Cerebral cortex (New York, N.Y. : 1991), Jan 16, 2018
Reduction in parvalbumin-positive (PV+) interneurons is observed in adult mice exposed to ethanol... more Reduction in parvalbumin-positive (PV+) interneurons is observed in adult mice exposed to ethanol at postnatal day 7 (P7), a late gestation fetal alcohol spectrum disorder model. To evaluate whether PV+ cells are lost, or PV expression is reduced, we quantified PV+ and associated perineuronal net (PNN)+ cell densities in barrel cortex. While PNN+ cell density was not reduced by P7 ethanol, PV cell density decreased by 25% at P90 with no decrease at P14. PNN+ cells in controls were virtually all PV+, whereas more than 20% lacked PV in ethanol-treated adult animals. P7 ethanol caused immediate apoptosis in 10% of GFP+ cells in G42 mice, which express GFP in a subset of PV+ cells, and GFP+ cell density decreased by 60% at P90 without reduction at P14. The ethanol effect on PV+ cell density was attenuated by lithium treatment at P7 or at P14-28. Thus, reduced PV+ cell density may be caused by disrupted cell maturation, in addition to acute apoptosis. This effect may be regionally specif...
Newly identified mitovesicles are a type of extracellular vesicles whose levels and cargo are alt... more Newly identified mitovesicles are a type of extracellular vesicles whose levels and cargo are altered in Down syndrome.
Ethanol induces neurodegeneration in the developing brain, which may partially explain the long-l... more Ethanol induces neurodegeneration in the developing brain, which may partially explain the long-lasting adverse effects of prenatal ethanol exposure in fetal alcohol spectrum disorders (FASD). While animal models of FASD show that ethanol-induced neurodegeneration is associated with glial activation, the relationship between glial activation and neurodegeneration has not been clarified. This review focuses on the roles of activated microglia and astrocytes in neurodegeneration triggered by ethanol in rodents during the early postnatal period (equivalent to the third trimester of human pregnancy). Previous literature indicates that acute binge-like ethanol exposure in postnatal day 7 (P7) mice induces apoptotic neurodegeneration, transient activation of microglia resulting in phagocytosis of degenerating neurons, and a prolonged increase in glial fibrillary acidic protein-positive astrocytes. In our present study, systemic administration of a moderate dose of lipopolysaccharides, which causes glial activation, attenuates ethanol-induced neurodegeneration. These studies suggest that activation of microglia and astrocytes by acute ethanol in the neonatal brain may provide neuroprotection. However, repeated or chronic ethanol can induce significant proinflammatory glial reaction and neurotoxicity. Further studies are necessary to elucidate whether acute or sustained glial activation caused by ethanol exposure in the developing brain can affect long-lasting cellular and behavioral abnormalities observed in the adult brain.
In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with ... more In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with changes in exosomes. We examined this linkage in the brain of mice that received cocaine injections for two weeks starting at 2.5 months of age. Cocaine caused a decrease in the number of both neuronal early and late endosomes and exosomes in the brains of male but not female mice. The response to cocaine in ovariectomized females mirrored male, demonstrating that these sex-differences in response to cocaine are driven by hormonal differences. Moreover, cocaine increased the amount of α-synuclein per exosome in the brain of females but did not affect exosomal α-synuclein content in the brain of males, a sex-difference eliminated by ovariectomy. Enhanced packaging of α-synuclein into female brain exosomes with the potential for propagation of pathology throughout the brain suggests a mechanism for the different response of females to chronic cocaine exposure as compared to males.
Hundreds of billions of commensal microorganisms live in and on our bodies, most of which coloniz... more Hundreds of billions of commensal microorganisms live in and on our bodies, most of which colonize the gut shortly after birth and stay there for the rest of our lives. In animal models, bidirectional communications between the central nervous system and gut microbiota (Gut–Brain Axis) have been extensively studied, and it is clear that changes in microbiota composition play a vital role in the pathogenesis of various neurodevelopmental and neurodegenerative disorders, such as Autism Spectrum Disorder, Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, anxiety, stress, and so on. The makeup of the microbiome is impacted by a variety of factors, such as genetics, health status, method of delivery, environment, nutrition, and exercise, and the present understanding of the role of gut microbiota and its metabolites in the preservation of brain functioning and the development of the aforementioned neurological illnesses is summarized in this rev...
A standard goal of medicinal chemists has been to discover efficient and potent drug candidates w... more A standard goal of medicinal chemists has been to discover efficient and potent drug candidates with specific enzyme-inhibitor abilities. In this regard, boron-based bioactive compounds have provided amphiphilic properties to facilitate interaction with protein targets. Indeed, the spectrum of boron-based entities as drug candidates against many diseases has grown tremendously since the first clinically tested boron-based drug, Velcade. In this review, we collectively represent the current boron-containing drug candidates, boron-containing retinoids, benzoxaboroles, aminoboronic acid, carboranes, and BODIPY, for the treatment of different human diseases.In addition, we also describe the synthesis, key structure–activity relationship, and associated biological activities, such as antimicrobial, antituberculosis, antitumor, antiparasitic, antiprotozoal, anti-inflammatory, antifolate, antidepressant, antiallergic, anesthetic, and anti-Alzheimer’s agents, as well as proteasome and lipog...
GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration i... more GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate
Results of recent studies have indicated an association between voluntary alcohol intake and acti... more Results of recent studies have indicated an association between voluntary alcohol intake and activities of kappa-opioid receptor systems in animal models. We assessed the possibility that genetic differences observed in alcohol preference among mouse strains are related to possible polymorphisms of the kappa-opioid receptor gene (Oprk1). We compared DNA sequences of the coding region and the promoter/regulatory region of Oprk1 among C57BL/6ByJ (B6, alcohol-preferring), BALB/cJ (alcohol-avoiding), CXBI (alcohol-avoiding), and six B6.C and B6.I Recombinant QTL Introgression (RQI) strains, which carry approximately 3% of the donor BALB/cJ genome in the background B6 genome and showed various alcohol preferences. Although there were no sequence differences in the coding region, BALB/cJ had a single nucleotide polymorphism (SNP) in the promoter region, which was not detected in other strains. The results indicate that the difference in alcohol preference between B6 and BALB/cJ is not cor...
A specific aminopeptidase localized exclusively in neurons of the central nervous system was iden... more A specific aminopeptidase localized exclusively in neurons of the central nervous system was identified with an automated continuous-flow aminopeptidase analyzer developed recently in this laboratory. The enzyme was purified from rat brain 4933-fold to homogeneity with 9.3% recovery by ammonium sulfate fractionation, followed by column chromatography successively on phenyl-Sepharose, Sephadex G-200, and twice on Mono Q FPLC. The purified single-chain enzyme was estimated to be 110 kDa in molecular mass. It has a pI of 5.25 and a pH optimum of 7.0. Only Mg(II) restores the activity of the apoenzyme. The neutral aminopeptidase hydrolyzes -naphthylamides of amino acids with aliphatic, polar uncharged, positively charged, or aromatic side chains. It has a K m of 95 M and a k cat of 7.8 s ؊1 on methionine-enkephalin, releasing only the N-terminal tyrosine. The thiol-dependent metallo-enzyme is most sensitive to amastatin inhibition with a K i of 0.04 M, and is the aminopeptidase most sensitive to puromycin. Its properties are different from those of the ubiquitous puromycin-sensitive aminopeptidase obtained from the same enzyme preparation. The blocked N terminus, substrate and inhibitor specificity, hydrolytic coefficiency, metal effects, pI, molecular weight, and catalytic site show that this enzyme is distinct from all other known aminopeptidases. Its enrichment in the synaptosomes suggests that this first reported neuron-specific peptidase plays a role in neurotransmission and synaptic differentiation.
Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegen... more Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.
Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on cul... more 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 microM 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 microM MK-801 or 100 microM 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 microM gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 microM 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 ...
Ethanol-induced neuronal death during a sensitive period of brain development is considered one o... more Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate. OPEN ACCESS Brain Sci. 2013, 3 671
Fetal Alcohol Spectrum Disorder (FASD) is a general diagnosis for those exhibiting long-lasting n... more Fetal Alcohol Spectrum Disorder (FASD) is a general diagnosis for those exhibiting long-lasting neurobehavioral and cognitive deficiencies as a result of fetal alcohol exposure. It is among the most common causes of mental deficits today. Those impacted are left to rely on advances in our understanding of the nature of early alcohol-induced disorders toward human therapies. Research findings over the last decade have developed a model where ethanol-induced neurodegeneration impacts early neural circuit development, thereby perpetuating subsequent integration and plasticity in vulnerable brain regions. Here we review our current knowledge of FASD neuropathology based on discoveries of long-lasting neurophysiological effects of acute developmental ethanol exposure in animal models. We discuss the important balance between synaptic excitation and inhibition in normal neural network function, and relate the significance of that balance to human FASD as well as related disease states. Finally, we postulate that excitation/inhibition imbalance caused by early ethanol-induced neurodegeneration results in perturbed local and regional network signaling and therefore neurobehavioral pathology.
Fetal alcohol exposure can cause developmental defects in offspring known as fetal alcohol spectr... more Fetal alcohol exposure can cause developmental defects in offspring known as fetal alcohol spectrum disorder (FASD). FASD symptoms range from obvious facial deformities to changes in neuroanatomy and neurophysiology that disrupt normal brain function and behavior. Ethanol exposure at postnatal day 7 in C57BL/6 mice induces neuronal cell death and long-lasting neurobehavioral dysfunction. Previous work has demonstrated that early ethanol exposure impairs spatial memory task performance into adulthood, and perturbs local and interregional brain circuit integrity in the olfacto-hippocampal pathway. Here we pursue these findings to examine whether lithium prevents anatomical, neurophysiological and behavioral pathologies that result from early ethanol exposure. Lithium has neuroprotective properties that have been shown to prevent ethanolinduced apoptosis. Here we show that mice co-treated with lithium on the same day as ethanol exposure, exhibit dramatically reduced acute neurodegeneration in the hippocampus and retain hippocampal-dependent spatial memory as adults. Lithium co-treatment also blocked ethanolinduced disruption in synaptic plasticity in slice recordings of hippocampal CA1 in the adult mouse brain. Moreover, long-lasting dysfunctions caused by ethanol in olfacto-hippocampal networks, including sensory-evoked oscillations and resting state coherence, were prevented in mice co-treated with lithium. Together, these results provide behavioral and physiological evidence that lithium is capable of preventing or reducing immediate and long-term deleterious consequences of early ethanol exposure on brain function.
Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken bra... more Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co2+-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.
We developed monoclonal antibodies (mAbs) against two isozymes of a cytosolic puromycin-sensitive... more We developed monoclonal antibodies (mAbs) against two isozymes of a cytosolic puromycin-sensitive aminopeptidase (PSA-I and PSA-II) purified from chicken brain. The isozymes could be distinguished using Ouchterlony double-immunodiffusion and Western immunoblot. Their distribution in neuronal and glial cells as visualized by indirect immunofluorescence with these mAbs was found to differ: PSA-I was confined mostly to glial lysosomes; PSA-II showed fibrillar distribution in both types of nerve cells, but in disparate patterns. These results and our findings of peptide structural differences suggest that the two PSA isozymes are expressed differently in the nervous system.
Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the thi... more Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces neuronal cell loss. However, the molecular mechanisms underlying these neuronal losses are still poorly understood. Here, we tested the possibility of histone methylation mediated by G9a (lysine dimethyltransferase) in regulating neuronal apoptosis in P7 mice exposed to ethanol. G9a protein expression, which is higher during embryogenesis and synaptogenic period compared to adult brain, is entirely confined to the cell nuclei in the developing brain. We found that ethanol treatment at P7, which induces apoptotic neurodegeneration in neonatal mice, enhanced G9a activity followed by increased histone H3 lysine 9 (H3K9me2) and 27 (H3K27me2) dimethylation. In addition, it appears that increased dimethylation of H3K9 makes it susceptible to proteolytic degradation by caspase-3 in conditions in which ethanol induces neurodegeneration. Further, pharmacological inhibition of G9a activity prior to ethanol treatment at P7 normalized H3K9me2, H3K27me2 and total H3 proteins to basal levels and prevented neurodegeneration in neonatal mice. Together, these data demonstrate that G9a mediated histone H3K9 and K27 dimethylation critically regulates ethanol-induced neurodegeneration in the developing brain. Furthermore, these findings reveal a novel link between G9a and neurodegeneration in the developing brain exposed to postnatal ethanol and may have a role in fetal alcohol spectrum disorders.
Cerebral cortex (New York, N.Y. : 1991), Jan 16, 2018
Reduction in parvalbumin-positive (PV+) interneurons is observed in adult mice exposed to ethanol... more Reduction in parvalbumin-positive (PV+) interneurons is observed in adult mice exposed to ethanol at postnatal day 7 (P7), a late gestation fetal alcohol spectrum disorder model. To evaluate whether PV+ cells are lost, or PV expression is reduced, we quantified PV+ and associated perineuronal net (PNN)+ cell densities in barrel cortex. While PNN+ cell density was not reduced by P7 ethanol, PV cell density decreased by 25% at P90 with no decrease at P14. PNN+ cells in controls were virtually all PV+, whereas more than 20% lacked PV in ethanol-treated adult animals. P7 ethanol caused immediate apoptosis in 10% of GFP+ cells in G42 mice, which express GFP in a subset of PV+ cells, and GFP+ cell density decreased by 60% at P90 without reduction at P14. The ethanol effect on PV+ cell density was attenuated by lithium treatment at P7 or at P14-28. Thus, reduced PV+ cell density may be caused by disrupted cell maturation, in addition to acute apoptosis. This effect may be regionally specif...
Newly identified mitovesicles are a type of extracellular vesicles whose levels and cargo are alt... more Newly identified mitovesicles are a type of extracellular vesicles whose levels and cargo are altered in Down syndrome.
Ethanol induces neurodegeneration in the developing brain, which may partially explain the long-l... more Ethanol induces neurodegeneration in the developing brain, which may partially explain the long-lasting adverse effects of prenatal ethanol exposure in fetal alcohol spectrum disorders (FASD). While animal models of FASD show that ethanol-induced neurodegeneration is associated with glial activation, the relationship between glial activation and neurodegeneration has not been clarified. This review focuses on the roles of activated microglia and astrocytes in neurodegeneration triggered by ethanol in rodents during the early postnatal period (equivalent to the third trimester of human pregnancy). Previous literature indicates that acute binge-like ethanol exposure in postnatal day 7 (P7) mice induces apoptotic neurodegeneration, transient activation of microglia resulting in phagocytosis of degenerating neurons, and a prolonged increase in glial fibrillary acidic protein-positive astrocytes. In our present study, systemic administration of a moderate dose of lipopolysaccharides, which causes glial activation, attenuates ethanol-induced neurodegeneration. These studies suggest that activation of microglia and astrocytes by acute ethanol in the neonatal brain may provide neuroprotection. However, repeated or chronic ethanol can induce significant proinflammatory glial reaction and neurotoxicity. Further studies are necessary to elucidate whether acute or sustained glial activation caused by ethanol exposure in the developing brain can affect long-lasting cellular and behavioral abnormalities observed in the adult brain.
In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with ... more In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with changes in exosomes. We examined this linkage in the brain of mice that received cocaine injections for two weeks starting at 2.5 months of age. Cocaine caused a decrease in the number of both neuronal early and late endosomes and exosomes in the brains of male but not female mice. The response to cocaine in ovariectomized females mirrored male, demonstrating that these sex-differences in response to cocaine are driven by hormonal differences. Moreover, cocaine increased the amount of α-synuclein per exosome in the brain of females but did not affect exosomal α-synuclein content in the brain of males, a sex-difference eliminated by ovariectomy. Enhanced packaging of α-synuclein into female brain exosomes with the potential for propagation of pathology throughout the brain suggests a mechanism for the different response of females to chronic cocaine exposure as compared to males.
Hundreds of billions of commensal microorganisms live in and on our bodies, most of which coloniz... more Hundreds of billions of commensal microorganisms live in and on our bodies, most of which colonize the gut shortly after birth and stay there for the rest of our lives. In animal models, bidirectional communications between the central nervous system and gut microbiota (Gut–Brain Axis) have been extensively studied, and it is clear that changes in microbiota composition play a vital role in the pathogenesis of various neurodevelopmental and neurodegenerative disorders, such as Autism Spectrum Disorder, Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, anxiety, stress, and so on. The makeup of the microbiome is impacted by a variety of factors, such as genetics, health status, method of delivery, environment, nutrition, and exercise, and the present understanding of the role of gut microbiota and its metabolites in the preservation of brain functioning and the development of the aforementioned neurological illnesses is summarized in this rev...
A standard goal of medicinal chemists has been to discover efficient and potent drug candidates w... more A standard goal of medicinal chemists has been to discover efficient and potent drug candidates with specific enzyme-inhibitor abilities. In this regard, boron-based bioactive compounds have provided amphiphilic properties to facilitate interaction with protein targets. Indeed, the spectrum of boron-based entities as drug candidates against many diseases has grown tremendously since the first clinically tested boron-based drug, Velcade. In this review, we collectively represent the current boron-containing drug candidates, boron-containing retinoids, benzoxaboroles, aminoboronic acid, carboranes, and BODIPY, for the treatment of different human diseases.In addition, we also describe the synthesis, key structure–activity relationship, and associated biological activities, such as antimicrobial, antituberculosis, antitumor, antiparasitic, antiprotozoal, anti-inflammatory, antifolate, antidepressant, antiallergic, anesthetic, and anti-Alzheimer’s agents, as well as proteasome and lipog...
GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration i... more GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate
Results of recent studies have indicated an association between voluntary alcohol intake and acti... more Results of recent studies have indicated an association between voluntary alcohol intake and activities of kappa-opioid receptor systems in animal models. We assessed the possibility that genetic differences observed in alcohol preference among mouse strains are related to possible polymorphisms of the kappa-opioid receptor gene (Oprk1). We compared DNA sequences of the coding region and the promoter/regulatory region of Oprk1 among C57BL/6ByJ (B6, alcohol-preferring), BALB/cJ (alcohol-avoiding), CXBI (alcohol-avoiding), and six B6.C and B6.I Recombinant QTL Introgression (RQI) strains, which carry approximately 3% of the donor BALB/cJ genome in the background B6 genome and showed various alcohol preferences. Although there were no sequence differences in the coding region, BALB/cJ had a single nucleotide polymorphism (SNP) in the promoter region, which was not detected in other strains. The results indicate that the difference in alcohol preference between B6 and BALB/cJ is not cor...
A specific aminopeptidase localized exclusively in neurons of the central nervous system was iden... more A specific aminopeptidase localized exclusively in neurons of the central nervous system was identified with an automated continuous-flow aminopeptidase analyzer developed recently in this laboratory. The enzyme was purified from rat brain 4933-fold to homogeneity with 9.3% recovery by ammonium sulfate fractionation, followed by column chromatography successively on phenyl-Sepharose, Sephadex G-200, and twice on Mono Q FPLC. The purified single-chain enzyme was estimated to be 110 kDa in molecular mass. It has a pI of 5.25 and a pH optimum of 7.0. Only Mg(II) restores the activity of the apoenzyme. The neutral aminopeptidase hydrolyzes -naphthylamides of amino acids with aliphatic, polar uncharged, positively charged, or aromatic side chains. It has a K m of 95 M and a k cat of 7.8 s ؊1 on methionine-enkephalin, releasing only the N-terminal tyrosine. The thiol-dependent metallo-enzyme is most sensitive to amastatin inhibition with a K i of 0.04 M, and is the aminopeptidase most sensitive to puromycin. Its properties are different from those of the ubiquitous puromycin-sensitive aminopeptidase obtained from the same enzyme preparation. The blocked N terminus, substrate and inhibitor specificity, hydrolytic coefficiency, metal effects, pI, molecular weight, and catalytic site show that this enzyme is distinct from all other known aminopeptidases. Its enrichment in the synaptosomes suggests that this first reported neuron-specific peptidase plays a role in neurotransmission and synaptic differentiation.
Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegen... more Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.
Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on cul... more 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 microM 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 microM MK-801 or 100 microM 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 microM gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 microM 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 ...
Ethanol-induced neuronal death during a sensitive period of brain development is considered one o... more Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate. OPEN ACCESS Brain Sci. 2013, 3 671
Fetal Alcohol Spectrum Disorder (FASD) is a general diagnosis for those exhibiting long-lasting n... more Fetal Alcohol Spectrum Disorder (FASD) is a general diagnosis for those exhibiting long-lasting neurobehavioral and cognitive deficiencies as a result of fetal alcohol exposure. It is among the most common causes of mental deficits today. Those impacted are left to rely on advances in our understanding of the nature of early alcohol-induced disorders toward human therapies. Research findings over the last decade have developed a model where ethanol-induced neurodegeneration impacts early neural circuit development, thereby perpetuating subsequent integration and plasticity in vulnerable brain regions. Here we review our current knowledge of FASD neuropathology based on discoveries of long-lasting neurophysiological effects of acute developmental ethanol exposure in animal models. We discuss the important balance between synaptic excitation and inhibition in normal neural network function, and relate the significance of that balance to human FASD as well as related disease states. Finally, we postulate that excitation/inhibition imbalance caused by early ethanol-induced neurodegeneration results in perturbed local and regional network signaling and therefore neurobehavioral pathology.
Fetal alcohol exposure can cause developmental defects in offspring known as fetal alcohol spectr... more Fetal alcohol exposure can cause developmental defects in offspring known as fetal alcohol spectrum disorder (FASD). FASD symptoms range from obvious facial deformities to changes in neuroanatomy and neurophysiology that disrupt normal brain function and behavior. Ethanol exposure at postnatal day 7 in C57BL/6 mice induces neuronal cell death and long-lasting neurobehavioral dysfunction. Previous work has demonstrated that early ethanol exposure impairs spatial memory task performance into adulthood, and perturbs local and interregional brain circuit integrity in the olfacto-hippocampal pathway. Here we pursue these findings to examine whether lithium prevents anatomical, neurophysiological and behavioral pathologies that result from early ethanol exposure. Lithium has neuroprotective properties that have been shown to prevent ethanolinduced apoptosis. Here we show that mice co-treated with lithium on the same day as ethanol exposure, exhibit dramatically reduced acute neurodegeneration in the hippocampus and retain hippocampal-dependent spatial memory as adults. Lithium co-treatment also blocked ethanolinduced disruption in synaptic plasticity in slice recordings of hippocampal CA1 in the adult mouse brain. Moreover, long-lasting dysfunctions caused by ethanol in olfacto-hippocampal networks, including sensory-evoked oscillations and resting state coherence, were prevented in mice co-treated with lithium. Together, these results provide behavioral and physiological evidence that lithium is capable of preventing or reducing immediate and long-term deleterious consequences of early ethanol exposure on brain function.
Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken bra... more Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co2+-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.
We developed monoclonal antibodies (mAbs) against two isozymes of a cytosolic puromycin-sensitive... more We developed monoclonal antibodies (mAbs) against two isozymes of a cytosolic puromycin-sensitive aminopeptidase (PSA-I and PSA-II) purified from chicken brain. The isozymes could be distinguished using Ouchterlony double-immunodiffusion and Western immunoblot. Their distribution in neuronal and glial cells as visualized by indirect immunofluorescence with these mAbs was found to differ: PSA-I was confined mostly to glial lysosomes; PSA-II showed fibrillar distribution in both types of nerve cells, but in disparate patterns. These results and our findings of peptide structural differences suggest that the two PSA isozymes are expressed differently in the nervous system.
Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the thi... more Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces neuronal cell loss. However, the molecular mechanisms underlying these neuronal losses are still poorly understood. Here, we tested the possibility of histone methylation mediated by G9a (lysine dimethyltransferase) in regulating neuronal apoptosis in P7 mice exposed to ethanol. G9a protein expression, which is higher during embryogenesis and synaptogenic period compared to adult brain, is entirely confined to the cell nuclei in the developing brain. We found that ethanol treatment at P7, which induces apoptotic neurodegeneration in neonatal mice, enhanced G9a activity followed by increased histone H3 lysine 9 (H3K9me2) and 27 (H3K27me2) dimethylation. In addition, it appears that increased dimethylation of H3K9 makes it susceptible to proteolytic degradation by caspase-3 in conditions in which ethanol induces neurodegeneration. Further, pharmacological inhibition of G9a activity prior to ethanol treatment at P7 normalized H3K9me2, H3K27me2 and total H3 proteins to basal levels and prevented neurodegeneration in neonatal mice. Together, these data demonstrate that G9a mediated histone H3K9 and K27 dimethylation critically regulates ethanol-induced neurodegeneration in the developing brain. Furthermore, these findings reveal a novel link between G9a and neurodegeneration in the developing brain exposed to postnatal ethanol and may have a role in fetal alcohol spectrum disorders.
Uploads
Papers by Mariko Saito