Geneviève Soucy

Circulating cell wall components derived from gram‐negative, not gram‐positive, bacteria cause a profound induction of the gene‐encoding Toll‐like receptor 2 in the CNS

The recent characterization of human homologs of Toll may be the missing link for the transduction events leading to nuclear factor‐κB (NF‐κB) activity and proinflammatory gene transcription during innate immune response. Mammalian cells may express as many as 10 distinct Toll‐like receptors (TLRs), although TLR2 is a key receptor for recognizing cell wall components of Gram‐positive bacteria. The present study investigated the effects of circulating bacterial cell wall components on the expression of the gene‐encoding TLR2 across the mouse brain. Surprisingly, while Gram‐negative components caused a robust increase in TLR2 transcription within the cerebral tissue, peptidoglycan (PGN) and lipoteichoic acid (LTA), either alone or combined, failed to modulate the receptor transcript. Indeed, the mRNA levels for TLR2 in the choroid plexus and few other regions of the brain remained similar between vehicle‐, LTA‐, PGN‐, and LTA/PGN‐administered mice at all the times evaluated (i.e. 30 min to 24 h post‐intraperitoneal injection). This contrasts with the profound de novo expression of TLR2 following a single systemic injection of the lipopolysaccharide (LPS). The signal was first detected in regions devoid of blood–brain barrier and few blood vessels and microcapillaries. A second wave of TLR2 expression was also detected from these structures to their surrounding parenchymal cells that stained for a microglial marker iba1. The rapid induction of IκBα (index of NF‐κB activity) and up‐regulation of the adaptor protein MyD88 suggest that LPS‐induced TLR2 transcription may be dependent on the NF‐κB pathway. These data provide the evidence that TLR2 is not only present in the brain, but its encoding gene is regulated by cell wall components derived from Gram‐negative, not Gram‐positive, bacteria. The robust wave of TLR2‐expressing microglial cells may have a determinant impact on the innate immune response that occurs in the brain during systemic infection by Gram‐negative, not Gram‐positive, bacteria.

Intracerebroventricular infusion of monoclonal antibody or its derived Fab fragment against misfolded forms of SOD1 mutant delays mortality in a mouse model of ALS.

J. Neurochem. (2010) 113, 1188–1199.

Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain

Although the neuroprotective effects of estrogens are well recognized, the exact mechanisms involved in the ability of these sex steroids to protect the cerebral tissue still remain unclear. We tested in our study the hypothesis that estradiol (E2) modulates the innate immune response and expression of genes encoding proteins that a provide survival signal to neurons during infection. Mice received a single systemic or cerebral injection of LPS to trigger a robust but transient inflammatory reaction in the brain. The endotoxin increased transcriptional activation of genes encoding TLR2, TNF-α, and IL-12 in microglial cells. Expression of these transcripts was largely inhibited in the brain of ovariectomized mice at time 24 h postchallenge. E2 replacement therapy totally rescued the ability of the endotoxin to trigger microglial cells and these permissive effects of E2 are mediated via the estrogen receptor (ER)α. Indeed, ERα-deficient mice exhibited an inappropriate reaction to LPS when compared with ERβ-deficient and wild-type mice. This defective innate immune response was also associated with a widespread viral replication and neurodegeneration in ovariectomized mice inoculated intranasally with HSV-2. These data provide evidence that interaction of E2 with their nuclear ERα plays a critical role in the control of cytokines involved in the transfer from the innate to adaptive immunity. This transfer is deviant in mice lacking E2, which allows pathogens to hide from immune surveillance and exacerbates neuronal damages during viral encephalitis.

Live imaging of Toll-like receptor 2 response in cerebral ischaemia reveals a role of olfactory bulb microglia as modulators of inflammation.

Activation of microglial cells in response to ischaemic injury, inflammatory and/or immune stimuli is associated with the marked induction of Toll-like receptor 2 (TLR2). At present, little is known about the spatial and temporal sequence of events, micro-regional specificities and the potential long term role of the TLR2 response to brain injuries. To investigate microglial activation/TLR2 response in real time, we generated a transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. In this model, transcriptional activation of TLR2 was visualized in the brains of live animals using biophotonic/bioluminescence molecular imaging and a high resolution/sensitivity charged coupled device camera. It was found that TLR2 induction/microglial activation has a marked chronic component after ischaemic injury and may last several months after the initial attack. The pro-inflammatory response was not restricted to the site of ischaemic injury but was also evident in the olfactory bulb. A significant TLR2 response was first seen in the olfactory bulb 6 h after stroke and several hours before the increase in photon emission over the site of infarction. This sequence of events was further confirmed by immunohistochemistry. A similar early TLR2 response from olfactory bulb microglia was observed in the brains immune response to pathogens. We therefore propose that, owing to their unique situation, receiving and translating numerous inputs from the brain as well as from the environment, olfactory bulb microglia may serve as sensors and/or modulators of brain inflammation.

Absence of tumor necrosis factor-α does not affect motor neuron disease caused by superoxide dismutase 1 mutations

An increase in the expression of the proinflammatory cytokine tumor necrosis factor α (TNF-α) has been observed in patients with amyotrophic lateral sclerosis (ALS) and in the mice models of the disease. TNF-α is a potent activator of macrophages and microglia and, under certain conditions, can induce or exacerbate neuronal cell death. Here, we assessed the contribution of TNF-α in motor neuron disease in mice overexpressing mutant superoxide dismutase 1 (SOD1) genes linked to familial ALS. This was accomplished by the generation of mice expressing SOD1G37R or SOD1G93A mutants in the context of TNF-α gene knock out. Surprisingly, the absence of TNF-α did not affect the lifespan or the extent of motor neuron loss in SOD1 transgenic mice. These results provide compelling evidence indicating that TNF-α does not directly contribute to motor neuron degeneration caused by SOD1 mutations.

Adeno-associated Virus-mediated Delivery of a Recombinant Single-chain Antibody Against Misfolded Superoxide Dismutase for Treatment of Amyotrophic Lateral Sclerosis

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)–mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1G93A mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1G93A mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

METHYLENE BLUE ADMINISTRATION FAILS TO CONFER NEUROPROTECTION IN TWO AMYOTROPHIC LATERAL SCLEROSIS MOUSE MODELS

Approximately 20% cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that methylene blue (MB) was efficient in conferring protection in several neurological disorders. MB was found to improve mitochondrial function, to reduce reactive oxygen species, to clear aggregates of toxic proteins, and to act as a nitric oxide synthase inhibitor. These pleiotropic effects of relevance to ALS pathogenesis led us to test MB in two models of ALS, SOD1(G93A) mice and TDP-43(G348C) transgenic mice. Intraperitoneal administration of MB at two different doses was initiated at the beginning of disease onset, at 90 days of age in SOD1(G93A) and at 6 months of age in TDP-43(G348C) mice. Despite its established neuroprotective properties, MB failed to confer protection in both mouse models of ALS. The lifespan of SOD1(G93A) mice was not affected by MB treatment. The declines in motor function, reflex score, and body weight of SOD1(G93A) mice remained unchanged. MB treatment had no effect on motor neuron loss and aggregation or misfolding of SOD1. A combination of MB with lithium also failed to provide benefits in SOD1(G93A) mice. In TDP-43(G348C) mice, MB failed to improve motor function. Cytosolic translocation of TDP-43, ubiquitination and inflammation remained also unchanged after MB treatment of TDP-43(G348C) mice.

IL-10 Controls Early Microglial Phenotypes and Disease Onset in ALS Caused by Misfolded Superoxide Dismutase 1

While reactive microgliosis is a hallmark of advanced stages of amyotrophic lateral sclerosis (ALS), the role of microglial cells in events initiating and/or precipitating disease onset is largely unknown. Here we provide novel in vivo evidence of a distinct adaptive shift in functional microglial phenotypes in preclinical stages of superoxide dismutase 1 (SOD1)-mutant-mediated disease. Using a mouse model for live imaging of microglial activation crossed with SOD1G93A and SOD1G37R mouse models, we discovered that the preonset phase of SOD1-mediated disease is characterized by development of distinct anti-inflammatory profile and attenuated innate immune/TLR2 responses to lipopolysaccharide (LPS) challenge. This microglial phenotype was associated with a 16-fold overexpression of anti-inflammatory cytokine IL-10 in baseline conditions followed by a 4.5-fold increase following LPS challenge. While infusion of IL-10R blocking antibody, initiated at day 60, caused a significant increase in markers of microglial activation and precipitated clinical onset of disease, a targeted overexpression of IL-10 in microglial cells, delivered via viral vectors expressed under CD11b promoter, significantly delayed disease onset and increased survival of SOD1G93A mice. We propose that the high IL-10 levels in resident microglia in early ALS represent a homeostatic and compensatory “adaptive immune escape” mechanism acting as a nonneuronal determinant of clinical onset of disease. SIGNIFICANCE STATEMENT We report here for the first time that changing the immune profile of brain microglia may significantly affect clinical onset and duration of disease in ALS models. We discovered that in presymptomatic disease microglial cells overexpress anti-inflammatory cytokine IL-10. Given that IL-10 is major homeostatic cytokine and its production becomes deregulated with aging, this may suggest that the capacity of microglia to adequately produce IL-10 may be compromised in ALS. We show that blocking IL-10 increased inflammation and precipitated clinical disease onset, whereas overexpression of IL-10 in microglia using a gene therapy approach significantly delayed disease onset and increased survival of ALS mice. Based on our results, we propose that targeted overexpression of IL-10 in microglia may have therapeutic potential in ALS.

Ablation of Proliferating Cells in the CNS Exacerbates Motor Neuron Disease Caused by Mutant Superoxide Dismutase

Proliferation of glia and immune cells is a common pathological feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, to investigate the role of proliferating cells in motor neuron disease, SOD1G93A transgenic mice were treated intracerebroventicularly (ICV) with the anti-mitotic drug cytosine arabinoside (Ara-C). ICV delivery of Ara-C accelerated disease progression in SOD1G93A mouse model of ALS. Ara-C treatment caused substantial decreases in the number of microglia, NG2+ progenitors, Olig2+ cells and CD3+ T cells in the lumbar spinal cord of symptomatic SOD1G93A transgenic mice. Exacerbation of disease was also associated with significant alterations in the expression inflammatory molecules IL-1β, IL-6, TGF-β and the growth factor IGF-1.

Loss of Glial Neurofascin155 Delays Developmental Synapse Elimination at the Neuromuscular Junction

Postnatal synapse elimination plays a critical role in sculpting and refining neural connectivity throughout the central and peripheral nervous systems, including the removal of supernumerary axonal inputs from neuromuscular junctions (NMJs). Here, we reveal a novel and important role for myelinating glia in regulating synapse elimination at the mouse NMJ, where loss of a single glial cell protein, the glial isoform of neurofascin (Nfasc155), was sufficient to disrupt postnatal remodeling of synaptic circuitry. Neuromuscular synapses were formed normally in mice lacking Nfasc155, including the establishment of robust neuromuscular synaptic transmission. However, loss of Nfasc155 was sufficient to cause a robust delay in postnatal synapse elimination at the NMJ across all muscle groups examined. Nfasc155 regulated neuronal remodeling independently of its canonical role in forming paranodal axo–glial junctions, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Rather, high-resolution proteomic screens revealed that loss of Nfasc155 from glial cells was sufficient to disrupt neuronal cytoskeletal organization and trafficking pathways, resulting in reduced levels of neurofilament light (NF-L) protein in distal axons and motor nerve terminals. Mice lacking NF-L recapitulated the delayed synapse elimination phenotype observed in mice lacking Nfasc155, suggesting that glial cells regulate synapse elimination, at least in part, through modulation of the axonal cytoskeleton. Together, our study reveals a glial cell-dependent pathway regulating the sculpting of neuronal connectivity and synaptic circuitry in the peripheral nervous system.