Longitudinal in vivo metabolic labeling reveals tissue-specific mitochondrial proteome turnover rates and proteins selectively altered by parkin deficiency.

Our study utilizes a longitudinal isotopic metabolic labeling approach in vivo in combination with organelle fraction proteomics to address the role of parkin in mitochondrial protein turnover in mice. The use of metabolic labeling provides a method to quantitatively determine the global changes in protein half-lives whilst simultaneously assessing protein expression. Studying two diverse mitochondrial populations, we demonstrated the median half-life of brain striatal synaptic mitochondrial proteins is significantly greater than that of hepatic mitochondrial proteins (25.7 vs. 3.5 days). Furthermore, loss of parkin resulted in an overall, albeit modest, increase in both mitochondrial protein abundance and half-life. Pathway and functional analysis of our proteomics data identified both known and novel pathways affected by loss of parkin that are consistent with its role in both mitochondrial quality control and neurodegeneration. Our study therefore adds to a growing body of evidence suggesting dependence on parkin is low for basal mitophagy in vivo and provides a foundation for the investigation of novel parkin targets.

Plasma gelsolin accumulates in macrophage nodules in brains of simian immunodeficiency virus infected rhesus macaques.

Plasma gelsolin (pGSN), an isoform 1, is secreted by various types of cells in the central nervous system (CNS) and periphery, but not by the liver. pGSN circulates in blood and cerebrospinal fluid (CSF); however, its concentration in CSF is approximately twenty times lower than in plasma. It has been shown that several types of cells such as oligodendrocytes, neurons, and/or astrocytes contribute to the overall pool of pGSN in the CNS. Further, it has been postulated that pGSN plays multiple roles during microbial infection and modulates inflammatory responses; however, the exact mechanism of regulation is not known. We previously showed that levels of pGSN in CSF of individuals with advanced neurocognitive impairment due to HIV infection of the brain are decreased. Here, we show that macrophages express significant amounts of pGSN in response to HIV infection in vitro. Using immunohistochemistry of simian immunodeficiency virus infected rhesus monkey brains, we show that increased levels of pGSN are present in macrophage nodules creating locally a high level of this protein within the brain. This may not be reflected by the overall decreased level in the distinct CSF compartment.

Deprenyl reduces inflammation during acute SIV infection.

In the era of antiretroviral therapy, inflammation is a central factor in numerous HIV-associated comorbidities, such as cardiovascular disease, cognitive impairment, and neuropsychiatric disorders. This highlights the value of developing therapeutics that both reduce HIV-associated inflammation and treat associated comorbidities. Previous research on monoamine oxidase inhibitors (MAOIs) suggests this class of drugs has anti-inflammatory properties in addition to neuropsychiatric effects. Therefore, we examined the impact of deprenyl, an MAOI, on SIV-associated inflammation during acute SIV infection using the rhesus macaque model of HIV infection. Our results show deprenyl decreased both peripheral and CNS inflammation but had no effect on viral load in either the periphery or CNS. These data show that the MAOI deprenyl may have broad anti-inflammatory effects when given during the acute stage of SIV infection, suggesting more research into the anti-inflammatory effects of this drug could result in a beneficial adjuvant for antiretroviral therapy.

Androgen treatment prevents diabetes in nonobese diabetic mice.

The nonobese diabetic (NOD) mouse strain provides a model system for human autoimmune diabetes. This disease model is extensively used not only to examine the etiology and pathogenesis of diabetes, but also as a means to evaluate therapies. In NOD mice, the disease progresses from insulitis to islet destruction and clinical diabetes in a high percentage of female mice. In this study, androgen therapy, begun after the onset of insulitis, was found to prevent islet destruction and diabetes without eliminating the islet inflammation in female NOD mice. However, diabetes can be adoptively transferred into such hormone-treated recipients. The prevention of disease onset by androgen is likely due to the hormonal alteration of the development or function of the immune cells necessary for islet destruction.

Myasthenia gravis-like syndrome induced by expression of interferon gamma in the neuromuscular junction.

Abnormal humoral responses toward motor end plate constituents in muscle induce myasthenia gravis (MG). To study the etiology of this disease, and whether it could be induced by host defense molecules, we examined the consequences of interferon (IFN) gamma production within the neuromuscular junction of transgenic mice. The transgenic mice exhibited gradually increasing muscular weakness, flaccid paralysis, and functional disruption of the neuromuscular junction that was reversed after administration of an inhibitor of acetylcholinesterase, features which are strikingly similar to human MG. Furthermore, histological examination revealed infiltration of mononuclear cells and autoantibody deposition at motor end plates. Immunoprecipitation analysis indicated that a previously unidentified 87-kD target antigen was recognized by sera from transgenic mice and also by sera from the majority of human MG patients studied. These results suggest that expression of IFN-gamma at motor end plates provokes an autoimmune humoral response, similar to human MG, thus linking the expression of this factor with development of this disease.

Antiviral treatment normalizes neurophysiological but not movement abnormalities in simian immunodeficiency virus-infected monkeys.

Simian immunodeficiency virus (SIV) infection of rhesus monkeys provides an excellent model of the central nervous system (CNS) consequences of HIV infection. To discern the relationship between viral load and abnormalities induced in the CNS by the virus, we infected animals with SIV and later instituted antiviral treatment to lower peripheral viral load. Measurement of sensory-evoked potentials, assessing CNS neuronal circuitry, revealed delayed latencies after infection that could be reversed by lowering viral load. Cessation of treatment led to the reappearance of these abnormalities. In contrast, the decline in general motor activity induced by SIV infection was unaffected by antiviral treatment. An acute increase in the level of the chemokine monocyte chemoattractant protein-1 (MCP-1) was found in the cerebrospinal fluid (CSF) relative to plasma in the infected animals at the peak of acute viremia, likely contributing to an early influx of immune cells into the CNS. Examination of the brains of the infected animals after return of the electrophysiological abnormalities revealed diverse viral and inflammatory findings. Although some of the physiological abnormalities resulting from SIV infection can be at least temporarily reversed by lowering viral load, the viral-host interactions initiated by infection may result in long-lasting changes in CNS-mediated functions.

Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers.

We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.

Nonhomologous pairing in mice heterozygous for a t haplotype can produce recombinant chromosomes with duplications and deletions.

We have investigated the structure and properties of a chromosomal product recovered from a rare recombination event between a t haplotype and a wild-type form of mouse chromosome 17. Our embryological and molecular studies indicate that this chromosome (twLub2) is characterized by both a deletion and duplication of adjacent genetic material. The deletion appears to be responsible for a dominant lethal maternal effect and a recessive embryonic lethality. The duplication provides an explanation for the twLub2 suppression of the dominant T locus phenotype. A reanalysis of previously described results with another chromosome 17 variant called TtOrl indicates a structure for this chromosome that is reciprocal to that observed for twLub2. We have postulated the existence of an inversion over the proximal portion of all complete t haplotypes in order to explain the generation of the partial t haplotypes twLub2 and TtOrl. This proximal inversion and the previously described distal inversion are sufficient to account for all of the recombination properties that are characteristic of complete t haplotypes. The structures determined for twLub2 and TtOrl indicate that rare recombination can occur between nonequivalent genomic sequences within the inverted proximal t region when wild-type and t chromosomes are paired in a linear, nonhomologous configuration.

Feline immunodeficiency virus as a model for study of lentivirus infection of the central nervous system.

Feline immunodeficiency virus infects the CNS and results in predictable pathophysiology strikingly similar to that seen with HIV-1 infection of humans. The observed pathophysiology is mimicked in several physiologically assessed modalities, further supporting the validity of the feline model. Peripheral and control evoked potential findings and the occurrence of the sleep architecture changes in both cat and human disease provide an intriguing focus for further investigation. Although structurally diverse in an absolute sense, FIV and HIV-1 share basic structural features and commonalities of their life cycle. It is likely that by understanding the common mechanisms by which these lentiviruses influence CNS function, a more complete understanding of the neurological deficits seen in HIV-1 infected patients will be obtained. The cat model is particularly valuable for study of CNS disease, since it allows detailed analyses of events during the acute phase of infection, under circumstances in which the nature and timing of the infection are carefully controlled. The availability of molecular clones for mutational analysis will facilitate mapping of genomic regions critical to the perturbation of CNS function. It is suggested that development of intervention strategies in the cat model will yield treatment modalities directly applicable to HIV-1 infection of humans.

Analysis of result variability from high-density oligonucleotide arrays comparing same-species and cross-species hybridizations.

There exists a significant limitation in the variety of organismsfor which microarrays have been developed because of a lack of genomic sequence data. A near-term solution to this limitation is to use microarrays designed for one species to analyze RNA samples from closely related species. The assumption is that conservation of gene sequences between species will be sufficient to generate a reasonable amount of good-quality data. While there have been relatively few published reports describing the use of microarrays for cross-species hybridizations, this technique is potentially a powerful tool for understanding genomics in model organisms such as nonhuman primates. Here we describe the analysis and comparison of hybridization characteristics and data variability from a set of cross-species (rhesus macaque) and same-species (human) hybridization experiments using human high-density Affymetrix oligonucleotide arrays. The data reveal that a large fraction of probe sets are effective at transcript detection in the cross-species hybridization, validating the application of cross-species hybridizations for nonhuman primate genomics research.

Cladosporium trichoides cerebral phaeohyphomycosis in a liver transplant recipient. Report of a case.

Cerebral phaeohyphomycosis (also referred to as cerebral chromomycosis), one of the diseases caused by the dematiaceous (black) fungi, is most commonly caused by Cladosporium trichoides (referred to by some as Xylohypha bantiana) and is a rare disease, with 31 culture-proven cases reported to date. Although most cases have occurred in immunocompetent hosts, recent experimental evidence suggests that host immunosuppression may predispose patients to the disease. The authors report a case of fatal cerebral phaeohyphomycosis in a liver transplant patient, the first to occur in a transplant patient of any type, to the best of the authors' knowledge. This case provides support for the hypothesis that immunosuppressed patients may be at increased risk for development of this disease.

The evolutionary young miR-1290 favors mitotic exit and differentiation of human neural progenitors through altering the cell cycle proteins.

Regulation of cellular proliferation and differentiation during brain development results from processes requiring several regulatory networks to function in synchrony. MicroRNAs are part of this regulatory system. Although many microRNAs are evolutionarily conserved, recent evolution of such regulatory molecules can enable the acquisition of new means of attaining specialized functions. Here we identify and report the novel expression and functions of a human and higher primate-specific microRNA, miR-1290, in neurons. Using human fetal-derived neural progenitors, SH-SY5Y neuroblastoma cell line and H9-ESC-derived neural progenitors (H9-NPC), we found miR-1290 to be upregulated during neuronal differentiation, using microarray, northern blotting and qRT-PCR. We then conducted knockdown and overexpression experiments to look at the functional consequences of perturbed miR-1290 levels. Knockdown of miR-1290 inhibited differentiation and induced proliferation in differentiated neurons; correspondingly, miR-1290 overexpression in progenitors led to a slowing down of the cell cycle and differentiation to neuronal phenotypes. Consequently, we identified that crucial cell cycle proteins were aberrantly changed in expression level. Therefore, we conclude that miR-1290 is required for maintaining neurons in a differentiated state.

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction.

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3'-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins.

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1-11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons.

MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological disease.

MicroRNAs (miRNAs) play important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both HIV associated dementia in humans and its monkey model SIV encephalitis we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. MiR-21 can be induced in neurons by prolonged N-methyl-D-aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease but also as a crucial effector of HIV induced neuronal dysfunction and neurodegeneration.

Protein kinase C expression in salivary gland acinar epithelial cells in non-obese diabetic mice, an experimental model for Sjögren's syndrome.

We planned to investigate the expression of protein kinase C (PKC) isoforms in acinar epithelial cells of salivary glands in the non-obese diabetic (NOD) mouse to find out if they develop changes of the PKC system like those seen in the human counterpart, i.e. in Sjögren's syndrome. Parotid, submandibular, and sublingual glands from NOD and control BALB/c mice were stained with a panel of monoclonal antibodies directed against conventional (alpha, beta, and gamma), novel (delta, epsilon, and theta), and atypical (lambda and iota) PKC isoforms using the streptavidin/HRP method. Similarly to human labial salivary glands, acinar epithelial cells of the healthy control BALB/c mice contained two of the conventional PKC isoforms, alpha and beta. Acinar and ductal epithelial cells also contained the atypical PKC isoforms lambda and iota. PKC isoforms gamma, delta, epsilon, and theta were not found. NOD mice which displayed focal sialadenitis contained the same conventional and atypical PKC isoforms. The acinar cells in NOD mice, in contrast to the Sjögren's syndrome patients, did not lack PKC alpha or beta. On the contrary, PKC alpha and beta staining was stronger than in the control BALB/c mice. The present results demonstrate that both conventional and atypical PKC isoforms participate in the salivary epithelial cell biology and that there are mouse strain-associated and/or disease state-associated changes in their expression. The lack of PKC alpha and beta isoforms found in Sjögren's syndrome was not reproduced in NOD mice, which discloses one more difference between the human disease and its NOD mouse model.

Sex steroids and the immune system.

Autoimmune diseases afflict women at a much higher rate than they do men. Although the differences in sex steroids probably play a role in this sexual dimorphism, the effects of sex steroids on the immune system and the mechanisms by which those effects occur are uncertain. We have begun studies to examine systematically these processes by examining the cells responsive to sex steroids, the genes regulated by these hormones, and the effect on the immune system and autoimmunity.

Interferon-gamma and the sexual dimorphism of autoimmunity.

The sexual difference in the incidence of autoimmune diseases has remained an enigma for many years. In the examination of the induction of autoimmunity in transgenic mice, evidence has been obtained further implicating the lymphokine interferon-gamma in the etiology of autoimmunity. Sex steroid regulation of the production of this molecule, as well as other cytokines, may help explain the gender-specific differences in the immune system, including autoimmunity.

Estrogen regulates the IFN-gamma promoter.

The greater immune reactivity of females has been attributed in part to the influence of sex steroid hormones, but the underlying mechanisms are unknown. Here we report evidence that expression of the IFN-gamma gene may be subject to direct hormonal control. In a transient expression assay, the sex steroid 17 beta-estradiol markedly increases activity of the IFN-gamma promoter in lymphoid cells that express the appropriate hormone receptor. This effect is mediated by sequences in the 5'-flanking region of the gene, and can augment the effect of T cell-activating agents. Short term exposure to estradiol also increases IFN-gamma mRNA expression in Con A-treated murine spleen cells. Hormonal regulation of this pleiotropic cytokine may account in part for the ability of estrogen to potentiate many types of immune responses, and for the disproportionate susceptibility of females to autoimmune disease.