Eugene Rogers

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.

Has enhanced folate status during pregnancy altered natural selection and possibly Autism prevalence? A closer look at a possible link

The inverse association between maternal folate status and incidence of infants born with neural tube defects (NTDs) was recognized over twenty years ago and led the US health agencies in the early 1990s to recommend that women of childbearing age consume 400 microg of folic acid each day. The FDA followed by mandating that certain foods be fortified with folic acid and this has resulted in a significant enhancement of maternal folate status to levels that are often difficult to otherwise achieve naturally. At least one study indicates that this has decreased the incidence of NTDs. However, this same time period directly coincides with what many feel is the apparent beginning and continuous increase in the prevalence of Autism and related Autism Spectrum Disorders (ASDs) in the US. Are these similar time frames of changes in maternal folate status and possible Autism prevalence a random event or has improved maternal (and fetal) folate status during pregnancy played a role? It is not only plausible but highly likely. A particular polymorphic form to a key enzyme required to activate folate for methylation in neurodevelopment, 5-methylenetetrahydrofolate reductase (MTHFR), demonstrates reduced activity under low or normal folate levels but normal activity under conditions of higher folate nutritional status. A consequence of the presence of the polymorphic form of this enzyme during normal or reduced folate status are higher plasma homocysteine levels than noncarriers and the combination of these factors have been shown in several studies to result in an increase rate of miscarriage via thrombotic events. However, the incidence of hyperhomocysteinemia in the presence of the polymorphism is reduced under the common condition of enhanced folate status and thereby masks the latent adverse effects of the presence of this enzyme form during pregnancy. Of great importance is that this polymorphism, although common in the normal population, is found in significantly higher frequency in Autisic individuals. It is hypothesized here that the enhancement of maternal folate status before and during pregnancy in the last 15 years has altered natural selection by increasing survival rates during pregnancy of infants possessing the MTHFR C677T polymorphism, via reduction in hyperhomocysteinemia associated with this genotype and thereby miscarriage rates. This also points directly to an increased rate of births of infants with higher postnatal requirements for folic acid needed for normal methylation during this critical neurodevelopmental period. If these numbers have increased then so have the absolute number of infants that after birth fail to maintain the higher folate status experienced in utero thus leading to an increased number of cases of developmental disorders such as Autism. Detection of the C677T polymorphism as well as other methionine cycle enzymes related to folate metabolism and methylation at birth as part of newborn screening programs could determine which newborns need be monitored and maintained on diets or supplements that ensure adequate folate status during this critical postnatal neurodevelopment period.

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.

Nanomaterials properties vs. biological oxidative damage: Implications for toxicity screening and exposure assessment

Biological oxidative damage (BOD) has been recognized as a key toxicity mechanism with potential as a novel global metric for nanomaterial (NM) exposure and rapid toxicity screening. A ‘Ferric reducing ability of serum (FRAS)’ assay, recently optimized by our group, was used to quantitate the degree of BOD induced by 19 diverse, commercially important NMs, including carbon blacks, fullerenes, carbon nanotubes, and titanium dioxide. This study investigated the relationship between several physico-chemical parameters of NMs and BOD and their relevance to exposure assessment and toxicity screening. FRAS-measured BOD strongly correlated with specific surface area and total content of select transition metals (especially Fe, Cr, Co, Mo and Mn). These two factors combined explained 93% of the BOD. The FRAS BOD potential of NMs appears to be a valid approach for screening purposes. These findings support the use of BOD as a metric for NM exposures.

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.

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.