Neuropathological signatures revealed by transcriptomic and proteomic analysis in Pten-deficient mouse models.

PTEN hamartoma tumour syndrome is characterised by mutations in the human PTEN gene. We performed transcriptomic and proteomic analyses of neural tissues and primary cultures from heterozygous and homozygous Pten-knockout mice. The somatosensory cortex of heterozygous Pten-knockout mice was enriched in immune response and oligodendrocyte development Gene Ontology (GO) terms. Parallel proteomic analysis revealed differentially expressed proteins (DEPs) related to dendritic spine development, keratinisation and hamartoma signatures. However, primary astrocytes (ASTs) from heterozygous Pten-knockout mice were enriched in the extracellular matrix GO term, while primary cortical neurons (PCNs) were enriched in immediate-early genes. In ASTs from homozygous Pten-knockout mice, cilium-related activity was enriched, while PCNs exhibited downregulation of forebrain neuron generation and differentiation, implying an altered excitatory/inhibitory balance. By integrating DEPs with pre-filtered differentially expressed genes, we identified the enrichment of traits of intelligence, cognitive function and schizophrenia, while DEPs in ASTs were significantly associated with intelligence and depression.

Investigating key factors underlying neurodegeneration linked to alpha-synuclein spread.

AIMS: It has long been considered that accumulation of pathological alpha-synuclein (aSyn) leads to synaptic/neuronal loss which then results in behavioural and cognitive dysfunction. To investigate this claim, we investigated effects downstream of aSyn preformed fibrils (PFFs) and 6-hydroxydopamine (6-OHDA), because aSyn PFFs induce spreading/accumulation of aSyn, and 6-OHDA rapidly causes local neuronal loss. METHODS: We injected mouse aSyn PFFs into the medial forebrain bundle (MFB) of Sprague-Dawley rats. We investigated spread of pathological aSyn, phosphorylation of aSyn and tau, oxidative stress, synaptic/neuronal loss and cognitive dysfunction 60, 90 and 120 days after injection. Similarly, we injected 6-OHDA into the MFB and examined the same parameters 1 and 3 weeks after injection. RESULTS: Following aSyn PFF injection, phosphorylated aSyn was found distant from the injection site in the hippocampus and frontal cortex. However, despite neuron loss being evident close to the site of injection in the substantia nigra at 120 days post injection, there were no other neurodegeneration-associated features associated with aSyn including synaptic loss. In contrast, 6-OHDA caused severe neuronal loss in the substantia nigra at 3 weeks post injection that was accompanied by phosphorylation of aSyn and tau, oxidative stress, loss of synaptic proteins, cognitive and motor dysfunction. CONCLUSIONS: Our results demonstrate that spread/replication and slow accumulation of pathological aSyn may not be sufficient to induce neurodegenerative changes. In contrast, oxidative stress responses in addition to aSyn accumulation were associated with other Parkinson's disease (PD)-associated abnormalities and cognitive dysfunction. Our results may be important when considering why only some PD patients develop dementia.

Dexras1 interacts with FE65 to regulate FE65-amyloid precursor protein-dependent transcription.

FE65 is an adaptor protein that binds to and forms a transcriptionally active complex with the gamma-secretase-derived amyloid precursor protein (APP) intracellular domain. The regulatory mechanisms of FE65-APP-mediated transcription are still not clear. In this report, we demonstrate that Dexras1, a Ras family small G protein, binds to FE65 PTB2 domain and potently suppresses the FE65-APP-mediated transcription. The suppression is not via competition for binding of FE65 between Dexras1 and APP because the two proteins can simultaneously bind to the FE65 PTB2 domain. Phosphorylation of FE65 tyrosine 547 within the PTB2 domain has been shown to enhance FE65-APP-mediated transcription but not to influence binding to APP. Here we find that this phosphorylation event reduces the binding between Dexras1 and FE65. We also demonstrate that Dexras1 inhibits the FE65-APP-mediated transcription of glycogen synthase kinase 3beta (GSK3 beta). Moreover, small interfering RNA knockdown of Dexras1 enhances GSK3 beta expression and increases phosphorylation of Tau, a GSK3 beta substrate. Thus, Dexras1 functions as a suppressor of FE65-APP-mediated transcription, and FE65 tyrosine 547 phosphorylation enhances FE65-APP-mediated transcription, at least in part, by modulating the interaction between FE65 and Dexras1. These findings reveal a novel regulatory mechanism for FE65-APP-mediated signaling.

Phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) is associated with neuronal degeneration in Alzheimer's disease.

Inhibition of protein translation is a mode of inducing neuronal apoptosis and neurodegeneration in Alzheimer's disease (AD). Phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) terminates global protein translation and induces apoptosis. We examined whether this signaling pathway occurs in degenerating neurons of AD. Brain sections from young individuals, age-matched control individuals and AD patients were examined for immunoreactivity of phosphorylated eIF2alpha by immunohistochemical analysis. While young brain sections did not display and age-matched brain sections have mild immunoreactive positive cells, AD brain sections revealed intense immunoreactivity for phosphorylated eIF2alpha. Most of the phosphorylated eIF2alpha immunoreactive positive neurons have high immunoreactivity for phosphorylated tau using AT8 antibody. Also, intense staining of phosphorylated eIF2alpha is associated vacuoles in degenerating neurons. This phenomenon was also observed for the immunohistochemical staining of phosphorylated PKR (double-stranded RNA-dependent protein kinase), the upstream kinase for eIF2alpha. Activation of PKR-eIF2alpha pathway is considered to be pro-apoptotic. In addition, formation of autophagy is regulated by eIF2alpha kinase. Therefore, it is concluded that phosphorylation of eIF2alpha is associated with the degeneration of neurons in AD.

p38 MAP kinase is involved in lipopolysaccharide-induced dopaminergic neuronal cell death in rat mesencephalic neuron-glia cultures.

Immune stimulants, such as the bacterial endotoxin, lipopolysaccharide (LPS), the human immunodeficiency virus-1 coat protein gp120, or beta-amyloid peptides, lead to glial activation and production of various immune mediators, such as nitric oxide (NO) and proinflammatory cytokines in the brain. These mediators appear to contribute to neuronal cell death in neurodegenerative diseases. However, the signaling pathways, which mediate the neurotoxic effect by the endotoxin, are not understood. The purpose of this study was to determine the role of mitogen-activated protein kinase (MAPK) in LPS-induced neurodegeneration using mesencephalic dopaminergic neuron/glia cultures. We have found that the p38 MAPK is important in LPS-induced death of mesencephalic neurons in rat neuron-glia mixed cultures. Upon treatment with 10 ng/ml LPS, the number of dopaminergic neurons decreased by 80% within 48 h, preceded by a significant production of NO by glia. Neuroprotection by selective inhibition of p38 MAPK activity paralleled a decrease in LPS-induced inducible nitric oxide synthase (iNOS) expression. These events were significantly reduced by the selective p38 MAPK inhibitor, SB202190, but not by the inactive analogue SB202474. Inhibition of iNOS activity and NO production by treatment with GW274150 was also neuroprotective. Although the p38 MAPK inhibitor afforded significant neuroprotection from LPS toxicity in the neuron-glia mixed culture, it failed to protect dopaminergic neurons from 6-hydroxy-dopamine-induced toxicity, which acts directly on dopaminergic neurons by inducing hydroxyl radical formation from the mitochondria. The results suggest that p38 MAPK in glia plays a significant role in the LPS-induced death of mesencephalic neurons through induction of nitric oxide synthase and resulting NO production.