Abstract List for 2013 meeting

#206: The Role for Shank3 in NMDAR-Mediated Deficits in Autism

Duffney, Lara J ¹; Wei, Jing ¹; Cheng, Jia ¹; Liu, Wenhua ¹; Smith, Katharine R²; Kittler, Josef T²; Yan, Zhen ¹;

¹Department of Physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Department of Neuroscience, University College London, London, UK;

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. Our work examines the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illness. Knockdown of Shank3 with a small interfering RNA (siRNA) causes a significant reduction of NMDAR-mediated ionic or synaptic current, as well as surface expression of NR1 subunits, in rat cortical cultures. Electrophysiological and immunochemical data, using various activators or inhibitors of molecules involved in regulation and stability of the actin cytoskeleton, suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with Rac1/PAK/cofilin/actin signaling. This is believed to lead to the loss of NMDAR membrane delivery or stability. These results have been further verified in a Shank3-deficient mouse model. Treatment in vivo with phosphorylated cofilin, a peptide that acts to stabilize actin, is able to recover social deficits and decreased NMDAR response present in Shank3 knockdown mice, while injection of an inhibitory PAK peptide acts to induce social and physiological deficits in wild type mice. This data provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

#198: Endogenous glucagon-like peptide-1 affects fluid intake in rats

McKay, Naomi J ; Daniels, Derek ;

Department of Psychology, SUNY University at Buffalo;

Glucagon-like peptide-1 (GLP-1) plays an important role in energy homeostasis. Injections of GLP-1 receptor (GLP-1R) agonists suppress food intake, and endogenous GLP-1 production and release are stimulated by the presence of nutrients in the gut. In addition to the established effects on energy balance, there is growing evidence that the GLP-1 system plays a role in maintaining body fluid homeostasis. Injection of GLP-1R agonists suppresses water and saline intake, but, the involvement of endogenous GLP-1 remains to be determined. To address this open question, we first tested whether blocking GLP-1 signaling would affect overnight water intake. To this end, rats were given a lateral ventricle (LV) injection of either the GLP-1R antagonist exendin (9-39) (Ex-9; 20 μg) or vehicle (1 μl 0.9% saline), food was removed, and overnight water intake was measured. Ex-9 injection was associated with more licking for water during the first 3 hr after injection (p<0.05). Next we used two approaches, injection of hypertonic saline or overnight water deprivation, to test if acutely stimulated drinking was affected by endogenous GLP-1. In each experiment, rats received either Ex-9 (100 μg) or vehicle (1 μl 0.9% saline) in the LV and fluid intake was measured. Regardless of the drinking stimulus, rats given an injection of Ex-9 drank more water or saline than did controls (p<0.05). Furthermore, analysis of the licking patterns during the testing periods found that the number of licking bursts were greater (p<0.05) in rats that received Ex-9 than in rats given vehicle, but burst size was not affected by the treatment condition (p=ns). Collectively, these results indicate that endogenous GLP-1 is part of the circuitry regulating drinking behavior and that it decreases fluid intake by enhancing post-ingestive feedback rather than by affecting orosensory feedback. Moreover, these data add to the growing evidence that the effect of GLP-1 is not specific to food intake.

#207: Activity-dependent regulation of the probability of neurotransmitter release at the endbulb of Held

Ngodup, Tenzin ; Xu-Friedman, Matthew A ;

Department of Biological Sciences, SUNY University at Buffalo, College of Arts and Sciences;

Different synapses vary in the probability of neurotransmitter release (P_r). At an individual synapse P_r is tightly regulated as it is crucial for the faithful transfer of information between neurons. However, it is unknown what the underlying mechanisms are for setting up of P_r. We hypothesized that neural activity could be an important factor in setting P_r. The role of activity in the development and plasticity of various higher sensory cortices has been well established. In the auditory system, acoustic trauma disrupts neuronal properties in the inferior colliculus and AI region. It could also lead to hearing loss and tinnitus. However, the role of sensory experience on the lower auditory nuclei is not well understood. We studied the effects of noise exposure on synaptic strength at the endbulb of Held. Endbulbs show strong depression, indicative of high P_r, but by rearing animals under constant, non-damaging noise, endbulbs change to low P_r. Surprisingly, there were no changes in the amplitude of first EPSC and quantal size (Q), suggesting the number of release sites (N) increases. Noise-reared bushy cells (BC) do not show significant morphological changes. Some animals were allowed to recover for 2-4 weeks following noise exposure. In this situation, P_r reverts from low to high, indicating the change is adaptive. Furthermore, noise-reared bushy cells have higher probability of spiking. These data indicate that even lower nuclei in the auditory pathway also undergo significant changes in physiology depending on sensory experience. Our results suggest that experience-dependent activity is a crucial factor in regulating P_r.

#196: Isolation of novel molecules involved in Schwann cell/axon recognition.

Poitelon, Yannick ¹; Wrabetz, Lawrence ¹; Bachi, Angela ²; Feltri, M. Laura ¹;

¹Hunter James Kelly Research Institute, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Division of Genetics and Cell Biology, Ospedale San Raffaele, Milan, Italy;

Schwann cells are the peripheral myelinating glia and provide mechanical protection, electric insulation and metabolic support to the axons. Defects in Schwann cells cause several acquired and inherited demyelinating neuropathies with important prevalence and disability. Schwann cell development and function strictly depend on contact with associated axons. Defects in the ability of glial cells to interact with axons during myelination or support them during regeneration and remyelination are at the origin of several of the aforementioned disorders. Thus, understanding the molecular mechanisms of axo-glial interactions is a high priority, yet only a few molecules involved in establishing and maintain axon-glia contact are known. To discover novel molecules in axo-glial interactions, we isolated the Schwann cell surface in contact with axons. To do this we adapted a widely used sub-fractionation method based on a modified Boyden chamber with a microporous filter, which allows us to isolate Schwann cell protrusions (“pseudopods”) formed in response to neuronal membranes. Schwann cells are placed in the top chamber while neuronal cell membranes are in the bottom to induce Schwann cell pseudopodial growth. Schwann cell proteins or RNA can be isolated from the top chamber where the cell bodies remain and from the bottom chamber where the polarized pseudopodia are growing. Both compartments have been analyzed by western blot and mass spectrometry and contain both known molecules appropriate to either compartment and novel molecules that when perturbed impair myelination. These include not only cytoplasmic but also mitochondrial proteins, raising the possibility that Schwann cell mitochondria are required for axo-glial interactions. These studies will identify new molecules important for normal Schwann cell-axon interactions and potentially relevant to demyelinating disorders.

#208: Transcription factor induction of human oligodendrocyte progenitors

Wang, Jing ¹; Pol, Suyog U ²; Keller, Alexa ¹; Wang, Chunming ¹; O'Bara, Melanie A ¹; Sim, Fraser J ¹;

¹Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Biomedical engineering, SUNY University at Buffalo;

Cell based therapies for demyelinating diseases require efficient generation of functional oligodendrocyte progenitors (OPCs) from human stem cells. However, such induction is hindered by poor understanding of the transcriptional regulation for human OPC fate determination. To directly address this question, we did genomic analysis which revealed that a discrete set of transcription factors (TFs) were differentially expressed by native human OPCs relative to neural progenitors(NPCs) (n=3-6). In this study, we asked if individual TF was sufficient to induce OPC fate from primary human NPCs. First, to determine if TFs could activate an OPC-specific enhancer, we infected freshly isolated NPCs with each TF with the Sox10-MCS5: GFP reporter. 3 of 8 candidate factors tested induced high levels of the OPC-specific enhancer activity 4 days post infection (n=4-9). Furthermore, 4 factors significantly increased OPC markers expression at day 7, suggesting OPC fate induction (n=3-5). When the genomic profile of those induced OPCs were examined, only SOX10 induced global changes that resembled the genomic signature of primary OPCs. Following growth factor withdrawal, enforced SOX10 expression resulted in a >10 fold increase in the production of O4⁺ immature and MBP⁺ mature oligodendrocytes (OLs) in vitro (n=3). To test if SOX10 overexpression was sufficient to induce myelinating OLs, we transplanted infected NPCs into hypomyelinating shiverer/rag2 mice. Compared with control NPCs, SOX10 significantly increased donor-derived CC1⁺ OLs in corpus callosum at 12 weeks and improved axonal myelination by mature MBP⁺ OLs (n=3). Such potential OL fate induction occurred at the cost of astrocytes as evidenced by >50% reduced GFAP⁺ cells in SOX10 group (n=3). No significant difference of nestin⁺ human NPCs or NG2⁺ OPCs was observed between groups. Thus, SOX10 was rate-limiting during OL lineage fate determination in human NPCs and is critical for reprogramming functional human OPCs.

#231: Withdrawal periods following extended access cocaine SA doesn't alter drug intake during a binge.

Adank, Danielle N ¹; Gancarz-Kausch, Amy M ²; Braunscheidel, Kevin M ¹; Pugliese, Jacob M ¹; Dietz, David M ³;

¹Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Research Institute on Addictions, ³Department of Pharmacology and Toxicology, SUNY University at Buffalo;

Drug addiction is characterized as a chronic and relapsing disorder which persists despite long periods of abstinence from the drug. Changes in subjective craving for the drug have been reported to increase with long periods of abstinence, a phenomenon identified as incubation. Incubation is a time-dependent progressive increase in drug-seeking behavior elicited by cues previously associated with cocaine availability. While there have been many studies that have illustrated that length of withdrawal period is highly correlated to drug craving and seeking, this phenomena does not seem to apply to traditional drug-induced relapse models. Here, we investigated the role of withdrawal periods on relapse to cocaine in a “binge” model of cocaine self-administration. Rats were trained to self-administer cocaine in an extended access self-administration paradigm (6 hr test sessions) for 10 days. After the final day of cocaine SA, rats were assigned to either a 1 or 14 days of withdrawal period, following which rats were exposed to a 12 hr binge session where they were allowed unlimited access to cocaine. Surprisingly, despite the imposed 14 day withdrawal period, animals self-administered an equal amount of cocaine during the 12 hr binge as animals with a brief, 1 day withdrawal period. Although these data do not support the incubation hypothesis, the findings are consistent with previous results of the stability of drug-induced reinstatement following variable periods of withdrawal. Furthermore, these data suggest the strength of learned self-administration behaviors in that there is no decay in responding despite periods of forced abstinence. These experiments are currently ongoing, and we are testing the effects of a 30 day withdrawal period of cocaine binge behavior. Finally, we are preparing to examine the transcriptional mechanisms that mediate such behavioral stability in an attempt to indentify novel molecular targets that may provide a therapeutic intervention.

#242: Human α-synuclein induces axonal transport and locomotion defects in a Drosophila model of PD

Anderson, Eric N ; White , Joseph ; Gunawardena, Shermali ;

Department of Biological Sciences, SUNY University at Buffalo, College of Arts and Sciences;

Parkinson’s disease (PD) is a common neurodegenerative disease that is characterized by loss of dopaminergic (DA) neurons, which results in severe locomotion dysfunction. The most common histopathologic characteristic of PD is the formation of intracytoplasmic α-synuclein containing inclusions called Lewy bodies and Lewy neuritis. α-Synuclein is proposed to have a major role in PD pathology. However, the exact function of α-synuclein (α-syn) remains elusive. Using a Drosophila model of PD, we tested the hypothesis that human α-syn have a role in axonal transport and that defects in transport can result in locomotion deficits. We found that expression of human α-syn (α-syn^WT) induces axonal defects in larval segmental nerves. Moreover, familial Parkinson’s disease (FPD) mutations in α-syn also show axonal blockages. Strikingly, WT and FPD mutant α-syn genetically interact with kinesin-1 and disrupt vesicular transport in vivo. Furthermore, biochemical evidence suggested that α-syn is membrane associated. In addition, we also found that both wild type and mutant forms of α-syn caused defects in larval locomotion. Together, our results indicate that α-syn may function in axonal transport and that defects in this pathway may result in locomotion deficits.

#223: Augmenting Neuregulin-1 Type III to Treat Congenital Hypomyelination Neuropathy

Belin, Sophie ; Wrabetz, Lawrence ;

Department of Neurology, SUNY University at Buffalo;

Protein Zero (P0) is the major structural protein in peripheral myelin. A dominant nonsense mutation, Q215X of the MPZ gene, truncates 33 amino acids from the intracellular domain of P0, and causes Congenital Hypomyelination (CH) neuropathy. We have previously shown that the mouse model of CH, Mpz^Q215X/+ manifests congenital hypomyelination due a combination of both toxic gain function and loss of function. To explore the hypothesis that increasing a key promyelinating axonal signal, NRG1 Type III, could ameliorate hypomyelination, we developed both genetic and pharmacological approaches in Mpz^Q215X/+ mice. We crossed Mpz^Q215X/+ mice with transgenic mice that overexpress an HA-Nrg1 Type III (HANI) fusion protein by 2-3 fold. We found that myelin thickness was increased in HANI/+// Mpz^Q215X/+ mice, with g-ratio and internodal lengths returned to normal. In addition, there was a trend towards normalization of the nerve conduction velocity and F-wave latency, and motor capacity as measured by grid walking. Myelin gene expression and protein levels were only mildly increased in HANI/+// Mpz^Q215X/+ nerves. One of the ADAM family of proteases, the a-secretase TACE, is known to negatively regulate NRG1 Type III and myelination in developing peripheral nerve. Preliminary experiments suggest that pharmacological treatment with a specific inhibitor of TACE improves myelination in dorsal root ganglion explant cultures from Mpz^Q215X/+. These data provide preliminary proof of principle that Nrg1 Type III may provide a therapeutic target in CH neuropathy.

#240: Ca_V1.2 Expression in Astrocytes

Benson, Courtney A ¹; Spreuer, Vilma ²; Santiago, Diara ¹; Cheli, Veronica ²; Paez, Pablo ¹;

¹Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Department of Pharmacology and Toxicology, SUNY University at Buffalo;

Astrocytes are important glial cells in the central nervous system (CNS) providing trophic support for neurons as well as aiding in the CNS immune response to damage. When an insult arises in the CNS astrocytes undergo astrogliosis, which is exhibited by changes in cellular morphology and protein expression profiles. Astrogliosis is necessary to stop damage from spreading to further areas within the CNS; however, chronic astrogliosis promotes the formation of scar tissue, which prevents undamaged neurons from reestablishing connections post-injury. Our lab has shown that an increased influx of calcium in astrocytes occurs during astrogliosis; however, the mechanism of calcium signaling is unknown. In this study we hypothesize that L-type voltage-operated calcium channels (VOCCs) expressed in astrocytes are essential for astrogliosis and induce a high influx of calcium in astrocytes. Our lab recently acquired two astrocyte cell lines and characterized the RNA and protein level expression of the VOCCs. To induce astrogliosis, astrocytes were treated with ATP, glutamate and LPS, or blocked pharmacologically. RT-PCR and Western blot analysis were performed to characterize the expression profiles. L-type channel isoform Ca_V1.2 displayed a significant increase in expression levels of protein compared to other VOCCs, suggesting this channel is important for astrogliosis. This in vitro model will deduce the importance of the upregulation of calcium signaling during astrogliosis. A mouse model will be created to conditionally knockout Ca_V1.2 channels in GFAP-expressing cells by utilizing the tamoxifen-inducible CRE. We will characterize expression through various methods and create injury models to determine how astrocytes behave with the Ca_V1.2 channel knockdown. This model will allow us to understand how the L-type calcium channel Ca_V1.2 is upregulated during astrogliosis and the potential benefits of knocking down this channel in an in vivo model.

#239: The Identification and Role of Novel Integrins at the Apical-like Surface of the Schwann Cell

Catignas, Kathleen K ¹; Pellegatta, Marta ²; Poitelon, Yannick ²; Wrabetz, Lawrence ³; Feltri, Maria Laura ¹;

¹Department of Biochemistry, ²San Rafaelle Scientific Institute, Milan, Italy, ³Department of Neurology, Hunter James Kelly Research Institute;

In the peripheral nervous system, Schwann cells (SCs) enwrap and synthesize myelin around an axon. Preceding myelination is a perinatal event – a prerequisite step – called “Axonal Sorting” where a SC extends its cytoplasmic process to make its first contact with the axon. This initial contact is the crucial first step of an axon selection process whereby a SC sorts through, then singles out one chosen axon from the bundle to be myelinated. We focus on this developmental step because Axonal Sorting is arrested in several human peripheral neuropathies. We therefore ask, “Which molecules mediate contact and subsequent signaling between axons and SCs during Axonal Sorting?” Our lab has shown that integrins localized on the ab-axonal (basal-like) side of the SC (the side interacting with its basal lamina) are required for Axonal Sorting. The ECM signals that the SC receives from its basal lamina are relayed by α6β1 and α7β1 integrins – these are laminin receptors which when doubly deleted specifically in mouse SCs, give rise to myelination defects. We and others have mainly studied the role of laminin receptor integrins in myelination. However we now investigate the opposite side of the SC, the ad-axonal (apical-like) side interacting with the axon, because 1) we found β1 integrins localizing here via immuno-EM and 2) conditional deletion of β1 specifically in SCs results in a more severe phenotype than our α6/α7 dKO mice. We now try to address the question this raises: What is attributing to the severity of the β1-null phenotype? Are there previously uncharacterized non-laminin integrin receptors localizing at the ad-axonal side of the SC that are important for the initial contact with the axon? We seek novel ad-axonal integrins that may be crucial for myelination. Characterization of the pathways of these integrins may lead to remyelinating therapies that target their signaling effectors.