operating-biocontainment-procedures-facility-for-laboratory-mice-with

Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Klinik und Hochschulambulanz für Neurologie

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

Einstein Center for Neuroscience

German Center for Cardiovascular Research (DZHK), partner site Berlin

Dietary deficiencies in folic acid result in elevated levels of plasma homocysteine, which has been associated with the development of dementia and other neurodegenerative disorders. Previously, we have shown that elevated levels of plasma homocysteine in mice deficient for a DNA repair enzyme, uracil-DNA glycosylase (UNG), result in neurodegeneration. The goal of this study was to evaluate how deficiencies in folic acid and UNG along with elevated levels of homocysteine affect vascular cognitive impairment, via chronic hypoperfursion in an animal model. Ung(+/+) and Ung(-/-) mice were placed on either control (CD) or folic acid deficient (FADD) diets. Six weeks later, the mice either underwent implantation of microcoils around both common carotid arteries. Post-operatively, behavioral tests began at 3-weeks, angiography was measured after 5-weeks using MRI to assess vasculature and at completion of study plasma and brain tissue was collected for analysis. Learning impairments in the Morris water maze (MWM) were observed only in hypoperfused Ung(-/-) FADD mice and these mice had significantly higher plasma homocysteine concentrations. Interestingly, Ung(+/+) FADD produced significant remodeling of the basilar artery and arterial vasculature. Increased expression of GFAP was observed in the dentate gyrus of Ung(-/-) hypoperfused and FADD sham mice. Chronic hypoperfusion resulted in increased cortical MMP-9 protein levels of FADD hypoperfused mice regardless of genotypes. These results suggest that elevated levels of homocysteine only, as a result of dietary folic acid deficiency, don't lead to memory impairments and neurobiochemical changes. Rather a combination of either chronic hypoperfusion or UNG deficiency is required.

Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil-DNA glycosylase impair learning in a mouse model of vascular cognitive impairment.

Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome.

Vascular signal transducer and activator of transcription-3 promotes angiogenesis and neuroplasticity long-term after stroke.

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 μg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice.

Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma (GBM), as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor interassay reproducibility as well as a weak correlation between methylation status and expression levels of MGMT. This study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas ( = 97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wild-type GBM. Tumor purity is an independent prognostic marker in glioblastoma and is associated with the extent of MGMT methylation. .

Prognostic Relevance of Tumor Purity and Interaction with MGMT Methylation in Glioblastoma.

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.

Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress.

In the adult hippocampal dentate gyrus (DG), the majority of newly generated cells are eliminated by apoptotic mechanisms. The apoptosis repressor with caspase recruitment domain (ARC), encoded by the Nol3 gene, is a potent and multifunctional death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. The aim of the present study was to parse the role of ARC in the development of new granule cell neurons. Nol3 gene expression as revealed by in situ hybridization is present in the entire dentate granule cell layer. Moreover, a comparison of Nol3 expression between FACS-sorted Sox2-positive neural stem cells and Doublecortin (DCX)-positive immature neurons demonstrates upregulation of Nol3 during neurogenesis. Using ARC-deficient mice, we show that proliferation and survival of BrdU birth-dated cells are strongly reduced in the absence of ARC while neuronal-glial fate choice is not affected. Both the number of DCX-positive cells and the number of calretinin (CR)-positive immature postmitotic neurons are reduced in the hippocampus of ARC mice. ARC knockout is not associated with increased numbers of microglia or with microglia activation. However, hippocampal brain-derived neurotrophic factor (BDNF) protein content is significantly increased in ARC mice, possibly representing a compensatory response. Collectively, our results suggest that ARC plays a critical cell-autonomous role in preventing cell death during adult granule cell neogenesis.

Reduced Hippocampal Neurogenesis in Mice Deficient in Apoptosis Repressor with Caspase Recruitment Domain (ARC).

Deep brain stimulation (DBS) is the preferred treatment for therapy-resistant movement disorders such as dystonia and Parkinson's disease (PD), mostly in advanced disease stages. Although DBS is already in clinical use for ~30 years and has improved patients' quality of life dramatically, there is still limited understanding of the underlying mechanisms of action. Rodent models of PD and dystonia are essential tools to elucidate the mode of action of DBS on behavioral and multiscale neurobiological levels. Advances have been made in identifying DBS effects on the central motor network, neuroprotection and neuroinflammation in DBS studies of PD rodent models. The phenotypic dt mutant hamster and the transgenic DYT-TOR1A (ΔETorA) rat proved as valuable models of dystonia for preclinical DBS research. In addition, continuous refinements of rodent DBS technologies are ongoing and have contributed to improvement of experimental quality. We here review the currently existing literature on experimental DBS in PD and dystonia models regarding the choice of models, experimental design, neurobiological readouts, as well as methodological implications. Moreover, we provide an overview of the technical stage of existing DBS devices for use in rodent studies.

Article	Year
Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil-DNA glycosylase impair learning in a mouse model of vascular cognitive impairment. Behavioural brain research\| PubMed ID: 25655513	2015
Vascular signal transducer and activator of transcription-3 promotes angiogenesis and neuroplasticity long-term after stroke. Circulation\| PubMed ID: 25794850	2015
Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 27488634	2016
Prognostic Relevance of Tumor Purity and Interaction with MGMT Methylation in Glioblastoma. Molecular cancer research : MCR\| PubMed ID: 28148826	2017
Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress. Scientific reports\| PubMed ID: 30816308	2019
Reduced Hippocampal Neurogenesis in Mice Deficient in Apoptosis Repressor with Caspase Recruitment Domain (ARC). Neuroscience\| PubMed ID: 31356897	2019
Experimental deep brain stimulation in rodent models of movement disorders. Experimental neurology\| PubMed ID: 34793784	2022

Christoph Harms

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