Biomedical subjects

Thomas B Shea

Publications and source records attributed to Thomas B Shea.

At least 37 records · Page 2Linked to original sources

PubMed2004

Neurofilament subunits undergo more rapid translocation within retinas than in optic axons.

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation, although recent studies have challenged this hypothesis. Our prior analyses of axonal transport in optic axons demonstrated two distinct NF transport rates that spatially and temporally correlated with changes in NF phosphorylation. In our prior studies, we focused on subunits already within axons. Re-examination of these data using additional approaches and examining additional earlier time points have allowed us to calculate rates at which subunits transport out of retinas and into optic axons. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the front of radiolabeled subunits immunoprecipitated from retinas and segments of optic axons, which demonstrated four distinct transport rates. Subunits within retinas exhibited the fastest rate, and underwent a 50% slowing upon exiting the retina and entering optic axons. While this slowing could be due to a regional caliber increase and/or regional increase in NF phosphorylation within the first segment, prior studies indicated that inhibition of phosphatase activities increased NF phosphorylation within retinas and slowed NF subunit exit from retinas to a degree similar to that normally observed within the first segment of axons, suggesting that regional phosphorylation played a major role in slowing of NF transport following their exit from the retina. These findings provide additional support for the notion that phosphorylation regulates NF axonal transport.

Animals↗

PubMed2004

Increased transcription and activity of glutathione synthase in response to deficiencies in folate, vitamin E, and apolipoprotein E.

Oxidative stress is a major contributing factor in neurodegeneration and can arise from dietary, environmental, and genetic sources. Here we examine the separate and combined impact of deprivation of folate and vitamin E, coupled with dietary iron as a prooxidant, on normal mice and transgenic mice lacking apolipoprotein E (ApoE-/- mice). Both mouse strains exhibited increased levels of glutathione when deprived of folate and vitamin E, but a substantial further increase was observed in ApoE-/- mice. To determine the mechanism(s) underlying this increase, we quantified transcription and activity of glutathione synthase (GS). Both normal and ApoE-/- mice demonstrated increased GS activity when deprived of folate and vitamin E. However, transcription was increased only in ApoE-/- mice deprived of folate and vitamin E. These findings demonstrate that deficiency in one gene can result in compensatory up-regulation in a second relevant gene and, furthermore, indicate that compensation for oxidative stress can occur in brain tissue at epigenetic and genetic levels depending on the nature and/or extent of oxidative stress.

Animals↗

PubMed2004

Cdk5 regulates axonal transport and phosphorylation of neurofilaments in cultured neurons.

Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, to influence axonal stability and their maturation to large-caliber axons. Cdk5, a serine/threonine kinase homologous to the mitotic cyclin-dependent kinases, phosphorylates NF subunits in intact cells. In this study, we used two different haptenized NF subunits and manipulated cdk5 activity by microinjection, transfection and pharmacological inhibition to monitor the effect of Cdk5-p35 on NF dynamics and transport. We demonstrate that overexpression of cdk5 increases NF phosphorylation and inhibits NF axonal transport, whereas inhibition both reduces NF phosphorylation and enhances NF axonal transport in cultured chicken dorsal-root-ganglion neurons. Large phosphorylated-NF 'bundles' were prominent in perikarya following cdk5 overexpression. These findings suggest that Cdk5-p35 activity regulates normal NF distribution and that overexpression of Cdk5-p35 induces perikaryal accumulation of phosphorylated-NFs similar to those observed under pathological conditions.

Animals↗

PubMed2004

Folate deficiency and homocysteine induce toxicity in cultured dorsal root ganglion neurons via cytosolic calcium accumulation.

Folate deficiency induces neurotoxicity by multiple routes, including increasing cytosolic calcium and oxidative stress via increasing levels of the neurotoxin homocysteine (HC), and inducing mitochondrial and DNA damage. Because some of these neurotoxic effects overlap with those observed in motor neuron disease, we examined the impact of folate deprivation on dorsal root ganglion (DRG) neurons in culture. Folate deprivation for 2 h increased cytosolic calcium and reactive oxygen species (ROS) and impaired mitochondrial function. Treatment with nimodipine [an L voltage-sensitive calcium channel (LVSCC) antagonist], MK-801 (an NMDA channel antagonist) and thapsigarin (an inhibitor of efflux of calcium from internal stores) indicated that folate deprivation initially induced calcium influx via the LVSCC, with subsequent additional calcium derived from NMDA channels and internal stores. These compounds also reduced ROS and mitochondrial degeneration, indicating that calcium influx contributed to these phenomena. Calcium influx was prevented by co-treatment with 3-deaza-adenosine, which inhibits HC formation, indicating that HC mediated increased cytosolic calcium following folate deprivation. Nimodipine, MK-801 and thapsigargin had similar effects following direct treatment with HC as they did following folate deprivation. These findings support the idea that folate deprivation and HC treatment can compromise the health of DRG neurons by perturbing calcium homeostasis.

Adenine↗

PubMed2004

The S-adenosyl homocysteine hydrolase inhibitor 3-deaza-adenosine prevents oxidative damage and cognitive impairment following folate and vitamin E deprivation in a murine model of age-related, oxidative stress-induced neurodegeneration.

Deficiencies in folate promote neurodegeneration and potentiate the influence of other risk factors for neurodegeneration. This is accomplished at least in part by increasing levels of the neurotoxin homocysteine (HC). The S-adenosyl homocysteine (SAH) hydrolase inhibitor 3-deaza-adenosine (DZA) prevents HC accumulation following folate deprivation. We tested the ability of dietary supplementation with DZA to counteract the deleterious influence of folate deprivation. Folate deficiency has previously been shown to potentiate the impact of apolipoprotein E (ApoE); ApoE-/- mice deprived of folate demonstrated increased oxidative damage in brain tissue and impaired cognitive performance as compared to normal mice or to ApoE-/- mice receiving folate. Herein, we demonstrate that dietary supplementation with DZA prevented both the increase in oxidative damage and impaired cognition characteristic of ApoE-/- mice following folate deprivation. These findings suggest that manipulation of the methionine cycle by DZA can counteract folate deficiency. Because folate deprivation, increased HC, and apolipoprotein E deficiency are all risk factors for Alzheimer's disease, these findings also underscore that DZA might be useful in a therapeutic approach to delay neurodegeneration in Alzheimer's disease.

Adenosylhomocysteinase↗

PubMed2004

Dietary supplementation with 3-deaza adenosine, N-acetyl cysteine, and S-adenosyl methionine provide neuroprotection against multiple consequences of vitamin deficiency and oxidative challenge: relevance to age-related neurodegeneration.

Folate deprivation induces neurotoxicity that is potentiated by additional nutritional and genetic deficiencies including vitamin E and apolipoprotein E deficiency. These deficiencies collectively induce oxidative damage, cognitive impairment, and compensatory alteration in glutathione generation. Treatment with agents that regulate distinct portions of the methionine cycle, including the S-adenosyl homocysteine hydrolase inhibitor, 3-deaza adenosine, the methyl donor S-adenosyl methionine, and the antioxidant N-acetyl cysteine, provide neuroprotection against various aspects of neurotoxicity in normal and apolipoprotein E-deficient mice and in cultured neuronal cells deprived of dietary folate and vitamin E and subjected to iron overload. Here it is demonstrated that simultaneous treatment with these agents provide superior neuroprotection by alleviating individual and overlapping neurotoxic consequences. These findings support combinatorial treatments with agents that compensate for differential insults in age-related neurodegenerative disorders.

Acetylcysteine↗

PubMed2004

Apple juice prevents oxidative stress induced by amyloid-beta in culture.

Increased oxidative stress contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Dietary supplementation with fruits and vegetables that are high in antioxidant potential have in some cases compensated for oxidative stress. Herein, we examined whether apple juice could alleviate the neurotoxic consequences of exposure of cultured neuronal cells to amyloid-beta (Abeta), since at least a portion of the neurotoxicity of Abeta is due to oxidative stress. Apple juice concentrate (AJC; 70 degree brix) was diluted into culture medium of SH-SY-5Y human neuroblastoma cells that had been differentiated for 7 days with 5 microM retinoic acid concurrent with the addition of 20 microM Abeta. AJC prevented the increased generation of reactive oxygen species (ROS) normally induced by Abeta treatment under these conditions. AJC also prevented Abeta-induced calcium influx and apoptosis, each of which results in part due to increased ROS. These findings suggest that the antioxidant potential of apple products can prevent Abeta-induced oxidative damage.

Amyloid beta-Peptides↗

PubMed2004

Differential susceptibity of transgenic mice lacking one or both apolipoprotein alleles to folate and vitamin E deprivation.

The E4 allele of apolipoprotein E (ApoE) is associated with neurodegeneration in part due to increased oxidative stress. Transgenic mice lacking ApoE (-/-) represent a model for the consequences of deficiencies in ApoE function. Dietary deficiency in folate and vitamin E has previously been shown to potentiate the impact of ApoE deficiency; ApoE-/- mice deprived of folate and vitamin E for 1 month demonstrated increased oxidative damage in brain tissue and impaired cognitive performance as compared to ApoE+/+ mice. Since individuals homozygous for E4 can demonstrate more increased risk for neurodegeneration and an earlier age of onset than individuals heterozygous for E4, we tested the impact of folate and vitamin E deprivation on ApoE+/- mice. Thiobarbituric acid-reactive substances in brain tissue of ApoE+/- were significantly increased compared to ApoE+/+ mice, but this increase was less than that observed in ApoE-/- mice. By contrast, livers of ApoE+/- and -/- mice displayed an identical increase over that of +/+ mice. ApoE-/- mice, but not +/- or +/+ mice, exhibited impaired cognitive performance in maze trials when deprived of folate and vitamin E. These findings support the notion that homozygous deficiency of ApoE function can be more severe than heterozygous deficiency. They further suggest that the impact of partial deficiency in ApoE function may present a latent risk that may manifest only when compounded by other factors such as dietary deficiency.

Alleles↗

PubMed2003

Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel.

Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimer's disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.

Antibodies, Monoclonal↗

PubMed2003

Quantification of antioxidant activity in brain tissue homogenates using the 'total equivalent antioxidant capacity'.

We demonstrate herein that the standard 'Trolox equivalent antioxidant capacity' (TEAC) assay, typically utilized to quantify total antioxidant levels within plasma, can also be utilized for tissue homogenates. Normal mice and transgenic mice lacking apolipoprotein E were subjected to a diet including iron as a generic pro-oxidant for 1 month (which has been shown to induce oxidative damage in our prior studies) and homogenates of brain tissue were subjected to the TEAC assay. Levels of the endogenous antioxidant glutathione levels were also monitored by HPLC. As described previously, ApoE-deficient mice expressed increased levels of glutathione; total antioxidant levels, as determined by TEAC, were increased to a similar extent. The increase in total antioxidant levels, as determined by TEAC, following dietary iron challenge paralleled the increase in glutathione levels, as determined by HPLC. These findings indicate that the TEAC assay may be useful for tissue homogenates. The rapid nature of this assay compared to HPLC, coupled with its lack of requirement for sophisticated equipment, makes it well suited for analyses of multiple tissue samples.

Animals↗

PubMed2003

Growth cones contain a dynamic population of neurofilament subunits.

Neurofilaments (NFs) are classically considered to transport in a primarily anterograde direction along axons, and to undergo bulk degradation within the synapse or growth cone (GC). We compared overall NF protein distribution with that of newly expressed NF subunits within NB2a/d1 cells by transfection with a construct encoding green fluorescent protein (GFP) conjugated NF-M subunits. GCs lacked phosphorylated NF epitopes, and steady-state levels of non-phosphosphorylated NF subunits within GC were markedly reduced compared to those of neurite shaft as indicated by conventional immunofluorescence. However, GCs contained significant levels of GFP-tagged subunits in the form of punctate or short filamentous structures that in some cases exceeded that visualized along the shaft itself, suggesting that GCs contained a relatively higher concentration of newly synthesized subunits. GFP-tagged NF subunits within GCs co-localized with non-phosphorylated NF immunoreactivity. GFP-tagged subunits were observed within GC filopodia in which steady-state levels of NF subunits were too low to be detected by conventional immunofluorescence. Selective localization of fluorescein versus rhodamine fluorescene was observed within GCs following expression of NF-M conjugated to DsRed1-E5, which shifts from fluorescein to rhodamine fluorescence within hours after expression; axonal shafts contained a more even distribution of fluorescein and rhodamine fluorescence, further indicating that GCs contained relatively higher levels of the most-recently expressed subunits. GFP-tagged structures were rapidly extracted from GCs under conditions that preserved axonal structures. These short filamentous and punctate structures underwent rapid bi-directional movement within GCs. Movement of GFP-tagged structures within GCs ceased following application of nocodazole, cytochalasin B, and the kinase inhibitor olomoucine, indicating that their motility was dependent upon microtubules and actin and, moreover, was due to active transport rather than simple diffusion. Treatment with the protease inhibitor calpeptin increased overall NF subunits, but increased those within the GC to a greater extent than those along the shaft, indicating that subunits in the GC undergo more rapid turnover than do those within the shaft. Some GCs contained coiled aggregates of GFP-tagged NFs that appeared to be contiguous with axonal NFs. NFs extended from these aggregates into the advancing GC as axonal neurites elongated. These data are consistent with the presence of a population of dynamic NF subunits within GCs that is apparently capable of participating in regional filament formation during axonal elongation, and support the notion that NF polymerization and transport need not necessarily occur in a uniform proximal-distal manner.

Actins↗

PubMed2003

Regulation of the transition from vimentin to neurofilaments during neuronal differentiation.

Vimentin (Vm) is initially expressed by nearly all neuronal precursors in vivo, and is replaced by neurofilaments (NFs) shortly after the immature neurons become post-mitotic. Both Vm and NFs can be transiently detected within the same neurite, and Vm is essential for neuritogenesis at least in culture. How neurons effect the orderly transition from expression of Vm as their predominant intermediate filament to NFs remains unclear. We examined this phenomenon within growing axonal neurites of NB2a/d1 cells. Transfection of cells with a construct expressing Vm conjugated to green fluorescent protein confirmed that axonal transport machinery for Vm persisted following the developmental decrease in Vm, but that the amount undergoing transport decreased in parallel to the observed developmental increase in NF transport. Immunoprecipitation from pulse-chase radiolabeled cells demonstrated transient co-precipitation of newly synthesized NF-H with Vm, followed by increasing co-precipitation with NF-L. Immunofluorescent and immuno-electron microscopic analyses demonstrated that some NF and Vm subunits were incorporated into the same filamentous profiles, but that Vm was excluded from the longitudinally-oriented "bundle" of closely-apposed NFs that accumulates within developing axons and is known to undergo slower turnover than individual NFs. These data collectively suggest that developing neurons are able to replace their Vm-rich cytoskeleton with one rich in NFs simply by down-regulation of Vm expression and upregulation of NFs, coupled with turnover of existing Vm filaments and Vm-NF heteropolymers.

Cell Differentiation↗

PubMed2003

Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders.

Folate is a cofactor in one-carbon metabolism, during which it promotes the remethylation of homocysteine -- a cytotoxic sulfur-containing amino acid that can induce DNA strand breakage, oxidative stress and apoptosis. Dietary folate is required for normal development of the nervous system, playing important roles regulating neurogenesis and programmed cell death. Recent epidemiological and experimental studies have linked folate deficiency and resultant increased homocysteine levels with several neurodegenerative conditions, including stroke, Alzheimer's disease and Parkinson's disease. Moreover, genetic and clinical data suggest roles for folate and homocysteine in the pathogenesis of psychiatric disorders. A better understanding of the roles of folate and homocysteine in neuronal homeostasis throughout life is revealing novel approaches for preventing and treating neurological disorders.

Aging↗

PubMed2003

Does neurofilament phosphorylation regulate axonal transport?

Phosphorylation of neurofilaments has long been considered to regulate their axonal transport rate and, in doing so, to provide stability to mature axons. Interpretation of data recently obtained following C-terminal deletion experiments has prompted a challenge to this hypothesis. We present evidence that these deletion studies remain consistent with, rather than refute, a role for C-terminal phosphorylation in regulation of neurofilament axonal transport.

Amino Acid Sequence↗

PubMed2003

Folate deprivation induces neurodegeneration: roles of oxidative stress and increased homocysteine.

Clinical studies suggest a relationship between folate deficiency and neurological and disorders including Alzheimer's disease (AD). To investigate mechanisms underlying this association, we examined the consequences of folate deprivation on neuronal cultures. Culturing embryonic cortical neurons and differentiated SH-SY-5Y human neuroblastoma cells in folate-free medium induced neurodegenerative changes characteristic of those observed in AD, including increased cytosolic calcium, reactive oxygen species (ROS), phospho-tau and apoptosis. In accord with clinical studies, generation of the neurotoxic amino acid homocysteine (HC) was likely to contribute to these phenomena, since (1) a significant increase in HC was detected following folate deprivation, (2) addition of the inhibitor of HC formation, 3-deazaadenosine, both prevented HC formation and eliminated the increase in ROS that normally accompanied folate deprivation, (3) direct addition of HC in the presence of folate induced the neurotoxic effects that accompanied folate deprivation, and (4) an antagonist of NMDA channels that blocks HC-induced calcium influx also blocked calcium influx following folate deprivation. Folate deprivation decreased the reduced form of glutathione, indicating a depletion of oxidative buffering capacity. This line of reasoning was supported by an increase in glutathione and reduction in ROS following supplementation of folate-deprived cultures with the cell-permeant glutathione precursor, N-acetyl-L-cysteine, or vitamin E. Folate deprivation potentiated ROS and apoptosis induced by amyloid-beta, while folate supplementation at higher concentrations prevented generation of ROS by amyloid-beta, suggesting that folate levels modulate the extent of amyloid-beta neurotoxicity. These findings underscore the importance of folate metabolism in neuronal homeostasis and suggest that folate deficiency may augment AD neuropathology by increasing ROS and excitotoxicity via HC generation.

Animals↗

PubMed2003

Folate and vitamin E deficiency impair cognitive performance in mice subjected to oxidative stress: differential impact on normal mice and mice lacking apolipoprotein E.

One factor contributing to the age-related decline in cognitive performance is increased oxidative stress, that can arise from environmental, nutritional, and/or genetic compromise. Folate deficiency has been linked to several age-related neurodegenerative conditions, including Alzheimer's disease (AD), at least in part by increasing oxidative stress. Folate deficiency also potentiates the impact of other known risk factors for AD. Adecrease in function of apolipoprotein E (ApoE), is associated with increased oxidative stress and is a risk factor for AD. We tested the combined impact of dietary deficiencies in folate and vitamin E, coupled with exposure to high dietary iron as a pro-oxidant, on cognitive performance in normal and ApoE-/- mice by monitoring the percent alternation in passive Y and T maze tests. Both normal and ApoE-/- mice displayed some cognitive impairment when deprived of folate and vitamin E and exposed to iron, but ApoE-/- mice were more severely affected. These findings highlight the potential combined impact of dietary deficiencies and genetic predisposition to neurodegeneration. They further leave open the possibility that one or more risk factors may remain latent, and neurodegeneration may ensue only following augmentation by one or more additional traumatic events or conditions.

Aging↗

PubMed2003

Vitamin E deficiency does not induce compensatory antioxidant increases in central nervous system tissue of apolipoprotein E-deficient mice.

Compensatory upregulation in endogenous antioxidants has been shown to accompany certain genetic and dietary deficiencies that promote oxidative stress, including that related to Alzheimer's disease. We compared antioxidant levels in brain tissue of normal and transgenic mice lacking apolipoprotein E following dietary deprivation of vitamin E or folate. As described previously, ApoE-deficient mice displayed increased levels of the endogenous antioxidant glutathione as compared to normal mice, and increased these levels further following folate deprivation. By contrast, glutathione was depleted following vitamin E deprivation in brain tissue of normal and ApoE-deficient mice. TBAR analyses confirmed increased oxidative damage following vitamin E deprivation. However, combined deprivation of folate and vitamin E resulted in levels of glutathione intermediate between those observed following deprivation of either agent, indicating that the lack of compensatory increase in glutathione following vitamin E deprivation was not due to overt neurotoxicity. Similar results were observed for total antioxidant levels in brain tissue. The differential response to vitamin E and folate deprivation is consistent with the possibility that specific differences in oxidative damage may result from deficiencies in either of these agents. The lack of a compensatory response to vitamin E deprivation highlights the importance of dietary vitamin E in prevention of chronic neurodegeneration.

Animals↗

PubMed2003

17 beta-estradiol alleviates synergistic oxidative stress resulting from folate deprivation and amyloid-beta treatment.

Oxidative stress is thought to be a pivotal factor in Alzheimer's disease (AD). Amyloid-beta (Abeta) induces oxidative damage, which is likely to be compounded by deficiencies in endogenous antioxidant capacity. Indeed, folate deprivation, which has been associated with AD, potentiates generation of reactive oxygen species (ROS) by Abeta. We examined whether the antioxidant 17-beta-estradiol could attenuate ROS generation following Abeta treatment in the presence and absence of folate using differentiated SH-SY-5Y human neuroblastoma cells. Folate-deprivation and Abeta treatment each induced an increase in ROS, and treatment of folate-deprived cultures with Abeta induced a synergistic increase in ROS. 17-beta-estradiol reduced ROS levels in beta-treated, folate-deprived cultures to ROS levels observed in cultures treated with Abeta in the presence of folate, suggesting that this antioxidant was able to prevent the synergistic impact of Abeta and folate deprivation on ROS generation. 17-beta-estradiol also completely prevented neuronal death induced by both Abeta and folate deprivation individually, and reduced neuronal death following Abeta treatment along with folate deprivation. These findings suggest that therapeutic approaches utilizing antioxidants may be particularly important in conditions such as AD, where multiple factors, including compromises in endogenous antioxidants, promote oxidative stress.

Alzheimer Disease↗