Hippocampal transcriptome-wide association study and pathway analysis of mitochondrial solute carriers in Alzheimer's disease.

The etiopathogenesis of late-onset Alzheimer's disease (AD) is increasingly recognized as the result of the combination of the aging process, toxic proteins, brain dysmetabolism, and genetic risks. Although the role of mitochondrial dysfunction in the pathogenesis of AD has been well-appreciated, the interaction between mitochondrial function and genetic variability in promoting dementia is still poorly understood. In this study, by tissue-specific transcriptome-wide association study (TWAS) and further meta-analysis, we examined the genetic association between mitochondrial solute carrier family (SLC25) genes and AD in three independent cohorts and identified three AD-susceptibility genes, including SLC25A10, SLC25A17, and SLC25A22. Integrative analysis using neuroimaging data and hippocampal TWAS-predicted gene expression of the three susceptibility genes showed an inverse correlation of SLC25A22 with hippocampal atrophy rate in AD patients, which outweighed the impacts of sex, age, and apolipoprotein E4 (ApoE4). Furthermore, SLC25A22 downregulation demonstrated an association with AD onset, as compared with the other two transcriptome-wide significant genes. Pathway and network analysis related hippocampal SLC25A22 downregulation to defects in neuronal function and development, echoing the enrichment of SLC25A22 expression in human glutamatergic neurons. The most parsimonious interpretation of the results is that we have identified AD-susceptibility genes in the SLC25 family through the prediction of hippocampal gene expression. Moreover, our findings mechanistically yield insight into the mitochondrial cascade hypothesis of AD and pave the way for the future development of diagnostic tools for the early prevention of AD from a perspective of precision medicine by targeting the mitochondria-related genes.

Traumatic Brain Injury Alters the Trajectory of Age-Related Mitochondrial Change.

Background: Some epidemiologic studies associate traumatic brain injury (TBI) with Alzheimer's disease (AD). Objective: To test whether a TBI-induced acceleration of age-related mitochondrial change could potentially mediate the reported TBI-AD association. Methods: We administered unilateral controlled cortical impact (CCI) or sham injuries to 5-month-old C57BL/6J and tau transgenic rTg4510 mice. In the non-transgenics, we assessed behavior (1-5 days, 1 month, and 15 months), lesion size (1 and 15 months), respiratory chain enzymes (1 and 15 months), and mitochondrial DNA copy number (mtDNAcn) (1 and 15 months) after CCI/sham. In the transgenics we quantified post-injury mtDNAcn and tangle burden. Results: In the non-transgenics CCI caused acute behavioral deficits that improved or resolved by 1-month post-injury. Protein-normalized complex I and cytochrome oxidase activities were not significantly altered at 1 or 15 months, although complex I activity in the CCI ipsilesional cortex declined during that period. Hippocampal mtDNAcn was not altered by injury at 1 month, increased with age, and rose to the greatest extent in the CCI contralesional hippocampus. In the injured then aged transgenics, the ipsilesional hippocampus contained less mtDNA and fewer tangles than the contralesional hippocampus; mtDNAcn and tangle counts did not correlate. Conclusions: As mice age their brains increase mtDNAcn as part of a compensatory response that preserves mitochondrial function, and TBI enhances this response. TBI may, therefore, increase the amount of compensation required to preserve late-life mitochondrial function. If TBI does modify AD risk, altering the trajectory or biology of aging-related mitochondrial changes could mediate the effect.

Increases in anterograde axoplasmic transport in neurons of the hyper-glutamatergic, glutamate dehydrogenase 1 (Glud1) transgenic mouse: Effects of glutamate receptors on transport.

The excitatory neurotransmitter glutamate has a role in neuronal migration and process elongation in the central nervous system (CNS). The effects of chronic glutamate hyperactivity on vesicular and protein transport within CNS neurons, that is, processes necessary for neurite growth, have not been examined previously. In this study, we measured the effects of lifelong hyperactivity of glutamate neurotransmission on axoplasmic transport in CNS neurons. We compared wild-type (wt) to transgenic (Tg) mice over-expressing the glutamate dehydrogenase gene Glud1 in CNS neurons and exhibiting increases in glutamate transmitter formation, release, and synaptic activation in brain throughout the lifespan. We found that Glud1 Tg as compared with wt mice exhibited increases in the rate of anterograde axoplasmic transport in neurons of the hippocampus measured in brain slices ex vivo, and in olfactory neurons measured in vivo. We also showed that the in vitro pharmacologic activation of glutamate synapses in wt mice led to moderate increases in axoplasmic transport, while exposure to selective inhibitors of ion channel forming glutamate receptors very significantly suppressed anterograde transport, suggesting a link between synaptic glutamate receptor activation and axoplasmic transport. Finally, axoplasmic transport in olfactory neurons of Tg mice in vivo was partially inhibited following 14-day intake of ethanol, a known suppressor of axoplasmic transport and of glutamate neurotransmission. The same was true for transport in hippocampal neurons in slices from Glud1 Tg mice exposed to ethanol for 2 h ex vivo. In conclusion, endogenous activity at glutamate synapses regulates and glutamate synaptic hyperactivity increases intraneuronal transport rates in CNS neurons.

Protection against Aß-induced neuronal damage by KU-32: PDHK1 inhibition as important target.

A feature of most neurodegenerative diseases is the presence of "mis-folded proteins" that form aggregates, suggesting suboptimal activity of neuronal molecular chaperones. Heat shock protein 90 (Hsp90) is the master regulator of cell responses to "proteotoxic" stresses. Some Hsp90 modulators activate cascades leading to upregulation of additional chaperones. Novobiocin is a modulator at the C-terminal ATP-binding site of Hsp90. Of several novobiocin analogs synthesized and tested for protection against amyloid beta (Aß)-induced neuronal death, "KU-32" was the most potent in protecting primary neurons, but did not increase expression of other chaperones believed to help clear misfolded proteins. However, KU-32 reversed Aß-induced superoxide formation, activated Complex I of the electron transfer chain in mitochondria, and blocked the Aß-induced inhibition of Complex I in neuroblastoma cells. A mechanism for these effects of KU-32 on mitochondrial metabolism appeared to be the inhibition of pyruvate dehydrogenase kinase (PDHK), both in isolated brain mitochondria and in SH-SY5Y cells. PDHK inhibition by the classic enzyme inhibitor, dichloroacetate, led to neuroprotection from Aß25-35-induced cell injury similarly to KU-32. Inhibition of PDHK in neurons would lead to activation of the PDH complex, increased acetyl-CoA generation, stimulation of the tricarboxylic acid cycle and Complex I in the electron transfer chain, and enhanced oxidative phosphorylation. A focus of future studies may be on the potential value of PDHK as a target in AD therapy.

A Mitochondrial DNA Haplogroup Defines Patterns of Five-Year Cognitive Change.

BACKGROUND: Mitochondrial DNA (mtDNA) may play a role in Alzheimer's disease (AD) and cognitive decline. A particular haplogroup of mtDNA, haplogroup J, has been observed more commonly in patients with AD than in cognitively normal controls. OBJECTIVE: We used two mtDNA haplogroups, H and J, to predict change in cognitive performance over five years. We hypothesized that haplogroup J carriers would show less cognitive resilience. METHODS: We analyzed data from 140 cognitively normal older adults who participated in the University of Kansas Alzheimer's Disease Research Center clinical cohort between 2011 and 2020. We used factor analysis to create three composite scores (verbal memory, attention, and executive function) from 11 individual cognitive tests. We performed latent growth curve modeling to describe trajectories of cognitive performance and change adjusting for age, sex, years of education, and APOE ɛ4 allele carrier status. We compared haplogroup H, the most common group, to haplogroup J, the potential risk group. RESULTS: Haplogroup J carriers had significantly lower baseline performance and slower rates of improvement on tests of verbal memory compared to haplogroup H carriers. We did not observe differences in executive function or attention. CONCLUSION: Our results reinforce the role of mtDNA in changes to cognitive function in a domain associated with risk for dementia, verbal memory, but not with other cognitive domains. Future research should investigate the distinct mechanisms by which mtDNA might affect performance on verbal memory as compared to other cognitive domains across haplogroups.

Exploratory analysis of mtDNA haplogroups in two Alzheimer's longitudinal cohorts.

INTRODUCTION: Inherited mitochondrial DNA (mtDNA) variants may influence Alzheimer's disease (AD) risk. METHODS: We sequenced mtDNA from 146 AD and 265 cognitively normal (CN) subjects from the University of Kansas AD Center (KUADC) and assigned haplogroups. We further considered 244 AD and 242 CN AD Neuroimaging Initiative (ADNI) subjects with equivalent data. RESULTS: Without applying multiple comparisons corrections, KUADC haplogroup J AD and CN frequencies were 16.4% versus 7.6% (P = .007), and haplogroup K AD and CN frequencies were 4.8% versus 10.2% (P = .063). ADNI haplogroup J AD and CN frequencies were 10.7% versus 7.0% (P = .20), and haplogroup K frequencies were 4.9% versus 8.7% (P = .11). For the combined 390 AD and 507 CN cases haplogroup J frequencies were 12.8% versus 7.3% (P = .006), odds ratio (OR) = 1.87, and haplogroup K frequencies were 4.9% versus 9.5% (P = .010), OR = 0.49. Associations remained significant after adjusting for apolipoprotein E, age, and sex. CONCLUSION: This exploratory analysis suggests inherited mtDNA variants influence AD risk.

Mitonuclear interactions influence Alzheimer's disease risk.

We examined the associations between mitochondrial DNA haplogroups (MT-hgs; mitochondrial haplotype groups defined by a specific combination of single nucleotide polymorphisms labeled as letters running from A to Z) and their interactions with a polygenic risk score composed of nuclear-encoded mitochondrial genes (nMT-PRS) with risk of dementia and age of onset (AOO) of dementia. MT-hg K (Odds ratio [OR]: 2.03 [95% CI: 1.04, 3.97]) and a 1 SD larger nMT-PRS (OR: 2.2 [95% CI: 1.68, 2.86]) were associated with elevated odds of dementia. Significant antagonistic interactions between the nMT-PRS and MT-hg K (OR: 0.45 [95% CI: 0.22, 0.9]) and MT-hg T (OR: 0.22 [95% CI: 0.1, 0.49]) were observed. Individual MT-hgs were not associated with AOO; however, a significant antagonistic interactions was observed between the nMT-PRS and MT-hg T (Hazard ratio: 0.62 [95% CI: 0.42, 0.91]) and a synergistic interaction between the nMT-PRS and MT-hg V (Hazard ratio: 2.28 [95% CI: 1.19, 4.35]). These results suggest that MT-hgs influence dementia risk and that variants in the nuclear and mitochondrial genome interact to influence the AOO of dementia.

Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions.

Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.

Effects of Ethanol Exposure on the Neurochemical Profile of a Transgenic Mouse Model with Enhanced Glutamate Release Using In Vivo 1H MRS.

Ethanol (EtOH) intake leads to modulation of glutamatergic transmission, which may contribute to ethanol intoxication, tolerance and dependence. To study metabolic responses to the hyper glutamatergic status at synapses during ethanol exposure, we used Glud1 transgenic (tg) mice that over-express the enzyme glutamate dehydrogenase in brain neurons and release excess glutamate (Glu) in synapses. We measured neurochemical changes in the hippocampus and striatum of tg and wild-type (wt) mice using proton magnetic resonance spectroscopy before and after the animals were fed with diets within which EtOH constituting up to 6.4% of total calories for 24 weeks. In the hippocampus, the EtOH diet led to significant increases in concentrations of EtOH, glutamine (Gln), Glu, phosphocholine (PCho), taurine, and Gln + Glu, when compared with their baseline concentrations. In the striatum, the EtOH diet led to significant increases in concentrations of GABA, Gln, Gln + Glu, and PCho. In general, neurochemical changes were more pronounced in the striatum than the hippocampus in both tg and wt mice. Overall neurochemical changes due to EtOH exposure were very similar in tg and wt mice. This study describes time courses of neurochemical profiles before and during chronic EtOH exposure, which can serve as a reference for future studies investigating ethanol-induced neurochemical changes.

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

Oxaloacetate enhances neuronal cell bioenergetic fluxes and infrastructure.

We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, whereas OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased cytochrome oxidase subunit 2, PGC1α, PGC1ß, and PGC1 related co-activator protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion. We examined how oxaloacetate (OAA) affects bioenergetic fluxes. To advance the understanding of how OAA mediates these changes, we compared the effects of OAA to malate, pyruvate, and glucose deprivation. We further examined how OAA affects levels of enzymes that facilitate its cytosolic metabolism, and found OAA increased the expression of malate dehydrogenase 1 (MDH1-cytosolic). We propose the following: OAA supports both glycolysis and respiration fluxes, shifts the cell redox balance toward a more oxidized state, and acts in an anaplerotic fashion. Abbreviations not defined in the text: MDH2, malate dehydrogenase 2 (mitochondrial).

Ischemic tolerance in an in vivo model of glutamate preconditioning.

Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. This study focuses on glutamate-mediated neuronal tolerance to ischemia-reperfusion. We employed an animal model of lifelong excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo glutamate preconditioning. Nine- and twenty-two-month-old Tg and wild-type (wt) mice were subjected to 90 min of middle cerebral artery occlusion, followed by 24 hr of reperfusion. The Tg mice suffered significantly reduced infarction and edema volume compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and microtubule-associated protein-2A (MAP2A) expression to understand the mechanism of neuroprotection observed in the Tg mice. We found that, in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes, whereas there was no significant difference in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising, significant increase was observed in MAP2A expression in neurons of the Tg hippocampus following ischemia-reperfusion compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia-reperfusion.

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice.

BACKGROUND: Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. RESULTS: During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. CONCLUSIONS: Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases.

Metabolism changes during aging in the hippocampus and striatum of glud1 (glutamate dehydrogenase 1) transgenic mice.

The decline in neuronal function during aging may result from increases in extracellular glutamate (Glu), Glu-induced neurotoxicity, and altered mitochondrial metabolism. To study metabolic responses to persistently high levels of Glu at synapses during aging, we used transgenic (Tg) mice that over-express the enzyme Glu dehydrogenase (GDH) in brain neurons and release excess Glu in synapses. Mitochondrial GDH is important in amino acid and carbohydrate metabolism and in anaplerotic reactions. We monitored changes in nineteen neurochemicals in the hippocampus and striatum of adult, middle aged, and aged Tg and wild type (wt) mice, in vivo, using proton ((1)H) magnetic resonance spectroscopy. Significant differences between adult Tg and wt were higher Glu, N-acetyl aspartate (NAA), and NAA + NAA-Glu (NAAG) levels, and lower lactate in the Tg hippocampus and striatum than those of wt. During aging, consistent changes in Tg and wt hippocampus and striatum included increases in myo-inositol and NAAG. The levels of glutamine (Gln), a key neurochemical in the Gln-Glu cycle between neurons and astroglia, increased during aging in both the striatum and hippocampus of Tg mice, but only in the striatum of the wt mice. Age-related increases of Glu were observed only in the striatum of the Tg mice.

Effects of gangliosides on the activity of the plasma membrane Ca2+-ATPase.

Control of intracellular calcium concentrations ([Ca(2+)]i) is essential for neuronal function, and the plasma membrane Ca(2+)-ATPase (PMCA) is crucial for the maintenance of low [Ca(2+)]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca(2+) homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca(2+) transporter.

Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines.

Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD+/NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

Decreases in plasma membrane Ca²âº-ATPase in brain synaptic membrane rafts from aged rats.

Precise regulation of free intracellular Ca(2+) concentrations [Ca(2+) ](i) is critical for normal neuronal function, and alterations in Ca(2+) homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca(2+) ](i) is the plasma membrane Ca(2+) -ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca(2+) . We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.

Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum.

We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50-60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders.

Selective neuronal vulnerability to oxidative stress in the brain.

Oxidative stress (OS), caused by the imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS), plays an important role in brain aging, neurodegenerative diseases, and other related adverse conditions, such as ischemia. While ROS/RNS serve as signaling molecules at physiological levels, an excessive amount of these molecules leads to oxidative modification and, therefore, dysfunction of proteins, nucleic acids, and lipids. The response of neurons to this pervasive stress, however, is not uniform in the brain. While many brain neurons can cope with a rise in OS, there are select populations of neurons in the brain that are vulnerable. Because of their selective vulnerability, these neurons are usually the first to exhibit functional decline and cell death during normal aging, or in age-associated neurodegenerative diseases, such as Alzheimer's disease. Understanding the molecular and cellular mechanisms of selective neuronal vulnerability (SNV) to OS is important in the development of future intervention approaches to protect such vulnerable neurons from the stresses of the aging process and the pathological states that lead to neurodegeneration. In this review, the currently known molecular and cellular factors that contribute to SNV to OS are summarized. Included among the major underlying factors are high intrinsic OS, high demand for ROS/RNS-based signaling, low ATP production, mitochondrial dysfunction, and high inflammatory response in vulnerable neurons. The contribution to the selective vulnerability of neurons to OS by other intrinsic or extrinsic factors, such as deficient DNA damage repair, low calcium-buffering capacity, and glutamate excitotoxicity, are also discussed.