Faculty Bibliography

P0 glycoprotein is an abundant product of terminal differentiation in myelinating Schwann cells. The mutant P0S63del causes Charcot-Marie-Tooth 1B neuropathy in humans, and a very similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum of Schwann cells, where it promotes unfolded protein stress and elicits an unfolded protein response (UPR) associated with translational attenuation. Ablation of Chop, a UPR mediator, from S63del mice completely rescues their motor deficit and reduces active demyelination by half. Here, we show that Gadd34 is a detrimental effector of CHOP that reactivates translation too aggressively in myelinating Schwann cells. Genetic or pharmacological limitation of Gadd34 function moderates translational reactivation, improves myelination in S63del nerves, and reduces accumulation of P0S63del in the ER. Resetting translational homeostasis may provide a therapeutic strategy in tissues impaired by misfolded proteins that are synthesized during terminal differentiation.

There is compelling evidence that oligodendrocyte apoptosis, in response to CNS inflammation, contributes significantly to the development of the demyelinating disorder multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Therefore, approaches designed to protect oligodendrocytes would likely have therapeutic value. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum (ER) stress increases cell survival under various cytotoxic conditions. Moreover, there is evidence that PERK signaling is activated in oligodendrocytes within demyelinating lesions in multiple sclerosis and EAE. Our previous study demonstrated that CNS delivery of the inflammatory cytokine interferon-gamma before EAE onset protected mice against EAE, and this protection was dependent on PERK signaling. In our current study, we sought to elucidate the role of PERK signaling in oligodendrocytes during EAE. We generated transgenic mice that allow for temporally controlled activation of PERK signaling, in the absence of ER stress, specifically in oligodendrocytes. We demonstrated that persistent activation of PERK signaling was not deleterious to oligodendrocyte viability or the myelin of adult animals. Importantly, we found that enhanced activation of PERK signaling specifically in oligodendrocytes significantly attenuated EAE disease severity, which was associated with reduced oligodendrocyte apoptosis, demyelination, and axonal degeneration. This effect was not the result of an altered degree of the inflammatory response in EAE mice. Our results provide direct evidence that activation of PERK signaling in oligodendrocytes is cytoprotective, protecting mice against EAE.

The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2-960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2-960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2-960), and homology modeling. Evolutionary analyses using the ScNup192(2-960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.

Animals across various phyla exhibit odor-evoked innate attraction behavior that is developmentally programmed. The mechanism underlying such behavior remains unclear because the odorants that elicit robust attraction responses and the neuronal circuits that mediate this behavior have not been identified. Here, we describe a functionally segregated population of olfactory sensory neurons (OSNs) and projection neurons (PNs) in Drosophila melanogaster that are highly specific to ammonia and amines, which act as potent attractants. The OSNs express IR92a, a member of the chemosensory ionotropic receptor (IR) family and project to a pair of glomeruli in the antennal lobe, termed VM1. In vivo calcium-imaging experiments showed that the OSNs and PNs innervating VM1 were activated by ammonia and amines but not by nonamine odorants. Flies in which the IR92a(+) neurons or IR92a gene was inactivated had impaired amine-evoked physiological and behavioral responses. Tracing neuronal pathways to higher brain centers showed that VM1-PN axonal projections within the lateral horn are topographically segregated from those of V-PN and DC4-PN, which mediate innate avoidance behavior to carbon dioxide and acidity, respectively, suggesting that these sensory stimuli of opposing valence are represented in spatially distinct neuroanatomic loci within the lateral horn. These experiments identified the neurons and their cognate receptor for amine detection, and mapped amine attractive olfactory inputs to higher brain centers. This labeled-line mode of amine coding appears to be hardwired to attraction behavior.

Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure-function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts.

SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.

PROBLEM: Harm reduction packages for people who inject illicit drugs, including those infected with human immunodeficiency virus (HIV), are cost-effective but have not been scaled up globally. In the north-eastern Indian states of Manipur and Nagaland, the epidemic of HIV infection is driven by the injection of illicit drugs, especially opioids. These states needed to scale up harm reduction programmes but faced difficulty doing so. APPROACH: In 2004, the Bill & Melinda Gates Foundation funded Project ORCHID to scale up a harm reduction programme in Manipur and Nagaland. LOCAL SETTING: In 2003, an estimated 10 000 and 16 000 people were injecting drugs in Manipur and Nagaland, respectively. The prevalence of HIV infection among people injecting drugs was 24.5% in Manipur and 8.4% in Nagaland. RELEVANT CHANGES: By 2012, the harm reduction programme had been scaled up to an average of 9011 monthly contacts outside clinics (80% of target); an average of 1709 monthly clinic visits (15% of target, well above the 5% monthly goal) and an average monthly distribution of needles and syringes of 16 each per programme participant. Opioid agonist maintenance treatment coverage was 13.7% and retention 6 months after enrolment was 63%. Antiretroviral treatment coverage for HIV-positive participants was 81%. LESSONS LEARNT: A harm reduction model consisting of community-owned, locally relevant innovations and business approaches can result in good harm reduction programme scale-up and influence harm reduction policy. Project ORCHID has influenced national harm reduction policy in India and contributed to the development of harm reduction guidelines.

G protein-coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering of native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized mGluRs (LimGluRs). The light-agonized LimGluR2, on which we focused, was fast, bistable and supported multiple rounds of on/off switching. Light gated two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. We found that the light-antagonized tool LimGluR2-block was able to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalized the optical control to two additional family members: mGluR3 and mGluR6. This system worked in rodent brain slices and in zebrafish in vivo, where we found that mGluR2 modulated the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.