The Genome-Wide Binding Profile for Human RE1 Silencing Transcription Factor Unveils a Unique Genetic Circuitry in Hippocampus.

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.SIGNIFICANCE STATEMENT The RE1 Silencing Transcription Factor (REST) repressor has served historically as a model for gene regulation during mouse neurogenesis. Recent studies of REST have also suggested a conserved role for REST repressor function across lower species during aging. However, direct genome-wide studies for REST have been lacking for human brain. Here, we perform the first genome-wide analysis of REST binding in both human and mouse hippocampus. The majority of REST-occupied genes in human hippocampus are distinct from those in mouse. Further, the REST-associated genes unique to human hippocampus represent a new set related to innate immunity and inflammation, where their gene dysregulation has been implicated in aging-related neuropathology, such as Alzheimer's disease.

The REST remodeling complex protects genomic integrity during embryonic neurogenesis.

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.

An RNA binding protein promotes axonal integrity in peripheral neurons by destabilizing REST.

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.

Lessons from development: A role for asymmetric stem cell division in cancer.

Asymmetric stem cell division has emerged as a major regulatory mechanism for physiologic control of stem cell numbers. Reinvigoration of the cancer stem cell theory suggests that tumorigenesis may be regulated by maintaining the balance between asymmetric and symmetric cell division. Therefore, mutations affecting this balance could result in aberrant expansion of stem cells. Although a number of molecules have been implicated in regulation of asymmetric stem cell division, here, we highlight known tumor suppressors with established roles in this process. While a subset of these tumor suppressors were originally defined in developmental contexts, recent investigations reveal they are also lost or mutated in human cancers. Mutations in tumor suppressors involved in asymmetric stem cell division provide mechanisms by which cancer stem cells can hyperproliferate and offer an intriguing new focus for understanding cancer biology. Our discussion of this emerging research area derives insight from a frontier area of basic science and links these discoveries to human tumorigenesis. This highlights an important new focus for understanding the mechanism underlying expansion of cancer stem cells in driving tumorigenesis.

Loss of AKAP150 perturbs distinct neuronal processes in mice.

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 context-dependent neuronal signaling events in vivo.

Reactivity of apolipoprotein E4 and amyloid beta peptide: lysosomal stability and neurodegeneration.

We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid beta (Abeta) 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 NH4Cl abolished the apoE4 potentiation of Abeta-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 Abeta, which forms multimers, and GM6 mutant Abeta, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Abeta-induced lysosomal leakage and apoptosis. Both apoE4 and Abeta1-42 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 Abeta on lysosomes and apoptosis. In cultured Neuro-2a cells, Abeta1-42 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 Abeta1-42 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.

Apolipoprotein (apo) E4 enhances amyloid beta peptide production in cultured neuronal cells: apoE structure as a potential therapeutic target.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid beta (Abeta) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Abeta production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Abeta 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 Abeta production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related 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 Abeta 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.

Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons.

The aim of this study was to evaluate whether the direct activation of the Wnt signaling pathway by its endogenous Wnt-3a ligand prevents the toxic effects induced by amyloid-beta-peptide (Abeta) in rat hippocampal neurons. We report herein that the Wnt-3a ligand was indeed able to overcome toxic effects induced by Abeta in hippocampal neurons, including a neuronal impairment on cell survival, an increase in glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation, a decrease in cytoplasmic beta-catenin and a decrease in the expression of the Wnt target gene engrailed-1. We further demonstrate that Wnt-3a protects hippocampal neurons from apoptosis induced by Abeta. Our results support the hypothesis that a loss of function of Wnt signaling may play a role in the progression of neurodegenerative diseases such as Alzheimer's disease.

Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease.

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.