Ruben Dagda - US grants

[unreadable] DESCRIPTION (provided by applicant): Heterotrimeric protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase that regulates many cellular signal transduction pathways. The neuron-specific B-beta regulatory subunit of PP2A appears to be a critical survival regulator, since a CAG repeat expansion in its promoter is responsible for the neurodegenerative disorder spinocerebellar ataxia type-12. Recently published work from our laboratory has demonstrated that B-beta2, a splice variant of B-beta, contains a N-terminal targeting sequences that directs PP2A to mitochondria to promote apoptosis in a neuronal cell line. Here, I propose to investigate the mechanism by which B-beta2 binds to mitochondria and antagonizes survival. In three aims, I will address the hypotheses that B-beta2 promotes apoptosis by (1) blocking the mitochondrial import receptor, (2) dephosphorylating Bcl-2 family survival regulators at the outer mitochondrial membrane, and by (3) interfering with mitochondrial metabolism. The findings in this proposal will be relevant for the development of novel therapies that target B-beta2 to prevent apoptosis during brain ischemia and neurodegeneration. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Dysregulated autophagy has been implicated in several human neurodegenerative diseases and their models. Macroautophagy is a regulated process for engulfment of cytoplasmic constituents into organelles termed autophagosomes, which deliver the cargo to lysosomes for degradation. Autophagic sequestration requires conjugation of components of the autophagy machinery to the pre-autophagosome membrane.Basal autophagy is critical for maintaining mitochondria) homeostasis in neurons, and either impaired or excessive mitochondria! turnover may contribute to neurodegeneration. However, mechanisms that regulate selective targeting of damaged organelles such as mitochondria are unknown. Our recent study indicates that alternative lipid signals are involved in signaling mitochondria! Autophagy during parkinsonian injury, and these signals are distinct from the commonly studied lipid signals that regulate nonselective starvation-induced autophagy. We hypothesize that oxidation and exposure of the mitochondria! inner membrane phospholipid cardlolipln (CL) triggers mitophagy through interactions with the autophagy machinery. Interdisciplinary approaches including mass spectrometry, high performance thin layer chromatography, lipid coated nanoparticles, live cell imaging, and mutational analysis will be employed to test this hypothesis through two specific aims that focus on the role of CL in triggering mitophagy and the mechanism of interaction with the autophagy machinery. The applicant is a postdoctoral fellow with experience in molecular neuropharmacology. To conduct the proposed studies, he will train under the multidisciplinary mentorship of experts in autophagic cell biology and Parkinson's disease mechanisms, oxidative lipidomics and nanotechnology, computational biology and specialized biologic imaging techniques. Regular meetings involving the applicant, his interdisciplinary mentoring team, and external advisors will be held to ensure effective integration of molecular cell biologic, biochemical and neurodegenerative considerations. The two-year research training plan will enable the applicant to derive career developmental skills to become an independent investigator in neurodegeneration research. Lay Summary. In addition to powering cellular energy needs, mitochondria actively regulate neuronal survival/death decisions. Mitochondria! alterations are implicated in neurodegenerative and [unreadable] psychiatric diseases and in aging. As autophagy is a key factor in mitochondria! quality control, scientific innovations from the proposed research can benefit the larger community by unveiling therapeutically relevant mechanisms in Parkinson's and other age-related neurodegenerative processes. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Mitochondrial dysfunction underlies the pathology of several diseases including Alzheimer's disease and diabetes. O-GlcNAcylation of mitochondrial proteins is increasingly being recognized as a regulatory mechanism contributing to these pathologies. O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) is an abundant, reversible and highly dynamic post-translational protein modification that regulates signal transduction, apoptosis, proteasome activity, transcription, translation and nuclear transport. Deregulated O-GlcNAcylation has been linked to diabetes-related complications, cancer progression, neurodegeneration and includes mitochondrial dysfunction. We have recently identified glycosylated isoforms of multiple essential mitochondrial proteins. The recent discovery of a specific mitochondrial isoform of OGT (mOGT) localized to the inner mitochondrial membrane raises the possibility that mitochondrial O-GlcNAcylation plays a role in oxidative phosphorylation, mitochondrial integrity, redox signaling, and cell survival pathways. While previous studies showed that stable or transient overexpression of mOGT results in apoptosis, the role of endogenous mOGT in mitochondrial protein O-GlcNAcylation and mitochondrial function remains largely unknown. This collaborative proposal will test the hypothesis that mitochondrial OGT activity is critical to mitochondrial structure and function. Unveiling novel physiological roles of mOGT will not only help us understand how O- GlcNAcylation regulates mitochondrial function but will help us to understand how dysregulated O-GlcNAcylation of key proteins contribute to disease pathogenesis of diabetes, cancer and neurodegenerative diseases.

The purpose of the proposed project, Community of Bilingual English-Spanish Speakers Exploring Issues in Science and Health (CBESS), is to increase linguistic diversity in science, technology, engineering, and math (STEM)-healthcare fields, including biomedical, behavioral, and clinical research careers. With support of the large group of Spanish-English bilingual (SEB), STEM-healthcare professionals that was formed during this proposal preparation, CBESS will contribute to the pipeline between K-12 and higher education/career. CBESS will recruit Spanish-English bilingual (SEB) high-school students at the end of tenth grade and implement several language-supported STEM-healthcare interventions during the eleventh and twelfth grade (17 months): family-engaged career exploration; Next Generation Science Standards (NGSS)- aligned, inquiry-based, youth-led summer research residential program; community outreach/ dissemination, internships, and mentoring. Applying methods that are known to be effective with the target population, CBESS will also train undergraduate, near-peer instructor-mentors--STEM-healthcare Leadership Trainees (LT)--in inquiry-based instruction and strategies for positioning K-12 bilingual students as ?insiders? in STEM- healthcare, as well as in the responsible conduct of research and mentoring skills, followed by practical application with SR. CBESS will develop and expand the nascent SEB STEM-healthcare community of practice (CoP) that was created during CBESS proposal preparation. Committed academic, clinical, research, and community partners will contribute to research and evaluation efforts, and support the pipeline between K- 12 and higher education/career through Community Based Participatory Research (CBPR), framing priority community health issues to be addressed by each cohort of SR from among issues identified by the SR during the application process. Finally, the CoP will target long-term institutional sustainability for linguistically diverse students in STEM-healthcare education and careers.

PROJECT SUMMARY Mutations in PTEN-induced kinase 1 (PINK1) are associated with autosomal recessive forms of Parkinson's disease (PD). In the mitochondrion, full-length PINK1 is proteolytically processed to lower molecular weight forms which are exported to the cytosol. While full-length PINK1 has been implicated in regulating mitophagy and mitochondrial function, the understanding of the functional role(s) of endogenous cleaved PINK1 (c- PINK1) in the brain is limited. Our recent research demonstrates a link of c-PINK1 and regulation of mitochondrial trafficking and dendrite outgrowth via PKA. This model is supported by preliminary and published data from our group, showing that loss of PINK1 function in vivo and in vitro impairs PKA-mediated dendrite connectivity and mitochondrial trafficking, but the connections between these two mechanisms are unresolved. The studies proposed will fill a critical void in our understanding of how c-PINK1 regulates PKA signaling to enhance dendrite connectivity and mitochondrial trafficking in PD models. In Aim 1, the molecular mechanisms by which PINK1 and dendrite-localized PKA protect dendrites from oxidative stress will be elucidated using image-based and molecular biology approaches. Aim 2 will elucidate the mechanisms by which PINK1 and PKA regulate mitochondrial trafficking in dendrites, and specifically test the hypothesis that PINK1 acts through PKA to increase mitochondrial content by phosphorylating the mitochondrial trafficking adaptor protein Miro2. Aim 3 will determine the mechanisms by which PINK1/PKA activation modulates neurite outgrowth. This work is expected to have an impact on health and human diseases in three areas. First, characterizing the PINK1-PKA signaling pathway will identify new protective mechanisms by which this novel signaling axis maintains dendrite homeostasis. Second, experiments proposed in Aims 1 and 2 will help us understand how PINK1 activates PKA signaling in mitochondria and dendrites regulate mitochondrial trafficking and protect dendrites from oxidative stress. Third, since dysregulation of PKA signaling, mitochondrial function, neurotrophic signaling, and loss of dendrites are implicated in multiple neurodegenerative diseases, identifying new dendrite-protective mechanisms can lead to new targeted, rational therapies via enhanced protective PKA signaling.

The broader impact/commercial potential of this I-Corps project is the improvement of the current standard of care for treating Parkinson's disease (PD), benefiting PD patients and healthcare practitioners. To date, there is no cure to reverse the course of PD and the current standard of care often focuses on managing motor symptoms (tremors, rigidity and loss of gait). A new drug delivery platform is developed to efficiently deliver nootropic agents to the brain in PD patients to alleviate symptoms and potentially reverse the course of disease. This new drug delivery platform was designed to reverse symptoms and cognitive decline while enhancing patient compliance though a non-invasive device that could complement or replace the current standard methods in an affordable manner without causing side effects. Beyond PD, the new drug delivery platform may be used to treat Alzheimer's disease and Lewy Body Dementia.

This I-Corps project develops a platform for reformulating oral forms of nootropic agents for intranasal delivery and use by Parkinson's disease (PD) patients. One of the major limitations of consuming oral versions of these nootropic agents is that these compounds do not efficiently reach the blood brain barrier and are extensively metabolized in the liver and gastrointestinal compartments. This inability to reach the target organs limits the drug's potential cognitive and brain-protective abilities in humans and limits drug efficacy in managing brain degenerative disorders such as PD. Prior preclinical research endeavors showed that the new intranasal formulations can deliver nootropic agents in high concentration in the brain in order to efficiently activate brain-protective signaling pathways and elevate energy output which can lead to better management of PD symptoms. For efficient and safe delivery to the brain via the nasal route, the intranasal formulations are compatible with a range of intranasal devices including nasal sprays, applicators, and atomizers.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.