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POSTER #40:
Phosphorylation of Rpt6 by CaMKII is responsible for localization of 26S proteasome at synapses
Carissa Chu
Dr. Gentry Patrick
The ubiquitin-proteasome system (UPS) is the main eukaryotic pathway for protein degradation. In neurons, degradation of synaptic proteins by the 26S proteasome is important for activity-induced plasticity of synaptic connections. In this study, we investigate the role of calmodulin-dependent kinase II (CaMKII) in regulating activity of the proteasome. CaMKII is a calcium-dependent protein kinase highly expressed in neurons, and it plays a central role in synaptic plasticity. Previous work in our lab determined that CaMKII regulates the function of the proteasome by phosphorylating Rpt6, a subunit of the 19S regulatory cap. Here, we show that CaMKII regulates proteasome function by increasing its distribution at synapses. Overexpression of a constitutively active CaMKII induces robust accumulation of Rpt6 along dendrites and at dendritic spines. This observation is consistent with our previous studies, which show increased proteasome trafficking in response to NMDA receptor activation and calcium influx. Using site-directed mutagenesis and immunofluorescence imaging, we also found that the Ser 120 residue on Rpt6, a target phosphorylation site for CaMKII, is involved in induced trafficking of proteasomes to dendritic spines. We compared the effects of phospho-mimicking (S120D) and phospho-dead (S120A) Rpt6 point mutants on Rpt6 localization in neurons. Preliminary data show a significant increase in proteasome concentration at synapses in neurons expressing phospho-mimicking (S120D) Rpt6, and no significant change in trafficking in neurons expressing phospho-dead (S120A) Rpt6. As a follow-up study, we will use time-lapse imaging to determine the effects of Rpt6 phospho-mutations on trafficking into dendritic spines upon stimulation. Altogether, our data suggest that CaMKII phosphorylation at Ser120 on the Rpt6 subunit is a key regulated step in activity-dependent protein degradation in neurons.
POSTER #41:
Characterization of Tagged Gap Junctional Proteins Innexin in the CNS of the Medicinal Leech
Constante Firme
Dr. Eduardo Macagno
Gap junctions (GJs) are channels between cells composed of either 12 innexin (invertebrate) or 12 connexin (vertebrate) subunit proteins. In each cell, subunits form hexameric structures that dock with a corresponding set from a different cell to form a pore that allows molecular signals to pass between the cells' cytoplasms. Non-coupled innexin hexamers, known as hemichannels, have been found to exist in some tissues, but little is known about their function. To get a better understanding of innexin hemichannel function, we have been studying the expression patterns of Inx 1, Inx2, and Inx6 in CNS neurons in the medicinal leech. Non-endogenous gap junctional proteins can be expressed in a neuron by transfection with plasmids coding for an enhanced green fluorescent protein (EGFP)tagged Innexin protein, so localization patterns can be mapped by fluorescent micro-imaging. Our preliminary data, from experiments in which the plasmids were delivered to neuronal nuclei on gold carrier particles shot with a miniature gene gun, suggest that neuronal expression of Inx2, a GJ protein that is normally is only expressed in the CNS by macroglia, is toxic to neurons, whereas Inx1 and Inx6 have no apparent effect on cellular morphology. To rule out the possibility that the toxic effects are due to the EGFP tag, a much smaller polyhistidine (HA) tag was used, and the results showed a similar neuronal pathology. These observations lead us to the hypothesis that Inx2 expressed in neurons may form leaky hemi-channels, resulting in the loss of cytoplasmic contents and cell death. Future molecular and electrophysiological experiments will test this hypothesis.
POSTER #42:
Determining the Composition and Regulation of ACR-2 Acetylcholine Receptor of C. elegans
Hannah Kang
Dr. Yishi Jin
Hannah Kang, Tamara Stawicki & Yishi Jin In our lab we use the nematode Caenorhabditis elegans as a model organism to investigate acetylcholine receptor regulation and function. Interestingly C. elegans contain twenty-seven acetylcholine receptor subunit genes compared to only seventeen such genes in vertebrates. Of those twenty-seven genes the best characterized are the muscle receptors. The C. elegans muscle acetylcholine receptors (AChRs) are the “LEV-UNC” and ACR-16 receptors. The LEV-UNC muscle receptor is a heteromeric receptor that is composed of five subunits: UNC-63, UNC-38, UNC-29, LEV-1, and LEV-8. Mutations in these receptor subunits genes cause the worms to exhibit a slow movement phenotype referred to as uncoordinated, or UNC, and resistance to the drug levamisole. On the other hand, the ACR-16 receptor is a homomeric receptor with no obvious phenotype when mutated. Although the C. elegans muscle AChRs are well characterized relatively little is known about the remaining AChR genes, most of which are believed to be expressed in neurons.. It has previously been shown that worms with a loss of GABA motor neuron function show a “shrinker” phenotype where they contract their body or “shrink” in response to touch. In a screen for additional GABAergic mutants we found worms with a unique phenotype, spontaneous shrinking. Mapping identified these worms as containing a mutation in the acr-2 gene. These acr-2(n2420) worms have a mutation that changes Valine to Methionine, 2nd transmembrane domain the pore lining TM2 domain. Previous studies have shown that mutations such as acr-2(n2420) lead to an overactive channel that increases ion flow into the cell. To better understand the regulation of the ACR-2 receptor we performed a suppressor screen. Genes identified in the screen include the acetylcholine receptor subunits acr-12, unc-38 and unc-63 as well as the trafficking proteins unc-50 and unc-74. Since the LEV-UNC muscle receptor also possess UNC-38 and UNC-63 subunits, we wanted to know if they were suppressing the shrinking because they are working in the muscle by receiving a chemical signal or acting in the neuron as coreceptors with ACR-2. Cell specific rescue shows that UNC-63 is functioning in the neuron. Interestingly, the suppressor screen turned up mutations in genes shared between the LEV-UNC muscle receptor and ACR-2R that eliminate shrinking while not showing the levamisole resistance and unc locomotion typically seen in LEV-UNC mutants. To determine the function, these subunit mutations in the ACR-2 receptor, pharmacology experiments were performed. The unc-50 (n2624) mutation is a weak allele of a trafficking protein that regulates trafficking from the golgi apparatus to the plasma membrane. We were curious if our other LEV-UNC weak alleles similarly effect trafficking. Consequently, we made several double mutants with unc-50(n2624) that are in the process of testing their resistance to levamisole and aldicarb looking for any double mutants that showed additive resistance. In the future we plan to look at surface labeling to further determine if the mutations effect trafficking and to perform recordings to observe channel properties.
POSTER #43:
Disrupted in Schizophrenia 1 and DumPY-19: Two Important Genes in Neuronal Development
Alex Ohlendorf
Dr. Yimin Zou
The Disrupted in Schizophrenia 1 (Disc1) gene was identified by linkage to schizophrenia and other major mental illnesses in a large Scottish pedigree. Disc1 is disrupted by a translocation between chromosomes 1 and 11, resulting in a truncated protein. The function of Disc1 in the cell is unknown, but it has been shown be involved with neurite outgrowth and neuronal migration. Binding partners of Disc1 include fasciculation and elongation protein zeta-1 (FEZ1), which responds to downstream signaling by a family of axon guidance molecules known as Wnts. To examine a possible role of Disc1 in axon guidance, this report addresses subcellular localization of Disc1. Dpy-19 is a gene known to be important for the migration of Q neuroblasts in C. elegans. Previous work on dpy-19 uncovered a mouse homologue, and showed that dpy-19 is important for neuronal cell migration in the mouse cortex. By causing in vivo knockdown of mouse dpy-19 expression, we examine the function of this gene in cortical neuron migration and morphology, and have found a significant phenotype.
POSTER # 44:
Quantitative PCR Measurements Reveal Increased Expression of Receptor Phosphatases during Neuroregeneration
Jasmine Sethi
Dr. Eduardo Macagno
In the medicinal leech, Hirudo medicinalis, the RPTPs HmLAR1 and HmLAR2 are abundantly expressed among central neurons during development. Various observations indicate that they play important roles in the development of the nervous system. Here, we have begun to explore whether these RPTPs might also be involved in adult neuronal regeneration. Selective lesions were administered to nerves in the CNS of adult leeches and the expression levels of the RPTPs between lesion and sham-operated ganglia were compared. Expression in dissected adult ganglia kept in tissue culture for 24 hours and 48 hours was also examined. The technique used to monitor gene expression, Q-PCR, was used to assay RPTP expression levels across the different experimental conditions and time courses, using several housekeeping genes (Actins, GAPDH, NADPH) to establish a baseline. In these experiments cDNA was synthesized from individual CNS ganglia. The measurements show that there is an increase in the levels of both HmLAR1 and HmLAR2 in the CNS as a result of either lesioning or culturing. We propose that mechanical trauma induces up-regulation of these RPTPs because they are involved in neuronal repair, a hypothesis we are planning to test by blocking their expression using RNAi.