Asma Zaidi

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Age-related decrease in brain synaptic membrane Ca2+-ATPase in F344/BNF1 rats.

We used Fisher 344/Brown Norway hybrid rats (F344/BNF1) to determine whether previously reported decreases in brain synaptic plasma membrane (SPM) Ca2+-ATPase activity in inbred F344 rats also occurred in the hybrids. Plasma membrane Ca2+-ATPase (PMCA) activity in SPMs from F344/BNF1 rat brains showed a progressive age-dependent decrease in Vmax from 60.9 +/- 3.7 nmol Pi/mg/min (n = 6) in 5-month rats to 32.4 +/- 3.6 nmol Pi/mg/min (n = 6) in 34-month animals, with no change in K (act) for Ca2+. Immunoreactive PMCA in SPMs also decreased by approximately 20% at 34 months, and the calmodulin (CaM) bound to membranes following extraction with EDTA also declined progressively with age. The effectiveness of CaM in stimulating PMCA activity was significantly lower when CaM was purified from the brains of old vs. young F344 rats and when CaM from 5-month rats was oxidized in vitro. These results indicate: 1) that PMCA activity in SPMs from longer lived F344/BNF1 hybrids also decreases with age; 2) that part of the reduction in PMCA activity is due to loss of PMCA from the membranes; and 3) that age-related structural changes in CaM may decrease its interaction with proteins in SPMs.

Genomic and biochemical approaches in the discovery of mechanisms for selective neuronal vulnerability to oxidative stress

BackgroundOxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches.ResultsIn this report, using in vitro neuronal cultures, ex vivo organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between in vivo vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons.ConclusionLow energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons.

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons : effects of cholesterol depletion

Spatial and temporal alterations in intracellular calcium [Ca2+]i play a pivotal role in a wide array of neuronal functions. Disruption in Ca2+ homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca2+‐ATPase (PMCA) is a high affinity Ca2+ transporter that plays a crucial role in the termination of [Ca2+]i signals and in the maintenance of low [Ca2+]i essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid‐ordered, cholesterol‐rich plasma membrane microdomains or ‘lipid rafts’ in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft‐associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft‐associated PMCA with no effect on the activity of the non‐raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca2+ signaling in the central nervous system.

Effects of paraquat-induced oxidative stress on the neuronal plasma membrane Ca2+-ATPase.

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca(2+)](i)) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in regulating [Ca(2+)](i). Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 microM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 microM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca(2+) transporter under prolonged oxidative stress could alter neuronal Ca(2+) signaling.

Decreased activity and increased aggregation of brain calcineurin during aging.

Age-related decline in strength of synaptic transmission and memory formation has been attributed to age-associated increases in the activity of calcineurin (Cn) in hippocampus neurons. In the present study, we examined how brain Cn activity, Cn subunit levels, and Cn protein oxidation were changing during the aging process. Cn activity decreased with advancing age in three brain subcellular fractions, homogenate, cytosol, and synaptic membranes, obtained from F344/BNF1 rats of 5-6, 22-24, and 34-36 months of age. Cn activity also decreased during aging in homogenate, cytosol, and a nerve ending-enriched fraction from the hippocampus. Cn protein levels in homogenate and cytosol, as determined by the immune reactivity of its subunits A and B, were not altered during aging. But, in synaptic membranes, there was an age-related decrease in CnA levels, but not of CnB. Another important observation was that of an oxidative modification of CnA, not CnB, with increasing age. Such modification caused the formation of large aggregates of CnA. Aggregate formation was due to SH-group oxidation as the monomeric form of CnA was recovered upon disulfide reduction of the proteins with dithiothreitol. The age-related formation of aggregates of the catalytic subunit of Cn was suggestive of a correlation between aggregate formation and diminished enzyme activity. The loss of Cn activity may alter signal transduction at synapses during the aging process.

RNAi– induced silencing of the plasma membrane Ca2+– ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca2+]i) regulation is altered in aging brain, possibly because of the changes in critical Ca2+ transporters. We previously reported that the levels of the plasma membrane Ca2+‐ATPase (PMCA) and the Vmax for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNAi techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH‐SY5Y neuroblastoma cells were transfected with in vitro– transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca2+]i revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca2+]i, slower recovery from KCl‐induced Ca2+ transients, and incomplete return to pre‐stimulation Ca2+ levels. Primary neurons and SH‐SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca2+ signaling and enhanced susceptibility to stresses that can alter the regulation of Ca2+ homeostasis.

Antisense oligonucleotide suppression of Na+/Ca2+exchanger activity in primary neurons from rat brain

An antisense (AS) oligodeoxynucleotide based on a conserved sequence in the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) was used to decrease expression of this Ca(2+) transporter in primary neuronal cultures. Two AS oligo applications decreased NCX activity by approximately 40% within 12-24 h, and neither sense (S) or missense (MS) oligos altered NCX activity. The reduced NCX expression was confirmed by immunoblots and enzyme-linked immunosorbent assays (ELISAs). Resting [Ca(2+)](i) levels were 20% higher in AS-treated neurons and showed a slower return to baseline levels following activation of Ca(2+) influx by N-methyl-D-aspartate (NMDA). These results suggest that NCX plays a significant role in maintaining neuronal Ca(2+) homeostasis and in restoring baseline Ca(2+) levels following depolarization.

Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1-Cn protein interactions occurring in vitro and in vivo

Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2*-). The effects of O2*- on Cn are still controversial. We found that O2*-, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1-rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2*- dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase K(m) without a change in V(max). A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

Decrease in Ca-ATPase activity in aged synaptosomal membranes is not associated with changes in fatty acyl chain dynamics.

We have examined lipid peroxidation (LPO) and fatty acid acyl chain dynamics in synaptosomal membranes isolated from aged rat (Fischer 344 x Brown Norway F1 hybrids) brains, correlating these results with measurements of enzymatic activity of the synaptic plasma membrane Ca2(+)-ATPase (PMCA). Calcium-dependent ATPase activity in these membranes exhibits progressive decreases with a maximal loss of activity with age of approximately 35%. The sensitivity of this membrane-bound ion transporter to the lipid composition of the surrounding membrane, as well as the high abundance of oxidatively sensitive polyunsaturated fatty acyl chains in synaptosomal membranes, suggests that this age-related loss in catalytic turnover may result from LPO-mediated protein modification and/or changes in the physical structure of the bilayer. However, high-performance liquid chromatography analysis of 2,4-dinitrophenylhydrazone derivatives reveals no significant age-related increases in the content of reactive aldehydes (malondialdehyde, formaldehyde, acetaldehyde or acetone) which comprise breakdown products of lipid peroxidation. Electron paramagnetic resonance measurements employing 5- and 12-stearic acid spin labels with the nitroxide reporter groups at two depths in the bilayer were used to assess the fatty acyl chain dynamics (fluidity) of synaptosomal membranes. The resulting spectra demonstrate anisotropic lipid dynamics of two populations of lipids, i.e. lipids in direct association with membrane proteins (boundary lipids) and bulk lipids that do not directly associate with proteins. The nanosecond dynamics of both lipid populations is unaltered with age indicating that any compositional changes occurring with age are insufficient to result in alterations in bilayer fluidity relevant to PMCA activity. Thus, the observed age-related decline in PMCA activity may be explained by direct modification of membrane protein.