Functional Genomics of Brain Aging and Alzheimer’s Disease: Focus on Selective Neuronal Vulnerability

Pivotal brain functions, such as neurotransmission, cognition, and memory, decline with advancing age and, especially, in neurodegenerative conditions associated with aging, such as Alzheimer’s disease (AD). Yet, deterioration in structure and function of the nervous system during aging or in AD is not uniform throughout the brain. Selective neuronal vulnerability (SNV) is a general but sometimes overlooked characteristic of brain aging and AD. There is little known at the molecular level to account for the phenomenon of SNV. Functional genomic analyses, through unbiased whole genome expression studies, could lead to new insights into a complex process such as SNV. Genomic data generated using both human brain tissue and brains from animal models of aging and AD were analyzed in this review. Convergent trends that have emerged from these data sets were considered in identifying possible molecular and cellular pathways involved in SNV. It appears that during normal brain aging and in AD, neurons vulnerable to injury or cell death are characterized by significant decreases in the expression of genes related to mitochondrial metabolism and energy production. In AD, vulnerable neurons also exhibit down-regulation of genes related to synaptic neurotransmission and vesicular transport, cytoskeletal structure and function, and neurotrophic factor activity. A prominent category of genes that are up-regulated in AD are those related to inflammatory response and some components of calcium signaling. These genomic differences between sensitive and resistant neurons can now be used to explore the molecular underpinnings of previously suggested mechanisms of cell injury in aging and AD

Sensitivity of the synaptic membrane Na+/Ca2+ exchanger and the expressed NCX1 isoform to reactive oxygen species

AbstractTwo plasma membrane proteins, the Na+/Ca2+ exchanger (NCX) and the Ca2+-ATPase, are major regulators of free intraneuronal Ca2+ levels as they are responsible for extrusion of Ca2+ from the intracellular to the extracellular medium. Because disruption of cellular Ca2+ regulation plays a role in damage occurring under conditions of oxidative stress, studies were conducted to assess the sensitivity of the NCX to reactive oxygen species (ROS). Exchanger activity in brain synaptic plasma membranes and in transfected CHO-K1 cells was inhibited following brief exposure to the peroxyl radical generating azo initiator 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) and to peroxynitrite. Incubation with hydrogen peroxide did not alter NCX activity, even at 800 μM concentration. In CHO-K1 cells transiently transfected with the NCX1 isoform of the exchanger, AAPH treatment decreased the maximal transport capacity (Vmax), whereas the Kact remained unchanged. Peroxynitrite led to an increase in Kact with no change in Vmax. Loss of activity following exposure to either AAPH or peroxynitrite was associated with the formation of high molecular weight aggregates of NCX, and AAPH also caused fragmentation of the exchanger protein. These findings suggest that the NCX is sensitive to biologically relevant ROS and could be involved in the loss of Ca2+ homeostasis observed under oxidative stress

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

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 \u3cem\u3eGlud1\u3c/em\u3e 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

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

(3R,5S,7as)-(3,5-bis(4-Fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol: A Novel Neuroprotective Agent

Compounds that interact with microtubules, such as paclitaxel, have been shown to possess protective properties against β-amyloid (Aβ)-induced neurodegeneration associated with Alzheimer's disease. In this work, the novel agent (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol was investigated for effectiveness in protecting neurons against several toxic stimuli and its interaction with the microtubule network. Exposure of neuronal cultures to Aβ peptide in the presence of 5 nM (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol resulted in a 50% increase in survival. Neuronal cultures treated with other toxic stimuli such as staurosporine, thapsigargin, paraquat and H2O2 showed significantly enhanced survival in the presence of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol. Microtubule binding and tubulin assembly studies revealed differences compared to paclitaxel, but confirmed the interaction of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol with microtubules. Furthermore, in vitro studies using bovine brain microvessel endothelial cells experiments suggest that (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol can readily cross the blood-brain barrier in a passive manner

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>In this report, using <it>in vitro </it>neuronal cultures, <it>ex vivo </it>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 <it>in vivo </it>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.</p> <p>Conclusion</p> <p>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.</p