Erythropoietin-derived peptide treatment reduced neurological deficit and neuropathological changes in a mouse model of tauopathy.

BACKGROUND: Prominent activation of microglial immune/inflammatory processes is a characteristic feature of brains of patients with tauopathies including Alzheimer's disease (AD), suggesting that neuroinflammation may be a critical factor in their pathogenesis. Strategies aimed at developing new therapeutics for tauopathies based on anti-inflammation or immunomodulation are likely to be promising avenues of research. We previously developed JM4-a 19'mer cyclic peptide derived from the first loop of human erythropoietin. This peptide possesses beneficial immune modulatory and tissue protective effects while lacking the undesirable side effects of full-length erythropoietin. In this preclinical study, we investigated the effect of chronic JM4 treatment on the PS19 mouse that carries the P301S mutant human tau gene, linked to a form of frontotemporal dementia. This transgenic mouse has been widely used as a model of tauopathies including AD and related dementias. METHODS: Daily subcutaneous treatment of female PS19 mice with JM4 was initiated before disease onset and continued on for the animals' lifespan. The progression of neurological deficit and the lifespan of these mice were assessed. To evaluate the effect of JM4 treatment on cognition of these animals, the PS19 mice underwent Barnes maze test and elevated plus maze test. In addition, neuronal loss, phosphorylated tau aggregation, and microglial activation were assessed using immunohistochemistry of PS19 mouse brain sections. RESULTS: JM4 treatment of PS19 mice initiated before disease onset reduced neurological deficit, prolonged lifespan, and rescued memory impairment. The beneficial effects of JM4 were accompanied by reductions in neuronal loss, phosphorylated tau aggregation, and microglial activation in the PS19 mouse brain. LIMITATIONS: Use of a single dose of JM4 and female mice only. CONCLUSION: JM4 is a potential novel therapeutic agent for the treatment of tauopathies including AD and related dementias.

Prolonged Beneficial Effect of Brief Erythropoietin Peptide JM4 Therapy on Chronic Relapsing EAE.

Potent beneficial immunomodulatory and anti-inflammatory effects of whole-molecule erythropoietin have been demonstrated in a variety of animal disease models including experimental autoimmune encephalomyelitis (EAE); however, excessive hematopoiesis limits its use in clinical applications. Our group previously generated an Epo-derived small peptide JM4 that is side-effect free and has strong neuroprotective activity without hematologic effects. Here, we investigated the long-term clinical effects of brief treatment with JM4 in chronic relapsing EAE using bioluminescence imaging (BLI) in transgenic mice containing the luciferase gene driven by the murine GFAP promoter. EAE mice treated with JM4 exhibited marked improvement in clinical scores and showed fewer disease flareups than control animals. JM4 therapy concomitantly led to markedly decreased GFAP bioluminescence in the brain and spinal cord in both acute and chronic relapsing EAE mouse models. We found a marker for toxic A1 astrocytes, complement component C3, that is upregulated in the brain and cord of EAE mice and sharply reduced in JM4-treated animals. In addition, an abnormally leaky neurovascular unit permeability was rapidly normalized within 5 days by JM4 therapy. The prolonged therapeutic benefit seen following brief JM4 treatment in EAE mice closely resemble that recently described in humans receiving pulsed immune reconstitution therapy with the disease-modifying compounds, alemtuzumab and cladribine. Our study suggests that JM4 therapy may have widespread clinical applicability for long-term treatment of inflammatory demyelinating diseases and that BLI is a useful noninvasive means of monitoring murine disease activity of the central nervous system.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development.

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

In vivo Bioluminescence Imaging Used to Monitor Disease Activity and Therapeutic Response in a Mouse Model of Tauopathy.

Many studies of tauopathy use transgenic mice that overexpress the P301S mutant form of tau. Neuronal damage in these mice is associated with astrogliosis and induction of glial fibrillary acidic protein (GFAP) expression. GFAP-luc transgenic mice express firefly luciferase under the GFAP promoter, allowing bioluminescence to be measured non-invasively as a surrogate biomarker for astrogliosis. We bred double transgenic mice possessing both P301S and GFAP-luc cassettes and compared them to control mice bearing only the GFAP-luc transgene. We used serial bioluminescent images to define the onset and the time course of astrogliosis in these mice and this was correlated with the development of clinical deficit. Mice containing both GFAP-luc and P301S transgenes showed increased luminescence indicative of astroglial activation in the brain and spinal cord. Starting at 5 months old, the onset of clinical deterioration in these mice corresponded closely to the initial rise in the luminescent signal. Post mortem analysis showed the elevated luminescence was correlated with hyperphosphorylated tau deposition in the hippocampus of double transgenic mice. We used this method to determine the therapeutic effect of JM4 peptide [a small peptide immunomodulatory agent derived from human erythropoietin (EPO)] on double transgenic mice. JM4 treatment significantly decreased the intensity of luminescence, neurological deficit and hyperphosphorylated tau in mice with both the P301S and GFAP-luc transgenes. These findings indicate that bioluminescence imaging (BLI) is a powerful tool for quantifying GFAP expression in living P301S mice and can be used as a noninvasive biomarker of tau-induced neurodegeneration in preclinical therapeutic trials.

An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity.

Transmission of dengue virus (DENV) requires successful completion of the infection cycle in the Aedes aegypti vector, which is initiated in the midgut tissue after ingestion of an infectious blood meal. While certain Ae. aegypti midgut-associated bacteria influence virus infection, little is known about the midgut-associated fungi (mycobiota), and how its members might influence susceptibility to DENV infection. We show that a Talaromyces (Tsp_PR) fungus, isolated from field-caught Ae. aegypti, render the mosquito more permissive to DENV infection. This modulation is attributed to a profound down-regulation of digestive enzyme genes and trypsin activity, upon exposure to Tsp_PR-secreted factors. In conclusion, we show for the first time that a natural mosquito gut-associated fungus can alter Ae. aegypti physiology in a way that facilitates pathogen infection.

Efficacy of nonselective optogenetic control of the medial septum over hippocampal oscillations: the influence of speed and implications for cognitive enhancement.

Optogenetics holds great promise for both the dissection of neural circuits and the evaluation of theories centered on the temporal organizing properties of oscillations that underpin cognition. To date, no studies have examined the efficacy of optogenetic stimulation for altering hippocampal oscillations in freely moving wild-type rats, or how these alterations would affect performance on behavioral tasks. Here, we used an AAV virus to express ChR2 in the medial septum (MS) of wild-type rats, and optically stimulated septal neurons at 6 Hz and 30 Hz. We measured the corresponding effects of these stimulations on the oscillations of the MS and hippocampal subfields CA1 and CA3 in three different contexts: (1) With minimal movement while the rats sat in a confined chamber; (2) Explored a novel open field; and (3) Learned and performed a T-maze behavioral task. While control yellow light stimulation did not affect oscillations, 6-Hz blue light septal stimulations altered hippocampal theta oscillations in a manner that depended on the animal's mobility and speed. While the 30 Hz blue light septal stimulations only altered theta frequency in CA1 while the rat had limited mobility, it robustly increased the amplitude of hippocampal signals at 30 Hz in both regions in all three recording contexts. We found that animals were more likely to make a correct choice during Day 1 of T-maze training during both MS stimulation protocols than during control stimulation, and that improved performance was independent of theta frequency alterations.

A natural Anopheles-associated Penicillium chrysogenum enhances mosquito susceptibility to Plasmodium infection.

Whereas studies have extensively examined the ability of bacteria to influence Plasmodium infection in the mosquito, the tripartite interactions between non-entomopathogenic fungi, mosquitoes, and Plasmodium parasites remain largely uncharacterized. Here we report the isolation of a common mosquito-associated ascomycete fungus, Penicillium chrysogenum, from the midgut of field-caught Anopheles mosquitoes. Although the presence of Pe. chrysogenum in the Anopheles gambiae midgut does not affect mosquito survival, it renders the mosquito significantly more susceptible to Plasmodium infection through a secreted heat-stable factor. We further provide evidence that the mechanism of the fungus-mediated modulation of mosquito susceptibility to Plasmodium involves an upregulation of the insect's ornithine decarboxylase gene, which sequesters arginine for polyamine biosynthesis. Arginine plays an important role in the mosquito's anti-Plasmodium defense as a substrate of nitric oxide production, and its availability therefore has a direct impact on the mosquito's susceptibility to the parasite. While this type of immunomodulatory mechanism has already been demonstrated in other host-pathogen interaction systems, this is the first report of a mosquito-associated fungus that can suppress the mosquito's innate immune system in a way that would favor Plasmodium infection and possibly malaria transmission.

Cytoplasmic actin is an extracellular insect immune factor which is secreted upon immune challenge and mediates phagocytosis and direct killing of bacteria, and is a Plasmodium Antagonist.

Actin is a highly versatile, abundant, and conserved protein, with functions in a variety of intracellular processes. Here, we describe a novel role for insect cytoplasmic actin as an extracellular pathogen recognition factor that mediates antibacterial defense. Insect actins are secreted from cells upon immune challenge through an exosome-independent pathway. Anopheles gambiae actin interacts with the extracellular MD2-like immune factor AgMDL1, and binds to the surfaces of bacteria, mediating their phagocytosis and direct killing. Globular and filamentous actins display distinct functions as extracellular immune factors, and mosquito actin is a Plasmodium infection antagonist.

Exploring Anopheles gut bacteria for Plasmodium blocking activity.

Malaria parasite transmission requires the successful development of Plasmodium gametocytes into flagellated microgametes upon mosquito blood ingestion, and the subsequent fertilization of microgametes and macrogametes for the development of motile zygotes, called ookinetes, which invade and transverse the Anopheles vector mosquito midgut at around 18-36 h after blood ingestion. Within the mosquito midgut, the malaria parasite has to withstand the mosquito's innate immune response and the detrimental effect of its commensal bacterial flora. We have assessed the midgut colonization capacity of five gut bacterial isolates from field-derived, and two from laboratory colony, mosquitoes and their effect on Plasmodium development in vivo and in vitro, along with their impact on mosquito survival. Some bacterial isolates activated the mosquito's immune system, affected the mosquito's lifespan, and were capable of blocking Plasmodium development. We have also shown that the ability of these bacteria to inhibit the parasites is likely to involve different mechanisms and factors. A Serratia marcescens isolate was particularly efficient in colonizing the mosquitoes' gut, compromising mosquito survival and inhibiting both Plasmodium sexual- and asexual-stage through secreted factors, thereby rendering it a potential candidate for the development of a malaria transmission intervention strategy.

Bacteria- and IMD pathway-independent immune defenses against Plasmodium falciparum in Anopheles gambiae.

The mosquito Anopheles gambiae uses its innate immune system to control bacterial and Plasmodium infection of its midgut tissue. The activation of potent IMD pathway-mediated anti-Plasmodium falciparum defenses is dependent on the presence of the midgut microbiota, which activate this defense system upon parasite infection through a peptidoglycan recognition protein, PGRPLC. We employed transcriptomic and reverse genetic analyses to compare the P. falciparum infection-responsive transcriptomes of septic and aseptic mosquitoes and to determine whether bacteria-independent anti-Plasmodium defenses exist. Antibiotic treated aseptic mosquitoes mounted molecular immune responses representing a variety of immune functions upon P. falciparum infection. Among other immune factors, our analysis uncovered a serine protease inhibitor (SRPN7) and Clip-domain serine protease (CLIPC2) that were transcriptionally induced in the midgut upon P. falciparum infection, independent of bacteria. We also showed that SRPN7 negatively and CLIPC2 positively regulate the anti-Plasmodium defense, independently of the midgut-associated bacteria. Co-silencing assays suggested that these two genes may function together in a signaling cascade. Neither gene was regulated, nor modulated, by infection with the rodent malaria parasite Plasmodium berghei, suggesting that SRPN7 and CLIPC2 are components of a defense system with preferential activity towards P. falciparum. Further analysis using RNA interference determined that these genes do not regulate the anti-Plasmodium defense mediated by the IMD pathway, and both factors act as agonists of the endogenous midgut microbiota, further demonstrating the lack of functional relatedness between these genes and the bacteria-dependent activation of the IMD pathway. This is the first study confirming the existence of a bacteria-independent, anti-P. falciparum defense. Further exploration of this anti-Plasmodium defense will help clarify determinants of immune specificity in the mosquito, and expose potential gene and/or protein targets for malaria intervention strategies based on targeting the parasite in the mosquito vector.

Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase.

Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.

Avian hemosporidian parasites from northern California oak woodland and chaparral habitats.

During spring-summer 2003-2004, the avian community was surveyed for hemosporidian parasites in an oak (Quercus spp.) and madrone (Arbutus spp.) woodland bordering grassland and chaparral habitats at a site in northern California, a geographic location and in habitat types not previously sampled for these parasites. Of 324 birds from 46 species (21 families) sampled (including four species not previously examined for hemosporidians), 126 (39%) were infected with parasites identified as species of one or more of the genera Plasmodium (3% of birds sampled), Haemoproteus (30%), and Leucocytozoon (11%). Species of parasite were identified by morphology in stained blood smears and were consistent with one species of Plasmodium, 11 species of Haemoproteus, and four species of Leucocytozoon. We document the presence of one of the parasite genera in seven new host species and discovered 12 new parasite species-host species associations. Hatching-year birds were found infected with parasites of all three genera. Prevalence of parasites for each genus differed significantly for the entire sample, and prevalence of parasites for the most common genus, Haemoproteus, differed significantly among bird families. Among families with substantial sample sizes, the Vireonidae (63%) and Emberizidae (70%) were most often infected with Haemoproteus spp. No evidence for parasite between-genus interaction, either positive or negative, was found. Overall prevalence of hemosporidians at the northern California sites and predominance of Haemoproteus spp. was similar to that reported in most other surveys for the USA, Canada, and the Caribbean islands.

Infection of murine oligodendroglial precursor cells with Human Herpesvirus 6 (HHV-6)--establishment of a murine in vitro model.

BACKGROUND: Human Herpesvirus 6 was previously demonstrated to infect human oligodendroglial precursor cells (OPCs) in vitro causing cell cycle arrest and premature differentiation with consequent loss of the precursor pool. OBJECTIVES: To develop an in vitro murine OPC model to study the cell cycle and differentiation effects of HHV-6 in more readily available, genetically well-defined cells free of the risk of contamination with human herpesviruses. STUDY DESIGN: Murine OPCs were exposed to infectious HHV-6A or HHV-6B and analyzed for production of viral transcripts, particles, and replicating virus. FACS analysis and specific markers were used to evaluate effects on cell cycling and differentiation. RESULTS: HHV-6 infection of murine OPCs resulted in production of both immediate-early and some late transcripts but no replicating virus by TaqMan quantitative PCR or electron microscopy. Both a specific G1/S cell cycle arrest and premature loss of OPCs through differentiation into oligodendrocytes as previously seen with human precursors were recapitulated. CONCLUSIONS: Infection of murine OPCs by HHV-6 reproduces the critical phenotypes of cell cycle arrest and altered differentiation seen in human cells. The murine system provides a highly defined, accessible, and reproducible source of cells permitting the elucidation of specific viral and cell cycle genes involved in CNS viral infections of OPCs.

Infection with an endemic human herpesvirus disrupts critical glial precursor cell properties.

Human herpesvirus 6 (HHV-6), a common resident virus of the human CNS, has been implicated in both acute and chronic inflammatory--demyelinating diseases. Although HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes, nothing is known about the susceptibility of glial precursors, the ancestors of myelin-producing oligodendrocytes, to viral infection. We show that HHV-6 infects human glial precursor cells in vitro. Active infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins, with subsequent formation of cell syncytia. Infection leads to alterations in cell morphology and impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by bromodeoxyuridine uptake, which was confirmed by blunting of the cell growth rate of infected cells compared with uninfected controls over time. The detailed analysis using novel, fluorescent-labeled HHV-6A or HHV-6B reagents demonstrated strong G1/S phase inhibition in infected precursor cells. Cell cycle arrest in HHV-6-infected cells was associated with a profound decrease in the expression of the glial progenitor cell marker A2B5 and a corresponding increase in the oligodendrocyte differentiation marker GalC. These data demonstrate for the first time that infection of primary human glial precursor cells with a neurologically relevant human herpesvirus causes profound alterations of critical precursor cell properties. In light of recent observations that repair of CNS demyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in demyelinating diseases and the disruption of normal glial maturation.

Human herpesvirus 6 genome and antigen in acute multiple sclerosis lesions.

Evidence for a candidate multiple sclerosis (MS) virus, human herpesvirus 6 (HHV-6), was sought in biopsy specimens of acute lesions that presented clinically as cerebral tumors obtained from 5 patients. Histopathology, magnetic resonance imaging, and clinical course confirmed the diagnosis of MS in each case. A sensitive in situ polymerase chain reaction (ISPCR) method was used to detect HHV-6 genome, in conjunction with immunocytochemical staining (ICC) to detect viral and cellular antigens. ISPCR revealed numerous oligodendrocytes, lymphocytes, and microglia containing HHV-6 genome within all lesions, whereas ICC showed only the HHV-6 glycoprotein 116 antigen in some reactive astrocytes and microglia. High frequencies of neuroglial and inflammatory cells containing HHV-6 genome were present in acute-phase lesion tissue from patients who were free of the effects of chronic MS and had not been received immunomodulatory therapy for MS. The prevalence of HHV-6 genome-containing cells, including oligodendrocytes, in each lesion suggests that HHV-6 plays a role in the demyelinative pathogenesis of MS; the significance of the discrepant expression of viral antigens remains uncertain.

Caspase-1 expression in multiple sclerosis plaques and cultured glial cells.

Caspase-1 is responsible for processing inflammatory cytokines and is associated with the induction of apoptosis. Using RT-PCR, we found that caspase-1 mRNA transcripts from frozen brain extracts were significantly elevated in multiple sclerosis (MS) compared to controls. Immunohistochemical staining using a specific antiserum confirmed the marked up regulation of caspase-1 within acute and chronic MS plaques, while little staining was seen in control brains. In addition to the expected caspase-1 expression in microglia and infiltrating perivascular mononuclear cells, we found that cytoplasmic caspase-1 expression was sharply increased in the resident oligodendrocytes of MS lesions. The TUNEL reaction for fragmented DNA co-localized over an occasional caspase-1-expressing cell and large numbers of caspase-1-positive "corpses" were observed within phagocytic macrophages of an acute evolving MS lesion. Studies using an immortalized human oligodendroglial hybrid cell line exposed to cytokine challenge showed that death induction was blocked by the caspase-1-like inhibitor Z-YVAD-fmk, while the caspase-3-like inhibitor Z-DEVD-fmk was less effective. Cellular levels of procaspase-1 were reduced compared to controls in oligodendroglia induced to die by cytokine challenge, as judged by Western immunoblotting. Our results suggest that caspase-1 may play a role in the inflammatory and apoptotic processes associated with MS pathogenesis.