PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves.

Mycobacterium leprae, an obligate intracellular bacillus, infects Schwann cells (SCs), leading to peripheral nerve damage, the most severe leprosy symptom. In the present study, we revisited the involvement of phenolic glycolipid I (PGL I), an abundant, private, surface M. leprae molecule, in M. leprae-SC interaction by using a recombinant strain of M. bovis BCG engineered to express this glycolipid. We demonstrate that PGL I is essential for bacterial adhesion and SC internalization. We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor (MR/CD206) in infected cells in a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent manner. Of note, blocking mannose recognition decreased bacterial entry and survival, pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve. Moreover, an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets (LDs) formation and prostaglandin E2 (PGE2), previously described as fundamental players in bacterial pathogenesis. Finally, this pathway was shown to induce IL-8 secretion. Altogether, our study provides evidence that the entry of live M. leprae through PGL I recognition modulates the SC phenotype, favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation.

Transcriptional profiling predicts overwhelming homology of Schwann cells, olfactory ensheathing cells, and Schwann cell-like glia.

Schwann cells (SCs), olfactory ensheathing cells (OECs), and central nervous system Schwann cell-like glia (SG) represent a group of nerve growth factor receptor p75 (NGFR)-positive cells, originating from different tissues. Because of their pro-regenerative capacities, these cells are subjects in experimental transplantation-based therapies of spinal cord trauma. The objective of this study was to compare the transcriptomes of uninfected and canine distemper virus-infected OECs, SCs, SG and fibroblasts (FBs) derived from four beagle dogs and cultured under identical conditions in vitro, employing canine genome 2.0 arrays (Affymetrix). Here, we observed a complete lack of transcriptional differerences between OECs and SG, a high similarity of OECs/SG to SCs, and a marked difference of SCs and OECs/SG towards FBs. Differentially expressed genes possibly involved in the maintenance of cell type-specific identity included an up-regulation of HOXD8 and HOXC4 in SCs, and an up-regulation of CNTNAP2 and EFEMP1 in OECs/SG. We identified cell type-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection. Thereby AQP1 and SCRG1 were predicted to be the most powerful biomarkers distinguishing SCs from OECs/SG. Immunofluorescence confirmed a higher expression of SCRG1 in OECs and SG, and conversely a higher expression of AQP1 in SCs in vitro. Furthermore, canine and murine olfactory nerves showed SCRG1-positive, AQP1-negative OECs and/or axons, whereas sciatic nerves displayed multifocal non-myelinated, AQP1-positive, SCRG1-negative cells. Conclusively, OECs/SG are suggested to be a uniform cell type differing only in the tissue of origin and highly related to SCs.

Context-Dependent Regulation of Peripheral Nerve Abundance by the PI3K Pathway in the Tumor Microenvironment of Head and Neck Squamous Cell Carcinoma.

Recent studies have highlighted neurons and their associated Schwann cells (SCs) as key regulators of cancer development. However, the mode of their interaction with tumor cells or other components of the tumor microenvironment (TME) remains elusive. We established an SC-related 43-gene set as a surrogate for peripheral nerves in the TME. Head and neck squamous cell carcinoma (HNSCC) from The Cancer Genome Atlas (TCGA) were classified into low, intermediate and high SC score groups based on the expression of this gene set. Perineural invasion (PNI) and TGF-ß signaling were hallmarks of SChigh tumors, whereas SClow tumors were enriched for HPV16-positive OPSCC and higher PI3K-MTOR activity. The latter activity was partially explained by a higher frequency of PTEN mutation and PIK3CA copy number gain. The inverse association between PI3K-MTOR activity and peripheral nerve abundance was context-dependent and influenced by the TP53 mutation status. An in silico drug screening approach highlighted the potential vulnerabilities of HNSCC with variable SC scores and predicted a higher sensitivity of SClow tumors to DNA topoisomerase inhibitors. In conclusion, we have established a tool for assessing peripheral nerve abundance in the TME and provided new clinical and biological insights into their regulation. This knowledge may pave the way for new therapeutic strategies and impart proof of concept in appropriate preclinical models.

The L-coding region of the DA strain of Theiler's murine encephalomyelitis virus causes dysfunction and death of myelin-synthesizing cells.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.

Large-Scale Gene Expression Signatures Reveal a Microbicidal Pattern of Activation in Mycobacterium leprae-Infected Monocyte-Derived Macrophages With Low Multiplicity of Infection.

Leprosy is a disease with a clinical spectrum of presentations that is also manifested in diverse histological features. At one pole, lepromatous lesions (L-pole) have phagocytic foamy macrophages heavily parasitized with freely multiplying intracellular Mycobacterium leprae. At the other pole, the presence of epithelioid giant cells and granulomatous formation in tuberculoid lesions (T-pole) lead to the control of M. leprae replication and the containment of its spread. The mechanism that triggers this polarization is unknown, but macrophages are central in this process. Over the past few years, leprosy has been studied using large scale techniques to shed light on the basic pathways that, upon infection, rewire the host cellular metabolism and gene expression. M. leprae is particularly peculiar as it invades Schwann cells in the nerves, reprogramming their gene expression leading to a stem-like cell phenotype. This modulatory behavior exerted by M. leprae is also observed in skin macrophages. Here, we used live M. leprae to infect (10:1 multiplicity of infection) monocyte-derived macrophages (MDMs) for 48 h and analyzed the whole gene expression profile using microarrays. In this model, we observe an intense upregulation of genes consistent with a cellular immune response, with enriched pathways including peptide and protein secretion, leukocyte activation, inflammation, and cellular divalent inorganic cation homeostasis. Among the most differentially expressed genes (DEGs) are CCL5/RANTES and CYP27B1, and several members of the metallothionein and metalloproteinase families. This is consistent with a proinflammatory state that would resemble macrophage rewiring toward granulomatous formation observed at the T-pole. Furthermore, a comparison with a dataset retrieved from the Gene Expression Omnibus of M. leprae-infected Schwann cells (MOI 100:1) showed that the patterns among the DEGs are highly distinct, as the Schwann cells under these conditions had a scavenging and phagocytic gene profile similar to M2-like macrophages, with enriched pathways rearrangements in the cytoskeleton, lipid and cholesterol metabolism and upregulated genes including MVK, MSMO1, and LACC1/FAMIN. In summary, macrophages may have a central role in defining the paradigmatic cellular (T-pole) vs. humoral (L-pole) responses and it is likely that the multiplicity of infection and genetic polymorphisms in key genes are gearing this polarization.

Knockdown of METTL3 inhibits enterovirus 71-induced apoptosis of mouse Schwann cell through regulation of autophagy.

Enterovirus A71 (EV-A71 or EV-71) is an RNA virus that causes hand, foot and mouse disease in children. The N6-methyladenosine (m6A) of RNA is a common RNA modification involved in various biological events. METTL3 is an m6A methyltransferase that regulates EV-71 replication. EV-71 infection induces autophagy, which also promotes EV-71 replication. In this study, we explored the role of METTL3 in EV-71 infection-induced autophagy. We constructed lentivirus expressing METTL3-specific shRNA and knocked down the endogenous METTL3 in mouse Schwann cells. We infected normal Schwann cells and METTL3 knockdown Schwann cells and compared the viral titer, expression of autophagy-related proteins and apoptosis-related protein. Transduction of lentivirus expressing METTL3 shRNA significantly decreased the endogenous METTL3. Knocking down METTL3 decreased the viral titer of EV-71 after infection. Knocking down METTL3 prevented EV-71-induced cell death and suppressed EV-71-induced expression of Bax while rescuing Bcl-2 expression after EV-71 infection. Knocking down METTL3 inhibited EV-71-induced expression of Atg5, Atg7 and LC3 II. Knocking down METTL3 inhibited EV-71-induced apoptosis and autophagy. In summary, our study describes the relationship of METTL3 and autophagy during EV-71 infection.

Neuroglia infection by rabies virus after anterograde virus spread in peripheral neurons.

The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.

Enterovirus 71 VP1 promotes mouse Schwann cell autophagy via ER stress­mediated PMP22 upregulation.

Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease­related deaths due to fatal neurological complications. EV71 structural viral protein 1 (VP1) promotes viral replication by inducing autophagy in neuron cells, but the effect of VP1 on myelin cells is unclear. The present study aimed to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells. An EV71 VP1­expressing vector (pEGFP­C3­VP1) was generated and transfected into mouse Schwann cells. Transmission electron microscopy and western blot analysis for microtubule­associated protein 1 light chain 3 α (LC3) II (an autophagy marker) were used to assess autophagy. Reverse transcription­quantitative PCR and immunofluorescence were performed to determine the expression of peripheral myelin protein 22 (PMP22). Small interfering RNA against PMP22 was used to investigate the role of PMP22 in mouse Schwann cell autophagy. Salubrinal [a selective endoplasmic reticulum (ER) stress inhibitor] was used to determine whether PMP22 expression was affected by ER stress. The present results indicated that VP1 promoted mouse Schwann cell autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3II and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by salubrinal. In conclusion, VP1 promoted mouse Schwann cell autophagy through upregulation of ER stress­mediated PMP22 expression. Therefore, the VP1/ER stress/PMP22 autophagy axis may be a potential therapeutic target for EV71 infection­induced fatal neuronal damage.

Human Schwann cells are susceptible to infection with Zika and yellow fever viruses, but not dengue virus.

Zika virus (ZIKV) is a re-emerged flavivirus transmitted by Aedes spp mosquitoes that has caused outbreaks of fever and rash on islands in the Pacific and in the Americas. These outbreaks have been associated with neurologic complications that include congenital abnormalities and Guillain-Barré syndrome (GBS). The pathogenesis of ZIKV-associated GBS, a potentially life-threatening peripheral nerve disease, remains unclear. Because Schwann cells (SCs) play a central role in peripheral nerve function and can be the target for damage in GBS, we characterized the interactions of ZIKV isolates from Africa, Asia and Brazil with human SCs in comparison with the related mosquito-transmitted flaviviruses yellow fever virus 17D (YFV) and dengue virus type 2 (DENV2). SCs supported sustained replication of ZIKV and YFV, but not DENV. ZIKV infection induced increased SC expression of IL-6, interferon (IFN)ß1, IFN-λ, IFIT-1, TNFα and IL-23A mRNAs as well as IFN-λ receptors and negative regulators of IFN signaling. SCs expressed baseline mRNAs for multiple potential flavivirus receptors and levels did not change after ZIKV infection. SCs did not express detectable levels of cell surface Fcγ receptors. This study demonstrates the susceptibility and biological responses of SCs to ZIKV infection of potential importance for the pathogenesis of ZIKV-associated GBS.

In vitro characterization and preferential infection by canine distemper virus of glial precursors with Schwann cell characteristics from adult canine brain.

AIMS: Canine distemper virus (CDV)-induced demyelinating leukoencephalomyelitis is a naturally occurring model for multiple sclerosis. The aim of this study was to establish primary glial cell cultures from adult canine brain for the analysis of CDV spread and cell tropism. METHODS: Cultures were inoculated with the CDV-R252 and a CDV-Onderstepoort strain expressing the green fluorescent protein (CDV-OndeGFP). CDV antigen expression was studied using cell type-specific antibodies at different days post infection. Glial cells expressing p75(NTR) were purified using antibody-based techniques and characterized with regard to antigen expression and proliferation. RESULTS: Three weeks after seeding, cultures contained spindle-shaped cells expressing p75(NTR), oligodendrocytic cells, astrocytes, microglia and fibroblasts. Both CDV strains induced a mild to moderate cytopathic effect that consisted of single necrotic and few syncytial giant cells, but displayed in part a differential cell tropism. Whereas CDV-OndeGFP expression in microglia and astrocytes did not exceed 1% and 50%, respectively, CDV-R252 infected 100% and 80% of both cell types, respectively. The cells most early infected by both CDV strains expressed p75(NTR) and may correlate to cells previously identified as aldynoglia. Treatment of p75(NTR+) cells with Schwann cell mitogens and serum deprivation increased proliferation and A2B5 expression, respectively, indicating common properties compared with Schwann cells and oligodendrocyte precursors. CONCLUSIONS: Infection of adult canine astrocytes and microglia revealed CDV strain-specific cell tropism. Moreover, this is the first identification of a glial cell type with Schwann cell-like properties in adult canine brain and, more importantly, these cells displayed a high susceptibility to CDV infection.

In Vivo Introduction of Transgenes into Mouse Sciatic Nerve Cells Using Viral Vectors.

Myelinated fibers are essential for the rapid and efficient propagation of nerve information throughout the body. These fibers result from an intimate crosstalk between myelinating glia and the myelinated axons and, because it is difficult to fully reproduce these interactions in vitro, the basic molecular mechanisms that regulate myelination, demyelination, and remyelination remain unclear. Schwann cells produce myelin in the peripheral nervous system (PNS) and remain associated with the axons of peripheral neurons throughout axonal migration to the target. In order to investigate more closely the biology of myelinated fibers, we developed a local transgenesis approach based on the injection of engineered viral vectors in the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach represents an alternative to germline modifications as it facilitates and speed up the investigation of peripheral nerve biology in vivo. Indeed the protocol we describe here requires just 3 weeks to complete. The injection of engineered viral vectors in the sciatic nerve of mice is a reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo in order to investigate specific molecular mechanisms.

Intrathecal Delivery of Viral Vectors for Gene Therapy.

Gene delivery to the peripheral nervous system for therapeutic applications remains technically challenging but could eventually have a significant impact on the development of innovative treatments not only for inherited but also for acquired peripheral neuropathies. Here we describe the method for lumbar intrathecal injection of viral vectors in experimental mice. This gene delivery route provides widespread and stable over time Schwann cell-targeted or ubiquitous gene expression in the peripheral nervous system.

Zika Virus Replication in Dorsal Root Ganglia Explants from Interferon Receptor1 Knockout Mice Causes Myelin Degeneration.

Zika virus (ZIKV) is a neurotropic agent that targets the developing fetal brain in women infected during pregnancy. In addition to the developing central nervous system, ZIKV has been recently shown to infect cells of the peripheral nervous system (PNS), highlighting its potential to cause acute peripheral neuropathies in adults, such as Guillain-Barré Syndrome (GBS). Here we show that myelinating dorsal root ganglia (DRG) explants obtained from interferon-alpha/beta receptor knock-out mice are productively infected by ZIKV. Virus replication is cytopathic in both peripheral neurons and myelinating Schwann cells leading to myelin disruption. These results confirm and extend previous observations suggesting that the PNS is indeed a potential site of ZIKV infection, replication and cytopathicity.

Efficient myelin repair in the macaque spinal cord by autologous grafts of Schwann cells.

Experimental transplantation in rodent models of CNS demyelination has led to the idea that Schwann cells may be candidates for cell therapy in human myelin diseases. Here we investigated the ability of Schwann cells autografts to generate myelin in the demyelinated monkey spinal cord. We report that monkey Schwann cells derived from adult peripheral nerve biopsies retain, after growth factor expansion and transduction with a lentiviral vector encoding green fluorescent protein, the ability to differentiate in vitro into promyelinating cells. When transplanted in the demyelinated nude mouse spinal cord, they promoted functional and anatomical repair of the lesions (n = 12). Furthermore, we obtained evidence by immunohistochemistry (n = 2) and electron microscopy (n = 4) that autologous transplantation of expanded monkey Schwann cells in acute lesions of the monkey spinal cord results in the repair of large areas of demyelination; up to 55% of the axons were remyelinated by donor Schwann cells, the remaining ones being remyelinated by oligodendrocytes. Autologous grafts of Schwann cells may thus be of therapeutic value for myelin repair in the adult CNS.

[Effects of AQP1 overexpression on morphology and water transport in mouse Schwann cells].

OBJECTIVE: To determine the effect of AQP1 gene on facial nerve edema following injury through investigation of the relationship between the expression of AQP1 gene and Schwann cells swelling. METHODS: The AQP1 expression in Schwann cells of mouse facial nerve tissues was detected by immunofluorescent staining. The transgenic protocol by lentivirus transduction was used to specifically upregulate AQP1 expression in Schwann cells. Lenti-AQP1 and CTRL (empty vector) transduced cells were observed during gene overexpression every 24 h for 6 days by using phase contrast microscopy. Cell volume of CTRL and Lenti-AQP1 treated cells was measured daily from the day of treatment, through day 6. RESULTS: Schwann cell primary cultures maintained a high level of AQP1 water channels, representing an ideal cell model to study the role of AQP1 in the facial nerve. The expression of AQP1 mRNA and protein in Schwann cells infected with the Lenti-AQP1 was increased significantly compared with CTRL lentivirus (P < 0.05). Lenti-AQP1 caused cell swelling in cultured Schwann cells, as validated by cell volume determinations (P < 0.01). CONCLUSIONS: AQP1 is an important factor responsible for the fast water transport of cultured Schwann cells. It plays an important role in facial nerve edema.

Human Endogenous Retrovirus and Neuroinflammation in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND: Human endogenous retroviruses HERV-W encode a pro-inflammatory protein, named MSRV-Env from its original identification in Multiple Sclerosis. Though not detected in various neurological controls, MSRV-Env was found in patients with chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). This study investigated the expression of MSRV in CIDP and evaluated relevant MSRV-Env pathogenic effects. METHODS: 50 CIDP patients, 19 other neurological controls (ONDs) and 65 healthy blood donors (HBDs) were recruited from two different countries. MSRV-env and -pol transcripts, IL6 and CXCL10 levels were quantified from blood samples. MSRV-Env immunohistology was performed in distal sensory nerves from CIDP and neurological controls biopsies. MSRV-Env pathogenic effects and mode of action were assayed in cultured primary human Schwann cells (HSCs). FINDINGS: In both cohorts, MSRV-env and -pol transcripts, IL6 positivity prevalence and CXCL10 levels were significantly elevated in CIDP patients when compared to HBDs and ONDs (statistically significant in all comparisons). MSRV-Env protein was detected in Schwann cells in 5/7 CIDP biopsies. HSC exposed to or transfected with MSRV-env presented a strong increase of IL6 and CXCL10 transcripts and protein secretion. These pathogenic effects on HSC were inhibited by GNbAC1, a highly specific and neutralizing humanized monoclonal antibody targeting MSRV-Env. INTERPRETATION: The present study showed that MSRV-Env may trigger the release of critical immune mediators proposed as instrumental factors involved in the pathophysiology of CIDP. Significant MSRV-Env expression was detected in a significant proportion of patients with CIDP, in which it may play a role according to its presently observed effects on Schwann cells along with previously known effects on immune cells. Experimental results also suggest that a biomarker-driven therapeutic strategy targeting this protein with a neutralizing antibody such as GNbAC1 may offer new perspectives for treating CIDP patients with positive detection of MSRV-Env expression. FUNDING: Geneuro-Innovation, France.

Retroviral inhibition of cAMP-dependent protein kinase inhibits myelination but not Schwann cell mitosis stimulated by interaction with neurons.

Schwann cells are the myelinating glia of the peripheral nervous system. Neuron-Schwann cell contact profoundly affects several aspects of Schwann cell phenotype, including stimulation of mitosis and myelin formation. Many reports suggest that neuronal contact exerts this influence on Schwann cells by elevating Schwann cell cAMP and activating cAMP-dependent protein kinase A (PKA). To elucidate the importance of Schwann cell PKA in neuronal stimulation of Schwann cell mitosis and myelination, the gene encoding the PKA inhibitory protein RIalphaAB or PKIEGFP was delivered to Schwann cells using retroviral vectors. PKA inhibitory retroviral vectors effectively blocked forskolin-stimulated Schwann cell mitosis and morphological change, demonstrating the ability of the vectors to inhibit PKA in infected Schwann cells. Treatment of dorsal root ganglia neuron-Schwann cell cocultures with H-89 (10 microm) or KT5720 (1-10 microm), chemical inhibitors selective for PKA, significantly inhibited neuronal stimulation of Schwann cell mitosis. In contrast, retrovirus-mediated inhibition of Schwann cell PKA had no effect on the ability of neurons to stimulate Schwann cell mitosis. However, markedly fewer myelin segments were formed by Schwann cells expressing PKA inhibitory proteins compared with controls. These results suggest that activation of Schwann cell PKA is required for myelin formation but not for Schwann cell mitosis stimulated by interaction with neurons.

Efficiency of adeno-associated virus type-2 vectors in non-human primate Schwann cells.

Adult macaque Schwann cells were infected using adeno-associated virus type-2-derived vectors expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus, the hybrid cytomegalovirus-betaactin, the myelin basic protein or the tetracycline-inducible promoters. On the basis of green fluorescent protein expression, gene transfer efficiency was compared in resting and dividing conditions following or not following hydroxyurea or etoposide treatment. Hydroxyurea allowed promoter-dependent expression of green fluorescent protein in infected Schwann cells. Etoposide treatment led to a high percentage of green fluorescent protein expressing cells (over 50%) with all promoters tested. When infected cells were grafted into demyelinated nude mice spinal cord, green fluorescent protein expression was only observed with the cytomegalovirus-betaactin and tetracycline-inducible promoters. In addition, adeno-associated virus type-2 infection reduced the grafted cell survival but increased their differentiation.

The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing.

Injury of peripheral nerves induces expression of several pro-protein convertases (PCs) involved in processing of precursor proteins into their diverse active end-products. In this study, the focus was on convertase PC1 which, although undetectable in control nerves, is strongly induced in injured nerves. High concentrations of PC1 mRNA of 9.0, 5.5, 3.0, 2.5 and 1.6 kb were observed on day 4 post-lesion in proximal and distal segments. By in situ hybridization PC1 mRNA was detected in most of endoneurial cells, which were further identified by immunocytochemistry as myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase containing Schwann cells. PC1 mRNA and protein were also present in cultured Schwann cells also containing convertases PC5, furin and PC7 as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Mostly unprocessed pro-NGF of 35 kDa and pro-BDNF of 35 kDa were found on Western blotting of Schwann cells. Expression of exogenous neurotrophins by infection with vaccinia virus vector showed that mouse pro-NGF and rat pro-BDNF are cleaved intracellularly on smaller forms of 13.5 kDa NGF and 14 kDa BDNF. Infection experiments demonstrated that Schwann cells contain active processing enzymes. In conclusion, this work provides in vivo evidence of the presence of several PCs in the injured rat sciatic nerve and ex vivo in cultured Schwann cells.

Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS.

In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied, following direct injection in several regions of the central and peripheral nervous system. The distribution of the cells expressing the transgene appears to be determined by natural anatomical boundaries and possibly by the degree of myelinization of a particular brain region. In highly myelinated areas with a compact cellular structure (e.g. the cortex and olfactory bulb) the spread of the viral vector is limited to the region close to the injection needle, while in areas with a laminar structure (e.g. the hippocampus and the eye) more widespread transgene expression is observed. Retrograde transport of the viral vector may serve as an attractive alternative route of transgene delivery. A time course of expression of beta-galactosidase in neural cells in the facial nucleus revealed high expression during the first week after AdLacZ injection. However, a significant decline in transgene expression during the second and third week was observed. This may be caused by an immune response against the transduced cells or by silencing of the cytomegalovirus promoter used to drive transgene expression. Taken together, the data underscore that for each application of adenoviral vectors as gene transfer agents in the nervous system it is important to examine vector spread in and infectability of the neural structure that is subject to genetic modification.