Basic Science Core I: Cell Culture and Neurotropic Viruses and Proteomics
This Core provides mammalian cell culture and virology services to investigators performing biomedical research on AIDS and the nervous system. Services include the preparation of CNS cell cultures, virus propagation, proteomics, biomarkers, and consultative expertise in gene editing. These services promote in uncovering mechanisms involved in the development of nervous system disorders associated with HIV-1 infection.
This section of the Core focuses on the study of HIV-1 infection at the molecular and cellular levels. Services include the purification, characterization, and maintenance of an assortment of central nervous system cells (neurons, astrocytes, oligodendrocytes, microglia, cerebral endothelial cells, progenitor and neural stem cells) and peripheral cells such as macrophages and T-cells.
For additional information, please contact Dianne Langford, PhD.
Targeted genome editing has been explosively fueled by the introduction of several novel DNA cleavage (nuclease) technologies including homing endonucleases or meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR-associated system 9 (Cas9) nucleases1. These nucleases utilize site-specific double-strand DNA break (DSB)-mediated DNA repair mechanisms allowing precise genome editing. Such strategy has enabled rapid, easy and efficient modification of endogenous genes/genomes in a wide variety of cell types and species. Clinically, it is being explored as a novel therapeutic approach for genetic disorders, viral infections and cancer. All ZFN, TALEN and Cas9 techniques have been used to disrupt HIV-1 entry co-receptors (CCR5, CXCR4) and proviral DNA-encoding viral proteins2. CCR5 gene-targeting ZFNs are in phase 2 clinical trials for HIV-1/AIDS treatment3. Also, ZFN and Cas9/gRNA have been recently shown to remove the proviral HIV-1 DNA from host cellular genome, by targeting its highly-conserved 5’ and 3’ long term repeats (LTRs)4. Our recent studies found that LTR-directed Cas9 eradicates the HIV-1 genome and effectively vaccinates target cells against HIV-1 reactivation and infection with high specificity and efficiency5. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses.
1 (Bedell et al., 2012; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013; Qi et al., 2013; Wang et al., 2012a)
Schematic illustration of strategy which can be adapted for cleaving HIV-1 genome at the 5’- and 3’- LTR, leading to excision of the proviral DNA from the host genome and protecting cells against new infection.
This Core provides state-of-the-art services in proteomics and data analysis, including novel methods for discovering biomarkers, innovative differential expression profiling and bioinformatics, interactomics (protein-protein interaction), and post-translational modifications (PTMs). In addition, this Core assists investigators with identification of discriminative proteins, protein function and pathway analysis, development of prognostic models, and modern methods for biomarker discovery from plasma and cerebral spinal fluid.
For additional information on proteomics, please contact Salim Merali, PhD.
Polyamine as a biomarker for HIV associated neurocognitive disorders
Soon after primary infection, HIV-1 is disseminated in the central nervous system (CNS) where productive replication in brain macrophages and microglia and limited expression of the viral genome in astrocytes may cause an array of toxic events that contribute to HIV-associated neurocognitive disorders (HAND). The presence of HIV-1 and expression of viral proteins, even at low levels, in the brains of HIV patients taking combined antiretroviral therapy (cART) is often associated with neurocognitive disorders. It is estimated that greater than 50% of HIV-1 infected individuals with low viral load and high CD4 lymphocyte cell counts, exhibit some form of HAND, and it is suggested that cART may be at least partially responsible for these impairments. At present, there are no molecular diagnostic biomarkers for different classes of HAND and diagnosis is mostly based on exclusion of other possible causes accompanied by neurological exam, neuropsychological tests, and brain MRI scan.
To begin to address this issue, Dr. Merali’s group performed a bioamine targeted metabolomics study in cerebral spinal fluid (CSF) from the HIV+ participants with and without HAND (n =99). Interestingly, their results showed a HAND-specific increase in the levels of acetylated polyamines in the CSF. The acetylated polyamines are the products of the catabolic enzyme spermidine/spermine acetyltransferase (SSAT), which tightly regulate the amounts of polyamines in the cell. Endogenous polyamines (putrescine, spermidine, and spermine) are known for modulating NMDA receptor function and early studies demonstrated that HIV-1 Tat-induced neurotoxicity involved interaction between polyamines and NMDA receptors. To further understand the origin and the implications of increased acetylated polyamines in the CSF of HIV+ individuals with HAND, the group showed that HIV-1 Tat activates polyamine flux in the astrocytes. This flux was generated upon activation of SSAT and was also increased in brain samples from HIV+ individuals with HAND as compared to those with normal cognition.
Enhanced levels of acetylated polyamine in HIV patients with more severe neurocognitive disorders suggest a potential role for these metabolites in the neurodegeneration process among HAND patients. Thus, polyamine may serve as a potential predictive diagnostic biomarker for different severities of HAND.
Kamel Khalili, PhD
Basic Science Core I Leader
Department of Neuroscience
Temple University School of Medicine
3500 N. Broad St., 7th Fl.
Philadelphia, PA 19140
T. Dianne Langford, PhD
Salim Merali, PhD
Wenhui Hu, MD, PhD