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Thomas B Shea

Publications and source records attributed to Thomas B Shea.

At least 19 recordsLinked to original sources
PubMed2006

Divergent effects of the MEKK-1/JNK pathway on NB2a/d1 differentiation: some activity is required for outgrowth and stabilization of neurites but overactivation inhibits both phenomena.

c-Jun N-terminal kinase (JNK), along with its upstream activator MEKK-1, is typically thought of as a stress-activated kinase that mediates apoptosis. However, additional studies indicate that the MEKK-1/JNK pathway mediates critical aspects of neuronal survival and differentiation. Herein, we demonstrate that transfection of differentiated NB2a/d1 cells with a construct expression constitutively activated (ca) MEKK-1 increases levels of phospho-dependent neurofilament (NF) immunoreactivity within perikarya, while expression of a dominant-negative (dn) form of MEKK-1 decreases it. Steady-state levels of perikaryal phospho-NF immunoreactivity are reduced and the increase resulting from expression of caMEKK-1 is prevented, by the JNK inhibitor SP600125, suggesting that JNK is a major downstream effector of MEKK-1 on NF phosphorylation. Unexpectedly, both caMEKK-1 and dnMEKK-1 inhibited neuritogenesis as well as translocation of NFs into newly elaborated neurites. The JNK inhibitor SP600125 also inhibited NF transport in a dose-dependent manner. caMEKK-1 also prevented the increase in NF transport otherwise mediated by MAP kinase. Finally, both caMEKK-1 and dnMEKK-1 prevented initial neuritogenesis. These findings indicate that the MEKK-1/JNK pathway regulates critical aspects of initial outgrowth, and subsequent stabilization of axonal neurites.

Animals↗
PubMed2006

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events.

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.

Animals↗
PubMed2006

Supplementation with apple juice attenuates presenilin-1 overexpression during dietary and genetically-induced oxidative stress.

Gain-of-function mutations in the presenilin-1 (PS-1) promote Alzheimer's disease (AD) by increasing reactive oxygen species, at least part of which is derived by an accompanying increase in generation of amyloid-beta (Abeta). Additional studies indicate that impaired Apolipoprotein E function, which also increases oxidative stress and is also associated with AD, potentiates the deleterious activity of PS-1. Folate deficiency is also associated with AD and potentiates the impact of both ApoE deficiency and beta exposure. More recently, folate deficiency has been shown to increase PS-1 expression. Since dietary supplementation with apple juice provides neuroprotection against ApoE deficiency, Abeta exposure and folate deficiency, we examined the impact of apple juice on PS-1 overexpression. Herein, we demonstrate that dietary deficiency in folate and vitamin E increased PS-1 expression in juvenile and adult normal C57B1/6J and ApoE-/- mice and in aged normal mice. Supplementation with apple juice concentrate (AJC) attenuated or prevent these increases. Prior studies demonstrate that impaired DNA methylation resulting from a deficiency in S-adenosylmethionine (SAM, which is rapidly depleted following folate deprivation) leads to PS-1 overexpression, and that direct supplementation with SAM attenuates PS-1 overexpression. We determined that AJC contained levels of SAM comparable to those capable of suppressing PS-1 overexpression, suggesting that the SAM content of AJC represents a potential mechanism for preventing PS-1 overexpression, and further highlighting the possibility that AJC provides neuroprotection by mechanisms in addition to its antioxidant potential.

Acetylcholine↗
PubMed2006

Apple juice concentrate maintains acetylcholine levels following dietary compromise.

Oxidative stress contributes to age-related cognitive decline. In some instances, consumption of fruits and vegetables rich in antioxidant can provide superior protection than supplementation with purified antioxidants. Our prior studies have shown that supplementation with apple juice concentrate (AJC) alleviates oxidative damage and cognitive decline in adult (9-12 months) mice lacking ApoE (as a model of increased oxidative stress) and in normal aged (2-2.5 years) mice when challenged with a vitamin-deficient, oxidative stress-promoting diet. Here, we demonstrate that AJC, administered in drinking water, maintains acetylcholine levels that otherwise decline when adult and aged mice are maintained on the above deficient diet. Normal mice aged either 9-10 months or 2-2.5 years and ApoE-/- mice aged 9-10 months were maintained for 1 month on a complete diet or a diet lacking folate and vitamin E and containing iron as a pro-oxidant, and additional groups received 0.5% AJC ad libitum in drinking water. Spectrophotometric assay of acetylcholine levels revealed a significant decline in homogenates of combined frontal cortex and hippocampus for all mice maintained on the deficient diet, and a prevention of this decline in mice maintained on the deficient diet when supplemented with AJC. These findings provide a likely mechanism by which consumption of antioxidant-rich foods such as apples can prevent the decline in cognitive performance that accompanies dietary and genetic deficiencies and aging.

Acetylcholine↗
PubMed2006

Dietary and genetically-induced oxidative stress alter tau phosphorylation: influence of folate and apolipoprotein E deficiency.

One hallmark of AD is the deposition of neurofibrillary tangles which are comprised of phosphorylated isoforms of the microtubule-associated protein tau. We demonstrate herein that dietary deprivation of folate and vitamin E, coupled with iron as a pro-oxidant, fosters an increase in nonphospho- and-phospho-tau within brain tissue of mice homozygously lacking apolipoprotein E as assayed by monoclonal antibodies Tau-1 and PHF-1, respectively. Tau immunoreactivity in mice homozygously expressing murine apolipoprotein E was not affected. Supplementation of this challenge diet with s-adenosylmethinone, known to be depleted following folate deprivation and further known to restore a portion of the oxidative buffering capactity of these mice when maintained under this challenge diet, alleviates the increase in nonphospho-tau but does not attenuate the increase in phospho-tau. These findings suggest that the combined deleterious impact of dietary- and genetically-induced oxidative stress fostered a specific increase in phospho-tau. While some studies consider that increased levels of phospho-tau represents a hallmark of neuropathology, the findings of the present study also remain consistent with the alternative viewpoint that accumulation of phospho-tau instead represents an index of antioxidant compensation.

Alzheimer Disease↗
PubMed2005

Folate, vitamin E, and acetyl-L-carnitine provide synergistic protection against oxidative stress resulting from exposure of human neuroblastoma cells to amyloid-beta.

Oxidative stress is an early and pivotal factor in Alzheimer's disease (AD). The neurotoxic peptide amyloid-beta (Abeta) contributes to oxidative damage in AD by inducing lipid peroxidation, which in turn generates additional downstream cytosolic free radicals and reactive oxygen species (ROS), leading to mitochondrial and cytoskeletal compromise, depletion of ATP, and ultimate apoptosis. Timely application of antioxidants can prevent all downstream consequences of Abeta exposure in culture, but in situ efficacy is limited, due in part to prior damage as well as difficulty in delivery. Herein, we demonstrate that administration of a combination of vitamin E (which prevents de novo membrane oxidative damage), folate (which maintains levels of the endogenous antioxidant glutathione), and acetyl-L-carnitine (which prevents Abeta-induced mitochondrial damage and ATP depletion) provides superior protection to that derived from each agent alone. These findings support a combinatorial approach in Alzheimer's therapy.

Acetylcarnitine↗
PubMed2005

The high and middle molecular weight neurofilament subunits regulate the association of neurofilaments with kinesin: inhibition by phosphorylation of the high molecular weight subunit.

Kinesin participates in axonal transport of neurofilaments (NFs), but the mode by which they attach to kinesin is unclear. We compared the association of NFs with kinesin in mice expressing or lacking NF-H or NF-M. In normal and M-/- mice, the leading edge of metabolically labeled NF subunits was selectively co-precipitated with kinesin. By contrast, the entire wave of radiolabeled subunits co-precipitated with kinesin in H-/- mice. Similar bulk levels of NFs co-precipitated with kinesin from normal and H-/- mice, but reduced levels co-precipitated from M-/- mice. These data suggest that both NF-H and NF-M regulate the association of NFs with kinesin. They further indicate that phosphorylation of NF-H dissociates NFs from kinesin and provides a mechanism by which NF-H phosphorylation can contribute to the slowing of NF axonal transport.

Animals↗
PubMed2005

Cdk5 inhibits anterograde axonal transport of neurofilaments but not that of tau by inhibition of mitogen-activated protein kinase activity.

Cyclin-dependent kinase 5 (cdk5) inhibits neurofilament (NF) anterograde axonal transport while p42/44 mitogen-activated protein kinase (MAPk) promotes it. Since cdk5 is known to inhibit MAP kinase activity, we examined whether or not cdk5 inhibits anterograde NF transport via inhibition of MAPk activity. To accomplish this, we manipulated the activity of these kinases in differentiated NB2a/d1 cells, and monitored anterograde axonal transport of green fluorescent protein-conjugated-NF-M (GFP-M) and cyan fluorescent protein-conjugated (CFP)-tau. The cdk5 inhibitor roscovitine increased anterograde axonal transport of GFP-M and CFP-tau; transfection with cdk5/p25 inhibited transport of both. Inhibition of MAPk activity by PD98059 or expression of dominant-negative MAPk inhibited anterograde GFP-M transport, while expression of constitutively active MAPk enhanced it; these treatments did not affect CFP-tau transport. PD98059 prevented roscovitine-mediated enhancement of GFP-M transport, but did not prevent enhancement of CFP-tau transport. Co-transfection with constitutively activated MAPk prevented the inhibition of GFP-M transport that normally accompanied transfection with cdk5/p25, but did not prevent inhibition of tau transport by cdk5/p25. Finally, the extent of inhibition of GFP-M axonal transport by PD98059 was not additive to that derived from transfection with cdk5/p35, and the increase in NF transport that accompanies roscovitine treatment was not additive to that derived from transfection with constitutively activated MAPk, suggesting that the influence of these kinases on NF transport was within the same, rather than distinct, pathways. These findings suggest that axonal transport of tau and NFs is under the control of distinct kinase cascades, and that cdk5 inhibits NF transport at least in part by inhibiting MAPk.

Animals↗
PubMed2005

Monitoring thiobarbituric acid-reactive substances (TBARs) as an assay for oxidative damage in neuronal cultures and central nervous system.

Oxidative stress is a pivotal factor in neuronal degeneration. A simple method to quantify oxidative damage in culture and in situ is therefore important for studies of neurodegeneration. We present herein modifications of the standard assay for thiobarbituric acid-reactive substances (TBARs) for analyses of both cell cultures and brain tissue homogenates. Since the TBAR assay measures end-point oxidative damage, it is useful to assess the overall impact of oxidative stress-inducing and neuroprotective agents; interpretation is not potentially confounded by the presence or absence of transient products of oxidative damage.

Animals↗
PubMed2005

Neurofilaments can undergo axonal transport and cytoskeletal incorporation in a discontinuous manner.

Neurofilaments (NFs) are thought to provide structural support for axons. Some NFs exhibit an extended residence time along axons, the nature of which remains unclear. In prior studies in NB2a/d1 cells, hypophosphorylated NFs were demonstrated to be dispersed throughout the axon and to undergo relatively rapid axonal transport, while extensively phosphorylated NFs organized into a "bundle" localized along the center of the axon. It was not conclusively determined whether bundled NFs underwent transport or instead underwent turnover via exchange with transporting individual NFs. Herein, using transfection with multiple constructs and regional photobleaching, we demonstrate that bundled NFs undergo relatively slow transport as well as exchange with surrounding individual NFs. We also demonstrate that newly synthesized NFs disperse nonhomogenously throughout axonal neurites and perikarya. These findings provide a mechanism by which some NFs exhibit extended residence time within axons, which lessens the metabolic burden of cytoskeletal turnover.

Animals↗
PubMed2005

N-acteyl cysteine alleviates oxidative damage to central nervous system of ApoE-deficient mice following folate and vitamin E-deficiency.

Oxidative stress is an early neurodegenerative insult in Alzheimer's disease (AD). Antioxidant mechanisms, including elements of the glutathione (GSH) pathway, undergo at least a transient compensatory increase that is apparently insufficient due to continued oxidative damage during disease progression. Mice deficient in apolipoprotein E, which provide a model for some aspects of AD, undergo increased oxidative damage to brain tissue and cognitive decline when maintained on a folate-free diet, despite a compensatory increase in glutathione synthase transcription and activity as well as increased levels of GSH. Dietary supplementation with N-acetyl cysteine (1 g/kg diet), a cell-permeant antioxidant and GSH precursor, alleviated oxidative damage and cognitive decline, and restored glutathione synthase and GSH levels in ApoE-deficient mice deprived of folate to those of normal mice maintained in the presence of folate. These data support the administration of antioxidant precursors to buffer oxidative damage in neurodegenerative disorders.

Acetylcysteine↗
PubMed2005

Nanosphere-mediated delivery of vitamin E increases its efficacy against oxidative stress resulting from exposure to amyloid beta.

Oxidative stress is an early hallmark of affected neurons in Alzheimer's disease (AD). The antioxidant vitamin E provided limited neuroprotection in AD, which may have derived from its lipophilic nature and resultant inability to quench cytosolic reactive oxygen species (ROS), including those generated from antecedent membrane oxidative damage. We examined herein whether or not encapsulation into polyethylene glycol (PEG)-based nanospheres, which can enter the cytosol, improved the efficacy of vitamin E against amyloid-beta(Abeta)-induced ROS. Unexcapsulated vitamin E prevented Abeta-induced ROS in cultured SH-SY-5Y human neuroblastoma cells only if present prior to, or applied simultaneously with, Abeta treatment. By contrast, encapsulated vitamin E was equally effective if administered 1 hr after Abeta exposure. These findings suggest suggests that nanosphere-mediated delivery methods may be a useful adjunct for antioxidant therapy in AD.

Aged↗
PubMed2005

Apple juice concentrate prevents oxidative damage and impaired maze performance in aged mice.

Oxidative stress contributes to age-related cognitive decline. In some instances, consumption of fruits and vegetables rich in antioxidant can provide superior protection than supplementation with purified antioxidants. Our prior studies have shown that supplementation with apple juice concentrate (AJC) alleviates oxidative damage and cognitive decline in a transgenic murine model compromised in endogenous antioxidant potential when challenged with a vitamin-deficient, oxidative stress-promoting diet. Herein, we demonstrate that AJC, administered in drinking water, is neuroprotective in normal, aged mice. Normal mice aged either 9-10 months or 2-2.5 years were maintained for 1 month on a complete diet or a diet lacking folate and vitamin E and containing iron as a pro-oxidant, after which oxidative damage was assayed by thiobarbituric acid-reactive substances and cognitive decline as assayed by performance in a standard Y-maze. Mice 9-12 months of age were unaffected by the deficient diet, while older mice demonstrated statistically-increased oxidative damage and poorer performance in a Y maze test. Supplementation with AJC prevented these neurodegenerative effects. These data are consistent with normal aged individuals being susceptible to neurodegeneration following dietary compromise such as folate deficiency, and a hastened onset of neurodegeneration in those individuals harboring a genetic risk factor such as ApoE deficiency. These findings also support the efficacy of antioxidant supplementation, including consumption of antioxidant-rich foods such as apples, in preventing the decline in cognitive performance that accompanies normal aging.

Age Factors↗
PubMed2004

Neurofilament transport is dependent on actin and myosin.

Real-time analyses have revealed that some newly synthesized neurofilament (NF) subunits translocate into and along axonal neurites by moving along the inner plasma membrane surface, suggesting that they may translocate against the submembrane actin cortex. We therefore examined whether or not NF axonal transport was dependent on actin and myosin. Perturbation of filamentous actin in NB2a/d1 cells with cytochalasin B inhibited translocation of subunits into axonal neurites and inhibited bidirectional translocation of NF subunits within neurites. Intravitreal injection of cytochalasin B inhibited NF axonal transport in optic axons in a dose-response manner. NF subunits were coprecipitated from NB2a/d1 cells by an anti-myosin antibody, and myosin colocalized with NFs in immunofluorescent analyses. The myosin light chain kinase inhibitor ML-7 and the myosin ATPase inhibitor 2,3-butanedione-2-monoxime perturbed NF translocation within NB2a/d1 axonal neurites. These findings suggest that some NF subunits may undergo axonal transport via myosin-mediated interactions with the actin cortex.

Actins↗
PubMed2004

Reexpression of vimentin in differentiated neuroblastoma cells enhances elongation of axonal neurites.

Vimentin (Vm) is initially expressed by early neuronal precursors in situ and in culture. Vm is essential for neuritogenesis at least in culture and is gradually replaced by neurofilaments (NFs) because of down-regulation of Vm expression. This period is accompanied by a slowing of axonal elongation. We examined whether continued expression of Vm could foster continued axonal elongation. NB2a/d1 cells differentiated with dibutyryl cAMP were transfected with constructs expressing Vm or the middle-molecular-weight NF subunit (NF-M) each conjugated to green fluorescent protein (GFP). Axonal neurites of cells expressing GFP-Vm were 30% longer than those of nonexpressing cells, or cells expressing GFP-M, and exhibited a decrease in neurite caliber. Expression of GFP-M did not enhance axonal neurite length but significantly increased caliber. These findings provide further evidence of a role for Vm in axonal outgrowth. Culturing of nontransfected cells on laminin increased neurite length, but cells expressing GFP-Vm demonstrated an equivalent increase whether cultured on laminin or culture plastic. Axonal neurites of cells expressing GFP-Vm turned to avoid a nonfavorable substrate (nitrocellulose), but culturing of these cells on nitrocellulose did not impair axonal outgrowth. These latter findings indicate that the more robust outgrowth following reexpression of Vm is independent of a favorable or nonfavorable substrate but that axonal neurites of these cells still interact with the substrate to the extent that the substrate can influence directionality.

Animals↗
PubMed2004

Mitogen-activated protein kinase regulates neurofilament axonal transport.

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.

Animals↗
PubMed2004

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress.

Cyclin-dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho-NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho-NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5-mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein-tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.

Animals↗