Altered Granger Causal Connectivity of Resting-State Neural Networks in Patients With Leukoaraiosis-Associated Cognitive Impairment-A Cross-Sectional Study.

Background: The purpose of this study was to provide an imaging reference for the measurement of disease progression, as well as to reveal the pathogenesis of leukoaraiosis (LA). Methods: Eighty-seven subjects were divided into three groups: LA patients with vascular dementia (LA-VaD) (20 subjects: 14 female, 6 male), LA patients with vascular cognitive impairment nondementia (LA-VCIND) (32 subjects: 14 male, 18 female), and normal controls (NC) (35 subjects: 14 male, 21 female). A multivariate Granger causality analysis (mGCA) was applied to the resting-state networks (RSNs) to evaluate the possible effective connectivity within the resting-state networks retrieved by independent component analysis (ICA) from resting-state functional magnetic resonance imaging (rs-fMRI) data. Results: Ten RSNs were identified: the primary visual network, secondary visual network, auditory network, sensorimotor network, anterior default mode network, posterior default mode network, salience network, dorsal attention network, left working memory network, and the right working memory network. Using independent component analysis, significant average Z scores were found in the anterior default mode network, salience network, dorsal attention network, and right working memory network between LA-VAD and NC groups. The functional connectivity (FC) strength of the networks was different between the NC, LA-VCIND, and LA-VaD groups. Effective connectivity between RSNs was compensated by either increased or decreased effective connectivity changes in these three groups. Conclusions: The components of resting-state networks kept changing as the disease progressed. Meanwhile, the activation intensity increased at the early stage of LA and decreased as patients' cognitive impairment aggravated. Furthermore, the direction and strength of connections between these networks changed and remodeled differently. These suggest that the human brain compensates for specific functional changes at different stages.

Abnormal Interactions of the Salience Network, Central Executive Network, and Default-Mode Network in Patients With Different Cognitive Impairment Loads Caused by Leukoaraiosis.

Leukoaraiosis (LA) is associated with cognitive impairment in the older people which can be demonstrated in functional connectivity (FC) based on resting-state functional magnetic resonance imaging (rs-fMRI). This study is to explore the FC changes in LA patients with different cognitive status by three network models. Fifty-three patients with LA were divided into three groups: the normal cognition (LA-NC; n = 14, six males), mild cognitive impairment (LA-MCI; n = 27, 13 males), and vascular dementia (LA-VD; n = 12, six males), according to the Mini Mental State Exam (MMSE) and Clinical Dementia Rating (CDR). The three groups and 30 matched healthy controls (HCs; 11 males) underwent rs-fMRI. The data of rs-fMRI were analyzed by independent components analysis (ICA) and region of interest (ROI) analysis by the REST toolbox. Then the FC was respectively analyzed by the default-mode network (DMN), salience networks (SNs) and the central executive network (CEN) with their results compared among the different groups. For inter-brain network analysis, there were negative FC between the SN and DMN in LA groups, and the FC decreased when compared with HC group. While there were enhanced inter-brain network FC between the SN and CEN as well as within the SN. The FC in patients with LA can be detected by different network models of rs-fMRI. The multi-model analysis is helpful for the further understanding of the cognitive changes in those patients.

A Neuroimaging Marker Based on Diffusion Tensor Imaging and Cognitive Impairment Due to Cerebral White Matter Lesions.

Background: The peak width of skeletonized mean diffusivity (PSMD) is a new, fully automated, robust imaging marker for cerebral small vessel disease (SVD), strongly associated with processing speed. However, it has never been applied to cerebral white matter lesions (WMLs). Our study aimed to investigate the correlation between PSMD and cognition, particularly in the executive function of patients with WMLs. Methods: A total of 111 WML patients and 50 healthy controls (HCs) were enrolled, and their demographic information and cardiovascular disease risk factors were recorded. Subjects were divided into three groups: WMLs with normal cognition (WMLs-NC), WMLs with vascular cognitive impairment (WMLs-VCI), and HCs. They underwent conventional head magnetic resonance imaging and diffusion tensor imaging (DTI), followed by neuropsychological and psychological examinations, including the Montreal Cognitive Assessment (MoCA), and the executive function tests. We compared executive function and PSMD among the three groups and analyzed the correlation between PSMD and cognitive function in all subjects. Results: There were no significant differences in demographic characteristics (age, sex, education level, and cardiovascular disease risk factors) among the three groups (P > 0.05), but there were significant differences in global cognition (P < 0.0001), executive function (P < 0.0001), and PSMD (P < 0.0001). The average PSMD value (×10-4 mm2/s) was 2.40 ± 0.23, 2.68 ± 0.30, and 4.51 ± 0.39 in the HC, WMLs-NC, and WMLs-VCI groups, respectively. There was no correlation between PSMD and cognition in the HC group, but PSMD was significantly correlated with MoCA scores (r = -0.3785, P < 0.0001) and executive function (r = -0.4744, P < 0.0001) in the WMLs-NC group and in the WMLs-VCI group (r = -0.4448, P < 0.0001 and r = -0.6279, P < 0.0001, respectively). Conclusions: WML patients have higher PSMD and worse cognitive performance than HCs, and PSMD is strongly associated with global cognition and executive functions in WML patients. This result provides new insights into the pathophysiology of cognitive impairment in WML patients. PSMD could be a surrogate marker for disease progression and could thus be used in therapeutic trials involving WML patients.

Effects of radio frequency assisted blanching on polyphenol oxidase, weight loss, texture, color and microstructure of potato.

This paper is focused on the effects of radio frequency (RF) heating on the relative activity of polyphenol oxidase (PPO), weight loss, texture, color, and microstructure of potatoes. The results showed that pure mushroom PPO was almost completely inactivated at 80 °C by RF heating. The relative activity of potato PPO reduced to less than 10% with increasing temperature (25-85 °C). Enzyme extract showed the lowest PPO relative activity at 85 °C after RF treatment, followed by the potato cuboids and mashed potato, about 0.19 ±â€¯0.017%, 3.24 ±â€¯0.19%, and 3.54 ±â€¯0.04%, respectively. Circular dichroism analysis indicated that RF heating changed the secondary structure of PPO, as α-helix content decreased. Both electrode gap and temperature had significant effect (P < .05) on weight loss, color, and texture of the potato cuboids. Microstructure analysis showed the changes of potato cell and starch during RF heating.

Resting-state functional magnetic resonance imaging in patients with leukoaraiosis-associated subcortical vascular cognitive impairment: a cross-sectional study.

OBJECTIVES: The aim of the study was to investigate the difference of resting-state default-mode network (DMN) between patients with leukoaraiosis (LA)-associated subcortical vascular cognitive impairment (SVCI) and control subjects, and to provide functional imaging evidence of SVCI. METHODS: All subjects (n = 58) were divided into two groups based on their clinical diagnosis: a LA-associated SVCI group (n = 31, 14 males) and a control group (n = 27, 14 males). Demographic information and resting-state functional MRI (rs-fMRI) data were obtained. These subjects were assessed using the Hamilton Anxiety Depression Scale, Clinical Dementia Rating, Mini Mental State Exam, and Montreal Cognitive Assessment. Experimental data and confounding factors were described with a General Liner Model. Independent components of fMRI data were analyzed with an fMRI toolbox. RESULTS: The active areas involved in DMN of LA-associated SVCI group were similar to those of the control group. However, several active areas of LA-associated SVCI group, especially the left anterior cingulate cortex and the right parahippocampal gyrus, showed significantly lower blood oxygen level-dependent (BOLD) signals compared with the control group (p ≤ 0.05); whereas the left caudate nucleus (p = 0.015), the right frontal lobe (p = 0.004), and the superior temporal gyrus/inferior parietal gyrus (p = 0.001) exhibited significantly higher BOLD signals compared with the control group. DISCUSSION: The present study provides neuroimaging evidence for the recognition of LA-associated SVCI. Moreover, the differences in the functional alterations of the resting-state DMN might be a distinguishing feature between SVCI and amnestic mild cognitive impairment patients.

Effects of two human chorionic gonadotropin doses administered to the ovarian states during the in vitro fertilization and embryo transfer program.

The aim of the present study was to examine the effects of the human chorionic gonadotropin (hCG) dose on the pulsatility indices (PI) of the intraovarian artery on the day of follicle aspiration and the oocyte quality, intrafollicular oxidative stress and luteinization. PI was also measured on the day of hCG administration. A total of 15 patients were undergoing the in vitro fertilization and embryo transfer (IVF-ET) program. To estimate whether there was any difference between the intraovarian artery blood flow and oocyte development of the same patients treated with 5,000 or 10,000 IU hCG, the intraovarian artery blood flow was measured by transvaginal color ultrasonography pulsed wave Doppler, and the follicular fluids and the granulosa cells were collected at follicle aspiration. There were statistically significant differences between the same patients undergoing the two different hCG-dose treatments in which the first protocol included 10,000 IU and the second protocol included 5,000 IU hCG treatment. These differences were apparent in the PI of intraovarian artery blood flow on the day of follicle aspiration (P=0.0023), in the incidence of apoptosis in cumulus (ApoC) and mural (ApoM) granulosa cells (ApoC, P=0.0077; ApoM, P=0.0128), in the total oocytes retrieved (P=0.0342) and in the follicle fluid progesterone concentration (P=0.0044). There were no significant differences between the two protocols in the PI of intraovarian artery blood flow on the day of hCG administration (P=0.4326), serum steroid on the day of follicle aspiration [serum P, P>0.9999; serum estradiol (E2), P=0.8589], follicle fluid E2 concentration (P=0.8939), mature oocyte rate (P=0.3743) and total mature oocytes retrieved (P=0.2026). In conclusion, the dose of hCG administration can significantly affect the intraovarian artery blood flow and the development of follicles and oocytes in an IVF-ET program.

Deficits in the mitochondrial enzyme α-ketoglutarate dehydrogenase lead to Alzheimer's disease-like calcium dysregulation.

Understanding the molecular sequence of events that culminate in multiple abnormalities in brains from patients that died with Alzheimer's disease (AD) will help to reveal the mechanisms of the disease and identify upstream events as therapeutic targets. The activity of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) in homogenates from autopsy brain declines with AD. Experimental reductions in KGDHC in mouse models of AD promote plaque and tangle formation, the hallmark pathologies of AD. We hypothesize that deficits in KGDHC also lead to the abnormalities in endoplasmic reticulum (ER) calcium stores and cytosolic calcium following K(+) depolarization that occurs in cells from AD patients and transgenic models of AD. The activity of the mitochondrial enzyme KGDHC was diminished acutely (minutes), long-term (days), or chronically (weeks). Acute inhibition of KGDHC produced effects on calcium opposite to those in AD, while the chronic or long-term inhibition of KGDHC mimicked the AD-related changes in calcium. Divergent changes in proteins released from the mitochondria that affect endoplasmic reticulum calcium channels may underlie the selective cellular consequences of acute versus longer term inhibition of KGDHC. The results suggest that the mitochondrial abnormalities in AD can be upstream of those in calcium.

Up-regulation of the mitochondrial malate dehydrogenase by oxidative stress is mediated by miR-743a.

These experiments reveal for the first time that microRNAs (miRNAs) mediate oxidant regulated expression of a mitochondrial tricarboxylic acid cycle gene (mdh2). mdh2 encoded malate dehydrogenase (MDH) is elevated by an unknown mechanism in brains of patients that died with Alzheimer's disease. Oxidative stress, an early and pervasive event in Alzheimer's disease, increased MDH activity and mRNA level of mdh2 by 19% and 22%, respectively, in a mouse hippocampal cell line (HT22). Post-transcriptional events underlie the change in mRNA because actinomycin D did not block the elevated mdh2 mRNA. Since miRNAs regulate gene expression post-transcriptionally, the expression of miR-743a, a miRNA predicted to target mdh2, was determined and showed a 52% reduction after oxidant treatment. Direct interaction of miR-743a with mdh2 was demonstrated with a luciferase based assay. Over-expression or inhibition of miR-743a led to a respective reduction or increase in endogenous mRNA and MDH activity. The results demonstrate that miR-743a negatively regulates mdh2 at post-transcriptional level by directly targeting the mdh2 3'UTR. The findings are consistent with the suggestion that oxidative stress can elevate the activity of MDH through miR-743a, and provide new insights into possible roles of miRNA in oxidative stress and neurodegeneration.

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex.

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.

Brain [U-13 C]glucose metabolism in mice with decreased α-ketoglutarate dehydrogenase complex activity.

The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), a mitochondrial enzyme complex that mediates the oxidative decarboxylation of α-ketoglutarate in the TCA cycle, is reduced in Alzheimer's disease. We investigated the metabolic effects of a partial KGDHC activity reduction on brain glucose metabolism using mice with disrupted expression of dihydrolipoyl succinyltransferase (DLST; gene encoding the E2k subunit of KGDHC). Brain tissue extracts from cortex and cerebellum of 6-week-old heterozygote DLST knockout mice (DLST+/-) and corresponding wild-type mice injected with [U-(13) C]glucose and decapitated 15 min later were analyzed. An increase in the concentration of glucose in cortex suggested a decrease in the cortical utilization of glucose in DLST+/- mice. Furthermore, the concentration and (13) C labelling of aspartate in cortex were reduced in DLST+/- mice. This decline was likely caused by a decrease in the pool of oxaloacetate. In contrast to results from cell culture studies, no indications of altered glycolysis or GABA shunt activity were found. Glucose metabolism in the cerebellum was unaffected by the decrease in KGDHC activity. Among metabolites not related to glucose metabolism, the concentration of taurine was decreased in the cortex, and that of tyrosine was increased in the cerebellum. These results imply that diminished KGDHC activity has the potential to induce the reduction in glucose utilization that is seen in several neurodegenerative diseases.

A mitocentric view of Alzheimer's disease suggests multi-faceted treatments.

Alzheimer's disease (AD) is defined by senile plaques made of amyloid-beta peptide (Abeta), neurofibrillary tangles made of hyperphosphorylated tau proteins, and memory deficits. Thus, the events initiating the cascade leading to these end points may be more effective therapeutic targets than treating each facet individually. In the small percentage of cases of AD that are genetic (or animal models that reflect this form of AD), the factor initiating AD is clear (e.g., genetic mutations lead to high Abeta1-42 or hyperphosphorylated tau proteins). In the vast majority of AD cases, the cause is unknown. Substantial evidence now suggests that abnormalities in glucose metabolism/mitochondrial function/oxidative stress (GMO) are an invariant feature of AD and occur at an early stage of the disease process in both genetic and non-genetic forms of AD. Indeed, decreases in brain glucose utilization are diagnostic for AD. Changes in calcium homeostasis also precede clinical manifestations of AD. Abnormal GMO can lead to plaques, tangles, and the calcium abnormalities that accompany AD. Abnormalities in GMO diminish the ability of the brain to adapt. Therapies targeting mitochondria may ameliorate abnormalities in plaques, tangles, calcium homeostasis, and cognition that comprise AD.

Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins.

The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

Mild reduction in the activity of the alpha-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis.

Diminished energy metabolism and reduced activity of brain alpha-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-13C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-13C]glucose, the E2k67 cells showed a greater increase in 13C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased 13C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer's mouse model.

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters.

Oxidant-induced changes in mitochondria and calcium dynamics in the pathophysiology of Alzheimer's disease.

Considerable data support the hypothesis that mitochondrial abnormalities link gene defects and/or environmental insults to the neurodegenerative process. The interaction of oxidants with calcium and the mitochondrial enzymes of the tricarboxylic acid cycle are central to that relationship. Abnormalities that were discovered in brains or fibroblasts from patients with Alzheimer's disease (AD) have been modeled in vitro and in vivo to assess their pathophysiological importance and to determine how they might be reversed. The conclusions are consistent with the hypothesis that the AD-related abnormalities result from oxidative stress. The selection of compounds for reversal is complex because the actions of the relevant compounds vary under different conditions, such as cell redox states and acute versus chronic changes. However, the models that have been developed are useful for testing the effectiveness of the potential medications. The results suggest that the reversal of mitochondrial deficits and a reduction in oxidative stress will reduce clinical and pathological changes and benefit patients.

Novel functions of the alpha-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease.

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H(2)O(2) and to probe the mechanism underlying these changes. Since the response to H(2)O(2) is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H(2)O(2) decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H(2)O(2) inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H(2)O(2) converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H(2)O(2) diminished glutathione to a similar level after 24 h. However, H(2)O(2), but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H(2)O(2) together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.

Oxidative stress and transcriptional regulation in Alzheimer disease.

Alzheimer disease (AD) is defined by progressive impairments in memory and cognition and by the presence of extracellular neuritic plaques and intracellular neurofibrillary tangles. However, oxidative stress and impaired mitochondrial function always accompany AD. Mitochondria are a major site of production of free radicals [ie, reactive oxygen species (ROS)] and primary targets of ROS. ROS are cytotoxic, and evidence of ROS-induced damage to cell membranes, proteins, and DNA in AD is overwhelming. Nevertheless, therapies based on antioxidants have been disappointing. Thus, alternative strategies are necessary. ROS also act as signaling molecules including for transcription. Thus, chronic exposure to ROS in AD could activate cascades of genes. Although initially protective, prolonged activation may be damaging. Thus, therapeutic approaches based on modulation of these gene cascades may lead to effective therapies. Genes involved in several pathways including antioxidant defense, detoxification, inflammation, etc, are induced in response to oxidative stress and in AD. However, genes that are associated with energy metabolism, which is necessary for normal brain function, are mostly down-regulated. Redox-sensitive transcription factors such as activator protein-1, nuclear factor-kappaB, specificity protein-1, and hypoxia-inducible factor are important in redox-dependent gene regulation. Peroxisome proliferators-activated receptor-gamma coactivator (PGC-1alpha) is a coactivator of several transcription factors and is a potent stimulator of mitochondrial biogenesis and respiration. Down-regulated expression of PGC-1alpha has been implicated in Huntington disease and in several Huntington disease animal models. PGC-1alpha role in regulation of ROS metabolism makes it a potential candidate player between ROS, mitochondria, and neurodegenerative diseases. This review summarizes the current progress on how oxidative stress regulates the expression of genes that might contribute to AD pathophysiology and the implications of the transcriptional modifications for AD. Finally, potential therapeutic strategies based on the updated understandings of redox state-dependent gene regulation in AD are proposed to overcome the lack of efficacy of antioxidant therapies.

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.

Reduction in the E2k subunit of the alpha-ketoglutarate dehydrogenase complex has effects independent of complex activity.

The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC) declines in brains of patients with several neurodegenerative diseases. KGDHC consists of multiple copies of E1k, E2k, and E3. E1k and E2k are unique to KGDHC and may have functions independent of the complex. The present study tested the consequences of different levels of diminished E2k mRNA on protein levels of the subunits, KGDHC activity, and physiological responses. Human embryonic kidney cells were stably transfected with an E2k sense or antisense expression vector. Sense control (E2k-mRNA-100) was compared with two clones in which the mRNA was reduced to 67% of control (E2k-mRNA-67) or to 30% of control (E2k-mRNA-30). The levels of the E2k protein in clones paralleled the reduction in mRNA, and E3 proteins were unaltered. Unexpectedly, the clone with the greatest reduction in E2k protein (E2k-mRNA-30) had a 40% increase in E1k protein. The activity of the complex was only 52% of normal in E2k-mRNA-67 clone, but was near normal (90%) in E2k-mRNA-30 clone. Subsequent experiments tested whether the physiological consequences of a reduction in E2k mRNA correlated more closely to E2k protein or to KGDHC activity. Growth rate, increased DCF-detectable reactive oxygen species, and cell death in response to added oxidant were proportional to E2k proteins, but not complex activity. These results were not predicted because subunits unique to KGDHC have never been manipulated in mammalian cells. These results suggest that in addition to its essential role in metabolism, the E2k component of KGDHC may have other novel roles.

Lysine 152 of MuLV reverse transcriptase is required for the integrity of the active site.

Comparison of the three-dimensional structure of the active sites of MuLV and HIV-1 reverse transcriptases shows the presence of a lysine residue (K152) in the substrate-binding region in MuLV RT, while its equivalent position in HIV-1 RT is occupied by a glycine (G112). To investigate the role of K152 in the mechanism of the polymerase reaction catalyzed by MuLV RT, four mutant RTs, namely, K152A, K152R, K152E, and K152G, were generated and biochemically characterized. All muteins exhibited reduced polymerase activity on both RNA and DNA template-primers with K152E being the most defective. The template-primer binding affinity and the processivity of DNA synthesis, however, remained unchanged. The steady-state kinetic characterization showed little change in K(m.dNTP) (except for that of K152E) and an approximately 3-10-fold decrease in k(cat) depending upon the template-primer and mutational substitutions. The ddNTP resistance patterns were unchanged for all muteins, suggesting no participation of K152 in ddNTP recognition. The ability of individual muteins to add dNTP on the covalently cross-linked enzyme-template-primer complex was significantly decreased. These results together with the analysis of the ion pairs in the catalytic apparatus of MuLV RT suggest that K152 participates in maintaining the integrity of the active site of MuLV RT. Examination of the prepolymerase ternary complex formation showed that neither the wild type nor any of the K152 muteins of MuLV RT are capable of forming stable ternary complexes. This property is in contrast to that of HIV-1 RT, which readily forms stable ternary complexes under similar conditions. These results further indicate that the catalytic mechanism of MuLV RT is significantly different from that of HIV-1 RT, despite the presence of a number of conserved motifs and amino acid residues.