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“Best” Posters from 2011-2009 SURP Poster Sessions Faulty Wiring: A Mutant With Disrupted Electrical Synapse Development Lisa Voelker, Adam Miller, Cecilia Moens Funded by: Fred Hutchinson Cancer Research Center, Seattle, WA Investigating the Disruption of Electrical Synapses in Dis2 Mutants Introduction Are Only M/CoLo Synapses Affected? Do Mauthner and CoLo develop the correct identity? Electrical synapses are also found on the M dendrite and cell body, as well as on other interneurons further posterior in the hindbrain WT Mut Dorsal Dorsal 3A-10 as a Marker of M Cell Fate Are the Number of CoLos Correctly Specified? Sensory perception, behavior, and even consciousness are generated by neural circuits. Such circuits are organized by the patterns and properties of their synapses, sites of adhesion and communication between neurons. The development of functional neural circuits involves cells developing the correct identity, guiding their processes (axons and dendrites) to the proper locale, and recognizing the appropriate targets with which to form synapses. Presence of CoLos 2011 Lee Hartwell Poster Award 2.0 Chemical and Electrical Synapses 1.5 1.0 Presynaptic Processes 0.5 0.0 Average CoLos per Somite The presence of approximately two CoLos in each somite (body segment) is typical of normal nervous system development 3A-10 Staining of M cell in 1 day old embryos M cell body 75 3A-10 stains neurofilaments, selectively marking M cells in the hindbrain during early development At one day it indicates normal identity formation CoLo cell bodies Dorsal 3A-10 staining No 3A-10 staining 50 M cell body other interneurons M cell body other interneurons 25 Dorsal Mut WT (n=18) (n=7) Actual (n=21) GFP 0 Expected if Dis2 confuses cell fate Chemical Synapse Electrical Synapse Sites of Cx35 puncta Lack of Cx35 puncta GFP Cx35 RMO44 Cx35 GFP Cx35 RMO44 Cx35 No, electrical synapses in the hindbrain are disrupted. Postsynaptic Processes Neurons communicate through chemical synapses, where they release neurotransmitters onto their partners, or electrical synapses, where electrical potential passes directly from one cell to another. While electrical synapses are known to be essential for development, little is known about their formation and maintenance. The Mauthner/CoLo Circuit Governs Auditory Evoked Fast Startle Response Turn toward stimulus inhibited by CoLo Excitatory Synapse Excitatory Synapse Inhibitory Synapse Propagation of Action Potential Acoustico/Vestibular input from ear Muscle Mauthner 147 CoLo Electrical Synapse Results in a stereotyped turn away from the aversive stimulus Is Connexin 35 the only gap junction protein missing? Using the larval zebrafish Mauthner (M) circuit, we have identified a mutation, termed Disconnect2 (Dis2), that disrupts electrical synapses between M and the Commissural Local interneurons (CoLos) in the spinal cord. My project this summer has been to understand why electrical synapses fail to form correctly in Dis2 mutant larvae and whether the defects lead to functional problems in the M-mediated escape response. In Dis2 Mutants the Electrical Synapse Protein Connexin 35 (Cx35) is no Longer Visible at M/CoLo Axon Crossings Dorsal ZO-1 Puncta at M/CoLo Axon Crossing Sites Colocalizaton of ZO-1 with Cx35 Puncta ZO-1 Puncta at M/CoLo axon crossings Lateral WT Colocalization of ZO-1 with Cx35 puncta Lateral WT GFP marks the M and CoLo cells Cx35 is a gap-junction protein present at electrical synapses RMO44 is a general marker of neurons Wild Type (WT) Mutant (Mut) Cx35 ZO-1 Cx35 ZO-1 Frequency of Phenotypes in Mutant Carrier Pair Crossings 75 75 GFP 100 50 GFP Cx35 RMO44 50 Cx35 75 25 ZO-1 25 % of population 100 % of population 100 % of population 100 M axon Yes, cells appear correctly fated. M cell body 3A-10 Are there behavioral consequences of the mutation? Do M and CoLo Guide Their Axons Correctly? Fast Escape Responses to Sound Closeups of the M/CoLo Axon Crossing Sites from Various Angles An auditory stimulus known to evoke an M-mediated startle responses was played to five day old larvae and subsequent behavior was recorded Larvae were then sorted by response prevalence and stained to determine Dis2 phenotype Mut Lateral Dorsal Xsec WT Lateral Dorsal Xsec In Dis2 mutants, M and CoLo axons still appear to come close to each other, indicating axon guidance signalling is not affected GFP Cx35 RMO44 Larval Escape Responses WT (n=117) Mut (n=47) 100 Cx35 75 50 Cx35 puncta Lack of Cx35 puncta GFP 25 % from each phenotype (wt or mut) 0 Yes, M and CoLo axons come into close proximity. High Med Low Each shape represents responses within a mutant carrier pair mating Yes, mutation correlates with reduced responsiveness. ZO-1 is an intracellular scaffolding protein that colocalizes with Cx35 at many electrical synapses Overall Results and Conclusions 50 ZO-1 puncta (n=15) 0 no ZO-1 puncta (n=5) Lateral Mut % larvae in population Lateral Cx35 25 In studying the Dis2 mutant, we have confirmed that the loss of Connexin 35 puncta... ...Is not as a result of incorrect M or CoLo cell fate development ...Is not as a result of faulty axon guidance ...Is not limited to Cx35 ...Is not limited to the M/CoLo junctions Which suggests the mutation affects electrical synapse building machinery. Additionally, disruption of electrical synapses correlated with reduced startle response Indicating properly functioning electrical synapses are required for normal behavior. 0 ZO-1 Future Directions Cx35 ZO-1 ZO-1 and Cx35 puncta (n=15) no ZO-1 or Cx35 puncta (n=5) Lateral Mut Cx35 ZO-1 GFP WT Mut Finer exploration of M/CoLo junctions with electron microscopy. Investigation of other sites of possible electrical synapse disruption (retina). Behavioral defects may not be limited to startle response, possible disruptions of general activity levels and time of emergence of other stereotypical behaviors (twitches, scoots, etc). What are the genetic and molecular bases of the mutation? Mapping and cloning the gene will give insight as to the molecular mechanisms underlying nervous system wiring. Sites of ZO-1 puncta Lack of ZO-1 puncta ZO-1 Cx35 0 No Cx35 Cx35 puncta puncta (Mut) (WT) (n=59) (n=160) An occurence of the mutant phenotype in approximately 25% of the population is indicative of a recessive genetic trait ZO-1/Cx35 puncta Lack of ZO-1/Cx35 puncta GFP Cx35 RMO44 ZO-1 No, mutation also affects ZO-1. Cx35 puncta Lack of Cx35 puncta Cx35
Analyzing the Antibody Response against H-Y Antigens Nathan Ord1 ; Takazu Kawase, M.D., Ph.D2 ; Edus Warren, M.D., Ph.D. 2 1 Whitman College, Walla Walla, WA; 2 Fred Hutchinson Cancer Research Center, Seattle, WA Results Abstract Background 2011 Lee Hartwell Poster Award XY Female to male (F→M) hematopoietic stem cell transplant (HSCT) exhibits: - Increased GVHD and GVL due to donor T-cell targeting mHAs - Recently demonstrated antibody against H-Y antigens (Miklos et al.) Hypothesis: KDM5D is the most important T-cell H-Y antigen and it will be highly immunogenic for B-cells as well To test this we obtained blood plasma from: - 39 HSCT F→M transplant patients - 16 HSCT patients with other donor/recipient gender combinations - 12 healthy female donors - 9 healthy male donors We tested for IgG antibody against: - 36 overlapping pentadecapeptide fragments representing residues 197-383 of KDM5D, peptides for a unique region of isoform 1, and several X-homologue peptides - Recombinant proteins DDX3Y, UTY, ZFY RPS4Y and E1F1AY, their X-homologues, and KDM5D-f (the one fragment currently purified) comprising residues 1320 to 1570 We found: - 15 of 39 (38%) F→M HSCT samples were reactive with ≥1 KDM5D peptide while 7 of 39 (18%) samples reacted with ≥3 KDM5D peptides. - 9 of 16 (56%) samples from other donor/recipient gender combinations were reactive with ≥1 KDM5D peptide and 2 of 16 (13%) were reactive with ≥3 KDM5D peptides. - Peptide specific results suggest that a potential target region for H-Y antibody exists around KDM5D281-375 - Strong antibody responses to KDM5D19 (VSSTLLKQHL--------SLEPC) were only observed in male recipients of female grafts (7 of 39) and responses to peptide 32 were also primarily observed in F→M transplant patients. - Recombinant protein responses were detected most frequently for UTY, followed by KDM5D-f. - Antibodies to UTY were observed in 11 of 39 (28%) F→M patients and in 1 of 16 (6%) patients with other donor/recipient gender combinations. Hematopoietic Stem Cell Transplant Donor Stem Cells GVHD No Rxn GVL The Bone marrow transplant replaces the blood forming cells of the patient with those of the donor. This reconstitutes the patient’s immune system and the engrafted T-cells interact with the host. Immune Cell Recognition 148 DNA polymorphisms result in differences in host/donor proteins, the donor immune system may recognize host peptide fragments displayed by MHC molecules as foreign resulting GVH or GVL effect against these minor histocompatibility antigens (mHAs). Minor Histocompatibility Antigens and the H-Y antigen system : XX XY Experimental Approach Coat 96 well plates with protein (protein fragments or peptides) in coating buffer Add plasma sample. If specific antibody exists in the plasma, the antibody will bind to the protein Add secondary antibody (anti human IgG antibody) conjugated with AP Add color development buffer and measure absorbance at 405 nm The female to male transplant scenario is a unique opportunity to study mHAs and their clinical significance because it restricts the search to just the Y-chromosome. Additionally, the gene must be expressed widely outside the testes, have a similar but non-identical X homolog, escape X inactivation, and reside outside the ampliconic region. Conclusions and Further Directions But what about the B cells? Conclusions 1. Results suggest that KDM5D is an important target for post-transplant antibody 2. There appears to be an antibody binding cluster near KDM5D281-375 3. The antibody response to H-Y antigens, particularly DDX3Y may not be as strong as previously suggested Further Directions 1. Assay more samples. It is interesting that the healthy samples exhibit high antibody responses, does everyone have similar antibody? 2. Does Rituximab benefit patients with ≥1 H-Y antibody response? 3. And what about the rest of SMCY? Currently we are synthesizing the full recombinant protein, does an antibody epitope reside outside the region we assayed? 4. How much antibody is present? Conduct sequential dilution to determine the antibody titer Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y YY Y Y Y YY Y Y YY Y Y GVHD No Rxn ? Acknowledgements and References GVL I would like thank Hootie and everyone in the Warren lab; David Miklos for supplying recombinant protein; Jim Russo, Doug Juers, and Marilyn Gotz for helping me here; Jennifer Anderson, Jorden Canas, and the countless people who supported the internship program; and the Howard Hughes Medical Institute and Whitman College along with Fred Hutchinson Cancer Research Center for sponsoring this opportunity. Miklos, D et al. 2004. “Antibody response to DBY minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors.” Blood. 103: 353- 359 Miklos, D et al. 2005. “Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission.” Blood. 105: 2973-2978 Murphy, K et al. Janeway’s Immuno Biology 7th Edition. New York: Garland Science, 2008. Although T-cells are the most relevant to both GVL and GVH, the efficacy of Rituximab in GVHD (a monoclonal CD20 antibody specific for B cells) and accumulating evidence has shown the presence and significance of antibodies in the H-Y transplant setting
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