Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons.

The research on human neural progenitor cells holds great potential for the understanding of the molecular programs that control differentiation of cells of glial and neuronal lineages, as well as pathogenetic mechanisms of neurological diseases. Stem cell technologies also provide opportunities for the pharmaceutical industry to develop new approaches for regenerative medicine. Here, we describe the protocol for the isolation and maintenance of neural progenitor cells and cortical neurons using human fetal brain tissue. This protocol can be successfully adapted for the preparation of rodent neural and oligodendrocyte progenitor cells. While several methods for isolating neural and oligodendrocyte progenitors from rodent brain tissue have been described, including techniques utilizing gene transfer and magnetic resonance beads, few methods are specifically focused on deriving human oligodendrocyte progenitor cells. Development of the human cultures provides the most physiologically relevant system for investigating mechanisms which regulate the function of oligodendrocytes, specifically of human origin.

Compartmentalized Neuronal Cultures.

The culturing of neurons results in formation of the layer of neurons with random extensive overlapping outgrowth. To understand specific roles of somas, axons, and dendrites in complex function of neurons and to identify molecular mechanisms of biological processes in these cellular compartments, various methods were developed. We utilized AXon Investigation System (AXIS™) manufactured by Millipore. This device provides an opportunity to orient neuronal outgrowth and spatially isolate neuronal processes from neuronal bodies. AXIS device is a slide-mounted microfluidic system, which consists of four wells. Two of the wells are connected by a channel on each side of the device. Channels are connected by microgrooves (approximately 120). The size of microgrooves (10µm in width and 5µm in height) does not permit passage of cell through while allowing extension of neurites. The microfluidic design also allows for an establishment of a hydrostatic gradient when the volume in one chamber is greater than that in the other (Park et al., Nat Protoc 1:2128-2136, 2006). This feature allows for studying of the effect of specific compounds on selected compartments of neurons.

Structure-based release analysis of the JC virus agnoprotein regions: A role for the hydrophilic surface of the major alpha helix domain in release.

Agnoprotein (Agno) is an important regulatory protein of JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) and these viruses are unable to replicate efficiently in the absence of this protein. Recent 3D-NMR structural data revealed that Agno contains two alpha-helices (a minor and a major) while the rest of the protein adopts an unstructured conformation (Coric et al., 2017, J Cell Biochem). Previously, release of the JCV Agno from the Agno-positive cells was reported. Here, we have further mapped the regions of Agno responsible for its release by a structure-based systematic mutagenesis approach. Results revealed that amino acid residues (Lys22, Lys23, Phe31, Glu34, and Asp38) located either on or adjacent to the hydrophilic surface of the major alpha-helix domain of Agno play critical roles in release. Additionally, Agno was shown to strongly interact with unidentified components of the cell surface when cells are treated with Agno, suggesting additional novel roles for Agno during the viral infection cycle.

Discovery and characterization of novel trans-spliced products of human polyoma JC virus late transcripts from PML patients.

Although the human neurotropic polyomavirus, JC virus (JCV), was isolated almost a half century ago, understanding the molecular mechanisms governing its biology remains highly elusive. JCV infects oligodendrocytes and astrocytes in the central nervous system (CNS) and causes a rare fatal brain disease known as progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals including AIDS. It has a small circular DNA genome (∼5 kb) and generates two primary transcripts from its early and late coding regions, producing several predicted alternatively spliced products mainly by cis-splicing. Here, we report the discovery and characterization of two novel open reading frames (ORF1 and ORF2) associated with JCV late transcripts, generated by an unusual splicing process called trans-splicing. These ORFs result from (i) the trans-splicing of two different lengths of the 5'-short coding region of VP1 between the coding regions of agnoprotein and VP2 after replacing the intron located between these two coding regions and (ii) frame-shifts occurring within the VP2 coding sequences terminated by a stop codon. ORF1 and ORF2 are capable of encoding 58 and 72 aa long proteins respectively and are expressed in infected cells and PML patients. Each ORF protein shares a common coding region with VP1 and has a unique coding sequence of their own. When the expression of the unique coding regions of ORFs is blocked by a stop codon insertion in the viral background, the mutant virus replicates less efficiently when compared to wild-type, suggesting that the newly discovered ORFs play critical roles in the JCV life cycle.

CRISPR Editing Technology in Biological and Biomedical Investigation.

The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy, and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future. J. Cell. Biochem. 118: 3586-3594, 2017. © 2017 Wiley Periodicals, Inc.

Nuclear Magnetic Resonance Structure of the Human Polyoma JC Virus Agnoprotein.

Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays critical roles during the viral replication cycle. It forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain, which is important for the stability and function of the protein. We recently resolved the partial 3D structure of this protein by NMR using a synthetic peptide encompassing amino acids Thr17 to Gln52, where the Leu/Ile/Phe- rich region was found to adopt a major alpha-helix conformation spanning amino acids 23-39. Here, we report the resolution of the 3D structure of full-length JCV agnoprotein by NMR, which not only confirmed the existence of the previously reported major α-helix domain at the same position but also revealed the presence of an additional minor α-helix region spanning amino acid residues Leu6 to lys13. The remaining regions of the protein adopt an intrinsically unstructured conformation. J. Cell. Biochem. 118: 3268-3280, 2017. © 2017 Wiley Periodicals, Inc.

Novel AIDS therapies based on gene editing.

HIV/AIDS remains a major public health issue. In 2014, it was estimated that 36.9 million people are living with HIV worldwide, including 2.6 million children. Since the advent of combination antiretroviral therapy (cART), in the 1990s, treatment has been so successful that in many parts of the world, HIV has become a chronic condition in which progression to AIDS has become increasingly rare. However, while people with HIV can expect to live a normal life span with cART, lifelong medication is required and cardiovascular, renal, liver, and neurologic diseases are still possible, which continues to prompt research for a cure for HIV. Infected reservoir cells, such as CD4+ T cells and myeloid cells, allow persistence of HIV as an integrated DNA provirus and serve as a potential source for the re-emergence of virus. Attempts to eradicate HIV from these cells have focused mainly on the so-called "shock and kill" approach, where cellular reactivation is induced so as to trigger the purging of virus-producing cells by cytolysis or immune attack. This approach has several limitations and its usefulness in clinical applications remains to be assessed. Recent advances in gene-editing technology have allowed the use of this approach for inactivating integrated proviral DNA in the genome of latently infected cells or knocking out HIV receptors. Here, we review this strategy and its potential to eliminate the latent HIV reservoir resulting in a sterile cure of AIDS.

The DNA damage response promotes polyomavirus JC infection by nucleus to cytoplasm NF- kappaB activation.

BACKGROUND: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or "inside-out" NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. METHODS: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. RESULTS: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. CONCLUSIONS: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.

Zika virus: An emergent neuropathological agent.

The emergence of Zika virus in the Americas has followed a pattern that is familiar from earlier epidemics of other viruses, where a new disease is introduced into a human population and then spreads rapidly with important public health consequences. In the case of Zika virus, an accumulating body of recent evidence implicates the virus in the etiology of serious pathologies of the human nervous system, that is, the occurrence of microcephaly in neonates and Guillain-Barré syndrome in adults. Zika virus is an arbovirus (arthropod-borne virus) and a member of the family Flaviviridae, genus Flavivirus. Zika virions are enveloped and icosahedral, and contain a nonsegmented, single-stranded, positive-sense RNA genome, which encodes 3 structural and 7 nonstructural proteins that are expressed as a single polyprotein that undergoes cleavage. Zika genomic RNA replicates in the cytoplasm of infected host cells. Zika virus was first detected in 1947 in the blood of a febrile monkey in Uganda's Zika Forest and in crushed suspensions of the Aedes mosquito, which is one of the vectors for Zika virus. The virus remained obscure, with a few human cases confined to Africa and Asia. There are two lineages of the Zika virus, African and Asian, with the Asian strain causing outbreaks in Micronesia in 2007 and French Polynesia in 2013-2014. From here, the virus spread to Brazil with the first report of autochthonous Zika transmission in the Americas in March 2015. The rapid advance of the virus in the Americas and its likely association with microcephaly and Guillain-Barré syndrome make Zika an urgent public health concern. Ann Neurol 2016;80:479-489.

Gene Editing for Treatment of Neurological Infections.

The study of neurological infections by viruses defines the field of neurovirology, which has emerged in the last 30 years and was founded upon the discovery of a number of viruses capable of infecting the human nervous system. Studies have focused on the molecular and biological basis of viral neurological diseases with the aim of revealing new therapeutic options. The first studies of neurovirological infections can be traced back to the discovery that some viruses have an affinity for the nervous system with research into rabies by Louis Pasteur and others in the 1880s. Today, the immense public health impact of neurovirological infections is illustrated by diseases such as neuroAIDS, progressive multifocal leukoencephalopathy, and viral encephalitis. Recent research has seen the development of powerful new techniques for gene editing that promise revolutionary opportunities for the development of novel therapeutic options. In particular, clustered regulatory interspaced short palindromic repeat-associated 9 system provides an effective, highly specific and versatile tool for targeting DNA viruses that are beginning to allow the development of such new approaches. In this short review, we discuss these recent developments, how they pertain to neurological infections, and future prospects.

HIV-1 Tat and Cocaine Impair Survival of Cultured Primary Neuronal Cells via a Mitochondrial Pathway.

Addictive stimulant drugs, such as cocaine, are known to increase the risk of exposure to HIV-1 infection and hence predispose towards the development of AIDS. Previous findings suggested that the combined effect of chronic cocaine administration and HIV-1 infection enhances cell death. Neuronal survival is highly dependent on the health of mitochondria providing a rationale for assessing mitochondrial integrity and functionality following cocaine treatment, either alone or in combination with the HIV-1 viral protein Tat, by monitoring ATP release and mitochondrial membrane potential (ΔΨm). Our results indicate that exposing human and rat primary hippocampal neurons to cocaine and HIV-1 Tat synergistically decreased both mitochondrial membrane potential and ATP production. Additionally, since previous studies suggested HIV-1 infection alters autophagy in the CNS, we investigated how HIV-1 Tat and cocaine affect autophagy in neurons. The results indicated that Tat induces an increase in LC3-II levels and the formation of Parkin-ring-like structures surrounding damaged mitochondria, indicating the possible involvement of the Parkin/PINK1/DJ-1 (PPD) complex in neuronal degeneration. The importance of mitochondrial damage is also indicated by reductions in mitochondrial membrane potential and ATP content induced by HIV-1 Tat and cocaine.

The Brd4 acetyllysine-binding protein is involved in activation of polyomavirus JC.

Brd4 is an epigenetic reader protein and a member of the BET (bromodomain and extra terminal domain) family of proteins with two bromodomains that recognize acetylated lysine residues. Brd4 specifically binds to acetylated transcription factor NF-κB p65 and coactivates transcription. Polyomavirus JC (JCV) is regulated by a noncoding control region (NCCR) containing promoter/enhancer elements for viral gene expression including a binding site for NF-κB, which responds to proinflammatory cytokines such as TNF-α, the DNA damage response, calcium signaling and acetylation of the NF-κB p65 subunit on lysine residues K218 and K221. Earlier studies indicated that NF-κB is involved in the reactivation of persistent/latent JCV in glial cells to cause progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the brain caused by replication of JCV in glial cells. To investigate the mechanism of action of NF-κB acetylation on JCV transcription, we examined Brd4 and found that JCV early transcription was stimulated by Brd4 via the JCV NF-κB site and that p65 K218 and K221 were involved. Treatment with the Brd4 inhibitor JQ1(+) or mutation of either K218 or K221 to glutamine (K218R or K221) inhibited this stimulation and decreased the proportion of p65 in the nucleus. We conclude that Brd4 is involved in the regulation of the activation status of JCV in glial cells.

Emerging From the Unknown: Structural and Functional Features of Agnoprotein of Polyomaviruses.

Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe-rich domain. Structural NMR studies revealed that this domain adopts an alpha-helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB-1, p53, Ku70, FEZ1, HP1α, PP2A, AP-3, PCNA, and α-SNAP; and viral proteins, including small t antigen, large T antigen, HIV-1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. J. Cell. Physiol. 231: 2115-2127, 2016. © 2016 Wiley Periodicals, Inc.

CRISPR/Cas9 and cancer targets: future possibilities and present challenges.

All cancers have multiple mutations that can largely be grouped into certain classes depending on the function of the gene in which they lie and these include oncogenic changes that enhance cellular proliferation, loss of function of tumor suppressors that regulate cell growth potential and induction of metabolic enzymes that confer resistance to chemotherapeutic agents. Thus the ability to correct such mutations is an important goal in cancer treatment. Recent research has led to the developments of reagents which specifically target nucleotide sequences within the cellular genome and these have a huge potential for expanding our anticancer armamentarium. One such a reagent is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, a powerful, highly specific and adaptable tool that provides unparalleled control for editing the cellular genome. In this short review, we discuss the potential of CRISPR/Cas9 against human cancers and the current difficulties in translating this for novel therapeutic approaches.

GRP78 Interacting Partner Bag5 Responds to ER Stress and Protects Cardiomyocytes From ER Stress-Induced Apoptosis.

Bag5 is a member of the BAG family of molecular chaperone regulators and is unusual in that it consists of five BAG domains, which function as modulators of chaperone activity. Bag family proteins play a key role in cellular as well as in cardiac function and their differential expression is reported in heart failure. In this study, we examined the importance of a Bag family member protein, Bag5, in cardiomyocytes during endoplasmic reticulum (ER) stress. We found that expression of Bag5 in cardiomyocytes is significantly increased with the induction of ER stress in a time dependent manner. We have taken gain-in and loss-of functional approaches to characterize Bag5 protein function in cardiomyocytes. Adenoviral mediated expression of Bag5 significantly decreased cell death as well as improved cellular viability in ER stress. Along with this, ER stress-induced CHOP protein expression is significantly decreased in cells that overexpress Bag5. Conversely, we found that siRNA-mediated knockdown of Bag5 caused cell death, increased cytotoxicity, and decreased cellular viability in cardiomyocytes. Mechanistically, we found that Bag5 protein expression is significantly increased in the ER during ER stress and that this in turn modulates GRP78 protein stability and reduces ER stress. This study suggests that Bag5 is an important regulator of ER function and so could be exploited as a tool to improve cardiomyocyte function under stress conditions. J. Cell. Biochem. 117: 1813-1821, 2016. © 2016 Wiley Periodicals, Inc.

Diagnostic assays for polyomavirus JC and progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at-risk population is growing larger and includes individuals with HIV-1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements.

Expression of Signaling Molecules in Progressive Multifocal Leukoencephalopathy.

INTRODUCTION: Progressive multifocal leukoencephalopathy (PML) is a debilitating demyelinating disease of the CNS caused by the infection and destruction of glial cells by JC virus (JCV) and is an AIDS-defining disease. Infection with JCV is common and most people acquire antibodies early in life. After initial infection, JCV remains in an asymptomatic persistent state and can be detected by PCR in many tissues including brain. A major question in PML pathogenesis is how the virus reactivates from persistence in HIV-1/AIDS. Our studies with primary cultures of glial cells have implicated transcription factors NF-κB and NFAT4, which bind to a unique site in the JCV noncoding control region and stimulate viral gene expression. Furthermore, these transcription factors are controlled by pathways downstream of proinflammatory cytokines, e.g., TNF-α activates NF-κB and stimulates JCV transcription. OBJECTIVES: We hypothesize that HIV-1/PML initiation may involve reactivation of JCV by cytokine disturbances in the brain such as occur in HIV-1/AIDS. In this study, the objective was to evaluate HIV-1/PML clinical samples for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 compared to non-PML controls. METHODS: We evaluated HIV-1/PML clinical samples and non-PML controls for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 using Western blot and immunohistochemistry. RESULTS: Consistent with our hypothesis, compared to non-PML controls, HIV-1/PML tissue has high levels of TNF-α and TNFR1 expression and NF-κB and NFAT4 were preferentially localized to the nucleus. CONCLUSION: The involvement of TNF-α/NF-κB/NFAT4 signaling in JCV regulation that we reported from experiments in cultured human glial cells may be clinically relevant in PML.

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.

Animal Models for Progressive Multifocal Leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the CNS caused by the human polyomavirus JC (JCV). JCV replication occurs only in human cells and investigation of PML has been severely hampered by the lack of an animal model. The common feature of PML is impairment of the immune system. The key to understanding PML is working out the complex mechanisms that underlie viral entry and replication within the CNS and the immunosurveillance that suppresses the virus or allows it to reactivate. Early models involved the simple inoculation of JCV into animals such as monkeys, hamsters, and mice. More recently, mouse models transgenic for the gene encoding the JCV early protein, T-antigen, a protein thought to be involved in the disruption of myelin seen in PML, have been employed. These animal models resulted in tumorigenesis rather than demyelination. Another approach is to use animal polyomaviruses that are closely related to JCV but able to replicate in the animal such as mouse polyomavirus and SV40. More recently, novel models have been developed that involve the engraftment of human cells into the animal. Here, we review progress that has been made to establish an animal model for PML, the advances and limitations of different models and weigh future prospects.