Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Caveats in Transneuronal Tracing with Unmodified Rabies Virus: An Evaluation of Aberrant Results Using a Nearly Perfect Tracing Technique.

Apart from the genetically engineered, modified, strains of rabies virus (RABV), unmodified 'fixed' virus strains of RABV, such as the 'French' subtype of CVS11, are used to examine synaptically connected networks in the brain. This technique has been shown to have all the prerequisite characteristics for ideal tracing as it does not metabolically affect infected neurons within the time span of the experiment, it is transferred transneuronally in one direction only and to all types of neurons presynaptic to the infected neuron, number of transneuronal steps can be precisely controlled by survival time and it is easily detectable with a sensitive technique. Here, using the 'French' CVS 11 subtype of RABV in Wistar rats, we show that some of these characteristics may not be as perfect as previously indicated. Using injection of RABV in hind limb muscles, we show that RABV-infected spinal motoneurons may already show lysis 1 or 2 days after infection. Using longer survival times we were able to establish that Purkinje cells may succumb approximately 3 days after infection. In addition, some neurons seem to resist infection, as we noted that the number of RABV-infected inferior olivary neurons did not progress in the same rate as other infected neurons. Furthermore, in our hands, we noted that infection of Purkinje cells did not result in expected transneuronal labeling of cell types that are presynaptic to Purkinje cells such as molecular layer interneurons and granule cells. However, these cell types were readily infected when RABV was injected directly in the cerebellar cortex. Conversely, neurons in the cerebellar nuclei that project to the inferior olive did not take up RABV when this was injected in the inferior olive, whereas these cells could be infected with RABV via a transneuronal route. These results suggest that viral entry from the extracellular space depends on other factors or mechanisms than those used for retrograde transneuronal transfer. We conclude that transneuronal tracing with RABV may result in unexpected results, as not all properties of RABV seem to be ubiquitously valid.

Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.

Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

Identification of feline panleukopenia virus proteins expressed in Purkinje cell nuclei of cats with cerebellar hypoplasia.

Parvoviruses depend on initiation of host cell division for their replication. Undefined parvoviral proteins have been detected in Purkinje cells of the cerebellum after experimental feline panleukopenia virus (FPV) infection of neonatal kittens and in naturally occurring cases of feline cerebellar hypoplasia. In this study, a parvoviral protein in the nucleus of Purkinje cells of kittens with cerebellar hypoplasia was shown by immunoprecipitation to be the FPV viral capsid protein VP2. In PCR-confirmed, FPV-associated feline cerebellar hypoplasia, expression of the FPV VP2 protein was demonstrated by immunohistochemistry in Purkinje cell nuclei in 4/10 cases and expression of the FPV non-structural protein NS1 was demonstrated in Purkinje cell nuclei in 5/10 cases. Increased nuclear ERK1 expression was observed in several Purkinje cells in 1/10 kittens. No expression of the G1 and S mitotic phase marker proliferating cell nuclear antigen (PCNA) was evident in Purkinje cell nuclei. These results support the hypothesis that FPV is able to proceed far into its replication cycle in post-mitotic Purkinje cells.

Modulation of lentiviral vector tropism in cerebellar Purkinje cells in vivo by a lysosomal cysteine protease cathepsin K.

We previously reported that vesicular stomatitis virus-derived glycoprotein (VSV-G)-pseudotyped lentiviral vectors harvested 2 days post-transfection preferred to infect Purkinje cells (PCs), whereas those harvested after a longer cultivation period exhibited Bergmann glia-preferential transduction. However, the mechanisms by which lentiviral tropism was altered remained unsolved. Here, we investigated whether proteases released from the cells during viral production affect lentiviral tropism. Enhanced green fluorescence protein-expressing lentiviral vectors were produced using human embryonic kidney (HEK) 293FT or 293 T cells and injected into the mouse cerebellum to examine tropism in PCs. We found that the addition of a protease inhibitor-in particular, the cathepsin K (CatK) inhibitor-into the culture medium significantly increased lentiviral tropism in PCs. Moreover, the concentration of CatK in the culture medium drastically increased upon prolonged cultivation, concomitant with the expression levels of CatK in HEK 293 T cells. An increase in CatK activity by the addition of recombinant CatK enzyme to PC-preferential viral solution, which was obtained 2 days post-transfection, shifted the viral tropism toward Bergmann glia. In contrast, a decrease in CatK activity in the Bergmann glia-preferential viral solution, which was obtained 6 days post-transfection by the addition of CatK inhibitor or by the removal of a CatK-containing fraction, restored the PC preference of viruses. These results suggest that the CatK released from deteriorated HEK 293 T cells plays a key role in reducing lentiviral tropism in PCs, presumably by affecting a receptor molecule for lentiviral VSV-G, resulting in the preferential transduction of Bergmann glia.

An efficient method for the long-term and specific expression of exogenous cDNAs in cultured Purkinje neurons.

We present a simple and efficient method for expressing cDNAs in Purkinje neurons (PNs) present in heterogeneous mouse cerebellar cultures. The method combines the transfection of freshly dissociated cerebellar cells via nucleofection with the use of novel expression plasmids containing a fragment of the L7 (Pcp2) gene that, within the cerebellum, drives PN-specific expression. The efficiency of PN transfection (determined 13 days post nucleofection) is approximately 70%. Double and triple transfections are routinely achieved at slightly lower efficiencies. Expression in PNs is obvious after one week in culture and still strong after three weeks, by which time these neurons are well-developed. Moreover, high-level expression is restricted almost exclusively to the PNs present in these mixed cultures, which greatly facilitates the characterization of PN-specific functions. As proof of principle, we used this method to visualize (1) the morphology of living PNs expressing mGFP, (2) the localization and dynamics of the dendritic spine proteins PSD-93 and Homer-3a tagged with mGFP and (3) the interaction of live PNs expressing mGFP with other cerebellar neurons expressing mCherry from a ß-Actin promoter plasmid. Finally, we created a series of L7-plasmids containing different fluorescent protein cDNAs that are suited for the expression of cDNAs of interest as N- and C-terminally tagged fluorescent fusion proteins. In summary, this procedure allows for the highly efficient, long-term, and specific expression of multiple cDNAs in differentiated PNs, and provides a favorable alternative to two procedures (viral transduction, ballistic gene delivery) used previously to express genes in cultured PNs.

Immunohistochemical detection of metalloproteinase-9 (MMP-9), anti-oxidant like 1 protein (AOP-1) and synaptosomal-associated protein (SNAP-25) in the cerebella of dogs naturally infected with spontaneous canine distemper.

In most viral infections of the central nervous system (CNS), the integrity of brain extracelluar matrix (ECM), oxidative stress and dysfunction in neuronal transmission may contribute to the observed pathology. The purpose of this study was to investigate the role of these factors in demyelinating canine distemper virus (CDV) infections. Regardless of ECM integrity, the expression of metalloproteinase-9 (MMP-9) was visualized in microglial-like cells, whereas the expression of anti-oxidant like-1 (AOP-1) and synaptosomal associated protein (SNAP-25) was frequently detected in Purkinje cells (r(2) = 0.989; p < 0.05), regardless of whether the lesions were classified as acute or chronic. Increased numbers of immunolabeled microglia-like cells and reactive gliosis were observed in advanced cases of demyelinating CDV, suggesting that the expression of AOP-1 and SNAP-25 is correlated with the ultimate death of affected cells. Our findings bring a new perspective to understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating chronic leukoencephalitis caused by CDV.

High transgene expression by lentiviral vectors causes maldevelopment of Purkinje cells in vivo.

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.

Lentiviral vector-mediated rescue of motor behavior in spontaneously occurring hereditary ataxic mice.

Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta2 glutamate receptor (GluRdelta2) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GluRdelta2 in Purkinje cells. Lentiviral vectors expressing GluRdelta2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluRdelta2 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GluRdelta2 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders.

Adeno-associated virus type 2 vectors: transduction and long-term expression in cerebellar Purkinje cells in vivo is mediated by the fibroblast growth factor receptor 1 : bFGFR-1 mediates AAV2 transduction of Purkinje cells.

The receptor for adeno-associated virus serotype 2 (AAV2) in Purkinje cells has not been identified, but based on work carried out in non-neuronal cell lines, heparan sulfate proteoglycan is thought to act as a primary receptor, with basic fibroblast growth factor receptor-1 being reported as a co-receptor. In this study, using antibody interference and protein competition strategies, we show specific reduction in Purkinje cell transduction by AAV2 vector in the presence of these inhibitors. We also demonstrate AAV2-mediated transgene expression in Purkinje cells in vivo that extends out to one year post-injection. These results provide new information on fibroblast growth factor receptor-1 involvement in AAV2-mediated transduction of Purkinje cells and have important implications for AAV-mediated treatment of various cerebellar disorders.

Purkinje-cell-preferential transduction by lentiviral vectors with the murine stem cell virus promoter.

Viral-vector-mediated gene delivery into Purkinje cells is a promising method for exploring the pathophysiology of the cerebellum; however, it is generally difficult to achieve sufficiently high levels of gene expression in Purkinje cells using viral vectors with a cell-type-specific promoter because of the weakness of transcriptional activity. In this study, we prepared lentiviral vectors that express GFP under the control of various ubiquitous promoters derived from murine stem cell virus (MSCV), cytomegalovirus (CMV), CMV early enhancer/chicken beta actin (CAG), and Rous sarcoma virus (RSV) and compared their potential to transduce Purkinje cells. Mice were sacrificed 7 days after lentiviral injection and the ratios of GFP(+) Purkinje cells to all transduced cells were determined. The highest transduction ratio was observed when we used lentivectors containing the MSCV promoter: approximately 70% of GFP(+) cells were Purkinje cells, the next highest ratio was for the CMV promoter (approximately 40%), then the CAG promoter (approximately 35%), and the lowest ratio was for the RSV promoter (approximately 10%). Moreover, the highest levels of GFP expression were also caused by the MSCV promoter. Thus, among the ubiquitous promoters examined, the MSCV promoter was the best for the expression of a foreign gene in Purkinje cells in vivo.

Progress in transduction of cerebellar Purkinje cells in vivo using viral vectors.

Expression of a foreign gene in cerebellar Purkinje cells in vivo is a powerful method for exploring the pathophysiology of the cerebellum. Although using developmental engineering many gene-modified mice have been generated, this approach is time-consuming and requires a lot of effort for crossing different lines of mice, genotyping and maintenance of animals. If a gene of interest can be transferred to and efficiently expressed in Purkinje cells of developing and mature animals, it saves much time, effort and money. Recent advances in viral vectors have markedly contributed to selective and efficient gene transfer to Purkinje cells in vivo. There are two approaches for selective gene expression in Purkinje cells: one is to take advantage of the viral tropism for Purkinje cells, which includes the tropism of adeno-associated virus and the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentivirus. Another method, which might be used in combination with the first one, is utilization of a Purkinje-cell-specific promoter. Focusing mainly on these points, recent progress in viral-vector-mediated transduction of Purkinje cells in vivo is reviewed.

Exposure of lentiviral vectors to subneutral pH shifts the tropism from Purkinje cell to Bergmann glia.

Cerebellar Purkinje cells play an important role in cerebellar function; lesions of Purkinje cells result in the disruption of motor coordination and motor learning. Although selective gene delivery to Purkinje cells would be a powerful technique for the study of pathophysiology in the cerebellum, a method for such a delivery has not yet been established. Here we employed human immunodeficiency virus-derived lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein to transduce Purkinje cells and examined factors that critically affect the viral tropism for Purkinje cells. Viral vectors encoding GFP were generated using different protocols, and were then injected into the mouse cerebellum. At 7 days and 2 months post-transduction, the relative proportions of transduced Purkinje cells were determined. Lentiviral vectors harvested from a medium of pH 7.2 preferentially transduced Purkinje cells (about half of the transduced cells). In contrast, when the viral vector was harvested from medium of

In vivo transduction of murine cerebellar Purkinje cells by HIV-derived lentiviral vectors.

Cerebellar Purkinje cells are key elements in motor learning and motor coordination, and therefore, it is important to clarify the mechanisms by which Purkinje cells integrate information and control cerebellar function. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum because adenovirus vectors, the vectors most commonly used for in vivo gene transfer, have very low affinity for Purkinje cells. In this study, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector carrying the GFP gene was injected into the cerebellar cortex. Seven days after the injection, Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.

Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum.

Establishing efficient gene transfer and expression in post-mitotic neurons is important in understanding the genetic basis of neural circuits with cellular complexity. This study evaluates the properties of exogenous green fluorescent protein (GFP) expression mediated by the Semliki forest virus (SFV) and adenovirus (Ad) vectors in dissociated and slice cultures of the mouse cerebellum. Infection with SFV-GFP resulted in early-onset and high-level GFP expression in about 90% of Purkinje cells and in about 40% of granule cells in dissociated cultures at 1 day after infection. Two days after infection, GFP-positive cells showed signs of SFV-derived cytotoxicity. Ad-GFP infected almost all astrocytes and granule cells in dissociated cultures, and showed a steady increase in GFP fluorescence with a plateau at around 2 days post-infection. Ad vector-mediated GFP expression lasted for several weeks with no significant cell damage. In the slice cultures, both viral vectors mainly infected astroglial cells, but also showed a similar cell preference as that in dissociated cultures. These data indicate that the use of different viral vectors and infection conditions offers a powerful means of expressing exogenous genes in cerebellar cultures with different cell-type specificity and timing and duration of expression.

Systemic FIV vector administration: transduction of CNS immune cells and Purkinje neurons.

The systemic effects of gene therapy have been previously described in a variety of peripheral organs following intravenous administration or intraperitoneal inoculation of viral vectors, as well as in the brain following intracranial administration. However, limited information is available on the ability of viral vectors to cross the blood-brain barrier and infect cells located within the central nervous system (CNS). We employed a VSV-G pseudotyped FIV(lacZ) vector capable of transducing dividing, growth-arrested, as well as post-mitotic cells with the reporter gene lacZ. Adult mice were injected intraperitoneally with FIV(lacZ), and the expression of beta-galactosidase was studied 5 weeks following treatment in the brain, liver, spleen and kidney by X-gal histochemistry and immunocytochemistry. Interestingly, relatively low doses of FIV(lacZ) administered intraperitoneally lead to beta-galactosidase detection in the brain and cerebellum. The identity of these cells was confirmed by double immunofluorescence, and included CD31-, CD3- and CD11b-positive cells. Fluorescent microspheres co-injected with FIV(lacZ) virus were identified within mononuclear cells in the brain parenchyma, suggesting infiltration of peripheral immune cells in the CNS. Cerebellar Purkinje neurons were also transduced in all adult-injected mice. Our observations indicate that relatively low doses of FIV(lacZ) administered intraperitoneally resulted in the transduction of immune cells in the brain, as well as a specific subset of cerebellar neurons.

Cerebellar loops with motor cortex and prefrontal cortex of a nonhuman primate.

We used transneuronal transport of neurotropic viruses to examine the topographic organization of circuits linking the cerebellar cortex with the arm area of the primary motor cortex (M1) and with area 46 in dorsolateral prefrontal cortex of monkeys. Retrograde transneuronal transport of the CVS-11 (challenge virus strain 11) strain of rabies virus in cerebello-thalamocortical pathways revealed that the arm area of M1 receives input from Purkinje cells located primarily in lobules IV-VI of the cerebellar cortex. In contrast, transneuronal transport of rabies from area 46 revealed that it receives input from Purkinje cells located primarily in Crus II of the ansiform lobule. Thus, both M1 and area 46 are the targets of output from the cerebellar cortex. However, the output to each area of the cerebral cortex originates from Purkinje cells in different regions of the cerebellar cortex. Anterograde transneuronal transport of the H129 strain of herpes simplex virus type 1 (HSV1) revealed that neurons in the arm area of M1 project via the pons to granule cells primarily in lobules IV-VI, whereas neurons in area 46 project to granule cells primarily in Crus II. Together, the findings from rabies and HSV1 experiments indicate that the regions of the cerebellar cortex that receive input from M1 are the same as those that project to M1. Similarly, the regions of the cerebellar cortex that receive input from area 46 are the same as those that project to area 46. Thus, our observations suggest that multiple closed-loop circuits represent a fundamental architectural feature of cerebrocerebellar interactions.

Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells.

The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral-mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN-EG-NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18-24 h postinfection. In most of GFP-positive cells, the expression of NR2B protein was detected by immunostaining with NR2B-specific antibodies. In Purkinje cells infected with SIN-EG-NR2B, the iontophoretic application of NMDA induced prominent NMDAR-mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR-mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.

Distribution of adenoviral vector in brain after intravenous administration.

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.