components of mitochondrial electron transport chain, which compromise bioenergetics and suggest a mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.

Cholesterol sulfate alters astrocyte metabolism and provides protection against oxidative stress.

Cholesterol sulfate (CS) is one of the most important known sterol sulfates in human plasma and it is present as a normal constituent in a variety of human tissues. In both the brain and periphery, CS serves as a substrate for the synthesis of sulfonated adrenal steroids such as pregnenolone sulfate and dehydroepiandrosterone (DHEA) sulfate and as a constituent of many biological membranes including red blood cells where it functions as a stabilizing agent. It also acts as an endogenous regulator of cholesterol synthesis. However, the role of CS in brain metabolism and neurological disorder is unclear. In the current study we investigated the neuroprotective action of CS as well as its role in brain energy metabolism. The neuroprotective effect of CS and its role on cell metabolism were determined in primary astrocyte prepared from the cortex of postnatal day 0-2 C57BL/6 pups and a hippocampal HT-22 cell line using Calcein AM and MTT cell viability assay, flow cytometry, Seahorse extracellular flux analysis, and metabolism assay kits. We found that CS attenuates glutamate and rotenone induced cell death in HT-22 cells, decrease glutamate induced mitochondria membrane potential collapse, and reactive oxygen species production. Additionally, CS activates the Akt/Bcl2 pathway. We observed that CS impacts astrocyte metabolism by increasing mitochondrial phosphorylation, ATP, and glycogen contents. Our study demonstrated that CS modulates brain energy metabolism and its neuroprotective effects might be due to the activation of Akt signaling or its ability to decrease reactive oxygen species production.

A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype.

BACKGROUND: Primary astrocyte cultures have been used for decades to study astrocyte functions in health and disease. The current primary astrocyte cultures are mostly maintained in serum-containing medium which produces astrocytes with a reactive phenotype as compared to in vivo quiescent astrocytes. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state. NEW METHOD: Serum free astrocyte base medium (ABM) supplemented with basic fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (ABM-FGF2-EGF) or serum supplemented DMEM (MD-10%FBS) was used to culture primary astrocytes isolated from cerebral cortex of postnatal day 1 C57BL/6 mice. RESULTS: Compared to astrocytes cultured in MD-10%FBS medium, astrocytes in ABM-FGF2-EGF had higher process bearing morphologies similar to in vivo astrocytes. Western blot, immunostaining, quantitative polymerase chain reaction and metabolic assays revealed that astrocytes maintained in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP expression, increased glutamine synthase, and glutamate transporter-1 mRNA levels as compared to astrocytes cultured in MD-10% FBS medium. COMPARISON TO EXISTING METHODS: These observations suggest that astrocytes cultured in ABM-FGF2-EGF media compared to the usual FBS media promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. CONCLUSION: This media provides a novel method for studying astrocytes functions in vitro under physiological and pathological conditions.

Hyperglycemia Alters Astrocyte Metabolism and Inhibits Astrocyte Proliferation.

Diabetes milieu is a complex metabolic disease that has been known to associate with high risk of various neurological disorders. Hyperglycemia in diabetes could dramatically increase neuronal glucose levels which leads to neuronal damage, a phenomenon referred to as glucose neurotoxicity. On the other hand, the impact of hyperglycemia on astrocytes has been less explored. Astrocytes play important roles in brain energy metabolism through neuron-astrocyte coupling. As the component of blood brain barrier, glucose might be primarily transported into astrocytes, hence, impose direct impact on astrocyte metabolism and function. In the present study, we determined the effect of high glucose on the energy metabolism and function of primary astrocytes. Hyperglycemia level glucose (25 mM) induced cell cycle arrest and inhibited proliferation and migration of primary astrocytes. Consistently, high glucose decreased cyclin D1 and D3 expression. High glucose enhanced glycolytic metabolism, increased ATP and glycogen content in primary astrocytes. In addition, high glucose activated AMP-activated protein kinase (AMPK) signaling pathway in astrocytes. In summary, our in vitro study indicated that hyperglycemia might impact astrocyte energy metabolism and function phenotype. Our study provides a potential mechanism which may underlie the diabetic cerebral neuropathy and warrant further in vivo study to determine the effect of hyperglycemia on astrocyte metabolism and function.

Margaritaria discoidea (Euphorbiaceae) stem bark extract attenuates allergy and Freund's adjuvant-induced arthritis in rodents.

BACKGROUND: Various parts of Margaritaria discoidea find use in traditional medicine in the treatment of pain and oedema. This study evaluated the anti-allergic, anti-inflammatory and anti-arthritic effects of a 70% (v/v) aqueous ethanol extract of the stem bark of Margaritaria discoidea, MDE in rodents. MATERIALS AND METHODS: Systemic anaphylaxis was induced by the injection of compound 48/80 into mice and their survival rate was monitored to evaluate the anti-allergic action of the extract. The effect of MDE assessed on the maximal and total oedema responses in the mouse carrageenan-induced paw oedema was used to evaluate the anti-inflammatory action of the extract while the Freund's adjuvant-induced arthritis model was employed to study the anti-arthritic effects of MDE. RESULTS: MDE dose-dependently increased the time for compound 48/80-induced mortality in mice. MDE suppressed the mean maximal swelling and the total paw swellings induced over 6 h in the carrageenan-induced paw oedema when administered either prophylactically or therapeutically. MDE caused a reduction in serum levels of TNFα and IL-6 and significantly suppressed Freund's adjuvant-induced arthritis. CONCLUSION: Margaritaria discoidea suppresses allergy and exhibits anti-inflammatory activity in mice. In addition it attenuates Freund's adjuvant-induced arthritis through a reduction in serum levels of TNFα and IL-6 in rats.