No abstract

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoformspecific effects on the deposition or clearance of amyloid ␤ (A␤) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on A␤ production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased A␤ production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in A␤ production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptorrelated protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and A␤ production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.Alzheimer's disease ͉ neurodegeneration

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate contextdependent neuronal signaling events in vivo.AMPA ͉ behavior ͉ KCNQ ͉ knockout S ophisticated systems have evolved to manage the spatial and temporal organization of signal transduction pathways. AKinase Anchoring Proteins (AKAPs) target various protein kinases and phosphatases to subcellular environments where they control the phosphorylation state of neighboring substrates (1). Movement of enzymes in and out of multiprotein complexes contributes to the temporal regulation of signaling. Hence genetic manipulation of AKAP expression impacts the specificity and magnitude of cellular regulation within the context of the whole organism. This is particularly evident in the central nervous system where the elongated and branched morphology of neurons creates many intracellular compartments where AKAPs synchronize neuronal events (2-4).AKAP79/150 is a family of three orthologs (human AKAP79, murine AKAP150, and bovine AKAP75) each initially defined on the basis of its ability to tether the type II PKA holoenzyme (4, 5). Additional binding partners were subsequently identified including PP2B and PKCs (6, 7). Thus, AKAP79/150 complexes can to respond to intracellular second messengers such as cAMP, calcium and phospholipids (7). Furthermore, the simultaneous anchoring of signal transduction and signal termination enzymes influences both forward and backward steps of a cellular event. For example, AKAP79/150 complexes can influence the phosphorylation and action of transmembrane proteins including G protein coupled receptors and adenylyl cyclases (8, 9). Loss of AKAP79/150 from heart cells contributes to the onset of angiotensin II-induced hypertension (10). Electrophysiological approaches have established a role for AKAP79...

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation.DOI: http://dx.doi.org/10.7554/eLife.09584.001

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4 -10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.The ⑀4 allele of the gene encoding apolipoprotein (apo) 1 E has been genetically linked to late-onset familial and sporadic Alzheimer's disease (AD) and has a gene-dose effect on the risk and age of onset of the disease (1-5). Individuals with two copies of the ⑀4 allele have a 50 -90% chance of developing AD by the age of 85, and those with one copy have about a 45% chance (1,6). Only about 20% of the general population develops AD by the age of 85 (1).ApoE is found in amyloid plaques and neurofibrillary tangles, two neuropathological hallmarks of AD (7-13), but its role in their pathogenesis is unclear. ApoE4 has several adverse effects that might explain the association between AD and the ⑀4 allele. It modulates the deposition and clearance of amyloid ␤ peptides and plaque formation (14 -21), impairs the antioxidative defense system (22), dysregulates neuronal signaling pathways (23), disrupts cytoskeletal structure and function (24,25), and alters the phosphorylation of tau and the formation of neurofibrillary tangles (26 -30). However, the mechanisms of these effects are still largely unknown, and it is not known which are the primary effects and which are subsequent or downstream effects.Initially, apoE was thought to be synthesized in the brain only by astrocytes, oligodendrocyte...

We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid ␤ (A␤) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH 4 Cl abolished the apoE4 potentiation of A␤-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of "Arctic" mutant A␤, which forms multimers, and GM6 mutant A␤, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate A␤-induced lysosomal leakage and apoptosis. Both apoE4 and A␤1-42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and A␤ on lysosomes and apoptosis. In cultured Neuro-2a cells, A␤1-42 increased lysosome formation to a greater extent in apoE3-or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and A␤1-42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration. Alzheimer disease (AD)2 is a debilitating neurodegenerative disease. With no effective treatment available, the incidence of AD is likely to increase as the population continues to age. Although in most cases the exact cause is unknown, two proteins have been implicated in its pathogenesis: apolipoprotein E4 (apoE4) and amyloid ␤ (A␤).ApoE has important functions in lipid transport in the blood and in the redistribution of lipids among cells in the brain (1). One of its three common isoforms (2, 3), apoE4, is the major genetic risk factor for AD (4 -6). Moreover, the ⑀4 allele is associated with impaired central nervous system repair after injury and in other neurodegenerative diseases (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).The A␤ peptide, typically 39 -43 amino acids, is derived from the proteolytic cleavage of the amyloid precursor protein (APP) (23,24). Amyloid plaques, a pathological hallmark of AD, are extracellular deposits of A␤ (25,26). Several findings suggest that apoE, another constituent of plaques, may modulate plaque formation or alter the availability of A␤ for plaque formation. Lipid-free apoE4 avidly complexes with A␤ (27), possibly inducing plaques, whereas lipidated apoE3 facilitates A␤ clearance (28 -32) and may reduce plaque formation. ApoE also modulates the cleavage of APP by ␥-secretase (33).S...

The RE1 Silencing Transcription Factor (REST) acts as a governor of the mature neuronal phenotype by repressing a large consortium of neuronal genes in non-neuronal cells. In the developing nervous system, REST is present in progenitors and downregulated at terminal differentiation to promote acquisition of mature neuronal phenotypes. Paradoxically, REST is still detected in some regions of the adult nervous system, but how REST levels are regulated, and whether REST can still repress neuronal genes, is not known. Here, we report that homeostatic levels of REST are maintained in mature peripheral neurons by a constitutive post-transcriptional mechanism. Specifically, using a three-hybrid genetic screen, we identify the RNA binding protein, ZFP36L2, associated previously only with female fertility and hematopoiesis, and show that it regulates REST mRNA stability. Dorsal root ganglia in Zfp36l2 knock-out mice, or wild-type ganglia expressing ZFP36L2 shRNA, show higher steady-state levels of Rest mRNA and protein, and extend thin and disintegrating axons. This phenotype is due, at least in part, to abnormally elevated REST levels in the ganglia because the axonal phenotype is attenuated by acute knockdown of REST in Zfp36l2 KO DRG explants. The higher REST levels result in lower levels of target genes, indicating that REST can still fine-tune gene expression through repression. Thus, REST levels are titrated in mature peripheral neurons, in part through a ZFP36L2-mediated post-transcriptional mechanism, with consequences for axonal integrity.

Early studies in mouse neurodevelopment led to the discovery of the RE1 Silencing Transcription Factor (REST) and its role as a master repressor of neuronal gene expression. Recently, REST was reported to also repress neuronal genes in the human adult brain. These genes were found to be involved in pro-apoptotic pathways; and their repression, associated with increased REST levels during aging, were found to be neuroprotective and conserved across species. However, direct genome-wide REST binding profiles for REST in adult brain have not been identified for any species. Here, we apply this approach to mouse and human hippocampus. We find an expansion of REST binding sites in the human hippocampus that are lacking in both mouse hippocampus and other human non-neuronal cell types. The unique human REST binding sites are associated with genes involved in innate immunity processes and inflammation signaling which, on the basis of histology and recent public transcriptomic analyses, suggest that these new target genes are repressed in glia. We propose that the increases in REST expression in mid-adulthood presage the beginning of brain aging, and that human REST function has evolved to protect the longevity and function of both neurons and glia in human brain.