GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABAAR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse-muscular insulin-intestinal innate immunity in vivo.

Sir Bernard Katz: 26 March 1911 - 20 April 2003.

Sir Bernard Katz established the cellular basis of synaptic transmission at the neuromuscular junction, the contact point between nerve and muscle. With his death, we lost one of the most distinguished biophysicists of our time. He laid the foundations for our understanding of almost every aspect of synaptic transmission. Bernard Katz revealed the existence of key molecules and formally described their interaction. With the benefit of his almost magical intuition, he formulated hypotheses that are now recognized as facts. During his career he pioneered research in three areas. He and Alan Hodgkin elucidated the ionic basis of the action potential overshoot, as formulated in the sodium hypothesis; he unravelled the biophysical mechanisms that generate the endplate potential; and he clarified mechanisms of transmitter release, as detailed in the quantal hypothesis and the vesicle hypothesis. In particular his work on the neuromuscular junction influenced and led several generations of neurophysiologists, and it continues to do so even though research focus has shifted to synapses in the central nervous system. Bernard Katz (or BK, as he was known to colleagues and students) trained several generations of young investigators who have been inspired by his hypotheses, by his impeccable thoroughness as an investigator, and by the straightforward, unpretentious style of his presentations - though some have been dismayed by his occasional unapproachability or his unforgiving nature when confronted with others' mistakes! Perhaps his most valuable and enduring legacy to collaborators and students is that, when data are difficult to interpret and we see only a faint glimmer of light at the end of a long tunnel, we ask ourselves, 'What would BK do now?'

Rabies virus infection of myotubes and neurons as elements of the neuromuscular junction.

The neuromuscular junction represents a site of transit for both fixed and street rabies viruses. Infection of cultured rat myotubes by fixed rabies virus was found to be restrictive, and although fluorescence observations showed that cells were infected, there were no infectious virus particles in the supernatant of infected myotubes. In contrast, infection of myotubes with street rabies virus produced infectious virus particles, and kinetic studies noted a growth cycle in these cells. Neurons derived from the rat spinal cord or from dorsal root ganglia were 10-100-fold more susceptible to infection with fixed rabies challenge virus strain (CVS) than were the myotubes, a finding that confirms the basically neurotropic nature of rabies virus. The abortive infection of CVS rabies in muscle cells may be one possible mechanism by which virus persists at the site of inoculation. In addition, competition-binding experiments show that alpha-bungarotoxin at 10(-5)-10(-7) M inhibits rabies virus infection of myotubes, a finding that suggests the involvement of low-affinity nicotinic acetylcholine receptors for rabies virus. Sialic acid was shown to be necessary for the attachment of rabies virus to the myotubes, a requirement confirming earlier data for other cell types. These data confirm observations in vivo. Infection of these cells in primary culture, which represent the natural target for rabies virus, mimics the situation in vivo. Such a model permits further investigation of virus-cell interactions at the neuromuscular junction.

Rabies virus binding at neuromuscular junctions.

Morphological, immunocytochemical, biochemical, and immunological techniques have been used to describe rabies virus binding to a sub-cellular unit and molecular complex at the neuromuscular junction (NMJ). Early after infection in vivo, virus antigen and virus particles were found by immunofluorescence, electron microscopy and immunoelectron microscopy in regions of high density acetylcholine receptors (AChR) at NMJs. One monoclonal antibody (alpha-Mab) to the alpha subunit of the AChR blocked attachment of radio-labeled rabies virus to cultured muscle cells bearing high density patches of AChR. A sub-cellular structure, resembling an array of AChR monomers, bound both rabies virus antigens and alpha-Mab. By immunoblotting with electrophoretically transferred motor endplate proteins, rabies virus proteins and alpha-Mab bound to two proteins of 43 000 and 110 000 daltons. A rabies virus glycoprotein antibody detected virus antigen bound to the 110 000 dalton protein. An auto-immune (anti-idiotypic) response followed immunization of mice with rabies virus glycoprotein antigen; the antibody was directed to the 110 000 dalton protein. This auto-antibody altered the kinetics of neutralization by rabies virus antibody and induced the formation of rabies virus antibody after inoculation of mice. These results define, at the neuromuscular junction, a rabies virus receptor which may be part of the acetylcholine receptor complex.

Herpes simplex virus infection of motor neurons: hypoglossal model.

Herpes simplex virus type 1 (HSV) was inoculated into the tongue muscle of A/J mice. Typical HSV vesicles developed on the tongue surface 4 days after HSV inoculation. Virus was isolated from hypoglossal nerve explants, and inflammatory cells appeared where the hypoglossal nerve exists from the ventral medulla. HSV viral capsids were present in astroglial cells near the point of nerve exit. A focal encephalitis ensued with immunoperoxidase staining of HSV antigens in neurons of the hypoglossal nucleus. These findings indicate that HSV can penetrate the neuromuscular junction, travel in a pure motor nerve, and produce a focal encephalitis in the corresponding central nervous system motor nucleus.

The acetylcholine receptor as a cellular receptor for rabies virus.

Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis.

Entry of rabies virus into the peripheral nerves of mice.

Young adult mice were inoculated in the hind limb with rabies virus or Sindbis virus. Rabies 1820B virus antigen was detected in leg sections by immuno-fluorescence at 1 h post-inoculation at sites comparable in form and distribution to cholinesterase-positive sites, which represent motor end-plates (MEPs). Sites which were rabies virus antigen-positive by immunofluorescence were also cholinesterase- positive on double-stained slides. Rabies CVS virus detected by autoradiography was similarly distributed at 6 h post-inoculation. Uptake of rabies virus at motor nerve endings was confirmed by the detection of rabies antigen by immunofluorescence in ventral horn cells in the spinal cord at 20 h post-inoculation before involvement of dorsal root ganglia. Rabies virus antigen could not be detected at MEPs if the virus had been inactivated by beta propiolactone or mixed with antibody prior to injection or if the sciatic nerve had been cut 7 days earlier, similarly treated groups of mice survived for the observation period of 6 weeks. Rabies virus antigen was found at MEPs in mice given antibody 24 h before virus injection, but virus antigen was not found in the spinal cord, and mice similarly treated survived. Sindbis virus strain Ar86, which like rabies virus is neurotropic in adult mice, was also found at MEPs and in peripheral nerves by autoradiography at 6 h post-inoculation. In contrast to results with rabies virus-infected mice, stimulation of the sciatic nerve for the first hour post-inoculation prevented mortality. Sindbis virus strain Ar339, which is not neurotropic in adult mice, could not be detected at MEP's by immunofluorescence or autoradiography and mice injected with virus survived. The results presented here suggest that rabies virus and perhaps other neurotropic viruses can use the motor axon terminal at the neuromuscular junction as a site of entry into the nervous system.

Lyssavirus infection of muscle spindles and motor end plates in striated muscle of hamsters.

Immunofluorescent, light, and electron microscopy were used to document lyssavirus infection of muscle spindles and motor end plates. Virus particles were seen in the narrow intercellular space between sensory nerve endings and intrafusal muscle fibers; they were also observed budding from intracellular and plasma membranes of the latter. Involvement of motor nerves and motor end plates could only be demonstrated by electron microscopy. In nature, rabies virus invasion of the peripheral nervous system must involve centripetal spread across these junctions.