Daniela Ortiz

Multiple aspects of homocysteine neurotoxicity: Glutamate excitotoxicity, kinase hyperactivation and DNA damage

Homocysteine (HC) is a neurotoxic amino acid that accumulates in several neurological disorders including Alzheimers 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.

Homocysteine potentiates β‐amyloid neurotoxicity: role of oxidative stress

The cause of neuronal degeneration in Alzheimers 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.

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

Clinical studies suggest a relationship between folate deficiency and neurological and disorders including Alzheimers 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.

Apolipoprotein E deficiency promotes increased oxidative stress and compensatory increases in antioxidants in brain tissue

The epsilon 4 allele of the apolipoprotein E gene (ApoE) is associated with Alzheimers disease (AD). The extent of oxidative damage in AD brains correlates with the presence of the E4 allele of ApoE, suggesting an association between the ApoE4 genotype and oxygen-mediated damage in AD. We tested this hypothesis by subjecting normal and transgenic mice lacking ApoE to oxidative stress by folate deprivation and/or excess dietary iron. Brain tissue of ApoE-deficient mice displayed increased glutathione and antioxidant levels, consistent with attempts to compensate for the lack of ApoE. Folate deprivation and iron challenge individually increased glutathione and antioxidant levels in both normal and ApoE-deficient brain tissue. However, combined treatment with folate deprivation and dietary iron depleted antioxidant capacity and induced oxidative damage in ApoE-deficient brains despite increased glutathione, indicating an inability to compensate for the lack of ApoE under these conditions. These data support the hypothesis that ApoE deficiency is associated with oxidative damage, and demonstrate a combinatorial influence of genetic predisposition, dietary deficiency, and oxidative stress on oxidative damage relevant to AD.

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 Alzheimers 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 Alzheimers therapy.

Acetyl-L-carnitine protects against amyloid-beta neurotoxicity: roles of oxidative buffering and ATP levels.

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 Alzheimers 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.

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.

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.

Amyloid-β promotes calcium influx and neurodegeneration via stimulation of L voltage-sensitive calcium channels rather than NMDA channels in cultured neurons

Exposure of cultured neurons and neuronal cells to aggregated amyloid-beta (Abeta) induces multiple neurodegenerative events including accumulation of cytosolic calcium, generation of reactive oxygen species, abnormal levels of phosphorylation of the microtubule-associated protein tau, and apoptosis. Prevention of accumulation of calcium within the cytosol also prevents all other events, suggesting that calcium accumulation is an early and pivotal event in Abeta neurotoxicity. Calcium influx has been suggested to occur via L voltage-sensitive calcium channels or NMDA channels. Calcium influx into differentiated human neuroblastoma cells has been previously attributed to the L voltage-sensitive calcium channel, but the contribution of the NMDA channel was not examined. In the present study, treatment of these cells with MK-801, an antagonist of NMDA channels, failed to attenuate Abeta-induced calcium influx or neurodegeneration, while nimopridine, an antagonist of the L voltage-sensitive calcium channel, blocked Abeta-induced calcium influx. Our findings suggest that NMDA channels do not contribute significantly to Abeta neurotoxicity in these acute cell culture analyses.

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.