Homocysteine potentiates β-amyloid neurotoxicity: role of oxidative stress

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 beta-amyloid fragment (Abeta) 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 Abeta neurotoxicity. HC potentiated the Abeta-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 Abeta, 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 Abeta.     

Safety and reactogenicity of canarypox ALVAC-HIV (vCP1521) and HIV-1 gp120 AIDSVAX B/E vaccination in an efficacy trial in Thailand

Background

A prime-boost vaccination regimen with ALVAC-HIV (vCP1521) administered intramuscularly at 0, 4, 12, and 24 weeks and gp120 AIDSVAX B/E at 12 and 24 weeks demonstrated modest efficacy of 31.2% for prevention of HIV acquisition in HIV-uninfected adults participating in a community-based efficacy trial in Thailand.

Methodology/Principal Findings

Reactogenicity was recorded for 3 days following vaccination. Adverse events were monitored every 6 months for 3.5 years, during which pregnancy outcomes were recorded. Of the 16,402 volunteers, 69% of the participants reported an adverse event any time after the first dose. Only 32.9% experienced an AE within 30 days following any vaccination. Overall adverse event rates and attribution of relatedness did not differ between groups. The frequency of serious adverse events was similar in vaccine (14.3%) and placebo (14.9%) recipients (p = 0.33). None of the 160 deaths (85 in vaccine and 75 in placebo recipients, p = 0.43) was assessed as related to vaccine. The most common cause of death was trauma or traffic accident. Approximately 30% of female participants reported a pregnancy during the study. Abnormal pregnancy outcomes were experienced in 17.1% of vaccine and 14.6% (p = 0.13) of placebo recipients. When the conception occurred within 3 months (estimated) of a vaccination, the majority of these abnormal outcomes were spontaneous or elective abortions among 22.2% and 15.3% of vaccine and placebo pregnant recipients, respectively (p = 0.08). Local reactions occurred in 88.0% of vaccine and 61.0% of placebo recipients (p<0.001) and were more frequent after ALVAC-HIV than AIDSVAX B/E vaccination. Systemic reactions were more frequent in vaccine than placebo recipients (77.2% vs. 59.8%, p<0.001). Local and systemic reactions were mostly mild to moderate, resolving within 3 days.

Conclusions/Significance

The ALVAC-HIV and AIDSVAX B/E vaccine regimen was found to be safe, well tolerated and suitable for potential large-scale use in Thailand.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00223080","term_id":"NCT00223080"}}NCT00223080     

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 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 M Abeta in the presence and absence of 50 M 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.     

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.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.