Transcriptome meta-analysis reveals a central role for sex steroids in the degeneration of hippocampal neurons in Alzheimer’s disease

BACKGROUND: Alzheimer’s disease is the most prevalent form of dementia. While a number of transcriptomic studies have been performed on the brains of Alzheimer’s specimens, no clear picture has emerged on the basis of neuronal transcriptional alterations linked to the disease. Therefore we performed a meta-analysis of studies comparing hippocampal neurons in Alzheimer’s disease to controls. RESULTS: Homeostatic processes, encompassing control of gene expression, apoptosis, and protein synthesis, were identified as disrupted during Alzheimer’s disease. Focusing on the genes carrying out these functions, a protein-protein interaction network was produced for graph theory and cluster exploration. This approach identified the androgen and estrogen receptors as key components and regulators of the disrupted homeostatic processes. CONCLUSIONS: Our systems biology approach was able to identify the importance of the androgen and estrogen receptors in not only homeostatic cellular processes but also the role of other highly central genes in Alzheimer’s neuronal dysfunction. This is important due to the controversies and current work concerning hormone replacement therapy in postmenopausal women, and possibly men, as preventative approaches to ward off this neurodegenerative disorder

Dynamic graph-based search in unknown environments

A novel graph-based approach to search in unknown environments is presented. A virtual geometric structure is imposed on the environment represented in computer memory by a graph. Algorithms use this representation to coordinate a team of robots (or entities). Local discovery of environmental features cause dynamic expansion of the graph resulting in global exploration of the unknown environment. The algorithm is shown to have O(k.nH) time complexity, where nH is the number of vertices of the discovered environment and 1 <= k <= nH. A maximum bound on the length of the resulting walk is given

Coexpression Network Analysis of miRNA-142 Overexpression in Neuronal Cells

MicroRNAs are small noncoding RNA molecules, which are differentially expressed in diverse biological processes and are also involved in the regulation of multiple genes. A number of sites in the 3′ untranslated regions (UTRs) of different mRNAs allow complimentary binding for a microRNA, leading to their posttranscriptional regulation. The miRNA-142 is one of the microRNAs overexpressed in neurons that is found to regulate SIRT1 and MAOA genes. Differential analysis of gene expression data, which is focused on identifying up- or downregulated genes, ignores many relationships between genes affected by miRNA-142 overexpression in a cell. Thus, we applied a correlation network model to identify the coexpressed genes and to study the impact of miRNA-142 overexpression on this network. Combining multiple sources of knowledge is useful to infer meaningful relationships in systems biology. We applied coexpression model on the data obtained from wild type and miR-142 overexpression neuronal cells and integrated miRNA seed sequence mapping information to identify genes greatly affected by this overexpression. Larger differences in the enriched networks revealed that the nervous system development related genes such as TEAD2, PLEKHA6, and POGLUT1 were greatly impacted due to miRNA-142 overexpression

Methamphetamine and Inflammatory Cytokines Increase Neuronal Na+/K+-ATPase Isoform 3: Relevance for HIV Associated Neurocognitive Disorders

Methamphetamine (METH) abuse in conjunction with human immunodeficiency virus (HIV) exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS), collectively termed HIV Associated Neurocognitive Disorders (HAND). Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV) differing by one regimen, METH treatment, we identified the neuron specific Na+/K+-ATPase alpha 1 isoform 3 (ATP1A3) to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK) pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

MicroRNA-142 reduces monoamine oxidase A expression and activity in neuronal cells by downregulating SIRT1

Aberrant expression of microRNAs (miRs) has been implicated in the pathogenesis of several neurodegenerative disorders. In HIV-associated neurocognitive disorders (HAND), miR-142 was found to be upregulated in neurons and myeloid cells in the brain. We investigated the downstream effects of chronic miR-142 upregulation in neuronal cells by comparing gene expression in stable clones of the human neuroblastoma cell line BE(2)M17 expressing miR-142 to controls. Microarray analysis revealed that miR-142 expression led to a reduction in monoamine oxidase (MAO) A mRNA, which was validated by qRT-PCR. In addition to the mRNA, the MAOA protein level and enzyme activity were also reduced. Examination of primary human neurons revealed that miR-142 expression indeed resulted in a downregulation of MAOA protein level. Although MAOA is not a direct target of miR-142, SIRT1, a key transcriptional upregulator of MAOA is, thus miR-142 downregulation of MAOA expression is indirect. MiR-142 induced decrease in MAOA expression and activity may contribute to the changes in dopaminergic neurotransmission reported in HAND

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism

<p>Abstract</p> <p>Background</p> <p>In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease.</p> <p>Results</p> <p>Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. <it>In vitro </it>overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis.</p> <p>Conclusions</p> <p>Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our <it>in vitro </it>studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.</p

Development and characterization of positively selected brain-adapted SIV

HIV is found in the brains of most infected individuals but only 30% develop neurological disease. Both viral and host factors are thought to contribute to the motor and cognitive disorders resulting from HIV infection. Here, using the SIV/rhesus monkey system, we characterize the salient characteristics of the virus from the brain of animals with neuropathological disorders. Nine unique molecular clones of SIV were derived from virus released by microglia cultured from the brains of two macaques with SIV encephalitis. Sequence analysis revealed a remarkably high level of similarity between their env and nef genes as well as their 3' LTR. As this genotype was found in the brains of two separate animals, and it encoded a set of distinct amino acid changes from the infecting virus, it demonstrates the convergent evolution of the virus to a unique brain-adapted genotype. This genotype was distinct from other macrophage-tropic and neurovirulent strains of SIV. Functional characterization of virus derived from representative clones showed a robust in vitro infection of 174xCEM cells, primary macrophages and primary microglia. The infectious phenotype of this virus is distinct from that shown by other strains of SIV, potentially reflecting the method by which the virus successfully infiltrates and infects the CNS. Positive in vivo selection of a brain-adapted strain of SIV resulted in a near-homogeneous strain of virus with distinct properties that may give clues to the viral basis of neuroAIDS