Daniela Ortiz

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.

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. Cell Motil. Cytoskeleton 62:166–179, 2005. © 2005 Wiley-Liss, Inc.

Abstract The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The b-amyloid fragment (Ab) of the amyloid precursor ...

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.

Acetyl-l-carnitine (ALCAR), normally produced in mitochondria, is a precursor of acetyl-CoA in the tricarboxylic (TCA) cycle. Since mitochondrial compromise and ATP depletion have been considered to play a role in neuronal degeneration in Alzheimer's disease (AD), we examined whether ALCAR attenuated oxidative stress and/or ATP depletion after exposure of cells to beta-amyloid (Abeta), a neurotoxic peptide that accumulates in AD brain. Differentiated SH-SY-5Y human neuroblastoma cells were exposed for 2–24 h to 20 μM Abeta in the presence and absence of 50 μM ALCAR. ALCAR attenuated oxidative stress and cell death induced by Abeta neurotoxicity. Abeta depleted ATP levels, suggesting Abeta may induce neurotoxicity in part by compromising neuronal energy. ALCAR prevented ATP depletion; therefore, ALCAR may mediate its protective effect by buffering oxidative stress and maintaining ATP levels.

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The β-amyloid fragment (Aβ) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Aβ neurotoxicity. HC potentiated the Aβ-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-l-cysteine blocked apoptosis following cotreatment with HC and Aβ, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Aβ.

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. A decrease 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.

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.

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.

Homocysteine (HC) is a neurotoxic amino acid that accumulates in several neurological disorders including Alzheimer's disease (AD). We examined the consequences of treatment of cultured murine cortical neurons with HC. Homocysteine-induced increases in cytosolic calcium, reactive oxygen species, phospho-tau immunoreactivity and externalized phosphatidyl serine (indicative of apoptosis). Homocysteine-induced calcium influx through NMDA channel activation, which stimulated glutamate excitotoxicity, as evidenced by treatment with antagonists of the NMDA channel and metabotropic glutamate receptors, respectively. The NMDA channel antagonist MK-801 reduced tau phosphorylation but not apoptosis after HC treatment, suggesting that HC-mediated apoptosis was not due to calcium influx. Apoptosis after HC treatment was reduced by co-treatment with 3-aminobenazmidine (3ab), an inhibitor of poly-ADP-ribosome polymerase (PARP), consistent with previous reports that ATP depletion by PARP-mediated repair of DNA strand breakage mediated HC-induced apoptosis. Treatment with 3ab did not reduce tau phosphorylation, however, therefore hyperphosphorylation of tau may not contribute to HC-induced apoptosis under these conditions. Inhibition of mitogen-activated protein kinase by co-treatment with the kinase inhibitor PD98059 inhibited tau phosphorylation but not apoptosis after HC treatment. HC accumulation reduces cellular levels of S-adenosyl methionine (SAM); co-treatment with SAM reduced apoptosis, suggesting that inhibition of critical methylation reactions may mediate HC-induced apoptosis. These findings indicate that HC compromises neuronal homeostasis by multiple, divergent routes. © 2002 Wiley-Liss, Inc.

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.

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. © 2004 Wiley-Liss, Inc.

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.