Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Mutations in presenilin-1 (<it>Psen1</it>) cause familial Alzheimer's disease (FAD). Both hypoxia and ischemia have been implicated in the pathological cascade that leads to amyloid deposition in AD. Here we investigated whether Psen1 might regulate hypoxic responses by modulating induction of the transcription factor hypoxia inducible factor 1-α (HIF-1α).</p> <p>Results</p> <p>In fibroblasts that lack Psen1 induction of HIF-1α was impaired in response to the hypoxia mimetic cobalt chloride, as well as was induction by insulin and calcium chelation. Reintroduction of human Psen1 using a lentiviral vector partially rescued the responsiveness of <it>Psen1-/- </it>fibroblasts to cobalt chloride induction. HIF-1α induction did not require Psen1's associated γ-secretase activity. In addition, the failure of insulin to induce HIF-1α was not explicable on the basis of failed activation of the phosphatidylinositol 3-kinase (PI3K/Akt) pathway which activated normally in <it>Psen1-/- </it>fibroblasts. Rather we found that basal levels of HIF-1α were lower in <it>Psen1-/- </it>fibroblasts and that the basis for lower constitutive levels of HIF-1α was best explained by accelerated HIF-1α degradation. We further found that Psen1 and HIF-1α physically interact suggesting that Psen1 may protect HIF-1α from degradation through the proteasome. In fibroblasts harboring the M146V Psen1 FAD mutation on a mouse Psen1 null background, metabolic induction of HIF-1α by insulin was impaired but not hypoxic induction by cobalt chloride. Unlike <it>Psen1-/- </it>fibroblasts, basal levels of HIF-1α were normal in FAD mutant fibroblasts but activation of the insulin-receptor pathway was impaired. Interestingly, in <it>Psen1-/- </it>primary neuronal cultures HIF-1α was induced normally in response to cobalt chloride but insulin induction of HIF-1α was impaired even though activation of the PI3K/Akt pathway by insulin proceeded normally in <it>Psen1-/- </it>neuronal cultures. Basal levels of HIF-1α were not significantly different in <it>Psen1-/- </it>neurons and HIF-1α levels were normal in <it>Psen1-/- </it>embryos.</p> <p>Conclusions</p> <p>Collectively these studies show that Psen1 regulates induction of HIF-1α although they indicate that cell type specific differences exist in the effect of Psen1 on induction. They also show that the M146V Psen1 FAD mutation impairs metabolic induction of HIF-1α, an observation that may have pathophysiological significance for AD.</p

FDG-PET imaging, EEG and sleep phenotypes as translational biomarkers for research in Alzheimer's disease

Peer reviewedPublisher PD

Unconventional animal models for traumatic brain injury and chronic traumatic encephalopathy

Traumatic brain injury (TBI) is one of the main causes of death worldwide. It is a complex injury that influences cellular physiology, causes neuronal cell death, and affects molecular pathways in the brain. This in turn can result in sensory, motor, and behavioral alterations that deeply impact the quality of life. Repetitive mild TBI can progress into chronic traumatic encephalopathy (CTE), a neurodegenerative condition linked to severe behavioral changes. While current animal models of TBI and CTE such as rodents, are useful to explore affected pathways, clinical findings therein have rarely translated into clinical applications, possibly because of the many morphofunctional differences between the model animals and humans. It is therefore important to complement these studies with alternative animal models that may better replicate the individuality of human TBI. Comparative studies in animals with naturally evolved brain protection such as bighorn sheep, woodpeckers, and whales, may provide preventive applications in humans. The advantages of an in-depth study of these unconventional animals are threefold. First, to increase knowledge of the often-understudied species in question; second, to improve common animal models based on the study of their extreme counterparts; and finally, to tap into a source of biological inspiration for comparative studies and translational applications in humans

DNA methylation determination by liquid chromatography–tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards

Methylation of the promoter CpG regions regulates gene transcription by inhibiting transcription factor binding. Deoxycytidine methylation may regulate cell differentiation, while aberrations in the process may be involved in cancer etiology and the development of birth defects (e.g. neural tube defects). Similarly, nutritional deficiency and certain nutragenomic interactions are associated with DNA hypomethylation. While LC-MS has been used previously to measure percentage genomic deoxycytidine methylation, a lack of a secure source of internal standards and the need for laborious and time-consuming DNA digestion protocols constitute distinct limitations. Here we report a simple and inexpensive protocol for the biosynthesis of internal standards from readily available precursors. Using these biosynthetic stable-isotopic [U-15N]-labeled internal standards, coupled with an improved DNA digestion protocol developed in our lab, we have developed a low-cost, high-throughput (>500 samples in 4 days) assay for measuring deoxycytidine methylation in genomic DNA. Inter- and intraassay variation for the assay (%RSD, n = 6) was <2.5%

Brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure

Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [18F]AV1451 (flortaucipir) tau ligand, we found that five of 10 veterans exhibited excessive retention of [18F]AV1451 at the white/gray matter junction in frontal, parietal, and temporal brain regions, a typical localization of CTE tauopathy. We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [18F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Clonal relatedness of Proteus mirabilis strains causing urinary tract infections in companion animals and humans

Research Areas: Microbiology. Veterinary SciencesABSTRACT - Proteus mirabilis is a major cause of urinary tract infection (UTI) in humans and companion animals. This study aimed to evaluate the antimicrobial resistance, virulence and clonal relatedness of P. mirabilis isolated from dogs, cats and humans with UTI. P. mirabilis isolated from companion animals (N = 107) and humans (N = 76) with UTI were compared by PFGE analysis after overnight Nod macro-restriction using Dice/UPGMA with a 1.5% tolerance. Strains were characterized for antimicrobial resistance by disk diffusion. Twenty-four resistance genes and four virulence genes were screened by PCR. Thirty-nine clusters (similarity > 80%) and 73 single pulse-types were detected. Nine clusters included P. mirabilis isolated from community and hospital patients, including strains with 100% similarity. A high number of clusters (43.6%, n = 17/39) included strains from companion animals and humans. Similarity between some companion animal and human strains varied between 80-100%. One strain from a dog was 100% similar to one human community-acquired P. mirabilis. One P. mirabilis from a cat was found to be 94.7% and 92.4% similar to community and hospital patient strains, respectively. P. mirabilis CMY-2-producers did not cluster all together. Nevertheless, cluster C36 included five P. mirabilis from companion animals (similarity 85.8%-95.7%), of which, four (80%) were multidrug-resistant CMY-2-producers. This study shows that companion animals and humans become infected with closely related P. mirabilis strains. The high number of clusters containing companion animals and human strains points to the zoonotic nature of P. mirabilis. These results underline the potential role of companion animals as reservoirs and in the dissemination of uropathogenic P. mirabilis to humans and vice versa.info:eu-repo/semantics/publishedVersio

Widely variable endogenous retroviral methylation levels in human placenta

It is generally assumed that transposable elements, including endogenous retroviruses (ERVs), are silenced by DNA methylation/chromatin structure in mammalian cells. However, there have been very few experimental studies to examine the methylation status of human ERVs. In this study, we determined and compared the methylation status of the 5′ long terminal repeats (LTRs) of different copies of the human endogenous retrovirus (HERV) family HERV-E, which are inserted in various genomic contexts. We found that three HERV-E LTRs which function as alternative gene promoters in placenta are unmethylated in that tissue but heavily methylated in blood cells, where these LTRs are not active promoters. This difference is not solely due to global hypomethylation in placenta, since two general measures of methylation levels of HERV-E and HERV-K LTRs suggest only 10–15% lower overall HERV methylation in placenta compared to blood. Comparisons between methylation levels of the LTR-derived gene promoters and six random HERV-E LTRs in placenta showed that the former display significantly lower methylation levels than random LTRs. Moreover, the differences in methylation between LTRs cannot always be explained by their genomic environment, since methylation of flanking sequences can be very different from methylation of the LTR itself

High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum

DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5′ flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer