Elevated hepatic and depressed renal cytochrome P450 activity in the Tg2576 transgenic mouse model of Alzheimer's disease.

Recent studies indicate that the Tg2576 transgenic mouse model of Alzheimer's disease [tg(hAPP)] demonstrates disturbances in plasma glucose and neuroendocrine function reminiscent of Alzheimer's disease (AD). Alterations in any one of these systems can have a profound effect on hepatic cytochrome P450 (CYP) expression. Additionally, the recent discovery that amyloid beta 1-42 can induce the expression of CYP reductase in neuronal cultures further suggests that hepatic CYP-related metabolism may be affected by the expression of mutant human amyloid precursor protein in these tg(hAPP) mice. Therefore, the current study was conducted to investigate the activity and protein content of several CYP isoforms in the livers and kidneys of aged (20-month-old) tg(hAPP) mice. tg(hAPP) mice exhibit significant elevations in hepatic CYP2B, CYP2E1-, CYP3A- and CYP4A-associated activities and CYP4A immunoreactive protein compared with wild-type. In contrast to the liver, a significant depression in renal CYP2E1- and CYP4A-associated activities were demonstrated in tg(hAPP) mice. The presence of the mutant hAPP protein was detected in the brain, kidney and livers of tg(hAPP) mice.

Corticotropin-releasing hormone protects neurons against insults relevant to the pathogenesis of Alzheimer's disease.

We previously reported that mice over-expressing the human amyloid precursor protein gene with the double Swedish mutation of familial Alzheimer's disease (mtAPP), which exhibit progressive deposition of amyloid beta-peptide in hippocampal and cortical brain regions, have an impaired ability to maintain a sustained glucocorticoid response to stress. Corticotropin releasing hormone (CRH), which initiates neuroendocrine responses to stress by activating the hypothalamic-pituitary-adrenal (HPA) axis, is expressed in brain regions prone to degeneration in Alzheimer's disease. We therefore tested the hypothesis that CRH can modify neuronal vulnerability to amyloid beta-peptide toxicity. In primary neuronal culture, CRH was protective against cell death caused by an amyloid-beta peptide, an effect that was blocked by a CRH receptor antagonist and by an inhibitor of cyclic AMP-dependent protein kinase. The increased resistance of CRH-treated neurons to amyloid toxicity was associated with stabilization of cellular calcium homeostasis. Moreover, CRH protected neurons against death caused by lipid peroxidation and the excitotoxic neurotransmitter glutamate. The level of mRNA encoding CRH was unchanged in mtAPP mouse brain, whereas the levels of mRNAs encoding glucocorticoid and mineralocorticoid receptors were subtly altered. Our results suggest that disturbances in HPA axis function can occur independently of alterations in CRH mRNA levels in Alzheimer's disease brain and further suggest an additional role for CRH in protecting neurons against cell death.

Impact of aging on stress-responsive neuroendocrine systems.

Throughout life organisms are challenged with various physiological and psychological stressors, and the ability to handle these stressors can have profound effects on the overall health of the organism. In mammals, the effects of stressors on the aging process and age-related diseases are complex, involving the nervous, endocrine and immune systems. Certain types of mild stress, such as caloric restriction, may extend lifespan and reduce the risk of diseases, whereas some types of psychosocial stress are clearly detrimental. We now have a basic understanding of the brain regions involved in stress responses, their neuroanatomical connections with neuroendocrine pathways, and the neuropeptides and hormones involved in controlling responses of different organ systems to stress. Not surprisingly, brain regions involved in learning and memory and emotion play prominent roles in stress responses, and monoaminergic and glutamatergic synapses play particularly important roles in transducing stressful sensory inputs into neuroendocrine responses. Among the neuropeptides involved in stress responses, corticotropin-releasing hormone appears to be a pivotal regulator of fear and anxiety responses. This neuropeptide is responsible for activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is critical for mobilizing energy reserves and immune responses, and improper regulation of the HPA axis mediates many of the adverse effects of chronic physical and psychosocial stress. In the brain, for instance, stress may contribute to disease processes by causing imbalances in cellular energy metabolism and ion homeostasis, and by inhibiting neuroprotective signaling pathways. There is considerable evidence that normal aging impacts upon neuroendocrine stress responses, and studies of the molecular and cellular mechanisms underlying the pathogenic actions of mutations that cause age-related neurological disorders, such as Alzheimer's disease, are revealing novel insight into the involvement of perturbed neuroendocrine stress responses in these disorders.

Neurodegenerative disorders and ischemic brain diseases.

Degeneration and death of neurons is the fundamental process responsible for the clinical manifestations of many different neurological disorders of aging, incuding Alzheimer's disease, Parkinson's disease and stroke. The death of neurons in such disorders involves apoptotic biochemical cascades involving upstream effectors (Par-4, p53 and pro-apoptotic Bcl-2 family members), mitochondrial alterations and caspase activation. Both genetic and environmental factors, and the aging process itself, contribute to intiation of such neuronal apoptosis. For example, mutations in the amyloid precursor protein and presenilin genes can cause Alzheimer's disease, while head injury is a risk factor for both Alzheimer's and Parkinson's diseases. At the cellular level, neuronal apoptosis in neurodegenerative disorders may be triggered by oxidative stress, metabolic compromise and disruption of calcium homeostasis. Neuroprotective (antiapoptotic) signaling pathways involving neurotrophic factors, cytokines and "conditioning responses" can counteract the effects of aging and genetic predisposition in experimental models of neurodegenerative disorders. A better understanding of the molecular underpinnings of neuronal death is leading directly to novel preventative and therapeutic approaches to neurodegenerative disorders.

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.

A mechanism for the neuroprotective effect of apolipoprotein E: isoform-specific modification by the lipid peroxidation product 4-hydroxynonenal.

Inheritance of the apolipoprotein E (apoE) epsilon4 allele increases the risk for Alzheimer's disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE-modified apoE in lumbar spinal cord samples from mice expressing an ALS-linked mutation in Cu/Zn-superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid beta-peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases.

No benefit of dietary restriction on disease onset or progression in amyotrophic lateral sclerosis Cu/Zn-superoxide dismutase mutant mice.

Amyotrophic lateral sclerosis (ALS) is a fatal disease in which spinal cord motor neurons degenerate resulting in progressive paralysis. Some cases of ALS are caused by mutations in the antioxidant enzyme Cu/Zn-superoxide dismutase (SOD). Transgenic mice expressing ALS-linked Cu/Zn-SOD mutations (SODMutM) exhibit a phenotype analogous to that of human ALS patients. Dietary restriction (DR) is a well-established means of extending lifespan in rodents. It may act by reducing levels of cellular oxidative stress. We therefore tested the hypothesis that DR will retard the development of the clinical phenotype and extend the lifespan of SODMutM. There was no significant difference in age of disease onset in mice placed on a DR regimen beginning at 6 weeks of age compared to mice fed ad libitum, and disease duration was shortened indicating that DR accelerates the clinical course. Histological analyses indicated a similar extent of lower motor neuron degeneration in SODMutM maintained on DR or ad libitum diets. We conclude that a dietary manipulation known to extend lifespan has no beneficial effect in an animal model of familial ALS.

Opposing actions of native and oxidized lipoprotein on motor neuron-like cells.

Lipoproteins are present in the central nervous system and surrounding vasculature and possibly mediate effects relevant to neuronal physiology and pathology. To determine the effects of lipoproteins on motor neurons, native low density lipoproteins (LDL) and oxidized LDL (oxLDL) were applied to a motor neuron cell line. Oxidized LDL, but not native LDL, resulted in a dose- and time-dependent increase in reactive oxygen species and neuron death. Oxidized LDL-induced toxicity was attenuated by a calcium chelator, antioxidants, caspase inhibitors, and inhibitors of macromolecular synthesis. In addition to being nontoxic, application of native LDL attenuated reactive oxygen species formation and neuron loss following glucose deprivation injury. Together, these data demonstrate a possible neuroprotective role for unmodified lipoproteins and suggest oxidized lipoproteins may amplify oxidative stress and neuron loss.

The lipid peroxidation product 4-hydroxynonenal impairs glutamate and glucose transport and choline acetyltransferase activity in NSC-19 motor neuron cells.

Both oxidative stress and excitotoxicity are implicated in the pathogenesis of a number of neurodegenerative disorders, such as amyotrophic lateral sclerosis. We previously reported increased modification of proteins by 4-hydroxynonenal (HNE), a product of membrane lipid peroxidation, in the spinal cords of patients with amyotrophic lateral sclerosis relative to controls. In the current study, we examined the functional consequences of protein modification by HNE in a cell line with a motor neuron phenotype, NSC-19. Treatment of NSC-19 cells with FeSO4, which catalyzes lipid peroxidation, or HNE induced concentration-dependent decreases in glucose and glutamate transport. Vitamin E and propyl gallate blocked the impairment of glucose and glutamate transport caused by FeSO4 in these cells, but not that caused by HNE, whereas glutathione blocked the effects of FeSO4 as well as HNE. Both FeSO4 and HNE caused an increase in the number of apoptotic nuclei in NSC-19 cultures, but this occurred subsequent to the impairment of glucose and glutamate transport. Reductions in choline acetyltransferase activity were also observed in FeSO4- or HNE-treated NSC-19 cells before induction of apoptosis. Our results suggest that, prior to cell death, oxidative stress and HNE down-regulate cholinergic markers and impair glucose and glutamate transport in motor neurons, the latter of which may lead to excitotoxic degeneration of the cells.

Cellular and molecular mechanisms underlying perturbed energy metabolism and neuronal degeneration in Alzheimer's and Parkinson's diseases.

Synaptic degeneration and death of nerve cells are defining features of Alzheimer's disease (AD) and Parkinson's disease (PD), the two most prevalent age-related neurodegenerative disorders. In AD, neurons in the hippocampus and basal forebrain (brain regions that subserve learning and memory functions) are selectively vulnerable. In PD dopamine-producing neurons in the substantia nigra-striatum (brain regions that control body movements) selectively degenerate. Studies of postmortem brain tissue from AD and PD patients have provided evidence for increased levels of oxidative stress, mitochondrial dysfunction and impaired glucose uptake in vulnerable neuronal populations. Studies of animal and cell culture models of AD and PD suggest that increased levels of oxidative stress (membrane lipid peroxidation, in particular) may disrupt neuronal energy metabolism and ion homeostasis, by impairing the function of membrane ion-motive ATPases and glucose and glutamate transporters. Such oxidative and metabolic compromise may there-by render neurons vulnerable to excitotoxicity and apoptosis. Studies of the pathogenic mechanisms of AD-linked mutations in amyloid precursor protein (APP) and presenilins strongly support central roles for perturbed cellular calcium homeostasis and aberrant proteolytic processing of APP as pivotal events that lead to metabolic compromise in neurons. Specific molecular "players" in the neurodegenerative processes in AD and PD are being identified and include Par-4 and caspases (bad guys) and neurotrophic factors and stress proteins (good guys). Interestingly, while studies continue to elucidate cellular and molecular events occurring in the brain in AD and PD, recent data suggest that both AD and PD can manifest systemic alterations in energy metabolism (e.g., increased insulin resistance and dysregulation of glucose metabolism). Emerging evidence that dietary restriction can forestall the development of AD and PD is consistent with a major "metabolic" component to these disorders, and provides optimism that these devastating brain disorders of aging may be largely preventable.

ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis.

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Aberrant stress response associated with severe hypoglycemia in a transgenic mouse model of Alzheimer's disease.

Patients with Alzheimer's disease (AD) exhibit alterations in glucose metabolism and dysregulation of the stress-responsive hypothalamic-pituitary-adrenal (HPA) neuroendocrine system. The mechanisms responsible for these alterations and their possible contributions to the neurodegenerative process in AD are unknown. We now report that transgenic mice expressing a mutant form of human amyloid precursor protein (APP) that causes inherited early-onset AD exhibit increased sensitivity to physiological stressors, which is associated with aberrancies in HPA function and regulation of blood glucose levels. Specifically, APP mutant mice exhibit severe hypoglycemia and death following food restriction, and sustained elevations of plasma glucocorticoid levels and hypoglycemia following restraint stress. The alterations in HPA function and glucose regulation were evident in relatively young mice prior to overt deposition of amyloid beta-peptide (A beta). However, diffuse accumulations of A beta were present in the hypothalamus of older mice, suggesting a role for soluble forms of A beta in dysregulation of HPA function. Our data demonstrate disturbances in neuroendocrine function in APP mutant mice similar to those seen in AD patients. These impairments in stress response, glucocorticoid signaling, and regulation of blood glucose should be considered in interpretations of data from past and future studies of APP mutant mice.

Protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients.

We report increased modification of proteins by 4-hydroxynonenal (HNE), a product of membrane lipid peroxidation, in the lumbar spinal cord of sporadic amyotrophic lateral sclerosis (ALS) patients versus that of neurologically normal controls. By immunohistochemistry, HNE-protein modification was detected in ventral horn motor neurons, and immunoprecipitation analysis revealed that one of the proteins modified by HNE was the astrocytic glutamate transporter EAAT2. Given that the function of proteins modified by HNE can be severely compromised as previously demonstrated for glutamate transporters in cortical synaptosome preparations, our findings suggest a scenario in which oxidative stress leads to the production of HNE, impairment of glutamate transport, and excitotoxic motor neuron degeneration in ALS.

Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease.

Many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding presenilin-1 (PS-1; chromosome 14) and presenilin-2 (PS-2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrites. PS-1 and PS-2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel-12, which is involved in Notch signaling, and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid beta-peptide (A beta1-42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid beta-peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.

Effects of amyloid precursor protein derivatives and oxidative stress on basal forebrain cholinergic systems in Alzheimer's disease.

The dysfunction and degeneration of cholinergic neuronal circuits in the brain is a prominent feature of Alzheimer's disease. Increasing data suggest that age-related oxidative stress contributes to degenerative changes in basal forebrain cholinergic systems. Experimental studies have shown that oxidative stress, and membrane lipid peroxidation in particular, can disrupt muscarinic cholinergic signaling by impairing coupling of receptors to GTP-binding proteins. Altered proteolytic processing of the beta-amyloid precursor protein (APP) may contribute to impaired cholinergic signaling and neuronal degeneration in at least two ways. First, levels of cytotoxic forms of amyloid beta-peptide (A beta) are increased; A beta damages and kills neurons by inducing membrane lipid peroxidation resulting in impairment of ion-motive ATPases, and glucose and glutamate transporters, thereby rendering neurons vulnerable to excitotoxicity. The latter actions of A beta may be mediated by 4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation that covalently modifies and inactivates the various transporter proteins. Subtoxic levels of A beta can also suppress choline acetyltransferase levels, and may thereby promote dysfunction of intact cholinergic circuits. A second way in which altered APP processing may endanger cholinergic neurons is by reducing levels of a secreted form of APP which has been shown to modulate neuronal excitability, and to protect neurons against excitotoxic, metabolic and oxidative insults. Mutations in presenilin genes, which are causally linked to many cases of early-onset inherited Alzheimer's disease, may increase vulnerability of cholinergic neurons to apoptosis. The underlying mechanism appears to involve perturbed calcium regulation in the endoplasmic reticulum, which promotes loss of cellular calcium homeostasis, mitochondrial dysfunction and oxyradical production. Knowledge of the cellular and molecular underpinnings of dysfunction and degeneration of cholinergic circuits is leading to the development of novel preventative and therapeutic approaches for Alzheimer's disease and related disorders.

Nerve growth factor-independent reduction in choline acetyltransferase activity in PC12 cells expressing mutant presenilin-1.

Mutations in the presenilin genes (PS-1 and PS-2) are linked to early onset familial Alzheimer's disease (AD), but the mechanisms by which these mutations cause the cognitive impairment characteristic of AD are unknown. Basal forebrain cholinergic neurons are involved in learning and memory processes, and reductions in choline acetyl-transferase (ChAT) activity are a characteristic feature of AD brain. We therefore hypothesized that presenilin mutations suppress expression of the cholinergic phenotype. In rat PC12 cells stably transfected with the human PS-1 gene containing the Leu --> Val mutation at codon 286 (L286V), we observed a drastic reduction (>90%) in basal ChAT activity compared with cells transfected with vector alone. By immunocytochemistry, a similar decrease in ChAT protein levels was found in the mutant transfectants. In cells differentiated with nerve growth factor, ChAT activity was again markedly lower in L286V-expressing cells than in control cells. We also observed reductions in ChAT activity in PC12 cells expressing the wild-type human PS-1 gene but to a lesser extent than in L286V-expressing cells. The viability of cells transfected with either the wild-type or the mutant PS-1 gene was not compromised. Our results suggest that PS-1 mutations may contribute to the cognitive impairment in AD by causing a nontoxic suppression of the cholinergic phenotype.

Characterization of the acetylcholine-reducing effect of the amyloid-beta peptide in mouse SN56 cells.

We previously reported that the amyloid-beta protein (Abeta) reduces the synthesis of acetylcholine (ACh) in a mouse septal cell line, SN56, without causing death of the cells. Here, we report that the ACh-reducing effect of either Abeta 1-28 or Abeta 1-42 (100 nM; 48 h) in SN56 cells can be prevented by a co-treatment with the tyrosine kinase inhibitors, genistein (75 microM) and tyrphostin A25 (50 microM). Treatment of the cells with either of these inhibitors alone increased ACh levels. An enhancement of the cellular ACh content was also obtained with aphidicolin, a compound which inhibits DNA synthesis. However, co-treatment of the cells for 48 h with aphidicolin (500 nM) and Abeta 1-42 (100 nM) did not prevent the reduction in ACh levels caused by the peptide. Furthermore, this effect could not prevented by a pre-treatment with vitamin E (50 microg/ml). These results suggest that the ACh-reducing effect of Abeta in SN56 cells is dependent on tyrosine phosphorylation, but is not dependent on DNA synthesis and may not be mediated by free radicals.

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain.

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.

All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha.

We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all-trans-retinol (vitamin A), all-trans-retinal, all-trans-retinoic acid (t-RA), 9-cis-retinoic acid (9c-RA), or 13-cis-retinoic acid caused time- and concentration-dependent increases in choline acetyltransferase activity (up to 3.4-fold) and in intracellular acetylcholine levels (up to 2.5-fold, with respective EC50 values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t-RA or 9c-RA at 1 microM for 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5' region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t-RA and 9c-RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t-RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor-alpha subtype, Ro 41-5253, we found that the effects of t-RA and 9c-RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor-alpha, Ro 40-6055, increased acetylcholine levels to a similar extent as t-RA and 9c-RA, and this effect was blocked by the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation by sphingolipids of calcium signals evoked by epidermal growth factor.

Receptor-activated breakdown of complex sphingolipids has been proposed as a mechanism for generating sphingoid base-containing putative second messenger molecules whose actions may modulate responses to extracellular signals. In human epidermoid carcinoma A431 cells, sphingosine (1-10 microM) by itself had no effect on intracellular free calcium concentrations ([Ca2+]i), yet within seconds, markedly enhanced the epidermal growth factor (EGF)-evoked Ca2+ influx (by up to 2-fold), but failed to alter Ca2+ release from the intracellular stores. Ca2+ signals evoked by serum were not affected by sphingosine. The response to sphingosine was dose-dependent and saturable, exhibiting an EC50 of 2.3 microM. In contrast, a ceramide, N-acetylsphingosine (10 microM), sphingosine 1-phosphate (10 microM), and sphingosylphosphorylcholine (10 microM) inhibited EGF-evoked elevations in [Ca2+]i. The latter two compounds by themselves transiently increased [Ca2+]i. N-Octanoylsphingosine, N,N-dimethylsphingosine, sphingomyelin, and stearylamine were inactive. The potentiation of calcium signals by sphingosine occurred at all concentrations of EGF tested (0.15-15 nM) and did not alter the EGF receptor protein kinase activity as determined by antiphosphotyrosine immunoblotting. Antiphosphoserine immunoblotting revealed that sphingosine (10 microM for 3 min) increased the phosphoserine content of two proteins with approximate molecular masses of 40 and 70 kDa. Serine hyperphosphorylation of the 40-kDa protein was also observed in cells treated with EGF alone, whereas the intensity of the 70-kDa band was highest in cells treated with both sphingosine and EGF. The modulation of growth factor receptor-regulated signaling, including changes in [Ca2+]i, may constitute a mechanism by which elevations in cellular levels of specific sphingolipids, which occur transiently upon activation of certain receptors and chronically in sphingolipid storage diseases, exert their physiological and pathophysiological effects.