MicroRNA-223 protects neurons from degeneration in experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.

MT3-MMP Promotes Excitatory Synapse Formation by Promoting Nogo-66 Receptor Ectodomain Shedding.

Cell-surface molecules are dynamically regulated at the synapse to assemble and disassemble adhesive contacts that are important for synaptogenesis and for tuning synaptic transmission. Metalloproteinases dynamically regulate cellular behaviors through the processing of cell surface molecules. In the present study, we evaluated the role of membrane-type metalloproteinases (MT-MMPs) in excitatory synaptogenesis. We find that MT3-MMP and MT5-MMP are broadly expressed in the mouse cerebral cortex and that MT3-MMP loss-of-function interferes with excitatory synapse development in dissociated cortical neurons and in vivo We identify Nogo-66 receptor (NgR1) as an MT3-MMP substrate that is required for MT3-MMP-dependent synapse formation. Introduction of the shed ectodomain of NgR1 is sufficient to accelerate excitatory synapse formation in dissociated cortical neurons and in vivo Together, our findings support a role for MT3-MMP-dependent shedding of NgR1 in regulating excitatory synapse development.SIGNIFICANCE STATEMENT In this study, we identify MT3-MMP, a membrane-bound zinc protease, to be necessary for the development of excitatory synapses in cortical neurons. We identify Nogo-66 receptors (NgR1) as a downstream target of MT3-MMP proteolytic activity. Furthermore, processing of surface NgR1 by MT3-MMP generates a soluble ectodomain fragment that accelerates the formation of excitatory synapses. We propose that MT3-MMP activity and NgR1 shedding could stimulate circuitry remodeling in the adult brain and enhance functional connectivity after brain injury.

Neurite outgrowth is differentially impacted by distinct immune cell subsets.

Axonal damage can occur in the central nervous system following trauma, during the course of autoimmune and neurodegenerative disease and during viral and bacterial infections. The degree of axonal damage and absence of spontaneous repair are major determinants of long-term clinical outcome. While inflammation is a common feature of these conditions, the impact of particular immune cell subsets and their products on injured axons is not fully known. To investigate the impact of immune cells on neuronal viability and axonal repair, we developed an in vitro culture system in which neurons are exposed to mixed or distinct immune cell subsets. We find that total peripheral blood mononuclear cells (PBMCs) have a significant inhibitory effect on neurite outgrowth that is independent of apoptosis. Using isolated immune cells subsets, we demonstrate that activated CD4+ T cells enhance neurite outgrowth while activated NK cells and CD8+ T cells inhibit neurite outgrowth. We find that NK cell inhibition of neuronal outgrowth is dependent on MAPK activity. Our findings describe heterogeneous effects of individual immune cell subsets on neuronal growth and offer important insights into the cellular and molecular mechanisms that may impact axonal repair in inflammatory CNS conditions.

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury.

In contrast to neurons in the CNS, damaged neurons from the peripheral nervous system (PNS) regenerate, but this process can be slow and imperfect. Successful regeneration is orchestrated by cytoskeletal reorganization at the tip of the proximal axon segment and cytoskeletal disassembly of the distal segment. Collapsin response mediator protein 4 (CRMP4) is a cytosolic phospho-protein that regulates the actin and microtubule cytoskeleton. During development, CRMP4 promotes growth cone formation and dendrite development. Paradoxically, in the adult CNS, CRMP4 impedes axon regeneration. Here, we investigated the involvement of CRMP4 in peripheral nerve injury in male and female Crmp4-/- mice following sciatic nerve injury. We find that sensory axon regeneration and Wallerian degeneration are impaired in Crmp4-/- mice following sciatic nerve injury. In vitro analysis of dissociated dorsal root ganglion (DRG) neurons from Crmp4-/- mice revealed that CRMP4 functions in the proximal axon segment to promote the regrowth of severed DRG neurons and in the distal axon segment where it facilitates Wallerian degeneration through calpain-dependent formation of harmful CRMP4 fragments. These findings reveal an interesting dual role for CRMP4 in proximal and distal axon segments of injured sensory neurons that coordinately facilitate PNS axon regeneration.

Nonmuscle myosin promotes cytoplasmic localization of PBX.

In the absence of MEIS family proteins, two mechanisms are known to restrict the PBX family of homeodomain (HD) transcription factors to the cytoplasm. First, PBX is actively exported from the nucleus via a CRM1-dependent pathway. Second, nuclear localization signals (NLSs) within the PBX HD are masked by intramolecular contacts. In a screen to identify additional proteins directing PBX subcellular localization, we identified a fragment of murine nonmuscle myosin II heavy chain B (NMHCB). The interaction of NMHCB with PBX was verified by coimmunoprecipitation, and immunofluorescence staining revealed colocalization of NMHCB with cytoplasmic PBX in the mouse embryo distal limb bud. The interaction domain in PBX mapped to a conserved PBC-B region harboring a potential coiled-coil structure. In support of the cytoplasmic retention function, the NMHCB fragment competes with MEIS1A to redirect PBX, and the fly PBX homologue EXD, to the cytoplasm of mammalian and insect cells. Interestingly, MEIS1A also localizes to the cytoplasm in the presence of the NMHCB fragment. These activities are largely independent of nuclear export. We show further that the subcellular localization of EXD is deregulated in Drosophila zipper mutants that are depleted of nonmuscle myosin heavy chain. This study reveals a novel and evolutionarily conserved mechanism controlling the subcellular distribution of PBX and EXD proteins.

Sequential histone modifications at Hoxd4 regulatory regions distinguish anterior from posterior embryonic compartments.

Hox genes are differentially expressed along the embryonic anteroposterior axis. We used chromatin immunoprecipitation to detect chromatin changes at the Hoxd4 locus during neurogenesis in P19 cells and embryonic day 8.0 (E8.0) and E10.5 mouse embryos. During Hoxd4 induction in both systems, we observed that histone modifications typical of transcriptionally active chromatin occurred first at the 3' neural enhancer and then at the promoter. Moreover, the sequential distribution of histone modifications between E8.0 and E10.5 was consistent with a spreading of open chromatin, starting with the enhancer, followed by successively more 5' intervening sequences, and culminating at the promoter. Neither RNA polymerase II (Pol II) nor CBP associated with the inactive gene. During Hoxd4 induction, CBP and RNA Pol II were recruited first to the enhancer and then to the promoter. Whereas the CBP association was transient, RNA Pol II remained associated with both regulatory regions. Histone modification and transcription factor recruitment occurred in posterior, Hox-expressing embryonic tissues, but never in anterior tissues, where such genes are inactive. Together, our observations demonstrate that the direction of histone modifications at Hoxd4 mirrors colinear gene activation across Hox clusters and that the establishment of anterior and posterior compartments is accompanied by the imposition of distinct chromatin states.

RhoA Proteolysis Regulates the Actin Cytoskeleton in Response to Oxidative Stress.

The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism.

Myeloid lineage cells inhibit neurite outgrowth through a myosin II-dependent mechanism.

The molecular mechanisms that underlie the axonal damage that accompanies CNS inflammation are largely unknown. Here, we investigate the effects of immune cells on neuronal viability and axonal growth and show that conditioned media from myeloid lineage cells inhibit neurite outgrowth without causing apoptosis. Treatment with monocyte conditioned medium enhances myosin light chain phosphorylation in neurons and the neurite outgrowth inhibitory effect of myeloid lineage cells can be attenuated with the myosin II inhibitor blebbistatin. Our results suggest that in the context of CNS inflammation myeloid cells may limit axonal repair in the CNS via a myosin II-dependent mechanism.

Myelin regulates immune cell adhesion and motility.

The etiology of multiple sclerosis (MS) has not been fully elucidated, however evidence supports an autoimmune disease model notable for the infiltration of pro-inflammatory immune cells into sites of active demyelination and axonal injury. Previous findings demonstrate that neutralization of Nogo, a protein originally identified as a myelin-associated inhibitor (MAI) of axon regeneration, ameliorates experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. More efficient axonal regeneration was suggested as a mechanism underlying the improved EAE outcome. However, neutralization of Nogo also led to an anti-inflammatory shift of T cell cytokines during EAE suggesting that another therapeutic mechanism may involve regulation of immune cell responses. Here we report that human immune cells from healthy individuals and MS patients express Nogo receptor1 (NgR1) indicating that they may be subject to regulation by MAIs. B cells, T cells and monocytes express NgR1 in a regulated fashion upon activation. While direct stimulation of human immune cells with an inhibitory fragment of Nogo does not impact their in vitro proliferation or cytokine production, the immune cells display reduced adhesion and enhanced motility in response to myelin, effects that are in part attenuated by antagonizing NgR1 signaling. We conclude that NgR1 alters the motility of immune cells exposed to myelin and may thus impact their behaviour within the CNS, particularly under conditions when immune cell activation is heightened.

Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity along the antero-posterior axis.

The antero-posterior (AP) and dorso-ventral (DV) patterning of the neural tube is controlled in part by HOX and PAX transcription factors, respectively. We have reported on a neural enhancer of Hoxd4 that directs expression in the CNS with the correct anterior border in the hindbrain. Comparison to the orthologous enhancer of zebrafish revealed seven conserved footprints including an obligatory retinoic acid response element (RARE), and adjacent sites D, E and F. Whereas enhancer function in the embryonic CNS is destroyed by separation of the RARE from sites D-E-F by a half turn of DNA, it is rescued by one full turn, suggesting stereospecific constraints between DNA-bound retinoid receptors and the factor(s) recognizing sites D-E-F. Alterations in the DV trajectory of the Hoxd4 anterior expression border following mutation of site D or E implicated transcriptional regulators active across the DV axis. We show that PAX6 specifically binds sites D and E in vitro, and use chromatin immunoprecipitation to demonstrate recruitment of PAX6 to the Hoxd4 neural enhancer in mouse embryos. Hoxd4 expression throughout the CNS is reduced in Pax6 mutant Sey(Neu) animals on embryonic day 8. Additionally, stage-matched zebrafish embryos having decreased pax6a and/or pax6b activity display malformed rhombomere boundaries and an anteriorized hoxd4a expression border. These results reveal an evolutionarily conserved role for Pax6 in AP-restricted expression of vertebrate Hoxd4 orthologs.

MEIS C termini harbor transcriptional activation domains that respond to cell signaling.

MEIS proteins form heteromeric DNA-binding complexes with PBX monomers and PBX.HOX heterodimers. We have shown previously that transcriptional activation by PBX.HOX is augmented by either protein kinase A (PKA) or the histone deacetylase inhibitor trichostatin A (TSA). To examine the contribution of MEIS proteins to this response, we used the chromatin immunoprecipitation assay to show that MEIS1 in addition to PBX1, HOXA1, and HOXB1 was recruited to a known PBX.HOX target, the Hoxb1 autoregulatory element following Hoxb1 transcriptional activation in P19 cells. Subsequent to TSA treatment, MEIS1 recruitment lagged behind that of HOX and PBX partners. MEIS1A also enhanced the transcriptional activation of a reporter construct bearing the Hoxb1 autoregulatory element after treatment with TSA. The MEIS1 homeodomain and protein-protein interaction with PBX contributed to this activity. We further mapped TSA-responsive and CREB-binding protein-dependent PKA-responsive transactivation domains to the MEIS1A and MEIS1B C termini. Fine mutation of the 56-residue MEIS1A C terminus revealed four discrete regions required for transcriptional activation function. All of the mutations impairing the response to TSA likewise reduced activation by PKA, implying a common mechanistic basis. C-terminal deletion of MEIS1 impaired transactivation without disrupting DNA binding or complex formation with HOX and PBX. Despite sequence similarity to MEIS and a shared ability to form heteromeric complexes with PBX and HOX partners, the PREP1 C terminus does not respond to TSA or PKA. Thus, MEIS C termini possess transcriptional regulatory domains that respond to cell signaling and confer functional differences between MEIS and PREP proteins.

Subcellular localization of multiple PREP2 isoforms is regulated by actin, tubulin, and nuclear export.

The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems.

Expression of the Wdr9 gene and protein products during mouse development.

Human WDR9 has been mapped to chromosome 21, within one of the Down syndrome (DS) critical regions. Here, we study the expression pattern of the murine Wdr9 gene and its protein product. We show that Wdr9 is broadly expressed in the mouse embryo by means of in situ hybridization and immunohistochemistry. Wdr9 expression levels are dynamic during embryonic development as revealed by Northern blot analysis. We further show that WDR9 is a nuclear protein associated with BRG1, a SWI/SNF complex component. We also demonstrate that a polyglutamine-containing region of the protein functions as a transcriptional activation domain. We propose that WDR9 is a transcriptional regulator involved in chromatin remodeling through the action of two bromodomains and contacts to the SWI/SNF complex. These results may provide a molecular basis for the association of WDR9 with DS.

Prep2: cloning and expression of a new prep family member.

We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes.