Timothy Vartanian

Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein

Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.

Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal death in vitro that depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived from tlr4 mutant mice. In an in vivo model of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration.

T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease, in which the role of inflammation is not well established. Innate and adaptive immunity were investigated in the CNS of the Superoxide Dismutase 1 (SOD1)G93A transgenic mouse model of ALS. CD4+ and CD8+ T cells infiltrated SOD1G93A spinal cords during disease progression. Cell-specific flow cytometry and gene expression profiling showed significant phenotypic changes in microglia, including dendritic cell receptor acquisition, and expression of genes linked to neuroprotection, cholesterol metabolism and tissue remodeling. Microglia dramatically up-regulated IGF-1 and down-regulated IL-6 expression. When mutant SOD1 mice were bred onto a TCRβ deficient background, disease progression was significantly accelerated at the symptomatic stage. In addition, microglia reactivity and IGF-1 levels were reduced in spinal cords of SOD1G93A (TCRβ−/−) mice. These results indicate that T cells play an endogenous neuroprotective role in ALS by modulating a beneficial inflammatory response to neuronal injury.

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR–NF-κB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

Hyaluronan blocks oligodendrocyte progenitor maturation and remyelination through TLR2

Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogen-derived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2+ oligodendrocytes and low-molecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.

Toll-like receptor 3 is a potent negative regulator of axonal growth in mammals.

Toll is a cell surface receptor with well described roles in the developmental patterning of invertebrates and innate immunity in adult Drosophila. Mammalian toll-like receptors represent a family of Toll orthologs that function in innate immunity by recognizing molecular motifs unique to pathogens or injured tissue. One member in this family of pattern recognition receptors, toll-like receptor 3 (TLR3), recognizes viral double-stranded RNA and host mRNA. We examined the expression and function of TLRs in the nervous system and found that TLR3 is expressed in the mouse central and peripheral nervous systems and is concentrated in the growth cones of neurons. Activation of TLR3 by the synthetic ligand polyinosine:polycytidylic acid (poly I:C) or by mRNA rapidly causes growth cone collapse and irreversibly inhibits neurite extension independent of nuclear factor κB. Mice lacking functional TLR3 were resistant to the neurodegenerative effects of poly I:C. Neonatal mice injected with poly I:C were found to have fewer axons exiting dorsal root ganglia and displayed related sensorimotor deficits. No effect of poly I:C was observed in mice lacking functional TLR3. Together, these findings provide evidence that an innate immune pattern recognition receptor functions autonomously in neurons to regulate axonal growth and advances a novel hypothesis that this class of receptors may contribute to injury and limited CNS regeneration.

A Mechanism for Neurodegeneration Induced by Group B Streptococci through Activation of the TLR2/MyD88 Pathway in Microglia

Group B Streptococcus (GBS) is a major cause of bacterial meningitis and neurological morbidity in newborn infants. The cellular and molecular mechanisms by which this common organism causes CNS injury are unknown. We show that both heat-inactivated whole GBS and a secreted proteinaceous factor from GBS (GBS-F) induce neuronal apoptosis via the activation of murine microglia through a TLR2-dependent and MyD88-dependent pathway in vitro. Microglia, astrocytes, and oligodendrocytes, but not neurons, express TLR2. GBS as well as GBS-F induce the synthesis of NO in microglia derived from wild-type but not TLR2−/− or MyD88−/− mice. Neuronal death in neuronal cultures complemented with wild-type microglia is NO-dependent. We show for the first time a TLR-mediated mechanism of neuronal injury induced by a clinically relevant bacterium. This study demonstrates a causal molecular relationship between infection with GBS, activation of the innate immune system in the CNS through TLR2, and neurodegeneration. We suggest that this process contributes substantially to the serious morbidity associated with neonatal GBS meningitis and may provide a potential therapeutic target.

The Epidermal Growth Factor Receptor Engages Receptor Interacting Protein and Nuclear Factor-κB (NF-κB)-inducing Kinase to Activate NF-κB IDENTIFICATION OF A NOVEL RECEPTOR-TYROSINE KINASE SIGNALOSOME

The transcription factor nuclear factor-κB (NF-κB) is activated by a diverse number of stimuli including tumor necrosis factor-α, interleukin-1, UV irradiation, viruses, as well as receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). NF-κB activation by the tumor necrosis factor receptor (TNFR) involves the formation of a multiprotein complex termed a signalosome. Although previous studies have shown that the activated EGFR can induce NF-κB, the mechanism of this activation remains unknown. In this study, we identify components of the signalosome formed by the activated EGFR required to activate NF-κB and show that, although the activated EGFR uses mechanisms similar to the TNFR, it recruits a distinct signalosome. We show the EGFR forms a complex with a TNFR-interacting protein (RIP), which plays a key role in TNFR-induced NF-κB activation, but not with TRADD, an adaptor protein which serves to recruit RIP to the TNFR. Furthermore, we show that the EGFR associates with NF-κB-inducing kinase (NIK) and provide evidence suggesting multiprotein complex formation between the EGFR, RIP, and NIK. Using a dominant negative NIK mutant, we show that NIK activation is required for EGFR-mediated NF-κB induction. We also show that a S32/36 IκBα mutant blocks EGFR-induced NF-κB activation. Our studies also suggest that a high level of EGFR expression, a frequent occurrence in human tumors, is optimal for epidermal growth factor-induced NF-κB activation. Finally, although protein kinase B/Akt has been implicated in tumor necrosis factor and PDGF-induced NF-κB activation, our studies do not support a role for this protein in EGFR-induced NF-κB activation.

The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes

Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2−/−), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2−/− oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.

Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease

We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin’s tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate.