Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege.

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

Comparison of two neurotrophic serpins reveals a small fragment with cell survival activity.

PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.

Aberrant production of tenascin-C in globoid cell leukodystrophy alters psychosine-induced microglial functions.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a rare and often fatal demyelinating disease caused by mutations in the galactocerebrosidase (galc) gene that result in accumulation of galactosylsphingosine (psychosine). We recently reported that the extracellular matrix (ECM) protease, matrix metalloproteinase-3, is elevated in GLD and that it regulates psychosine-induced microglial activation. Here, we examined central nervous system ECM component expression in human GLD patients and in the twitcher mouse model of GLD using immunohistochemistry. The influence of ECM proteins on primary murine microglial responses to psychosine was evaluated using ECM proteins as substrates and analyzed by quantitative real-time polymerase chain reaction, immunocytochemistry, and ELISA. Functional analysis of microglial cytotoxicity was performed on oligodendrocytes in coculture, and cell death was measured by lactose dehydrogenase assay. Tenascin-C (TnC) was expressed at higher levels in human GLD and in twitcher mice versus controls. Microglial responses to psychosine were enhanced by TnC, as determined by an increase in globoid-like cell formation, matrix metalloproteinase-3 mRNA expression, and higher toxicity toward oligodendrocytes in culture. These findings were consistent with a shift toward the M1 microglial phenotype in TnC-grown microglia. Thus, elevated TnC expression in GLD modified microglial responses to psychosine. These data offer a novel perspective and enhance understanding of the microglial contribution to GLD pathogenesis.

MMP-3 mediates psychosine-induced globoid cell formation: implications for leukodystrophy pathology.

Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.

Astrocyte regulation of CNS inflammation and remyelination.

Astrocytes regulate fundamentally important functions to maintain central nervous system (CNS) homeostasis. Altered astrocytic function is now recognized as a primary contributing factor to an increasing number of neurological diseases. In this review, we provide an overview of our rapidly developing understanding of the basal and inflammatory functions of astrocytes as mediators of CNS responsiveness to inflammation and injury. Specifically, we elaborate on ways that astrocytes actively participate in the pathogenesis of demyelinating diseases of the CNS through their immunomodulatory roles as CNS antigen presenting cells, modulators of blood brain barrier function and as a source of chemokines and cytokines. We also outline how changes in the extracellular matrix can modulate astrocytes phenotypically, resulting in dysregulation of astrocytic responses during inflammatory injury. We also relate recent studies describing newly identified roles for astrocytes in leukodystrophies. Finally, we describe recent advances in how adapting this increasing breadth of knowledge on astrocytes has fostered new ways of thinking about human diseases, which offer potential to modulate astrocytic heterogeneity and plasticity towards therapeutic gain. In summary, recent studies have provided improved insight in a wide variety of neuroinflammatory and demyelinating diseases, and future research on astrocyte pathophysiology is expected to provide new perspectives on these diseases, for which new treatment modalities are increasingly necessary.