HDAC2 Negatively Regulates Memory Formation and Synaptic Plasticity

Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment

Identification of Ischemic Regions in a Rat Model of Stroke

Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia.Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed.TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

Overview of differently affected regions following 30 min, 120 min, and permanent MCAO (black: infarct core; striped: perilesional regions; light grey: remote areas).

<p>The maximum expansion of each region is displayed, whereby 120 min and permanent MCAO revealed the same maxima. Additionally staining procedures used in this study are displayed relating to their applicability to detect these differently affected regions at various reperfusion times (2 h, 1 d, 7 d, 30 d).</p

Histological (thionine) and immunohistochemical (MAP2, HSP72, HSP27) characterizations of infarcts induced by a transient (30 and 120 min) and permanent MCAO.

<p>Occlusion of the MCA for 30 min always leads to an irreversible injury (infarct core) in the striatum and frequently in parts of the neocortex. The neocortex is also part of the perilesional region which also might include hippocampus, amygdala, thalamus, and hypothalamus. An irregular impairment of remote areas also occurs (cingulate and retrosplenial cortex, septum, and all contralateral affected regions). Following 120 min MCAO striatum, neocortex, amygdala, and hypothalamus are part of the infarct core. Impaired perilesional zones include hippocampus, thalamus, and hypothalamus. The ipsi- and contralateral cingulate and retrosplenial cortex and the contralateral hippocampus as well as the septal area were identified as remote affected regions. Permanent MCAO leads to infarct cores always comprising the striatum, neocortex, amygdala, and hypothalamus. Further impaired regions are similar as identified following 120 min MCAO. Rare cerebrovascular anatomies like a unilateral origin of the ACA could also lead to ischemic changes in usually remote areas like cingulate and retrosplenial cortex (1 d following 30 min MCAO). Black dotted lines indicate the infarct core and perilesional affected regions, whereas red dotted lines display remote effects. Please note that a cellular resolution can not be provided by this overview, for this purpose please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004764#pone-0004764-g002" target="_blank">Fig. 2</a>. Scale bar: 2 mm.</p

TTC-stained brain sections at various reperfusion times following transient (30 min and 120 min) and permanent MCAO.

<p>TTC method reliably delineates the infarct core at 1 d after 30 min MCAO. After 120 min and permanent MCAO the infarct core was clearly detectable at all investigated times. Scale bar: 5 mm.</p

Thionine staining (A–D) of the contralateral (A) and the ipsilateral striatum after 30 min MCAO (B–D).

<p>Cells displaying aberrant morphology after 2 h reperfusion (B), major cell loss was found at 1 d (C) and a massive infiltration of microglia and astrocytes occurs at 7 d after reperfusion (D). MAP2 immunoreactivity (E–H) in the contralateral striatum (E) and neocortex (F). Absent MAP2 immunoreactivity in the infarct core at 1 d after 30 min MCAO (G). Attenuated MAP2 immunoreactivity in the ipsilateral neocortex at 1 d after 30 min MCAO (H). HSP72 immunoreactivity (I–L) in the contralateral (I), the ipsilateral striatum (infarct core) at 1 d after 30 min MCAO with HSP72-positive bipolar (arrows) and endothelial cells (arrowhead) (J) and HSP72-positive neurons in the ipsilateral neocortex (perilesional zone) at 1 d after 30 min (K) and 120 min MCAO (L). HSP27 immunoreactivity after 30 min MCAO (M–P) in the contralateral striatum with HSP27 expression in endothelial cells (M), the ipsilateral striatum (infarct core) at 1 d after reperfusion with an upregulated HSP27 expression in endothelial cells (arrows) and microglia (arrowhead) (N), HSP27-positive astrocytes (arrow) and neurons (arrowheads) in the ipsilateral neocortex 1 d after reperfusion (O). HSP27-positive astrocytes at 7 d after reperfusion in the ipsilateral neocortex (perilesional zone, P). Co-localization of HSP72 and HSP27 at 1 d after 30 min MCAO (Q, R), revealing co-expression of both proteins in most neurons. The framed area is enlarged in R. Scale bar A–Q: 50 µm, R: 20 µm.</p

The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

International audienceBackground : The hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency.Methods : We generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients. Results : Homozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established. Conclusions : Our data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models

Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

<div><h3>Background</h3><p>Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear.</p> <h3>Methods and Results</h3><p>As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed.</p> <h3>Conclusions</h3><p>Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.</p> </div