Mladen-Roko Rasin - US grants

The corticospinal system is the principal motor pathway for controlling voluntary movements in mammals. This system is formed by long-range descending axonal projections from layer 5 neurons in sensorimotor neocortical areas to terminal targets in the brain stem and spinal cord. As the axons from cortex descend into the spinal cord, they also form collateral connections with intermediate targets involved in sensorimotor integration, such as the striatum and thalamus. How corticospinal neurons differentiate, navigate their axons and what is the importance of synaptic contacts with the intermediate subcortical targets is still poorly understood. We and others have recently shown that the transcription factor fez family zinc finger 2 (FEZF2;also known as FEZL or ZFP312) is required for the formation of corticospinal axonal projections, dendritic arborization, and the expression of multiple genes previously implicated in the development of neuronal circuits for sensorimotor integration. However, FEZF2 downstream signaling effectors are still unknown. In my preliminary studies, I have found that FEZF2-mediated transcriptional activation of two axon guidance molecules, is required for the formation of the corticospinal tract and its thalamic collaterals. In addition, my preliminary observations indicate that regionally restricted expression is not only transcriptionally regulated by FEZF2, but suggest that it depends on the formation of subcortical projections. Here, I propose to directly test the central hypothesis of this proposal: that retrograde signaling controls gene expression and differentiation of layer 5 neurons constituting the corticospinal system. Results should have a significant impact on the understanding of sensorimotor integration and treatment approaches for spinal cord injury and the regeneration of severed corticospinal tract axons in adulthood.

DESCRIPTION (provided by applicant): Neocortical neurons show admirable diversity in function, transcriptional programs, dendritic morphology and axonal targets. To specify this remarkable diversity, the intrinsic molecular pathways defining transition from genome to proteome in developing neocortical neurons must be precisely regulated in space and time. RNA- binding proteins (RBPs) are known to be at the cross-road of the genome to proteome axis through the splicing, export, stabilization and translation of transcribed mRNAs. By regulating these post-transcriptional events, RBPs are implicated in control of protein expression levels and neuritogenesis by interfacing with distinct morphogen and/or trophic extracellular signals. Bound mRNAs can include distinct subsets of transcribed mRNAs and form what we call RNA-operons. These RNA-operons also respond to external cues providing immediate translation to protein, allowing for rapid adjustments of protein levels for a crucial cellular event. These RNA-operons then respond to environmental signals allowing prompt translation of the given mRNA to protein necessary for a particular cellular event. We identified a neocortical RNA-operon that consists of an RBP that binds six neocortical mRNAs characterized by forkhead transcription factor domains at least five days before their translation. To the best of our knowledge this is the first evidence that an RBP binds a subset of six forkhead mRNAs in any system, including developing neocortex. Moreover, we identified a thalamic morphogen signal that affects the phosphorylation state of this neocortical RBP and increases the protein levels of a neocortical forkhead transcription factor. This cascade of events happens before neocortical neurons finish their neurite development. Indeed, both neocortical RBP and thalamic morphogen affect neocortical neuritogenesis. Therefore, our central hypothesis is that this mechanism of thalamic morphogenic regulation of neocortical RNA-operons is crucial for the spatiotemporal specification of subtypes of neocortical neurons and their neurite differentiation. Therefore, we will determine (1) the mechanisms of the neocortical RNA operon in the specification of neocortical projection neurons and their dendrite differentiation; and (2) the role of a thalamic morphogen on neocortical RNA-operon-dependent specifications. To do this, we will use an elegant combination of neuroanatomical, cellular, molecular and genetic approaches. Findings from this proposal will provide previously unrecognized molecular mechanisms of post-transcriptional control in the overall specification of subtypes of neocortical neurons that may open new avenues for treatment of distinct neurodevelopmental disorders.

DESCRIPTION (provided by applicant): The current proposal is to support a Summer Undergraduate Research Program (SURP) in Molecular and Developmental Neurobiology mentored by UMDNJ/Robert Wood Johnson Medical School and Rutgers University research faculty. The program has three primary goals: (1) To increase student knowledge and appreciation of basic biological research by providing a closely-mentored, hands-on research experience; (2) To increase student knowledge and interest in pursuing careers in research through career development and educational activities; (3) To provide continued advice, support and guidance to program alumni to facilitate post-program career planning and implementation. Twelve undergraduates will be admitted to our program each year beginning 2013. Special consideration will be given to students from economically and/or socially disadvantaged backgrounds including under-represented minority applicants, first-generation college students, and those with limited research experience or opportunities at their present colleges to generate a deep and diverse pool of potential future scientists. The research component of the proposed SURP involves hands-on experimentation in labs with active, nationally recognized and federally funded research programs in Molecular and Developmental Neurobiology on the Busch Campus of UMDNJ/RWJMS and Rutgers Universities. The infrastructure and general organization of the proposed NIH Summer Research Experience program will be based on an existing program that has been in place for 16 years. Students will work on their own independent research projects involving cutting-edge molecular, genetic and genomic approaches under the close guidance and supervision of mentors, postdocs or senior graduate students in their host labs. The proposed SURP is designed to integrate student research experiences and mentoring with career development and also includes training in research ethics. Student activities include weekly meetings attended by scientists from our campus and from the professional community to discuss their education and careers including traditional and non-traditional paths. Each year students will present the results of their summer research projects in a symposium that is open to scientists on campus and members of the general public. Several of the mentors who will be supervising the students study mental disorders including depression, bipolar disorder, autism, and schizophrenia. The multidisciplinary research focus of the other faculty mentors on the cellular, molecular, and genetic processes underlying developmental and degeneration of the nervous system also has implications for the etiology, pathogenesis and progression of mental disorders, many of which have developmental origins.

Abstract: The long-term goal of this competing renewal is to understand how the previously underappreciated regulation of protein synthesis (mRNA translation) in space and time drives neuronal diversity. Neuronal diversity relies on intricate steps of functional gene expression. It has been established that spatio-temporal expression of transcription factors drive neuronal and dendritic differences. While unidentified molecular mechanisms of post- transcriptional control like mRNA translation have strong potential to drive neuronal diversity, they have been thus far understudied. mRNA translation is the final essential step in the functional gene expression. We showed in the previous funding period that regulation of this process is of the key molecular mechanisms in neocortical neuronal development. Our published and preliminary studies, supported by this grant, have led to five important discoveries. First, we described that genes associated with mRNA translation show temporal dynamics in both expression and activity during neurogenesis in developing neocortices. Second, we reported that mRNA translation and the core components of the ribosome in the neocortex ? the ?neocortical ribosome signature?? are developmentally regulated by the intrinsic Elav RNA binding proteins (RBPs). Third, we published that timed ingrowth of thalamocortical axons secrete WNT morphogen and extrinsically define temporally dynamic mRNA translation and the ribosome signature in the developing neocortex. Fourth, that both Elav RBPs and thalamocortical WNT signaling dictate identities of developing neocortical glutamatergic neurons and maturation of oligodendrocytes. Finally, we reported that prenatal deletion of an Elav RBP results in abnormal neocortical dendritogenesis and behavior. However, there are still critical gaps in our knowledge regarding how timed mRNA translation and ribosome signature dictate development of distinct neocortical glutamatergic neurons. In this proposal, we hypothesize that layer-specific ribosome signatures and RBPs dictate timed mRNA translation in distinct subpopulations of developing glutamatergic neurons, thus governing their neurite development. Therefore, we will determine (1) how RBP-defined ribosome signatures dictate mRNA translation specificity and dendrite and axon development within distinct layer specific subpopulations of neocortical glutamatergic neurons; and (2) how WNT-mediated Frizzled signaling dictates RBP-defined assembly of the neocortical layer- specific ribosome signature, mRNA translation and dendrite and axon development in distinct glutamatergic neurons. To do this, we will use an elegant combination of neuroanatomical, cellular, molecular, and genetic approaches. We have produced all of the preliminary data necessary to demonstrate feasibility of the proposed approaches. Findings from this proposal will reveal previously unrecognized molecular mechanisms of post- transcriptional control in the overall specification of neocortical glutamatergic neurons and dendrite development, which can open new avenues for treatment of neurological and neuropsychiatric disorders involving these.

Project Summary The current proposal is to support a Summer Undergraduate Research Program (SURP) in Neuroscience mentored by Rutgers University research faculty. The program has three primary goals: (1) To increase student knowledge and appreciation of basic biological research by providing a closely-mentored, hands-on research experience; (2) To increase student knowledge and interest in pursuing careers in research through career development and educational activities; (3) To provide continued advice, support and guidance to program alumni to facilitate post-program career planning and implementation. Twelve undergraduates will be admitted to our program each year beginning 2018. Special consideration will be given to students from economically and/or socially disadvantaged backgrounds including under- represented minority applicants, first-generation college students, and those with limited research experience or opportunities at their present colleges to generate a deep and diverse pool of potential future scientists. The research component of the proposed SURP involves hands-on experimentation in labs with active, nationally recognized and funded research programs in Neuroscience on the Piscataway and New Brunswick campuses of Rutgers Universities. The infrastructure and general organization of the proposed NIH Summer Research Experience program will be based on an existing program that has been in place for 21 years. Students will work on their own independent research projects involving cutting-edge molecular, genetic and genomic approaches under the close guidance and supervision of mentors, postdocs or senior graduate students in their host labs. The proposed SURP is designed to integrate student research experiences and mentoring with career development and also includes training in research ethics. Student activities include weekly meetings attended by scientists from our campus and from the professional community to discuss their education and careers including traditional and non-traditional paths. Each year students will present the results of their summer research projects in a symposium that is open to scientists on campus and members of the general public. The mentors who will be supervising the students study neurological and neurodegenerative disease including Alzheimer?s, Multiple Sclerosis, traumatic brain injury, spinal cord injury, lysosomal disease, sensory loss, epilepsy, and stroke. The multidisciplinary research focus of the faculty mentors on the cellular, molecular, and genetic processes underlying developmental and degeneration of the nervous system has implications for the etiology, pathogenesis and progression of neurological disorders.