Satyabrata Kar

Amyloid β peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures

The neuropathological features associated with Alzheimers disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta protein (Abeta), intracellular neurofibrillary tangles containing phosphorylated tau protein and the loss of basal forebrain cholinergic neurons which innervate regions such as the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Abeta accumulation in vivo may initiate phosphorylation of tau protein, which by disrupting neuronal network may trigger the process of neurodegeneration observed in AD brains. However, the underlying cause of degeneration of the basal forebrain cholinergic neurons and their association, if any, to Abeta peptides or phosphorylated tau remains mostly unknown. In the present study, using rat primary septal cultures, we have shown that aggregated Abeta peptides, in a time (18-96 h)- and concentration (0.7-60 microM)-dependent manner, induce toxicity and decrease choline acetyltransferase enzyme activity in cultured neurons. Using immunocytochemistry and immunoblotting, we have also demonstrated that Abeta treatment can significantly increase the phosphorylation of tau protein in septal cultures. At the cellular level, hyperphosphorylated tau is mostly apparent in the somatodendritic compartment of the neurons. Abeta peptide (10 microM), in addition to tau phosphorylation, also activates mitogen-activated protein kinase and glycogen synthase kinase-3beta, the two kinases which are known to be involved in the formation of hyperphosphorylated tau in the AD brain. Exposure to specific inhibitors of the mitogen-activated protein kinase (i.e. PD98059) or glycogen synthase kinase-3beta (i.e. LiCl) attenuated the hyperphosphorylation of the tau protein in cultured neurons. Given the evidence that tau phosphorylation can induce cell loss by disrupting neuronal cytoskeleton, it is likely that aggregated Abeta peptide triggers degeneration of septal neurons, including those expressing the cholinergic phenotype, by phosphorylation of the tau protein activated by mitogen-activated protein kinase and glycogen synthase kinase-3beta. These results, taken together, suggest that cultured septal cholinergic neurons are vulnerable to Abeta-mediated toxicity and tau phosphorylation may play an important role in Abeta-induced neurodegeneration.

β-Amyloid peptides as direct cholinergic neuromodulators: a missing link?

Beta-Amyloid peptide (Abeta) is found in diffuse and focal deposits throughout the brain from Alzheimers disease (AD) patients. Another feature of AD is the widespread degeneration and dysfunction of the basal-forebrain cholinergic system. Until now, it has been unclear how these features of AD might be related. Recent reports, however, suggest that Abeta can potently inhibit various cholinergic neurotransmitter functions independently of apparent neurotoxicity. This capacity of Abeta might contribute to the vulnerability of selected cholinergic neuronal populations in AD. Moreover, the high potency (picomolar to nanomolar concentrations) of these effects and the secretion of Abeta by brain cells indicate that Abeta-induced cholinergic hypoactivity might have physiological in addition to pathological significance.

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimers disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β‐peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ‐related peptides, under acute conditions, potently inhibit K+‐evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ‐mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high‐affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ1–40 (10−8M) potently inhibited veratridine‐evoked endogenous ACh release from rat hippocampal slices and also decreased the K+‐evoked release potentiated by the nitric oxide‐generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10−14 to 10−6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10−6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.

Insulin-like Growth Factor-1-induced Phosphorylation of the Forkhead Family Transcription Factor FKHRL1 Is Mediated by Akt Kinase in PC12 Cells

The Forkhead family transcription factor FKHRL1, a mammalian homolog of DAF16 in the nematodeCaenorhabditis elegans, is an inducer of apoptosis in its unphosphorylated form and was recently reported as a substrate of Akt kinases. Insulin-like growth factor (IGF-1) is a potent stimulant of Akt kinase, leading to inhibition of the apoptotic pathway. In this study, we characterized the phosphorylation of FKHRL1 induced by IGF-1 in PC12 cells and various neuronal cell types and examined the potential role of Akt in this regard. IGF-1 rapidly induced the phosphorylation of Akt and FKHRL1 in PC12 cells. The phosphorylation of Akt and FKHRL1 induced by 10 nm IGF-1 was inhibited by the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (0.25–2 μm) and LY294002 (12.5–100 μm), but not by the MEK inhibitor PD98059 (50 μm) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the phosphorylation of FKHRL1 induced by IGF-1 is mediated by the PI3K pathway. As observed for IGF-1, an in vitro kinase assay with purified active Akt kinase demonstrated that the kinase is capable of directly phosphorylating FKHRL1 at Thr32 and Ser253, leading to inhibition of its pro-apoptotic properties. Moreover, transient expression of constitutively active Akt (MS-Akt, where MS is a myristylation signal) increased the phosphorylation of FKHRL1, whereas the expression of kinase-dead Akt (M179A Akt) attenuated the phosphorylation of FKHRL1 induced by 10 nm IGF-1 in PC12 cells. Interestingly, FKHRL1 co-immunoprecipitated with Akt in PC12 cells, indicating that these two proteins can associate in these cells. As IGF-1 also induced the phosphorylation of FKHRL1 in primary cortical and cerebellar neuronal cultures, these data, taken together, demonstrate that IGF-1, acting via the PI3K/Akt kinase pathway, can regulate the phosphorylation of FKHRL1, leading to inhibition of this apoptotic transcription factor in neuronal cells.

Memantine protects rat cortical cultured neurons against β-amyloid-induced toxicity by attenuating tau phosphorylation

It has been suggested that accumulation of beta‐amyloid (Aβ) peptide triggers neurodegeneration, at least in part, via glutamate‐mediated excitotoxicity in Alzheimer’s disease (AD) brain. This is supported by observations that toxicity induced by Aβ peptide in cultured neurons and in adult rat brain is known to be mediated by activation of glutamatergic N‐methyl‐d‐aspartate (NMDA) receptors. Additionally, recent clinical studies have shown that memantine, a noncompetitive NMDA receptor antagonist, can significantly improve cognitive functions in some AD patients. However, very little is currently known about the potential role of memantine against Aβ‐induced toxicity. In the present study, we have shown that Aβ1–42‐induced toxicity in rat primary cortical cultured neurons is accompanied by increased extracellular and decreased intracellular glutamate levels. We subsequently demonstrated that Aβ toxicity is induced by increased phosphorylation of tau protein and activation of tau kinases, i.e. glycogen synthase kinase‐3β and extracellular signal‐related kinase 1/2. Additionally, Aβ treatment induced cleavage of caspase‐3 and decreased phosphorylation of cyclic AMP response element binding protein, which are critical in determining survival of neurons. Memantine treatment significantly protected cultured neurons against Aβ‐induced toxicity by attenuating tau‐phosphorylation and its associated signaling mechanisms. However, this drug did not alter either conformation or internalization of Aβ1–42 and it was unable to attenuate Aβ‐induced potentiation of extracellular glutamate levels. These results, taken together, provide new insights into the possible neuroprotective action of memantine in AD pathology.

Internalization of β-Amyloid Peptide by Primary Neurons in the Absence of Apolipoprotein E

Extracellular accumulation of β-amyloid peptide (Aβ) has been linked to the development of Alzheimer disease. The importance of intraneuronal Aβ has been recognized more recently. Although considerable evidence indicates that extracellular Aβ contributes to the intracellular pool of Aβ, the mechanisms involved in Aβ uptake by neurons are poorly understood. We examined the molecular mechanisms involved in Aβ-(1–42) internalization by primary neurons in the absence of apolipoprotein E. We demonstrated that Aβ-(1–42) is more efficiently internalized by axons than by cell bodies of sympathetic neurons, suggesting that Aβ-(1–42) uptake might be mediated by proteins enriched in the axons. Although the acetylcholine receptor α7nAChR, previously suggested to be involved in Aβ internalization, is enriched in axons, our results indicate that it does not mediate Aβ-(1–42) internalization. Moreover, receptors of the low density lipoprotein receptor family are not essential for Aβ-(1–42) uptake in the absence of apolipoprotein E because receptor-associated protein had no effect on Aβ uptake. By expressing the inactive dynamin mutant dynK44A and the clathrin hub we found that Aβ-(1–42) internalization is independent of clathrin but dependent on dynamin, which suggests an endocytic pathway involving caveolae/lipid rafts. Confocal microscopy studies showing that Aβ did not co-localize with the early endosome marker EEA1 further support a clathrin-independent mechanism. The lack of co-localization of Aβ with caveolin in intracellular vesicles and the normal uptake of Aβ by neurons that do not express caveolin indicate that Aβ does not require caveolin either. Instead partial co-localization of Aβ-(1–42) with cholera toxin subunit B and sensitivity to reduction of cellular cholesterol and sphingolipid levels suggest a caveolae-independent, raft-mediated mechanism. Understanding the molecular events involved in neuronal Aβ internalization might identify potential therapeutic targets for Alzheimer disease.

Distribution and levels of [125I]IGF-I, [125I]IGF-II and [125I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats

The insulin-like growth factors (IGF-I and IGF-II) and insulin are localized within distinct brain regions and their respective functions are mediated by specific membrane receptors. High densities of binding sites for these growth factors are discretely and differentially distributed throughout the brain, with prominent levels localized to the hippocampal formation. IGFs and insulin, in addition to their growth promoting actions, are considered to play important roles in the development and maintenance of normal cell functions throughout life. We compared the anatomical distribution and levels of IGF and insulin receptors in young (five month) and aged (25 month) memory-impaired and memory-unimpaired male Long Evans rats as determined in the Morris water maze task in order to determine if alterations in IGF and insulin activity may be related to the emergence of cognitive deficits in the aged memory-impaired rat. In the hippocampus, [125I]IGF-I receptors are concentrated primarily in the dentate gyrus (DG) and the CA3 sub-field while high amounts of [125I]IGF-II binding sites are localized to the pyramidal cell layer, and the granular cell layer of the DG. [125I]insulin binding sites are mostly found in the molecular layer of the DG and the CA1 sub-field. No significant differences were found in [125I]IGF-I. [125I]IGF-II or [125I]insulin binding levels in any regions or laminae of the hippocampus of young vs aged rats. and deficits in cognitive performance did not relate to altered levels of these receptors in aged memory-impaired vs aged memory-unimpaired rats. Other regions. including various cortical areas, were also examined and failed to reveal any significant differences between the three groups studied. It thus appears that IGF-I, IGF-II and insulin receptor sites are not markedly altered during the normal ageing process in the Long Evans rat, in spite of significant learning deficits in a sub-group (memory-impaired) of aged animals. Hence. recently reported changes in IGF-I receptor messenger RNA levels in aged memory-impaired rats are apparently not reflected at the level of the translated protein.

Insulin-like growth factor-1 (IGF-1): a neuroprotective trophic factor acting via the Akt kinase pathway

Insulin-like growth factor-I (IGF-I) is a pleiotropic polypeptide with a wide range of actions in both central and peripheral nervous sytems. Over the past few years, we studied the trophic as well as neuromodulatory roles of IGF-I in the brain. Accumulated evidence indicates that IGF-I, apart from regulating growth and development, protects neurons against cell death induced by amyloidogenic derivatives, glucose or serum deprivation via the activation of intracellular pathways implicating phosphatidylinositide 3/Akt kinase, winged-helix family of transcription factor FKHRL1 phosphorylation or production of free radicals. The effects of IGF-I on neuroprotection, glucose metabolism and activity-dependent plasticity suggest the potential usefulness of this growth factor or related mimetics in the treatment of Alzheimers disease and other neurodegenerative disorders.

Estrogen effects on object memory and cholinergic receptors in young and old female mice

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [(3)H]pirenzepine/M(1)-like and [(3)H]AFDX384/M(2)-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors.

FKHRL1 and its homologs are new targets of nerve growth factor Trk receptor signaling

We report that the Forkhead family of transcription factors, FKHRL1, FKHR and AFX are novel components of neurotrophin receptor signaling. NGF rapidly induced the phosphorylation of FKHRL1 in PC12 cells. This effect is mediated by high‐affinity TrkA receptor as nerve growth factor (NGF) induced the phosphorylation of FKHRL1 only in TrkA expressing cells and not p75‐expressing cells. Additional experiments with various kinase inhibitors, the transient expression of constitutively active and dominant‐negative Akt, and in vitro kinase assay revealed that phosphatidylinositol‐3 (PtdIns3)/Akt kinase mediated the actions of NGF. Similar data were obtained for brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and neurotrophin‐4 (NT‐4) in primary cortical cultured neurons. These findings demonstrate for the first time that the phosphorylation of the Forkhead family of transcription factors can be modulated by neurotrophins via Trk receptors and PtdIns3K/Akt kinase (but not MAP or S6p70 kinases) in neuronal and non‐neuronal cells. Moreover, survival assays with the PtdIns3 kinase inhibitor LY294002, active and dominant‐negative forms of Akt indicate that the phosphorylation of FKHRL1 plays a role in neurotrophins‐mediated cell survival.