Rosiglitazone attenuates learning and memory deficits in Tg2576 Alzheimer mice.

The thiazolidinediones, such as rosiglitazone, increase peripheral insulin sensitivity and their use is proposed for the treatment of Alzheimer's disease. However, the mechanisms underlying the potential beneficial effects of rosiglitazone in Alzheimer's disease remain unclear. In previous studies, we observed that Tg2576 Alzheimer mice develop peripheral insulin resistance with age and have much higher serum corticosterone levels than wild-type mice when fasted overnight. We further showed that both of these defects can be ameliorated by rosiglitazone administration. Here, we report that during behavioral testing which involves repetitive overnight fasting, Tg2576 mice administered rosiglitazone exhibited better spatial learning and memory abilities and had lower serum corticosterone levels than untreated Tg2576 mice. When untreated Tg2576 mice were administered metyrapone, a drug that blocks glucocorticoid production, their spatial learning and memory abilities and serum corticosterone levels were similar to those of rosiglitazone-treated mice. We further report here that rosiglitazone attenuated reductions in insulin-degrading enzyme (IDE) mRNA and activity, and reduced amyloid beta-peptide (Abeta)42 levels without affecting amyloid deposition, in the brains of Tg2576 mice. These results demonstrate that rosiglitazone attenuates learning and memory deficits in Tg2576 mice and suggest that the effects of the drug on learning and memory, brain IDE levels, and brain Abeta42 levels in the mice may be due to its glucocorticoid-lowering actions.

Insulin resistance contributes to aberrant stress responses in the Tg2576 mouse model of Alzheimer's disease.

We previously reported aberrant stress responses and impaired glucose tolerance in transgenic Tg2576 mice, a model of Alzheimer's disease (AD). Here we report that by 8 months of age, Tg2576 mice had lower basal serum insulin concentrations and exhibited a delayed insulin-induced reduction in blood glucose levels relative to wild-type mice. However, the basal levels of blood glucose and percent glycosylated hemoglobin (%HbA1c) were similar between the two groups of mice. While the basal levels of serum corticosterone were similar between Tg2576 and wild-type mice, an overnight fasting caused a greater rise in serum corticosterone levels and an excessive reduction in serum insulin concentrations in the transgenics. At 9 months of age, we began administering Tg2576 mice rosiglitazone, an agonist of peroxisome proliferator-activated receptor-gamma that increases peripheral insulin sensitivity, and after 6 weeks of administration the Tg2576 mice had the same response to insulin and increase in serum corticosterone levels after an overnight fast as did wild-type mice. By 13 months of age, untreated Tg2576 mice had become hyperinsulinemic, in contrast to Tg2576 mice administered rosiglitazone for 4 months where the serum insulin concentrations were maintained at levels observed in wild-type mice. These results provide evidence for a relationship between insulin resistance, impaired regulation of insulin and glucose levels, and aberrant stress responses in Tg2576 mice.

Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C-mediated mechanism.

Arachidonic acid (AA) plays an important role as a signaling factor in the CNS. Therefore, exposure to AA may affect cholinergic neurons in the spinal cord. To test this hypothesis, mRNA expression and activity of choline acetyltransferase (ChAT) was measured in cultured spinal cord neurons treated with increasing concentrations (0.1-10 microm) of AA. Exposure to AA increased mRNA levels and activity of ChAT in dose- and time-dependent manners. The most marked effect of AA on ChAT expression was observed in spinal cord neurons treated with 10 microm AA for 1 h. To study the mechanisms associated with these effects, ChAT mRNA levels and activity were measured in cultured spinal cord neurons exposed to AA and inhibitors of protein kinase C (PKC), such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dichloride (H-7) and chelerythrine. Inhibition of PKC completely prevented an AA-induced increase in ChAT expression. In addition, exposure of spinal cord neurons to phorbol-12-myristate-13-acetate (PMA), an activator of PKC, mimicked AA-induced stimulation of ChAT activity. The AA-mediated increase in ChAT mRNA levels and activity was also prevented by treatments with EGTA, indicating the role of calcium metabolism in induction of this enzyme. In contrast, treatments with 7-nitroindazole (7-NI, a specific inhibitor of neuronal nitric oxide synthase), sodium vanadate (NaV, a non-specific inhibitor of phosphatases), and N-acetyl-cysteine (NAC, an antioxidant) had no effect on AA-induced changes in ChAT activity. The protein synthesis inhibitor cycloheximide completely blocked AA-mediated increase in ChAT activity. These results indicate that the AA-evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, presumably at the transcriptional level.

Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease.

Alzheimer's disease (AD) is an age-related disorder characterized by deposition of amyloid beta-peptide (Abeta) and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction. The pathogenesis of AD is tightly linked to Abeta deposition and oxidative stress, but it remains unclear as to how these factors result in neuronal dysfunction and death. We report alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brains of AD patients that result in accumulation of long-chain ceramides and cholesterol. Membrane-associated oxidative stress occurs in association with the lipid alterations, and exposure of hippocampal neurons to Abeta induces membrane oxidative stress and the accumulation of ceramide species and cholesterol. Treatment of neurons with alpha-tocopherol or an inhibitor of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Abeta. Our findings suggest a sequence of events in the pathogenesis of AD in which Abeta induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers a neurodegenerative cascade that leads to clinical disease.

Numb isoforms containing a short PTB domain promote neurotrophic factor-induced differentiation and neurotrophic factor withdrawal-induced death of PC12 Cells.

The development of the nervous system is regulated by trophic signals that control cell proliferation, differentiation, and survival. Numb is an evolutionarily conserved protein identified by its ability to control cell fate in the nervous system of Drosophila. Mammals express four isoforms of Numb that differ in the length of a phosphotyrosine-binding (PTB) domain and a proline-rich region (PRR). Using PC12 cells stably expressing each of the human isoforms, we show that Numb regulates sensitivity of the cells to neurotrophic factor-induced differentiation and neurotrophic factor withdrawal-induced death in an isoform-specific manner. Numb isoforms containing a short PTB domain enhance the differentiation response to NGF and enhance apoptosis upon NGF withdrawal; Numb isoforms containing a long PTB domain exhibit the same sensitivity to NGF as vector-transfected cells. These effects of Numb were found to be independent of the length of the PRR. In undifferentiated conditions, the levels of full-length TrkA and of phosphorylated p44/p42 mitogen-activated protein kinase (MAPK) are increased in cells expressing Numb isoforms with a short PTB domain, indicating an up-regulation of NGF signaling pathways. Furthermore, we provide evidence that the mechanism whereby short PTB domain Numb isoforms sensitize cells to trophic factor deprivation-induced apoptosis involves elevations in intracellular calcium concentrations. Our results suggest that Numb sensitizes cells to neurotrophin responses in an isoform-specific manner, an effect that may play an important role in the development and plasticity of the nervous system.

Evidence that accumulation of ceramides and cholesterol esters mediates oxidative stress-induced death of motor neurons in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the spinal cord resulting in progressive paralysis and death. The pathogenic mechanism of ALS is unknown but may involve increased oxidative stress, overactivation of glutamate receptors, and apoptosis. We report abnormalities in sphingolipid and cholesterol metabolism in the spinal cords of ALS patients and in a transgenic mouse model (Cu/ZnSOD mutant mice), which manifest increased levels of sphingomyelin, ceramides, and cholesterol esters; in the Cu/ZnSOD mutant mice, these abnormalities precede the clinical phenotype. In ALS patients and Cu/Zn-SOD mutant mice, increased oxidative stress occurs in association with the lipid alterations, and exposure of cultured motor neurons to oxidative stress increases the accumulation of sphingomyelin, ceramides, and cholesterol esters. Pharmacological inhibition of sphingolipid synthesis prevents accumulation of ceramides, sphingomyelin, and cholesterol esters and protects motor neurons against death induced by oxidative and excitotoxic insults. These findings suggest a pivotal role for altered sphingolipid metabolism in the pathogenesis of ALS.

Neurotrophic and neuroprotective effects of milk thistle (Silybum marianum) on neurons in culture.

Herbal products are being increasingly used as dietary supplements and therapeutic agents. However, much more research must be performed in order to determine the biological basis for their putative clinical effects. We tested the effects of milk thistle (Silybum marianum) extract on the differentiation and survival of cultured neural cells. Milk thistle enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC-12 neural cells and prolonged their survival in culture. Milk thistle extract also protected cultured rat hippocampal neurons against oxidative stress-induced cell death. Our data demonstrate that milk thistle extract can promote neuronal differentiation and survival, suggesting potential benefits of chemicals in this plant on the nervous system.

Numb modifies neuronal vulnerability to amyloid beta-peptide in an isoform-specific manner by a mechanism involving altered calcium homeostasis: implications for neuronal death in Alzheimer's disease.

Increased production of neurotoxic forms of amyloid beta-peptide (Abeta) and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch, a membrane receptor that controls cell-fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase enzyme activity that involves the presenilin-1 (PS1) protein in which mutations cause early-onset inherited AD. The actions of Notch can be antagonized by Numb, an evolutionarily conserved protein that exists in four isoforms that differ in two functional domains: a phosphotyrosine-binding (PTB) domain and a proline-rich region (PRR). We now report that Numb isoforms containing a short PTB domain increase the vulnerability of PC12 cells to death induced by Abeta1-42 and by 4-hydroxynonenal, a lipid peroxidation product previously shown to mediate neurotoxic effects of Abeta. Dysregulation of cellular calcium homeostasis occurs in cells expressing Numb isoforms with a short PTB domain, and the death-promoting effect of Numb is abolished by pharmacological inhibition of calcium release. The levels of Numb are increased in cultured primary hippocampal neurons exposed to Abeta, suggesting a role for endogenous Numb in the neuronal death process. Furthermore, higher levels of Numb were detected in the cortex of mice expressing mutant amyloid precursor protein (APP) relative to age-matched wild-type mice. Our data identify a novel isoform-specific effect of Numb on neuronal life and death cell fate decisions potentially relevant to the pathogenesis of AD. Our findings also suggest that the effects of Numb on cell fate decisions, both during development of the nervous system and in neurodegenertive disorders, are mediated by changes in cellular calcium homeostasis.

Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease.

Recent epidemiological and clinical data suggest that persons with low folic acid levels and elevated homocysteine levels are at increased risk of Alzheimer's disease (AD), but the underlying mechanism is unknown. We tested the hypothesis that impaired one-carbon metabolism resulting from folic acid deficiency and high homocysteine levels promotes accumulation of DNA damage and sensitizes neurons to amyloid beta-peptide (Abeta) toxicity. Incubation of hippocampal cultures in folic acid-deficient medium or in the presence of methotrexate (an inhibitor of folic acid metabolism) or homocysteine induced cell death and rendered neurons vulnerable to death induced by Abeta. Methyl donor deficiency caused uracil misincorporation and DNA damage and greatly potentiated Abeta toxicity as the result of reduced repair of Abeta-induced oxidative modification of DNA bases. When maintained on a folic acid-deficient diet, amyloid precursor protein (APP) mutant transgenic mice, but not wild-type mice, exhibited increased cellular DNA damage and hippocampal neurodegeneration. Levels of Abeta were unchanged in the brains of folate-deficient APP mutant mice. Our data suggest that folic acid deficiency and homocysteine impair DNA repair in neurons, which sensitizes them to oxidative damage induced by Abeta.

Urocortin, but not urocortin II, protects cultured hippocampal neurons from oxidative and excitotoxic cell death via corticotropin-releasing hormone receptor type I.

Urocortin and urocortin II are members of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. Two high-affinity G-protein-coupled receptors have been identified that bind CRH and/or urocortin I and II, designated CRHR1 and CRHR2, both of which are present in hippocampal regions of mammalian brain. The hippocampus plays an important role in regulating stress responses and is a brain region in which neurons are vulnerable during disease and stress conditions, including cerebral ischemia, Alzheimer's disease, and anxiety disorders. Here we report that urocortin exerts a potent protective action in cultured rat hippocampal neurons with concentrations in the range of 0.5-5.0 pm, increasing the resistance of the cells to oxidative (amyloid beta-peptide, 4-hydroxynonenal, ferrous sulfate) and excitotoxic (glutamate) insults. We observed that urocortin is 10-fold more potent than CRH in protecting hippocampal neurons from insult, whereas urocortin II is ineffective. RT-PCR and sequencing analyses revealed the presence of both CRHR1 and CRHR2 in the hippocampal cultures, with CRHR1 being expressed at much higher levels than CRHR2. Using subtype-selective CRH receptor antagonists, we provide evidence that the neuroprotective effect of exogenously added urocortin is mediated by CRHR1. Furthermore, we provide evidence that the signaling pathway that mediates the neuroprotective effect of urocortin involves cAMP-dependent protein kinase, protein kinase C, and mitogen-activated protein kinase. This is the first demonstration of a biological activity of urocortin in hippocampal neurons, suggesting a role for the peptide in adaptive responses of hippocampal neurons to potentially lethal oxidative and excitotoxic insults.