2009 — 2010 |
Paradis, Suzanne |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Identification of the Protein Kinases Which Regulate Mammalian Synapse Formation
DESCRIPTION (provided by applicant): Synapses are specialized sites of cell-cell contact that mediate communication between neurons in the nervous system. Much remains to be discovered about the molecular mechanisms that underlie formation of these critical structures in the mammalian central nervous system. Previously, we developed a novel, forward genetic, RNA-interference (RNAi)-based approach to identifying new molecules that regulate synapse formation. Currently, we are in the process of applying this technology to understanding the cell biology of drug addiction. Thus far, a number of kinases have been implicated in regulating synapse formation or function, lending support to the hypothesis that protein kinases have critical functions at the synapse. Further, activation of protein kinase signaling has been hypothesized to underlie changes in neuronal structure and synapses in response to drug exposure. Therefore, further investigation into the role of protein kinases in synapse formation is warranted. To this end, we propose to take a genome-wide approach to identify the full complement of protein kinases that are expressed at the time that synapses are forming in cultured mammalian neurons. Next, we will utilize our RNAi-based screening approach to ask which kinases are required for the formation of functional glutamatergic and/or GABAergic synapses. PUBLIC HEALTH RELEVANCE: A current hypothesis to explain the persistent features of drug addiction, including drug cravings and relapse, posits that changes in synaptic structure and neuronal connectivity underlie these features of the disease. Further, the function of protein kinases has been implicated in these changes in synaptic structure. Thus, a genome-wide approach to understanding the role of protein kinases in synapse formation and function as outlined in this proposal has the potential to yield important insights into the underlying causes of a subset of features of drug addiction.
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2010 — 2014 |
Paradis, Suzanne |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Elucidating the Function of Class 4 Semaphorins in Gabaergic Synapse Formation
DESCRIPTION (provided by applicant): Synapses are specialized sites of cell-cell contact that mediate communication between neurons in the nervous system. Much remains to be discovered about the molecular mechanisms that underlie formation of these critical structures in the mammalian central nervous system. While recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. In addition, it is hypothesized that aberrant synapse formation and function contributes to neurological disorders such as mental retardation, autism spectrum disorders and epilepsy. To appreciate how synapse dysfunction contributes to these widespread neurological impairments, it is important to first understand how synapses are formed, maintained, and function in the non-pathological state. To this end, we developed a novel, forward genetic RNA interference (RNAi)-based screen in cultured hippocampal neurons that has identified new molecules required for synapse formation. Using this technique, we discovered that RNAi-mediated knockdown of a class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Thus, Sema4D is one of only a few molecules identified thus far that preferentially regulates inhibitory synapse formation. Further, Sema4D appears to be playing a specific role in assembling the postsynaptic specialization at inhibitory synapses. Therefore, understanding the mechanism of action of Sema4D in this process promises to yield key insights into the assembly of inhibitory synapses in the mammalian central nervous system. PUBLIC HEALTH RELEVANCE: Numerous studies now point to defects in synapse formation as a possible cause for neurological disorders such as autism, mental retardation, and epilepsy. To appreciate how aberrant synapse formation contributes to these widespread neurological impairments, it is important to first understand how synapses are formed, maintained, and function in the non-pathological state. Thus, in-depth study of the mechanism of action of Sema4D in synapse formation as outlined in this proposal has the potential to yield important insights into the underlying cause of some of these disorders.
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2015 — 2018 |
Paradis, Suzanne |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Elucidating the Function of Class 4 Semaphorins in Gabaergic Synapse Formation.
? DESCRIPTION (provided by applicant): The balance between excitation and inhibition in neuronal networks (E/I balance) regulates overall network function; disruptions to this balance are thought to underlie neurodevelopmental disorders such as Autism Spectrum Disorders and epilepsy. To understand how E/I balance is established and regulated, it is first necessary to define the genes and signaling pathways that instruct excitatory and inhibitory synaptic connections between neurons. During the last funding cycle, we identified a novel ligand-receptor pair, Sema4D and PlexinB1, which bi-directionally regulates GABAergic synapse formation on an unprecedentedly fast time-scale. We also discovered that Sema4D could be used to rapidly (within 2 hrs) drive inhibition and suppress neuronal hyperactivity in organotypic hippocampal slice cultures. Intriguingly, our preliminary data also demonstrate that in vivo application of Sema4D can reduce seizure severity in a mouse model of epilepsy, consistent with a Sema4D-dependent increase in inhibition in the nervous system of these animals. In addition, preliminary studies indicate that a relatively short time window of Sema4D treatment (e.g. 2 hrs) promotes the formation of functional GABAergic synapses that persist for days. The overall goal of this proposal is to elucidate how the Sema4D-dependent signaling pathway acts to re-set E/I balance in neuronal circuits through the promotion of GABAergic, inhibitory synapse development. In particular, we propose a set of experiments to understand how Sema4D and PlexinB1 mediate the rapid formation of GABAergic synapses using a combination of molecular biology, biochemistry, electrophysiology, and cutting-edge, time-lapse microscopy both in vitro and in vivo. Further, this Sema4D-dependent, rapid formation of GABAergic synapses leads us to hypothesize that, in the long term, harnessing the synaptogenic potential of Sema4D/PlexinB1 signaling could translate into an effective therapeutic for neurological conditions in which disruptions to the E/I balance is a salient feature.
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2017 — 2018 |
Marr, Michael Thomas Paradis, Suzanne |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Defining the Mechanism of Camkii Inhibition by the Non-Canonical Gtpase Rem2
Project Summary Previously, we identified the small, Ras-like GTPase Rem2 as a critical regulator of neuronal morphology, synapse formation and neuronal plasticity in the vertebrate nervous system. Rem2 is a member of the RGK family of non-canonical Ras-like GTPases and is primarily expressed in the brain. Our published studies demonstrated that Rem2 functions in a CaMK signaling pathway that restricts dendritic branching. In fact, Rem2 is itself a substrate of CaMKII, and phosphorylation of Rem2 by CaMKII is required for Rem2 to regulate dendritic branching. CaMKII is an abundant protein kinase that serves many functions in diverse tissues including regulation of activity-dependent dendritic remodeling, Long Term Potentiation (LTP), which is the biological correlate of learning and memory, and regulation of hypertrophy and Ca2+ homeostasis in heart muscle. To better understand Rem2 signaling, we took an unbiased, proteomics approach to identify Rem2 interacting proteins and found that Rem2 interacts with all four CaMKII isoforms. Using an in vitro kinase assay with purified proteins, we demonstrated that Rem2, while a substrate of CaMKII, also potently inhibits its kinase activity. This suggests that Rem2 is a direct, endogenous inhibitor of CaMKII activity, a previously un-described function for this protein. While many pathways that activate CaMKII have been identified, there is only one other molecule described thus far in mammalian cells that inhibits CaMKII signaling (CaMKIIN). Interestingly, other RGK family members influence CaMKII activity in different cell types, although the exact mechanism of this regulation has not been reported. We propose experiments that will provide a detailed, mechanistic understanding of CaMKII inhibition by Rem2 and further, determine if inhibition of CaMKII activity is a conserved function of the RGK family.
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2017 — 2020 |
Paradis, Suzanne Lovett, Susan [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Reu Site: Cell and Molecular Visualization At Brandeis University
This REU Site award to Brandeis University, located in Waltham, MA will support the training of 10 students for 10 weeks during the summers of 2017- 2019 in biological research employing modern cell and molecular visualization techniques. This program, encompassing over 50 faculty in the Life Sciences, will introduce undergraduate students to a broad range of topics concerning biological structure and function. Students will conduct full-time research guided by their mentors and will participate in weekly lunch seminars, which will include faculty research presentations and professional development activities such as panel discussion with students and postdoctoral fellows from the Greater Boston area concerning careers in biotechnology, research, education, and policy. There will be group discussions of ethical issues and mentoring in science. Students will write a synopsis of their summer project, with feedback and editing, and will participate in a capstone symposium including poster presentations. Participants will be selected from a nation-wide pool based on academic record, recommendations, and potential for research in biology and should be current freshmen, sophomores or juniors at a college or university.
It is anticipated that a total of 30 students, primarily from schools with limited research opportunities, will be trained in the program which welcomes students from underrepresented groups in science. Training will take place in a supportive and interactive environment, and the participating faculty have a strong record of mentoring undergraduates in research and publishing with student co-authors. Students will have an opportunity to interact with scientists with diverse interests, at different stages in their careers, to learn how research is done. Many students will present their work at scientific conferences.
A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after the program in order to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.bio.brandeis.edu/undergrad/summerResearch/ or by contacting the PI (Dr. Susan Lovett at lovett@brandeis.edu) or the co-PI (Dr. Paradis at paradis@brandeis.edu).
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2020 — 2021 |
Paradis, Suzanne |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Semaphorin-Dependent Gabaergic Synapse Formation: a Novel Approach to Increasing Inhibition in the Intact Brain
SUMMARY We propose to harness the synaptogenic potential of Sema4D signaling to increase GABAergic synapse number, thus enhancing inhibition in neural circuits and suppressing seizures. This approach could be beneficial to preventing the establishment of epilepsy, halting its progression, or suppressing hyperexcitability during a seizure event. Previously my lab discovered that the secreted protein Semaphorin 4D (Sema4D) drives inhibitory synapse formation on a remarkably fast time scale (i.e. minutes) in hippocampal neurons and slice cultured from the pre-natal and neonatal hippocampus. We also demonstrated that intra-hippocampal infusion of the extracellular domain of Sema4D into the adult hippocampus rapidly promotes the formation of new GABAergic synapses. Importantly, these data demonstrate that the molecular machinery regulating GABAergic synaptogenesis in the young hippocampus remains functional in the adult and that this machinery can be harnessed to modulate network excitability. We directly addressed this point by demonstrating that Sema4D treatment protects against seizures induced by direct electrical stimulation of the hippocampus or by intravenous infusion of the proconvulsant drug pentylenetetrazol. Further, we found that Sema4D treatment restored the efficacy of diazepam in a rodent model of refractory status epilepticus. Given the success of these studies, my laboratory has undertaken a new experimental direction to determine if Sema4D treatment has therapeutic potential for human epilepsies. We will focus our work on the translatability of Sema4D as an anti-seizure therapeutic for treating status epilepticus (SE). Unfortunately, approximately 30% of patients with SE are refractory to treatment with current medications including benzodiazepines. One hypothesis about the origin of refractoriness in status epilepticus is that prolonged neural depolarization leads to internalization of cell-surface GABAA receptors, thus reducing total inhibitory current in response to GABAergic signaling (Joshi & Kapur, 2012). We hypothesize that by acutely increasing the number of inhibitory synapses using Sema4D treatment, we could maintain or re-establish benzodiazepine sensitivity in the brains of these individuals. Second, in order to further our in vivo mouse studies of Sema4D-dependent seizure suppression, and to further the translatability of our findings, we will explore alternative methods of administering Sema4D (e.g. intravenous injection of virus encoding Sema4D) to mice. Lastly, we will test the translatability of our findings with Sema4D in rodents by asking if Sema4D promotes inhibitory synapse formation in human neurons.
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