Mitsuo Saito

Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimers disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimers disease.

Tau Phosphorylation and Cleavage in Ethanol-Induced Neurodegeneration in the Developing Mouse Brain

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3β (GSK-3β) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3β and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3β inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3β and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.

Gangliosides attenuate ethanol-induced apoptosis in rat cerebellar granule neurons.

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 μM MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 μM MK-801 or 100 μM glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 μM gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 μM LIGA20 (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.

Involvement of ceramide in ethanol-induced apoptotic neurodegeneration in the neonatal mouse brain

J. Neurochem. (2010) 115, 168–177.

Glucosylceramide synthase decrease in frontal cortex of Alzheimer brain correlates with abnormal increase in endogenous ceramides: Consequences to morphology and viability on enzyme suppression in cultured primary neurons

Abnormal increase in native long-chain ceramides (lcCer) in AD implicates roles in neuronal atrophy and cognitive dysfunction especially in view of divergent roles this second messenger plays in cell function. Since clearance is mediated by glucosylceramide synthase (GCS, EC 2.4.1.80) levels of the enzyme were compared for 18 samples of AD Brodmann area 9/10 frontal cortex with 11 age-matched controls. Western analysis for (ir)GCS showed a significant decrease in AD brain (p<0.01) consistent with the hypothesis that enzyme dysfunction contributes to neuronal decay. To examine kinetics and consequences to morphology, cerebellar granule cells were treated in vitro with d-threo-P4 (P4). This potent inhibitor of GCS induced a time- and concentration-dependent increase in lcCer parallel to loss of viability and dramatic changes in neuron/neurite morphology via caspase-independent pathways distinct from those of apoptosis or necrosis. Fluorescent labeling with NBD-sphingolipids or immunostaining with anti-synaptic or cytoskeletal markers showed unusual formation of globular swellings along neurites rich in synaptophysin that may resemble formation of dystrophic neurites in AD. Effects of the inhibitor were verified by changes in lcCer mass and turnover of (14)[C]-acetate and -galactose or NBD-labeled anabolic products. Addition of a panel of inhibitors of other pathways confirms GCS as the major route for clearance in the present model. Pretreatment with GM(1) whose turnover is compromised was protective and pointed to useful therapeutic applications by supplementing existing membrane stores prior to GSC dysfunction.

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3beta (GSK-3beta), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3beta, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3beta, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

Brain damage results in down-regulation of N-acetylaspartate as a neuronal osmolyte

N-acetyl-l-aspartate (NAA) is present in the vertebrate brain, where its concentration is one of the highest of all free amino acids. Although NAA is synthesized and stored primarily in neurons, it is not hydrolyzed in these cells. However, after its regulated release into extracellular fluid, neuronal NAA is hydrolyzed by amidohydrolase II that is present in oligodendrocytes. About 30% of neurons do not contain appreciable amounts of NAA, but its prominence in 1H nuclear magnetic resonance spectroscopic (MRS) studies has led to its wide use as a neuronal marker in diagnostic human medicine as both an indicator of brain pathology, and of disease progression in a variety of central nervous system (CNS) diseases. Loss of NAA has been interpreted as indicating either loss of neurons, or loss of neuron viability. In this investigation, the upregulation of NAA in early stages of construction of the CNS, and its downregulation in experimentally induced damage models of the CNS is reported. The results of this study indicate that the buildup of NAA is not required for viability of neurons in monocellular cultures, and that NAA is lost from multicellular cultured brain slice explants that contain viable neurons. Thus, loss of NAA does not necessarily indicate either loss of neurons or their function. The NAA system, when present in the brain, appears to reflect a high degree of cellular integration, and therefore may be a unique metabolic construct of the intact vertebrate brain.

Ethanol alters lipid profiles and phosphorylation status of AMP-activated protein kinase in the neonatal mouse brain.

Previously, we have shown that ethanol‐induced apoptosis in cultured neurons is accompanied by changes in cellular lipid profiles. In the present study, the effects of ethanol on brain lipid metabolism were studied using 7‐day‐old C57BL/6ByJ mice, which display apoptotic neurodegeneration upon exposure to ethanol. The brain lipids were extracted 4–24 h after the ethanol or saline treatment, and analyzed by TLC. We found that the levels of triglyceride, cholesterol ester, ceramide, and N‐acylphosphatidylethanolamine increased significantly in the brains of ethanol‐treated mice compared to those of saline‐treated mice. Concomitantly, ethanol reduced Thr172 phosphorylation of AMP‐activated protein kinase (AMPK) α subunits. Ethanol also reduced phosphorylation of acetyl‐CoA carboxylase, a substrate of AMPK and a lipogenic enzyme known to be activated by dephosphorylation. In contrast, lipid profiles of 19‐day‐old mouse brains, which scarcely manifested neurodegeneration upon ethanol exposure, were not significantly affected by ethanol. Also, the basal levels of Thr172‐phosphorylated AMPK α were lower in these brains than in 7‐day‐old mouse brains, and no detectable changes in the phosphorylation status were observed by ethanol treatment. Our findings indicate that the ethanol‐induced apoptotic neurodegeneration observed in mice during restricted developmental periods is accompanied by alterations in both the lipid content and the activity of AMPK in the brain.

Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain

J. Neurochem. (2012) 121, 649–661.

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

J. Neurochem. (2012) 121, 806–817.