Quantitative in vivo measurement of early axonal transport deficits in a triple transgenic mouse model of Alzheimer’s disease using manganese-enhanced MRI

Impaired axonal transport has been linked to the pathogenic processes of Alzheimer’s disease (AD) in which axonal swelling and degeneration are prevalent. The development of non-invasive neuroimaging methods to quantitatively assess in vivo axonal transport deficits would be enormously valuable to visualize early, yet subtle, changes in the AD brain, to monitor the disease progression and to quantify the effect of drug intervention. A triple transgenic mouse model of AD closely resembles human AD neuropathology. In this study, we investigated age-dependent alterations in the axonal transport rate in a longitudinal assessment of the triple transgenic mouse olfactory system, using fast multi-sliced T1 mapping with manganese-enhanced MRI. The data show that impairment in axonal transport is a very early event in AD pathology in these mice, preceding both deposition of Aβ plaques and formation of Tau fibrils

Effects of Paraquat-induced Oxidative Stress on the Neuronal Plasma Membrane Ca2+-ATPase

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium [Ca2+]i are not known. The calmodulin (CaM) -stimulated plasma membrane Ca2+-ATPase (PMCA) plays a critical role in regulating [Ca2+]i. Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would likely occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 μM) increased PMCA basal activity by 2-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 μM) inhibited both components of PMCA activity. Immunoblots showed the formation of high molecular weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca2+ transporter under prolonged oxidative stress could alter neuronal Ca2+ signaling

Decreases in Plasma Membrane Ca2+-ATPase in Brain Synaptic Membrane Rafts from Aged Rats

Precise regulation of free intracellular Ca2+ concentrations [Ca2+]i is critical for normal neuronal function, and alterations in Ca2+ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca2+]i is the plasma membrane Ca2+-ATPase (PMCA), the high affinity transporter that fine tunes the cytosolic nanomolar levels of Ca2+. 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 the present 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 of 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 CaM 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

Genomic and biochemical approaches in the discovery of mechanisms for selective neuronal vulnerability to oxidative stress

Background: Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches. Results: In this report, using in vitro neuronal cultures, ex vivo organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between in vivo vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons. Conclusion: Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons

Oxaloacetate Enhances Neuronal Cell Bioenergetic Fluxes and Infrastructure

"This is the peer reviewed version of the following article: Wilkins, Heather M. et al. “Oxaloacetate Enhances Neuronal Cell Bioenergetic Fluxes and Infrastructure.” Journal of neurochemistry 137.1 (2016): 76–87., which has been published in final form at 10.1111/jnc.13545. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."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 (MDH1) 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, while OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased COX2, PGC1α, PGC1β, and PRC 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

Effects of Gangliosides on the Activity of the Plasma Membrane Ca2+-ATPase

Control of intracellular calcium concentrations ([Ca2+]i) is essential for neuronal function, and the plasma membrane Ca2+-ATPase (PMCA) is crucial for the maintenance of low [Ca2+]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca2+ 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 Ca2+ transporter

Overcoming the blood-brain barrier to taxane delivery for brain tumors and neurodegenerative diseases and brain tumors

The original publication is available at www.springerlink.comThe blood-brain barrier (BBB) effectively prevents microtubule stabilizing drugs from readily entering the central nervous system (CNS). A major limiting factor for microtubule stabilizing drug permeation across the BBB is the active efflux back into the circulation by the over-expression of the multidrug resistant gene product (MDR1) or P-glycoprotein (P-gp). This study has focused on strategies to overcome P-gp-mediated efflux of taxol analogues, microtubule (MT) stabilizing agents that could be used to treat brain tumors and, potentially, neurodegenerative diseases such as Alzheimer’s disease. However, taxol is a strong P-gp substrate which limits its distribution across the BBB and therapeutic potential in the CNS. We have found that addition of a succinate group to the C-10 position of taxol results in an agent, Tx-67, with reduced interactions with P-gp and enhanced permeation across the BBB in both in vitro and in situ models. Our studies demonstrate the feasibility of making small chemical modifications to taxol to generate analogues with reduced affinity for the P-gp but retention of MT-stabilizing properties, i.e., a taxane that may reach and treat therapeutic targets in the CNS

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.This work was supported by the NSF (National Science Foundation) [grant number EEC-0310723]; NIH/NIDA (National Institutes of Health/National Institute on Drug Abuse) [grant number DA017186]; CEBRA, Phase II, NIA, [grant number AG12993]; NIAAA (National Institute of Alcohol Abuse and Alcoholism) [grant numbers AA11419, AA04732, AA12276]; NSF [grant numbers DBI-9987807, DBI-0352848]; NIDA [grant number DA017186]; NINDS (National Institute of Neurological Disorders and Strokes) [grant number NS39787]; NIMH (National Institute of Mental Health) [grant number MH58414]; NIDA Training [grant number DA022738]; NIDA [grant number DA015088], The Kansas Technology Enterprise Corporation, The Miller, Hedwig and Wilbur Fund, and The University of Kansas Research Development Fund

Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as ‘lipid rafts’ contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p < 0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

This is the published version. Copyright 2009 Society for Neuroscience.The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes