Barry Kaplan - US grants

This competing renewal application describes an interrelated series of clinical and preclinical studies of an alteration in platelet membrane fluidity associated with primary dementia in the elderly. Increased platelet membrane fluidity (PMF), as measured by a decrease in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) in labeled membranes, identifies a subgroup of patients with Alzheimer's disease (AD) who have distinct clinical features. This phenotype appears to be a stable, familial trait that is vertically transmitted in families of patients with Alzheimer's disease. Segregation analysis of data from these families indicates that increased PMF results from the inheritance of a single major locus that controls at least 80% of the variance in this membrane phenotype. At the cellular level, evidence from ultrastructural and biochemical studies suggests that increased PMF results from an accumulation of abnormal internal membranes resembling smooth endoplasmic reticulum that may be functionally abnormal. We hypothesize that this inherited abnormality is expressed in the brain and contributes to the formation of senile plaques and neurofibrillary tangles, the histologic hallmarks of AD. During the requested award period, we propose to prospectively evaluate increased PMF as a risk factor for AD, to further define the cellular and molecular basis for increased PMF, and to use this information to direct hypothesis-driven studies of post-mortem brain tissue aimed at elucidating the pathophysiology of AD.

Axons and nerve terminals are unique subcellular structures of the neuron that play a critical role in the development and maintenance of neural connectivity. One of the central tenets in neuroscience is that the protein constituents of these distal neuronal compartments are synthesized in the nerve cell body and subsequently transported to their ultimate sites of function. Hence, the structure and function of these highly specialized distal domains of the neuron are totally dependent on slow anterograde axoplasmic transport. Although the majority of neuronal mRNAs are indeed transcribed and translated in the neuronal cell soma, it is now well- established that there exists a diverse population of mRNAs in the distal structural/functional domains of the neuron to include the axon and presynaptic nerve terminal. It has also become well- accepted that proteins synthesized from these mRNAs play a key role in the development of the neuron and the function of the axon and nerve terminal, including navigation of the axonal growth cone, synthesis of membrane receptors employed as axon guidance molecules, axon transport, synapse formation, and in activity-dependent synaptic plasticity. In previous studies, we reported the surprising finding that several nuclear-encoded mitochondrial mRNAs were present in the axon and that approximately 25% of the total protein synthesized locally in the nerve terminal was destined for the mitochondria. Based upon these findings, we hypothesized that the local protein synthetic system played a critical role in the maintenance of the mitochondrial population and ultimately, the function of the axon and presynaptic nerve terminal. Currently, we are testing this working hypothesis using rat primary sympathetic neurons cultured in multi-compartment Campenot chambers. Results of these studies established that acute inhibition of the local protein synthetic system significantly diminished the membrane potential of mitochondria, and also reduced the ability of mitochondria to maintain basal levels of axonal ATP and restore levels of axonal ATP after prolonged neural activity (e.g., stress). Moreover, the inhibition of local protein synthesis for more than six hours significantly reduced the viability of the axon, as judged by the structure's ability to grow and thrive in the cell culture system. Most recently, we have discovered that Cytochrome c oxidase IV (COXIV) and ATP synthase mRNAs are present in the axon. These proteins are key components of Complexes IV and V of the oxidative phosphorylation chain and are rate-limiting factors in the mitochondrion's ability to generate ATP in the cell. Inhibition of the local synthesis of these proteins results in marked decrements in axon respiration, elevation in axon levels of reactive oxygen species (ROS), and attenuation of the growth of the axon. Interestingly, the deleterious effects on axonal metabolism derived from the inhibition of the local synthesis of these two proteins were additive, suggesting that ATP generation in the axon was controlled by the local translation of multiple nuclear-encoded mitochondrial mRNAs. These new findings are now being prepared for publication. During this past year, we also completed a structural/functional analysis of the 3'untranslated region (3'UTR) of the COXIV mRNA. Our results revealed that the region contained three putative regulatory domains which comprised three hairpin-loop structures. Findings derived from a deletion mutation analysis of the 3'UTR revealed that the first putative regulatory stem-loop structure contained sequences that controlled the trafficking of this mRNA to the axon (i.e., the "zip-code" element). The second and third regulatory domains identified by the secondary structural analysis contained target sequences for two different brain-specific microRNAs. These RNAs comprise a family of small noncoding RNA molecules that regulate the posttranscriptional expression of genes that are involved in various fundamental biological processes, such as cell growth and differentiation. Results of a microarray expression analysis indicated that there were approximately 130 different microRNAs present in the axon, several of which were highly abundant and appeared selectively transported into and/or were being preferentially stabilized this subcellular compartment of the neuron. Surprisingly, the expression of COXIV in the axon is being regulated by two different brain-specific microRNAs which bind to their respective signal sequences present in the second and third hair-pin loop structures located in the 3'untranslated region (3'UTR) of the mRNA. Modulation of axonal COXIV expression by these microRNAs has marked effects on the axon's metabolic activity and its capacity to generate energy. Most recently, we have identified a second nuclear-encoded mitochondrial mRNA which is regulated by these two microRNAs and also contains the "zip-code" element in its 3'UTR. This finding is important because it raises the possibility that the trafficking and translation of several of the mRNAs that encode key proteins in the oxidative phosphorylation chain present in mitochondria are being co-ordinately regulated in the axon. Last, we have discovered that mRNAs encoding two translation initiation factors are present in the axon. The results generated from this portion of our investigation establish that the local expression of these factors regulate the activity of the intra-axonal protein synthetic system. The inhibition of local expression of these key proteins has a profound inhibitory effect on axon growth, as well as the long-term viability of the axon. A manuscript describing these recent results is now in preparation. Taken together, these results indicate that the local protein synthetic system plays a key role in the regulation of mitochondrial activity and the growth and maintenance of the axon. We anticipate that this line of investigation will augment our understanding of the molecular mechanisms that underlie neuronal development, regeneration, and plasticity and generate new avenues of research into the pathophysiology of mental illness.

The Office of Fellowship Training (OFT) organized and managed several educational events and programs for the DIRP and the extramural research communities. These events, geared to service the needs of a multidisciplinary basic and clinical training program, encompassed the participation of approximately 262 Fellows and 119 Special Volunteers OFT continued the DIRP professional development activities for the fellows and staff which entailed major teaching commitments. The DIRP grantwriting training program was continued in conjunction with Grant Writers Seminars and Workshops. Training was provided in three phases, gradually transitioning from didactic introductory material (Phase I) to an intensive grant-writing experience in the form of an individualized tutorial (Phase III). Phase I provided extensive training in Grantsmanship to 25 NIMH/DIRP fellows, 30 NINDS fellows, and 5 fellows from NEI and NIDCD. Twelve fellows participated in Phase II of the training, which focused on the development of an innovative research idea and the formulation of a compelling set of Specific Aims. Phase III provided an intensive grantwriting experience to a subset of senior fellows. This tutorial, was planned to be conducted over the course of 10 weeks, encompassed small, weekly group meetings with the OFT Director and covered all components of the grant application including the experimental approach, candidate information, biosketch, facilities, and abstract. This year, no DIRP fellows were awarded a K99/R00 grant. We anticipate that about four NIMH fellows will resubmit their applications for the K99/R00 award this October and February 2014. Ten Fellows submitted applications to the Brain & Behavior Research Foundations for the NARSAD Young Investigators Award in January 2013: three fellows received an award. In addition, 7 NIMH fellows received the NIH Fellows Award for Research Excellence (FARE). OFT organized the recruitment program and interview process, and continued to provide administrative services for the University College London-NIMH/NINDS Joint Doctoral Training Program in Neuroscience. Two offers of acceptance were made; however, no new students were recruited into the program. For the fourth year OFT provided administrative support including recruitment and interview programs for the Karolinska-NIH Program for Graduate Training in the Neurosciences. The OFT maintained interactive websites for both programs. The OFT also continued management of the PhD program between the NIMH/DIRP and the Departments of Epidemiology and Biostatistics of the Johns Hopkins Bloomberg School of Public Health. At present, there is one student participating in this graduate program. The OFT provided weekly orientations and administrative support for students coming to the NIMH to participate in the NIH Summer Research Training Program. The NIMH DIRP accommodated 63 high school students, college undergraduates, and medical and graduate students. Twenty-three of these summer interns were from the minority educational community. This year, 34 summer interns participated in the NIH Summer Student Poster Day. This year, aggressive efforts to enhance the diversity of the DIRP training program continued with staff attending as an exhibitor and/or providing talks at several national student conferences. One indicator of the success of these efforts is that approximately 36% of the students in this year's Summer Research Program derived from underrepresented minority educational communities. In collaboration with the Office of the Clinical Director, OFT organized the 25th Annual NIMH National Outstanding Resident Awards Program, honoring the academic achievements of 12 PGY-III residents selected from leading psychiatry departments. The award honors psychiatry residents who show outstanding scientific and leadership potential. The program provides a vehicle to recruit the best and brightest to the DIRP Clinical Research Training Program. The two-day award program consists of presentations by the clinical faculty, tours of the Clinical Center and DIRP research facilities, interviews with branch chiefs and investigators, lunch with the DIRP clinical fellows and an awards dinner honoring the award recipients. The DIRP Investigator Seminar Series, designed to familiarize DIRP fellows and faculty with their colleagues' research, featured five DIRP investigators and was well-attended. The Ethics for Lunch seminar series ran for three months as part of the NIH mandate that each institute provide Scientific Research Ethics Training to the Scientific Staff. This Brown Bag seminar was attended by Fellows (to include Predocs, Postbacs, Postdoctoral IRTAS, Visiting Fellows and Research Fellows,) Clinical Fellows, Principal Investigators (Tenured and Tenure-Track), Staff Scientists and Staff Clinicians, Laboratory Technicians, as well as contractors whose duties fall under the above listed categories. Forty-five fellows attended the Tax Seminar OFT provides annually. This seminar is viewed as an essential service given that NIH IRTAs are required to file their taxes quarterly. The tax specialist invited to present the seminar covers the unique tax requirement specific to the NIH IRTA, and for two weeks after the seminar, is available by email to answer questions from the NIMH IRTA fellows. A key function of the DIRP/OFT is to provide a vehicle for fellow/mentor grievance resolution and career counseling. This year the OFT Director participated in the successful resolution of two fellow/mentor disputes and provided formal career counseling for 20-25 fellows. The OFT Director also provided consultation services for DIRP investigators on topics related to training and/or career development. The Office affected the laboratory transfer and/or departure of six fellows in response to investigator retirements, departures or supervisor-trainee incompatibilities. OFT staff continues to meet with all fellows to evaluate their fellowship experience as they leave for PhD programs, medical schools, academic positions and/or industry. From an administrative perspective, the OFT continued its collaboration with the Administrative Services Branch (ASB), providing quarterly training to DIRP laboratory/branch secretarial and administrative support staff in personnel case management. This training has proven advantageous since the OFT reviews and works with ASB and administrative staff in preparing nearly 750 personnel cases annually. The OFT Specialists continue to provide orientations to all newly appointed trainees. The ASB, the SD and the OFT coordinated these orientations to coincide with the bi-monthly NIH HR orientations. The orientation covers information relevant to the NIH and NIMH organization, function, and mission, and provides fellows with reference materials on ethical conduct of research and mentorship. All materials presented during the summer and bi-monthly orientations were provided on a memory stick in an effort to go green in the OFT. In its third year, the Special Volunteer and Student IRTA online application and the subsequent processing and online approval continues to work smoothly. All new and current fellowship cases passing through the OFT continue to be scanned and stored in SharePoint. (as of the spring of 2011, all OFT fellowship cases are stored electronically.)

Although once highly controversial, it is now well- established that there exists a diverse population of mRNAs and non-coding microRNAs in the distal structural/functional domains of the neuron which include the dendrite, axon, and presynaptic nerve terminal. It has also become well- accepted that proteins synthesized from these mRNA templates play a key role in the development and long-term viability of the axon. The findings derived from this new area of investigation have recently been reviewed (Scott et al., 2015). In regard to this past years research activities, members of the Section were committed to the conduct of two major projects. The progress effected in each of these projects is summarized, briefly, below: A third project which continued our investigation of axonal microRNA was conducted in the form of an international collaboration and employed only minimal laboratory personnel and resources . Project #1. Regulation of the axonal trafficking of RNA. This past year, the protein constituents of the axonal trafficking granule associated with Cytochrome c-oxidase (Cox IV) mRNA, a messenger that codes for a key component of the oxidative phosphorylation chain, have been tentatively identified by a combination of RNA-affinity purification and mass spectroscopy. This work was conducted in collaboration with Dr. J.A. Kowalak, a staff scientist in the new NIMH/NINDS Mass Spectroscopy Core Facility. Previously the local translation of two of these nuclear-encoded mitochondrial mRNAs was found to play a key role in the regulation of local energy metabolism, modulating the synthesis of ATP and ultimately the production of harmful reactive oxygen species (ROS) (reviewed in Scott et al., 2015; Gale et al., 2018). Dysregulation of the local translation of this mRNA resulted in reductions in the levels of ATP in the axon and an elevation in the production of ROS, two factors which negatively affected the growth and health of the axon. Surprisingly, there are approximately 30-50 different proteins that associate with the RNA sequences that regulate the transport of this mRNA to the axon (i.e., zipcode), a finding that suggests that the zipcode serves as a nucleation site for the formation of an RNA-protein trafficking complex ( see Kar et al., 2017). In addition, the proteins associated with the zipcode, regulating the trafficking of TH mRNA to the axon were identified. The protein composition of the TH mRNA trafficking complex are now being prepared for publication (Aschrafi et al., Manuscript in preparation). Project # 2. MicroRNAs present in the axon Previously, we had reported, that in addition to a highly diverse population of mRNAs, that the axon contained over 100 different small, noncoding RNAs One of these miRNAs, miRNA338, was found to coordinately regulate the local expression of several nuclear-encoded mitochondrial mRNAs that coded for key components of the oxidative phosphorylation chain and regulated local energy metabolism (e,g., Cytochrome-c oxidase IV and ATP synthase mRNAs).. Interestingly, the precursor of the mature, functional form of miRNA 338 was observed to be associated with the organelle, itself. This finding suggested that this precursor miRNA was bound to the mitochondria and served as a reservoir in the activity-dependent activation of the mature miRNA 338 (Vargas et al., 2016). The results of affinity purification and mass spectroscopy experiments indicated that the precursor miRNA338 associated with a large number of proteins that regulated its transport to the axon, as well as its binding to the mitochondria (Vargas et al., 2016). The results of gene ontology analysis indicated that these proteins included mitochondrial, cytoskeletal, and axonal motor proteins, as well as a number of well-known RNA-binding proteins. Additionally, Members of our international collaborative research team have found that miRNA338 also regulates the expression of several axon guidance genes that markedly affect the migration and differentiation of cortical neurons during development. Project #3. Local Regulation of Neurotransmitter Synthesis. This year members of the laboratory reported that mRNAs encoding the enzymes that comprise the catecholamine biosynthetic pathway were present in the axon of primary sympathetic neurons and were being actively translated. (Gervasi et al., 2016; Aschrafi et al., 2017). Importantly the introduction of the mRNA encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, directly into the axon by transfection, markedly increased the synthesis of the catecholamine neurotransmitters, dopamine and norepinephrine. Stimulation of these sympathetic neurons resulted in significant increases in the release of these two neurotransmitters into the cell culture media (Aschrafi et al., 2017). Conversely, removal of the zipcode from the 3UTR of the TH mRNA, by gene editing techniques, blocked the transport of the mRNA to the axon and significantly diminished the local synthesis of protein, as well as decreased the axonal levels and release of DA and NE. This year it was also demonstrated that Angiotensin II ( Ang II) augmented the axonal trafficking of TH and DBH mRNAs and enhanced the synthesis of DA and NE. These effects were abolished by the pretreatment of the neurons with an Angiotensin II antagonist. Surprisingly, the affinity purification of RNP trafficking granules, using the TH zipcode, resulted in the enrichment of the relative abundance of both the TH and DBH mRNAs, suggesting that both of these mRNAs were being transported to the axon together, perhaps in the same RNP granule. Taken together, these results point to a novel mechanism by which Ang II participates in the regulation of the axonal synthesis of NE by modulating the local expression of TH and DBH, two enzymes involved in the catecholamine biosynthetic pathway.