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James W Geddes

Publications and source records attributed to James W Geddes.

16 recordsLinked to original sources
PubMed2006

Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria.

Mitochondria in nerve terminals are subjected to extensive Ca2+ fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders.

Adenosine Triphosphate↗
PubMed2005

Mitochondrial localization of mu-calpain.

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80kDa mu-calpain large subunit and 28kDa small subunit. In rat cortex, antibodies against domains II and III of the large mu-calpain subunit also detected a 40kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte mu-calpain with Ca(2+). Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of mu-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca(2+) released from mitochondrial stores.

Animals↗
PubMed2005

Nicotine attenuates morphological deficits in a contusion model of spinal cord injury.

Protection against the progression of secondary injury appears to be an effective therapeutic strategy in spinal cord injury (SCI). Evidence indicates that nicotine can induce potent neuroprotective effects against injury to spinal cord neurons. Therefore, the present study was focused on the effects of nicotine on the behavioral and morphological recovery associated with SCI. Adult male Long-Evans rats were subjected to a moderate contusion model of SCI and received subcutaneous injections of nicotine for 14 days at the dose of 0.35 or 7 mg/kg/day. The rats were examined using the BBB locomotor rating scale for 6 weeks. At the end of the BBB recording, spinal cords were examined for the volumetric tissue sparing of gray and white matters. All SCI rats demonstrated a loss of hindlimb function followed by a recovery phase that peaked at 2-3 weeks after the trauma. Compared to untreated SCI rats, chronic nicotine administration appeared to improve the recovery of the locomotor functions. Indeed, nicotine-treated animals scored consistently higher on the BBB scale indicating that the treatment altered animal behavior. However, when taking under consideration correction factors for multiple comparisons, these data did not reach significance at overall experimental levels of significance 0.05. Nevertheless, nicotine administration was effective in sparing tissue at injury epicenter and a lower dose of nicotine also resulted in significant sparing of white matter of the injured spinal cord. These results suggest that agonists of neuronal nicotinic receptors can be attractive candidates for SCI therapy.

Animals↗
PubMed2005

Proteasome or calpain inhibition does not alter cellular tau levels in neuroblastoma cells or primary neurons.

Impaired tau catabolism may contribute to tau accumulation and aggregation in Alzheimer's disease and neurofibrillary tangle formation. This study examined the effects of proteasome and calpain inhibition on tau levels and turnover in primary rat hippocampal neurons and differentiated SH-SY5Y human neuroblastoma cells. Administration of proteasome (MG-115, lactacystin) or calpain (MDL28170) inhibitors for up to 24 hours did not alter tau levels in differentiated SH-SY5Y cells or rat hippocampal neurons. Addition of 1 microM and 10 microM MG-115 did not change total tau levels, but did result in increased reactivity of phosphorylation-dependent tau antibodies (PHF-1, CP-13) and decreased Tau-1 immunoreactivity. Administration of cycloheximide to inhibit de novo protein synthesis also did not alter tau levels in the presence or absence of lactacystin. These results demonstrate that although the proteasome and calpain protease systems are capable of degrading tau in cell-free assays, their inhibition does not alter cellular tau levels in primary neurons or differentiated neuroblastoma cells.

Alzheimer Disease↗
PubMed2004

Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.

Mitochondria are known to be localized in synaptic and non-synaptic compartments in the brain. Synaptoneurosomes, which contain high numbers of mitochondria, may act as a major contaminant of currently used isolation techniques. Currently, there is no method employed to successfully disrupt synaptoneurosomes and isolate both synaptic and non-synaptic mitochondria without structural or functional damage. A novel method is reported here for disruption of synaptoneurosomes and isolation of total brain mitochondria from synaptic and non-synaptic sources using a nitrogen decompression technique. Nitrogen gas was dissolved into crude mitochondrial preparations and maintained under constant, moderate pressure. After a short incubation, the pressure was released causing the nitrogen to come out of solution as growing bubbles, which ruptures cellular and synaptoneurosomal membranes. Mitochondria isolated using this rapid technique were bioenergetically competent and exhibited functional characteristics comparable to mitochondria isolated using traditional techniques. This nitrogen decompression technique will allow for further characterization of synaptic pools of mitochondria, which are almost exclusively neuronal in origin.

Animals↗
PubMed2004

Nicotine attenuates oxidative stress, activation of redox-regulated transcription factors and induction of proinflammatory genes in compressive spinal cord trauma.

Pathophysiology of neurodegeneration following spinal cord injury (SCI) involves alterations of cellular redox status, activation of transcription factors and induction of proinflammatory genes. In addition, recent evidence indicates that nicotine can induce potent neuroprotective effects. To study the influence of nicotine on the redox signaling pathways in relationship to SCI, moderate contusions of spinal cords at the level of T-10 were induced in rats treated or untreated with nicotine. Cellular oxidative stress, DNA binding activity of redox-responsive transcription factors (AP-1, NF-kappaB and CREB) as well as mRNA levels of inflammatory genes (MCP-1 and TNF-alpha) were determined in the thoracic and lumbar regions of the spinal cords. Nicotine was administrated 2 h after the SCI in a single i.p. injection at the dose of 0.35, 3.5 or 7 mg/kg, and rats were sacrificed 3 h following such an injection. Spinal cord trauma was associated with a significant increase in oxidative stress, and activation of NF-kappaB, AP-1 and CREB, as well as overexpression of MCP-1 and TNF-alpha in both the thoracic and lumbar regions. Nicotine administration following the SCI markedly attenuated, especially in the lumbar region, these oxidative and proinflammatory responses. These protective effects of nicotine were fully reversed by inhibition of neuronal nicotinic receptors by mecamylamine. The present results indicate that nicotine administration can attenuate the oxidative injury to spinal cords and suggest that neuronal nicotinic receptors can be attractive targets for neuroprotective therapy.

Animals↗
PubMed2004

Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses.

The pathogenesis of incipient Alzheimer's disease (AD) has been resistant to analysis because of the complexity of AD and the overlap of its early-stage markers with normal aging. Gene microarrays provide new tools for addressing complexity because they allow overviews of the simultaneous activity of multiple cellular pathways. However, microarray data interpretation is often hindered by low statistical power, high false positives or false negatives, and by uncertain relevance to functional endpoints. Here, we analyzed hippocampal gene expression of nine control and 22 AD subjects of varying severity on 31 separate microarrays. We then tested the correlation of each gene's expression with MiniMental Status Examination (MMSE) and neurofibrillary tangle (NFT) scores across all 31 subjects regardless of diagnosis. These well powered tests revealed a major transcriptional response comprising thousands of genes significantly correlated with AD markers. Several hundred of these genes were also correlated with AD markers across only control and incipient AD subjects (MMSE > 20). Biological process categories associated with incipient AD-correlated genes were identified statistically (ease program) and revealed up-regulation of many transcription factor/signaling genes regulating proliferation and differentiation, including tumor suppressors, oligodendrocyte growth factors, and protein kinase A modulators. In addition, up-regulation of adhesion, apoptosis, lipid metabolism, and initial inflammation processes occurred, and down-regulation of protein folding/metabolism/transport and some energy metabolism and signaling pathways took place. These findings suggest a new model of AD pathogenesis in which a genomically orchestrated up-regulation of tumor suppressor-mediated differentiation and involution processes induces the spread of pathology along myelinated axons.

Algorithms↗
PubMed2004

Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity.

Excessive activation of calpains (calcium-activated neutral proteases) is observed following spinal cord contusion injury, traumatic brain injury, stroke, and in neurodegenerative disorders including Alzheimer's disease. Calpain inhibition represents an attractive therapeutic target, but current calpain inhibitors possess relatively weak potency, poor specificity, and in many cases, limited cellular and blood-brain barrier permeability. We developed novel calpain inhibitors consisting of the endogenous inhibitor, calpastatin or its inhibitory domain I, fused to the protein transduction domain of the HIV trans-activator (Tat) protein (Tat(47-57)). The Tat-calpastatin fusion proteins were potent calpain inhibitors in a cell-free activity assay, but did not inhibit cellular calpain activity in primary rat cortical neurons when applied exogenously at concentrations up to 5 microM. The fusion proteins were able to transduce neurons, but were localized within endosome-like structures. A similar endosomal uptake was observed for Tat-GFP. Together, the results suggest that endosomal uptake of the Tat-calpastatin prevents its interaction with calpain in other cellular compartments. Endosomal uptake of proteins fused to the Tat protein transduction domain severely limits the applications of this methodology.

Animals↗
PubMed2004

Brain region-specific, age-related, alterations in mitochondrial responses to elevated calcium.

An age-related Ca(2+) dysregulation and increased production of reactive oxygen species (ROS) may contribute to late-onset neurodegenerative disorders. These alterations are often attributed to impaired mitochondrial function yet few studies have directly examined mitochondria isolated from various regions of the aged brain. The purpose of this study was to examine Ca(2+)-buffering and ROS production in mitochondria isolated from Fischer 344 rats ranging in age from 4 to 25 months. Mitchondria isolated from the cortex of the 25 month rat brain exhibited greater rates of ROS production and mitochondrial swelling in response to increasing Ca(2+) loads as compared to mitochondria isolated from younger (4, 13 month) animals. The increased swelling is indicative of opening of the mitochondrial permeability transition pore indicating impaired Ca(2+) buffering/cycling in aged animals. These age-related differences were not observed in mitochondria isolated from cerebellum. Together, these results demonstrate region specific, age-related, alterations in mitochondrial responses to Ca(2+).

Adaptation, Physiological↗
PubMed2003

Glutamate regulates caveolin expression in rat hippocampal neurons.

Caveolae are cholesterol-rich, membrane microdomains that appear critical to signaling between extracellular and intracellular macromolecules as well as cholesterol homeostasis. Caveolae formation is modulated by caveolin, a protein family that is the proteinaceous hallmark of caveolae. Very little is known regarding the events that modulate caveolin expression and regulation in neurons. To detect caveolin expression in neurons, primary rat hippocampal neurons were harvested at embryonic day 18, maintained for 7 days in vitro, and then analyzed for caveolin immunofluorescence. Caveolin-1 immunoreactivity was detected in cells that were identified as neurons by morphology and concurrent microtubule-associated protein (MAP2) staining. Changes in caveolin-1 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of RNA isolated from hippocampal neurons treated with glutamate receptor agonists. Glutamate induced a concentration-dependent increase in caveolin-1 mRNA. The largest increases in caveolin-1 mRNA were detected after 6 hours of treatment. Kainate and AMPA both mimicked glutamate effects on caveolin-1 mRNA expression. Western blot analyses revealed that caveolin was induced at the protein level as well. Taken together, these data suggest that glutamate can regulate caveolin expression through kainate and AMPA ionotropic glutamate receptors.

Animals↗
PubMed2003

Influence of cytosolic and mitochondrial Ca2+, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid.

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.

Adenosine Triphosphate↗
PubMed2003

Evaluation of conditions for calpain inhibition in the rat spinal cord: effective postinjury inhibition with intraspinal MDL28170 microinjection.

Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased axonal damage and neurofilament proteolysis with postinjury intravenous administration of relatively low concentrations of the calpain inhibitors leupeptin, E-64-D, and calpeptin. We sought to determine if conditions under which calpain inhibitors were administered in previous studies resulted in effective calpain inhibition, and to identify conditions that result in significant calpain inhibition following spinal cord injury. Contusive spinal cord injury was produced in female Long-Evans rats using the NYU impactor at the 12.5-25-mm height setting. The results demonstrate that intravenous administration of 1 mg/kg E-64-D or 250 micro g/kg calpeptin does not inhibit total calpain activity in the rat spinal cord, measured using a BODIPY-FL labeled casein assay. Intravenous administration of MDL28170 (20 mg/kg) resulted in mild calpain inhibition and a modest decrease in the proteolysis of calpain substrates alpha-spectrin and MAP2. Intraspinal microinjection of 50 nmoles/19 micro g MDL28170, either 30 min prior to or 20 min following contusion injury, resulted in a more robust inhibition of total calpain activity and greater attenuation of alpha-spectrin breakdown and MAP2 proteolysis. The decreased proteolysis persisted 24 h postinjury. Together, the results demonstrate that direct microinjection of the calpain inhibitor MDL28170 is more effective than intravenous infusion in reducing calpain activity and decreasing the injury-induced proteolysis of calpain substrates.

Animals↗
PubMed2003

Calpain facilitates the neuron death induced by 3-nitropropionic acid and contributes to the necrotic morphology.

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.

Amino Acid Chloromethyl Ketones↗
PubMed2002

3-nitropropionic acid-induced changes in bilayer fluidity in synaptosomal membranes: implications for Huntington's disease.

The use of 3-nitropropionic acid (3-NP) and other mitochondria inhibitors to effectuate animal models of Huntington's disease has been well established. 3-NP administration has been shown to lead to pathology similar to that of HD, including massive loss of striatal neurons associated with oxidative stress. Oxidative stress induced by 3-NP also extends to the cortex, an area where little neuron loss occurs. No mechanism as of yet accounts for selective loss of striatal neurons while sparing cortical neurons. In the present study, a nitroxide stearate lipid bilayer-specific spin-label was utilized to probe 3-NP-induced fluidity changes in striatal and cortical synaptosomal membranes. In cortical synaptosomes, membrane fluidity increased in animals previously treated with 3-NP when compared to controls injected with saline vehicle, while in striatal synaptosomes, membrane fluidity decreased in animals treated with 3-NP when compared to controls. The results of the present study suggest that oxidatively-induced changes in membrane fluidity may be involved in mechanisms by which selective striatal neuronal loss occurs in this animal model of Huntington's disease.

Cell Membrane↗
PubMed2002

Increased protein oxidation and decreased creatine kinase BB expression and activity after spinal cord contusion injury.

Traumatic injury to the spinal cord triggers several secondary effects, including oxidative stress and compromised energy metabolism, which play a major role in biochemical and pathological changes in spinal cord tissue. Free radical generation and lipid peroxidation have been shown to be early events subsequent to spinal cord injury. In the present study, we demonstrated that protein oxidation increases in rat spinal cord tissue after experimental injury. As early as h after injury, the level of protein carbonyls at the injury epicenter was significantly higher than in control (169%, p < 0.05) and increased gradually over the next 4 weeks to 1260% of control level. Both caudal and rostral parts of the injured spinal cord demonstrated a mild increase of protein carbonyls by 4 weeks postinjury (135-138%, p < 0.05). Immunocytochemical analysis of protein carbonyls in the spinal cord cross-sections showed increased protein carbonyl immunoreactivity in the epicenter section compared to rostral and caudal sections of the same animal or control laminectomy animals. Increased protein carbonyl formation in damaged spinal cord tissue was associated with changes in activity and expression of an oxidative sensitive enzyme, creatine kinase BB, which plays an important role in the maintenance of ATP level in the CNS tissue. Damage to CK function in the CNS may severely aggravate the impairment of energy metabolism. The results of our study indicate that events associated with oxidative damage are triggered immediately after spinal cord trauma but continue to occur over the subsequent 4 weeks. These results suggest that antioxidant therapeutic strategies may be beneficial to lessen the consequences of the injury and potentially improve the restoration of neurological function.

Animals↗