Oxidative stress is associated with region-specific neuronal death during thiamine deficiency.

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism and selective neuronal loss. TD leads to region-specific neuronal death and elevation of inducible nitric oxide synthase (iNOS) in macrophages/microglia in mouse brain. Identification of the initial site of neuronal death in the submedial thalamic nucleus allowed us to test the role of iNOS and oxidative stress in TD-induced neuronal death. The pattern of neuronal loss, which begins after 9 days of TD, overlapped with induction of the oxidative stress marker heme oxygenase-1 (HO-1) in microglia. Neuronal death and microglial HO-1 induction spread to engulf the whole thalamus after 11 days of TD. As in past studies, reactive iron and ferritin accumulated in microglia beginning on day 10. The lipid peroxidation product, 4-hydroxynonenal (HNE) accumulated in the remaining thalamic neurons only after 11 days of TD. These responses were not likely mediated by iNOS because HO-1 induction and HNE accumulation were comparable in iNOS knockout mice and wild-type controls. These results show that region and cell specific oxidative stress is associated with selective neurodegeneration during TD. Thus, TD is a useful model to help elucidate neuron-microglial interaction in neurodegenerative diseases associated with oxidative stress.     

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood–brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (−60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (−30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.     

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood-brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (-60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (-30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.     

CD40L deletion delays neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism

Inflammatory/immune processes are important in the pathogenesis of neurodegenerative diseases. Thiamine deficiency (TD) models the region selective neuronal loss in brain that accompanies mild impairment of oxidative metabolism. TD induces well-defined alterations in neurons, microglia, astrocytes, and endothelial cells. To test the role of inflammatory/immune mechanisms in TD-induced neurodegeneration, the temporal profile of neurodegeneration was compared to the activation of CD68-positive microglia and ICAM-1-positive endothelial cells during TD in wild type mice and in CD40L-/- mice. CD40L-/- delayed the onset of TD-induced neuronal death as well as the activation of microglia and endothelial cells. The current results suggest that CD40L-mediated immune and inflammatory responses have a role in TD-induced neuronal death.     

Reversal of thiamine deficiency-induced neurodegeneration

Neurodegenerative diseases are characterized by abnormalities in oxidative processes, region-selective neuron loss, and diminished thiamine-dependent enzymes. Thiamine deficiency (TD) diminishes thiamine dependent enzymes, alters mitochondrial function, impairs oxidative metabolism, and causes selective neuronal death. In mice, the time course of TD-induced changes in neurons and microglia were determined in the brain region most sensitive to TD. Significant neuron loss (29%) occurred after 8 or 9 days of TD (TD8-9) and increased to 90% neuron loss by TD10-11. The number of microglia increased 16% by TD8 and by nearly 400% on TD11. Hemeoxygenase-1 (HO-1)-positive microglia were not detectable at TD8, yet increased dramatically coincident with neuron loss. To test the duration of TD critical for irrevocable changes, mice received thiamine after various durations of TD. Thiamine administration on TD8 blocked further neuronal loss and induction of HO-1-positive microglia, whereas other microglial changes persisted. Thiamine only partially reversed effects on TD9, and was ineffective on TD10-11. These studies indicate that irreversible steps leading to neuronal death and induction of HO-1-positive microglia occur on TD9. The results indicate that TD induces alterations in neurons. endothelial cells, and microglia contemporaneously. This model provides a unique paradigm for elucidating the molecular mechanisms involved in neuronal commitment to neuronal death cascades and contributory microglial activity.     

Changes in inflammatory processes associated with selective vulnerability following mild impairment of oxidative metabolism

Abnormalities in oxidative metabolism and reductions of thiamine-dependent enzymes accompany many age-related neurodegenerative diseases. Thiamine deficiency (TD) produces a cascade of events including mild impairment of oxidative metabolism, activation of microglia, astrocytes and endothelial cells that leads to neuronal loss in select brain regions. The earliest changes occur in a small, well-defined brain region, the submedial thalamic nucleus (SmTN). In the present study, a micropunch technique was used to evaluate quantitatively the selective regional changes in mRNA and protein levels. To test whether this method can distinguish between changes in vulnerable and non-vulnerable regions, markers for neuronal loss (NeuN) and endothelial cells (eNOS) and inflammation (IL-1beta, IL-6 and TNF-alpha) in SmTN and cortex of control and TD mice were assessed. TD significantly reduced NeuN and increased CD11b, GFAP and ICAM-1 immunoreactivity in SmTN as revealed by immunocytochemistry. When assessed on samples obtained by the micropunch method, NeuN protein declined (-49%), while increased mRNA levels were observed for eNOS (3.7-fold), IL-1beta (43-fold), IL-6 (44-fold) and TNF-alpha (64-fold) in SmTN with TD. The only TD-induced change that occurred in cortex with TD was an increase in TNF-alpha (22-fold) mRNA levels. Immunocytochemical analysis revealed that IL-1beta, IL-6 and TNF-alpha protein levels increased in TD brains and colocalized with glial markers. The consistency of these quantitative results with immunocytochemical measurements validates the micropunch technique. The results demonstrate that TD induces quantitative, distinct inflammatory responses and oxidative stress in vulnerable and non-vulnerable regions that may underlie selective vulnerability.     

Heme Oxygenase in the Experimental ALS Mouse

Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4–5 months of age. All mice were killed at 7–8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP⁺cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.     

Distinct roles of oxidative stress and antioxidants in the nucleus dorsalis and red nucleus following spinal cord hemisection

Oxidative stress plays an important role in the pathogenesis of neurodegeneration after the acute central nervous system injury. We reported previously that increased nitric oxide (NO) production following spinal cord hemisection tends to lead to neurodegeneration in neurons of the nucleus dorsalis (ND) that normally lacks expression of neuronal NO synthase (nNOS) in opposition to those in the red nucleus (RN) that constitutively expresses nNOS. We wondered whether oxidative stress could be a mechanism underlying this NO involved neurodegeneration. In the present study, we examined oxidative damage evaluated by the presence of 4-hydroxynonenal (HNE) and iron accumulation and expression of putative antioxidant enzymes heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in neurons of the ND and RN after spinal cord hemisection. We found that HNE expression was induced in neurons of the ipsilateral ND from 1 to 14 days following spinal cord hemisection. Concomitantly, iron staining was seen from 7 to 14 days after lesion. HO-1, however, was only transiently induced in ipsilateral ND neurons between 3 and 7 days after lesion. In contrast to the ND neurons, HNE was undetectable and iron level was unaltered in the RN neurons after spinal cord hemisection. HO-1, SOD-Cu/Zn and SOD-Mn were constitutively expressed in RN neurons, and lesion to the spinal cord did not change their expression. These results suggest that oxidative stress is involved in the degeneration of the lesioned ND neurons; whereas constitutive antioxidant enzymes may protect the RN neurons from oxidative damage.     

Induction of inducible heme oxygenase (HO-1) in the central nervous system: is HO-1 helpful or harmful?

Heme oxygenase (HO) produced biliverdin and bilirubin, which are powerful antioxidants, therefore, it has been proposed as helpful against oxidative stress. In contrast, HO also produces iron, and it could increase oxidative stress if not handled properly. To clarify the effect of HO, i.e., helpful or harmful, we examined the expression, localization, and induction mechanism of HO-1 in the rat hippocampus after transient forebrain ischemia and injection of kainic acid (KA). Following ischemia, HO-1 expression was observed early but transiently in the CA1 pyramidal neurons and later but continuously in glial cells. In addition, HO-1 expressing pyramidal neurons were colocalized well with phosphorylated c-Jun, which is a critical step in neuronal apoptosis. After injection of KA, HO-1 expression was observed only in glial cells but not neurons, and HO-1 expression was observed in predominantly ameboid microglia, along with a few astrocytes. HO-1 expressing ameboid microglia expressed major histocompatibility complex class II antigen, suggesting strong activation. These results suggested that HO-1 may have double-edged effects, and its effects may depend on the cell type. This short review is intended to highlight on the effect of HO-1 in neurodegeneration.     

Phosphorylation of c-Jun and its localization with heme oxygenase-1 and cyclooxygenase-2 in CA1 pyramidal neurons after transient forebrain ischemia.

Accumulating evidence on the molecular and cellular basis of ischemia/reperfusion-induced neurodegeneration suggests that oxidative stress is involved. Heme oxygenase (HO) and cyclooxygenase (COX) play physiologically important roles in the CNS. Conversely, HO and COX also can increase oxidative stress. Recent studies suggest that c-Jun phosphorylation is an important step in some forms of stress-induced neuronal apoptosis. In this study, the authors tried to clarify the association of HO and COX with c-Jun phosphorylation. Inducible forms of HO and COX (HO-1 and COX-2, respectively) were transiently induced in CA1 pyramidal neurons after ischemia. c-Jun also was induced in pyramidal neurons throughout the hippocampal formation, but its phosphorylation was limited to CA1. In contrast, these molecules were constitutively expressed at low levels. Most (84%) of the CA1 pyramidal neurons examined expressed HO-1, COX-2, or both, and such expression showed good co-localization with c-Jun phosphorylation. These results suggest the following: (1) c-Jun phosphorylation was associated with ischemia/reperfusion-induced neuronal apoptosis; (2) HO-1 and COX-2 were induced in CA1 pyramidal neurons, which undergo cell death; and (3) most CA1 pyramidal neurons expressed HO-1, COX-2, or both, which strongly suggests that these are candidates for neuron killers.     

血红素氧合酶-2基因缺失对氧化应激性脑损伤的保护作用

目的 探讨血红素氧合酶-2基因缺失对血红素诱导氧化应激性脑损伤的保护作用.方法 分别将6 μl (8 μmol/L)灭菌氯高铁血红素定向注入野生型小鼠和基因(HO-2)敲除小鼠的纹状体内,72 h后分别检测纹状体细胞生存率,蛋白和脂类的氧化作用.用蛋白质印迹法检测血红素氧合酶-1,2(HO-1)的表达.结果 与野生型相比,基因(HO-2)敲除小鼠纹状体内蛋白和脂类的氧化作用显著降低,而纹状体细胞的存活率显著增加;HO-1的表达在两种小鼠注射前后没有明显差异.结论 结果提示,血红素氧合酶-2基因缺失对血红素诱导的氧化应激性脑损伤具有保护作用;选择性抑制神经元血红素氧合酶-2基因的表达可减轻氧化应激性脑损伤.     

Heme oxygenase-2 protects against glutathione depletion-induced neuronal apoptosis mediated by bilirubin and cyclic GMP

Heme oxygenase (HO) enzymes catalyze the breakdown of heme to iron, carbon monoxide (CO), and biliverdin, which is rapidly converted to bilirubin. HO-2 has been implicated in protection against oxidative stress, ischemia, and traumatic brain injury. The neuroprotective effects of HO-2 have been attributed to the generation of bilirubin, which is an important radical scavenger. However, the mechanism by which HO-2 provides protection is unclear. We utilized the olfactory system as a model to define the roles of HO-2 in glutathione depletion-induced oxidative injury, since olfactory receptor neurons (ORNs) express high levels of HO isoforms. We demonstrated that L-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione biosynthesis, lowered glutathione levels and induced apoptosis of ORNs. Despite the presence of HO-1 in ORNs, HO-2 null animals displayed increased levels of neuronal death after BSO treatment compared to wild type mice. Levels of bilirubin and cGMP were also reduced in HO-2 null mice. Primary cultures of ORNs confirmed that the neuroprotective role of HO-2 was mediated by bilirubin and cGMP. Taken together, these results suggest that HO-2 plays a major role in neuroprotection from oxidative stress, an effect that is mediated by cGMP and bilirubin.     

Participation of prostate apoptosis response-4 in degeneration of dopaminergic neurons in models of Parkinson's disease.

Dysfunction and death of midbrain dopaminergic neurons underlies the clinical features of Parkinson's disease (PD). Increasing evidence suggests roles for oxidative stress and a form of cell death called apoptosis in the pathogenesis of PD. We recently identified a 38-kd protein called prostate apoptosis response-4 (Par-4), which is rapidly induced in cultured neurons after exposure to apoptotic insults, and appears to play a necessary role in the cell death process. We now report that Par-4 levels increase dramatically in midbrain dopaminergic neurons of monkeys and mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The increase in Par-4 levels occurs in both neuronal cell bodies in the substantia nigra and their axon terminals in the striatum, and precedes loss of tyrosine hydroxylase immunoreactivity and cell death. In the monkey model, Par-4 levels were also increased in several brain regions (red nucleus, lateral geniculate nucleus, and cerebral cortex) in which functional alterations have previously been documented in PD patients and MPTP-treated monkeys. Exposure of cultured human dopaminergic neural cells to the complex I inhibitor rotenone, or to Fe2+, resulted in Par-4 induction, mitochondrial dysfunction, and subsequent apoptosis. Blockade of Par-4 induction by antisense treatment prevented rotenone- and Fe2+-induced mitochondrial dysfunction and apoptosis demonstrating a critical role for Par-4 in the cell death process. The data suggest that Par-4 may be involved in the neurodegenerative process in PD.     

Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo

Trimethyltin (TMT), an organotin with potent neurotoxic effects by selectively damaging to hippocampus, is used as a tool for creating an experimental model of neurodegeneration. In the present study, we investigated the protective effects of magnolol, a natural biphenolic compound, on TMT-induced neurodegeneration and glial activation in vitro and in vivo. In HT22 murine neuroblastoma cells, TMT induced necrotic/apoptotic cell death and oxidative stress, including intracellular reactive oxygen species (ROS), protein carbonylation, induction of heme oxygenase-1 (HO-1), and activation of all mitogen-activated protein kinases (MAPKs) family proteins. However, magnolol treatment significantly suppressed neuronal cell death by inhibiting TMT-mediated ROS generation and activation of JNK and p38 MAPKs. In BV-2 microglial cells, magnolol efficiently attenuated TMT-induced microglial activation via suppression of ROS generation and activation of JNK, p38 MAPKs, and nuclear factor-κB (NF-κB) signaling. In an in vivo mouse study, TMT induced massive neuronal damage and enhanced oxidative stress at day 2. We also observed a concomitant increase in glial cells and inducible nitric oxide synthase (iNOS) expression on the same day. These features of TMT toxicity were reversed by treatment of magnolol. We observed that p-JNK and p-p38 MAPK levels were increased in the mouse hippocampus at day 1 after TMT treatment and that magnolol blocked TMT-induced JNK and p38 MAPK activation. Magnolol administration prevented TMT-induced hippocampal neurodegeneration and glial activation, possibly through the regulation of TMT-mediated ROS generation and MAPK activation.     

Cultured astrocytes from heme oxygenase-1 knockout mice are more vulnerable to heme-mediated oxidative injury

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO-2, the isoform expressed predominantly in neurons, accelerates heme-mediated neuronal injury, inhibitor studies suggest that HO-1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO-1 knockout and wild-type astrocytes to hemin. Consistent with prior observations, exposure of wild-type cultures to hemin for 24 hr resulted in protein carbonylation and concentration-dependent cell death between 10 and 60 microM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO-1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO-1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO-1 gene prior to hemin exposure. HO-2 expression was observed in both knockout and wild-type cultures and was not altered by HO-1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7-fold higher in knockout cells. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries.     

Involvement of the endothelin receptor subtype A in neuronal pathogenesis after traumatic brain injury

Endothelin-1 (ET-1) is a 21 amino acid peptide that has been closely linked to cerebral vasospasm and more recently to oxidative stress after traumatic brain injury. In this study, we have examined the effects of the endothelin receptor subtype A antagonist, Ro 61-1790, on acute cortical neuronal injury and delayed neuronal death in the cerebellum after mild traumatic brain injury. Rats were administered Ro 61-1790 or vehicle for 24 h after injury and euthanized at 1 day, 3 days, or 7 days. Heat shock protein70 (HSP70), a marker of neuronal stress/injury, was immunolocalized in the cortex. Induction of heme oxygenase-1 (HO-1) and enhanced immunoexpression of the complement C3bi receptor, both of which are indicators of cerebellar glial reactivity, and Purkinje cell loss were evaluated in the cerebellum. There was maximal induction of HSP70 in cortical neurons at 24 h postinjury in all animals. Drug treated animals showed significantly fewer HSP70 labeled cortical neurons at this time point. There were fewer reactive glia in the cerebellum of drug treated animals as compared to vehicle controls at 3 days postinjury. However, at 7 days postinjury glial reactivity and Purkinje cell loss were similar in both groups. These findings demonstrate that Ro 61-1790, when administered for the first 24 h postinjury, limits acute neuronal injury in the cortex, transiently influences glial reactivity in the cerebellum, and does not attenuate delayed Purkinje cell death. The latter finding may reflect the duration of infusion of the drug.     

Chronic and cyclical neuronal loss in hippocampal slice cultures following transient inhibition of the type 1 isoform of superoxide dismutase

Increased oxidative stress contributes to chronic neurodegenerative diseases, yet the underlying mechanisms are poorly understood. Hippocampal slice cultures prepared from 20-30-day-old mice or rats were used to model chronic neuronal loss following oxidative stress. Neuronal loss was initiated by inhibition of the antioxidant enzyme, superoxide dismutase type 1 (SOD1), using the copper chelator diethyldithiocarbamate (DDC). Continuous DDC treatment of slice cultures induced delayed neuronal loss beginning at 9 days of treatment that lasted for over 4 weeks. Neuronal loss was not uniform, rather it was cyclic: peaking at days 9-13 and at days 19-21 after DDC exposure. Neuronal loss was significantly attenuated in slice cultures that overexpress SOD1, suggesting that SOD1 inhibition was responsible. Inhibitors of nitric oxide synthase also attenuated DDC-induced neuronal loss. Chronic neuronal loss, however, did not require continuous SOD1 inhibition. Application of DDC for 13 days resulted in loss of SOD1 activity. Removal of DDC restored SOD1 activity, yet the cycles of cell loss continued until no neurons remained. Astrocyte activation was observed following the second peak of neuronal loss. Media conditioned by cultures following DDC removal induced neuronal loss and microglial activation in recipient cultures. These data suggest that slice cultures released soluble neurotoxic factor(s) following DDC removal. These data also suggest that a transient reduction of SOD1 activity leads to chronic loss of hippocampal neurons. This neuronal loss may be mediated by soluble neurotoxic factor(s) and microglial activation. Cyclical neuronal loss may also underlie chronic neurodegeneration in vivo.     

Disruption of dopamine homeostasis underlies selective neurodegeneration mediated by alpha-synuclein

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.     

Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice: protective effects of R-alpha-lipoic acid

Chronic systemic exposure of D-galactose to mice, rats, and Drosophila causes the acceleration of senescence and has been used as an aging model. However, the underlying mechanism is as yet unclear. To investigate the mechanisms of neurodegeneration in this model, we studied cognitive function, hippocampal neuronal apoptosis and neurogenesis, and peripheral oxidative stress biomarkers and also the protective effects of the antioxidant R-alpha-lipoic acid. Chronic systemic exposure of mice to D-galactose (100 mg/kg, s.c., 7 weeks) induced a spatial memory deficit, an increase in cell karyopyknosis, apoptosis, and caspase-3 protein levels in hippocampal neurons, a decrease in the number of new neurons in the subgranular zone in the dentate gyrus, a reduction of migration of neural progenitor cells, and an increase in death of newly formed neurons in the granular cell layer. The D-galactose exposure also induced an increase in peripheral oxidative stress, including an increase in malondialdehyde and decreases in total antioxidative capabilities (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) activities. A concomitant treatment with lipoic acid ameliorated cognitive dysfunction and neurodegeneration in the hippocampus and also reduced peripheral oxidative damage by decreasing malondialdehyde and increasing T-AOC and T-SOD, without an effect on GSH-Px. These findings suggest that chronic D-galactose exposure induces neurodegeneration by enhancing caspase-mediated apoptosis and inhibiting neurogenesis and neuron migration, as well as increasing oxidative damage. In addition, D-galactose-induced toxicity in mice is a useful model for studying the mechanisms of neurodegeneration and neuroprotective drugs and agents.