Ved Chauhan

Interaction of amyloid beta-protein with anionic phospholipids: possible involvement of Lys28 and C-terminus aliphatic amino acids.

Fibrillar amyloid beta-protein (Aβ) is the major protein of amyloid plaques in the brains of patients with Alzheimers disease (AD). The mechanism by which normally produced soluble Aβ gets fibrillized in AD is not clear. We studied the effect of neutral, zwitterionic, and anionic lipids on the fibrillization of Aβ 1-40. We report here that acidic phospholipids such as phosphatidic acid, phosphatidylserine, phosphatidylinositol (PI), PI 4-phosphate, PI 4,5-P2 and cardiolipin can increase the fibrillization of Aβ, while the neutral lipids (diacylglycerol, cholesterol, cerebrosides), zwitterionic lipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) and anionic lipids lacking phosphate groups (sulfatides, gangliosides) do not affect Aβ fibrillization. Aβ was found to increase the fluorescence of 1-acyl-2-[12-[ (7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-glycero-3-phosphate (NBD-PA) in a concentration-dependent manner, while no change was observed with 1-acyl-2-[12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-glycero-3-phosphoethanolamine (NBD-PE). Under similar conditions, other proteins such as apolipoprotein E, gelsolin and polyglutamic acid did not interact with NBD-PA. The order of interaction of amyloid β-peptides with NBD-PA was Aβ 1-43 = Aβ 1-42 = Aβ 17-42 > Aβ 1-40 = Aβ 17-40. Other Aβ peptides such as Aβ 1-11, Aβ 1-16, Aβ 1-28, Aβ 1-38, Aβ 12-28, Aβ 22-35, Aβ 25-35, and Aβ 31-35 did not increase the NBD-PA fluorescence. These results suggest that phosphate groups, fatty acids, and aliphatic amino acids at the C-terminus end of Aβ 1-40/Aβ 1-42 are essential for the interaction of Aβ with anionic phospholipids, while hydrophilic Aβ segment from 1-16 amino acids does not participate in this interaction. Since positively charged amino acids in Aβ are necessary for the interaction with negatively charged phosphate groups of phospholipids, it is suggested that Lys28 of Aβ may provide anchor for the phosphate groups of lipids, while aliphatic amino acids (Val-Val-Ile-Ala) at the C-terminus of Aβ interact with fatty acids of phospholipids.

Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils

Amyloid beta-protein (Abeta) is present in soluble form in the plasma and cerebrospinal fluid (CSF) of normal people and patients with Alzheimers disease (AD). However, in AD patients, Abeta gets fibrillized as the main constituent of amyloid plaques in the brain. Soluble synthetic Abeta also forms amyloid-like fibrils when it is allowed to age. The mechanism that prevents soluble Abeta from fibrillization in biological fluids is not clear. We recently reported that gelsolin, a secretory protein, binds to Abeta, and that gelsolin/Abeta complex is present in the plasma [V.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Biochem. Biophys. Res. Commun. 258 (1999) 241-246.]. We now studied the effect of gelsolin on Abeta fibrillization. Congo red staining and electron microscopic examination in negative staining of aged samples of Abeta alone and Abeta incubated with gelsolin showed that gelsolin inhibits the fibrillization of synthetic Abeta 1-40 and Abeta 1-42 at gelsolin to Abeta molar ratio of 1:40. In addition, gelsolin also defibrillized the preformed fibrils of Abeta 1-40 and Abeta 1-42 in a time-dependent manner. These results suggest that gelsolin functions as an anti-amyloidogenic protein in the plasma and CSF, where it prevents Abeta from fibrillization, and helps to maintain it in the soluble form.

BDNF‐Akt‐Bcl2 antiapoptotic signaling pathway is compromised in the brain of autistic subjects

Although the pathogenesis of autism is not understood, emerging evidence points to apoptotic mechanisms being involved in this disorder. However, it is not known whether apoptosis signaling is deregulated in the brain of autistic subjects. This study investigates how the apoptosis‐related proteins are regulated in the autistic brain. Our studies show that Bcl2 is significantly decreased, whereas the expression of p53 is increased, in the brain of autistic subjects in comparison with age‐matched controls. We also found that the expression and phosphorylation/activation of Akt kinase that regulates Bcl2 are significantly decreased in the autistic brain. The down‐regulation of Akt may result from a decreased concentration of brain‐derived neurotrophic factor (BDNF), the growth factor that modulates Akt activities. These results suggest that down‐regulation of the BDNF‐Akt‐Bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism.

Action of amyloid beta-protein on protein kinase C activity.

Amyloid beta-protein (A beta), the major protein of cerebrovascular and plaque amyloid in Alzheimer disease, is considered a primary factor in the pathology of this disease. The effect of synthetic A beta (1-40) on the activity of protein kinase C (PKC) was studied with histones for a substrate in a mixed micellar assay, and with calmodulin-depleted soluble brain proteins in a liposomal system. We report here that A beta affects PKC activity in a biphasic manner. An initial stimulation of PKC was noted at low concentrations of A beta (less than 2.5 microM); while PKC-inhibition was observed in a concentration-dependent manner at higher concentrations of A beta. The in vitro phosphorylation of 20, 47, and 87 kDa brain proteins (known PKC substrates) was significantly reduced by 60 microM A beta. The role of 20 kDa in memory storage, of 87 kDa in neurotransmission and neurosecretory processes, and of 47 kDa in long-term potentiation or memory is well recognized, and A beta is known to have both neurotrophic and neurotoxic effects. Since PKC plays an important role in neuronal function, it is suggested that dual modulation of PKC by A beta may be linked to its neurotrophic and neurotoxic effects. We propose that at low concentrations A beta, by stimulating PKC, may contribute to neurites generation; and at higher concentrations A beta, by inhibiting PKC activity, might lead first to memory impairment, and then to neuronal loss.

Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C.

Phosphatidylserine/calcium-dependent protein kinase C (PKC) from rat brain is activated fifty times more efficiently by phosphatidylinositol-4,5-bisphosphate (PIP2) (Kapp = 0.04 mole% in Triton-lipid micelles) than by diacylglycerol (DG) (Kapp = 2 mole%). Both effector lipids appear to bind to the same site but PIP2 may confer a narrower substrate specificity on the kinase. DG, which together with inositol trisphosphate (IP3) is generated by hydrolysis from PIP2 after cell stimulation, has been considered the natural activator of the kinase but it is likely to be anteceded in this function by PIP2; DG may perhaps retain the function of a back-up activator. The lack of PKC-activation by phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP) opens the possibility that the Inositide Shuttle, PI reversible PIP reversible PIP2, has a role in controlling the activity of the kinase.

Anti-amyloidogenic, anti-oxidant and anti-apoptotic role of gelsolin in Alzheimer's disease.

Fibrillar amyloid beta-protein (Aβ) is a major component of amyloid plaques in the brains of individuals with Alzheimer’s disease (AD) and of adults with Down syndrome (DS). Gelsolin, a cytoskeletal protein, is present both intracellularly (cytoplasmic form) and extracellularly (secretory form in biological fluids). These two forms of gelsolin differ from each other in length and in cysteinyl thiol groups. Previous studies from our and other groups have identified the anti-amyloidogenic role of gelsolin in AD. Our studies showed that both plasma and cytosolic gelsolin bind to Aβ, and that gelsolin inhibits the fibrillization of Aβ and solubilizes preformed fibrils of Aβ. Other studies have shown that peripheral administration of plasma gelsolin or transgene expression of plasma gelsolin can reduce amyloid load in the transgenic mouse model of AD. Our recent studies showed that gelsolin expression increases in cells in response to oxidative stress. Oxidative damage is considered a major feature in the pathophysiology of AD. Aβ not only can induce oxidative stress, but also its generation is increased as a result of oxidative stress. In this article, we review evidence of gelsolin as an anti-amyloidogenic agent that can reduce amyloid load by acting as an inhibitor of Aβ fibrillization, and as an antioxidant and anti-apoptotic protein.

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.

Amyloid β-protein stimulates casein kinase I and casein kinase II activities

Abstract Amyloid β-protein (Aβ) is the major protein of cerebrovascular and plaque amyloid in Alzheimers disease (AD). Extensive evidence has demonstrated abnormal protein phosphorylation in this disease. We investigated the effect of synthetic Aβ with the amino-acid sequence corresponding to cerebrovascular Aβ and plaque Aβ on the activities of casein kinase I (CK I) and casein kinase II (CK II). These enzymes were purified from bovine brain and casein was used as a substrate. Aβ was found to stimulate markedly CK I- and CK II-mediated phosphorylation of casein in a concentration-dependent manner. The effect of plaque Aβ was considerably higher than that of cerebrovascular Aβ. Heparin, which is known to be a specific inhibitor of CK II, completely inhibited Aβ-stimulated CK II activity. Aβ itself was not a substrate for casein kinases. These findings were confirmed using other substrates for CK I and CK II. The experiments with synthetic CK II-substrate peptide (Leu-Glu-Leu-Ser-Asp-Asp-Asp-Asp-Glu) and the phosphorylation of erythrocyte membrane proteins by intrinsic membrane-bound CK I in erythrocytes showed marked stimulation in activities of casein kinases in the presence of Aβ 1–40 or blocked Aβ. We propose that Aβ, by stimulating casein kinases, may contribute to abnormal protein phosphorylation in AD, in particular to increased phosphorylation of microtubule-associated proteins, leading to the neurofibrillary tangles formation and neurodegeneration in this disease. Interaction of Aβ with protein kinases, thus, may characterize the beginning of the disease.

Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

Bisphenol A induces oxidative stress and mitochondrial dysfunction in lymphoblasts from children with autism and unaffected siblings.

Autism is a behaviorally defined neurodevelopmental disorder. Although there is no single identifiable cause for autism, roles for genetic and environmental factors have been implicated in autism. Extensive evidence suggests increased oxidative stress and mitochondrial dysfunction in autism. In this study, we examined whether bisphenol A (BPA) is an environmental risk factor for autism by studying its effects on oxidative stress and mitochondrial function in the lymphoblasts. When lymphoblastoid cells from autistic subjects and age-matched unaffected sibling controls were exposed to BPA, there was an increase in the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential in both groups. A further subdivision of the control group into two subgroups-unaffected nontwin siblings and twin siblings-showed significantly higher ROS levels without any exposure to BPA in the unaffected twin siblings compared to the unaffected nontwin siblings. ROS levels were also significantly higher in the autism vs the unaffected nontwin siblings group. The effect of BPA on three important mtDNA genes-NADH dehydrogenase 1, NADH dehydrogenase 4, and cytochrome b-was analyzed to observe any changes in the mitochondria after BPA exposure. BPA induced a significant increase in the mtDNA copy number in the lymphoblasts from the unaffected siblings group and in the unaffected twin siblings group vs the unaffected nontwin siblings. In all three genes, the mtDNA increase was seen in 70% of the subjects. These results suggest that BPA exposure results in increased oxidative stress and mitochondrial dysfunction in the autistic subjects as well as the age-matched sibling control subjects, particularly unaffected twin siblings. Therefore, BPA may act as an environmental risk factor for autism in genetically susceptible children by inducing oxidative stress and mitochondrial dysfunction.