Stefan Strack - US grants

DESCRIPTION (provided by applicant): The roles of protein phosphatases in neuronal function and dysfunction are poorly understood. Protein phosphatase 2A (PP2A) is a ubiquitous and abundant enzyme with roles in most cellular processes that involve reversible Ser/'rhr phosphorylation. In neurons, PP2A dephosphorylates a staggering array of substrates include cytoskeletal proteins, receptors and ion channels, neurotransmitter-synthesizing enzymes, kinases, and transcription factors. PP2A is a trimeric holoenzyme of two core subunits (a catalytic and a scaffold subunit) complexed to a variable, regulatory subunit. The identity of the regulatory subunit determines where the PP2A holoenzyme is localized, which substrates it dephosphorylates, and how its activity is modulated by intracellular signaling. None of this has been investigated in any detail. Some variable, regulatory subunits are only expressed in brain, suggesting that brain-specific PP2A holoenzymes have unique roles in neuronal physiology and development. A trinucleotide repeat expansion in the promoter region of one of these neuron-specific subunits, B[beta], has recently been linked to spinocerebellar ataxia type 12 (SCA12). Thus, aberrant PP2A subunit expression may be a hallmark of certain human neurodegenerative disorders. In this proposal, we explore the roles of the brain regulatory subunit B[gamma] in subcellular localization of PP2A holoenzymes and developmental signal transduction cascades. Aim 1 investigates the sequence determinants for differential subcellular localization of B subunits, and for association with the two core PP2A subunits. Proteins that interact with the B[gamma] subunit are identified in Aim 2 using yeast two-hybrid and protein purification techniques. Aim 3 investigates the function of neuronal PP2A regulatory subunits in Ras-MAP kinase signaling and neurite outgrowth of PC12 cells and primary neurons. These studies will significantly advance our understanding of how the ubiquitous PP2A enzyme regulates specifically neuronal functions, and may lead to the development of drugs that target neuronal PP2A isoforms.

[unreadable] DESCRIPTION (provided by PI): Ischemic stroke kills brain cells by causing massive depolarization and glutamate release, which in turn disrupts calcium homeostasis and generates free radicals. Mitochondria are central to these events, as well as to the wave of delayed cell death that follows the ischemic injury. Attention has focused on neuroprotective drugs that prevent the initial calcium overload, while interventions into later stages of neuronal damage have been largely neglected despite the promise of a wider treatment window. Protein phosphatase 2A (PP2A) is an essential and ubiquitous Ser/Thr phosphatase that predominately exists as a heterotrimer of two core subunits (a catalytic and a scaffolding subunit) complexed to a third, regulatory subunit Bp2 is an alternative splice variant of a PP2A regulatory subunit gene that is expressed only in the adult brain. A non-coding CAG repeat expansion in the B(3 gene causes the neurodegenerative disorder spinocerebellar ataxia type 12. The alternative N terminus of B(32 promotes translocation of PP2A to the outer mitochondrial membrane (OMM) to induce apoptosis in PC 12 cells (JBC 278:24976; JBC 280:27375), whereas interfering with endogenous mitochondrial PP2A protects neurons against ischemic insults in vitro. The proposal is centered on the observation that mitochondrial shape transitions underlie the pro- apoptotic activity of PP2A/Bp2, with overexpression of Bp2 fragmenting mitochondria and Bp2 silencing causing mitochondrial elongation in hippocampal neurons. Opposing the fission activity of the phosphatase is protein kinase A (PKA) anchored to the OMM via A-kinase anchoring protein D-AKAP1. Aim 1 test the hypothesis that OMM-localized PP2A and PKA control neuronal survival by reciprocally modulating mitochondrial fission and fusion, which in turn alters free radical and calcium homeostasis. Aim 2 explores the fission GTPase Drp1 as an effector substrate for outer-mitochondrial PP2A and PKA. In Aim 3, in vivo delivery of viruses expressing three kinds of PP2A/Bp2 antagonists is intended to provide proof-of-concept evidence that this neuron-specific pro-apoptotic protein is a promising target for stroke therapy. Finally, Aim 4 characterizes Bp2 null mice for mitochondrial changes and resistance to ischemic injury. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mitochondria provide energy, buffer calcium, and sequester cell death-inducing molecules, and mitochondrial dysfunction is implicated in various neuropathologies. Especially in neurons, mitochondria are highly dynamic organelles that constantly move, divide, and fuse. This proposal investigates the regulation of mitochondrial fission and fusion in neurons, which are antagonistic processes carried out by large GTPases similar to dynamin. Mutations in two of these enzymes, Opal and Mfn2, are responsible for hereditary neurological diseases. While a proper balance of mitochondrial fusion and fragmentation is clearly important for neuronal survival, some fragmentation is necessary for axonal and dendritic transport of mitochondria, and consequently for the development and function of synapses. We have found that shape changes of mitochondria are controlled by an opposing protein kinase and phosphatase that are localized to the outer mitochondrial membrane via specific targeting/regulatory subunits. On the phosphatase side, Bp2 is a neuron-specific, postnatally induced protein phosphatase 2A (PP2A) regulatory subunit mutated in spinocerebellar ataxia type 12. The alternatively spliced N terminus of Bp2 mediates translocation of the PP2A holoenzyme to the mitochondrial surface, where PP2A accelerates cell death, apparently by fragmenting mitochondria. The kinase opposing PP2A/Bp2's effect on mitochondrial morphology and survival is cAMP-dependent protein kinase (PKA) anchored to the OMM via A kinase anchoring protein (AKAP)121. Aim 1 investigates the mechanism by which outer-mitochondrial PP2A and PKA control neuronal survival. In Aim 2, we will identify the relevant substrates and phosphorylation sites among mitochondrial fission/fusion enzymes. Aim 3 addresses the role of PP2A/PKA-dependent mitochondrial restructuring in the delivery of mitochondria to and development of dendritic spines. Finally, Aim 4 characterizes PP2A/Bp2 knockout mice in terms of mitochondria and synapse morphology and resistance to ischemic injury. These studies will advance our understanding of how shape transitions of mitochondria are regulated, and how this affects vulnerability of neurons and the establishment of functional connections between them. Our studies may ultimately lead to better therapies for stroke and neurodegenerative disorders.

DESCRIPTION (provided by applicant): This is the second competitive renewal of a training program whose main goal is to promote the interdisciplinary training of graduate students in Pharmacological Sciences. Its secondary goal is to foster interactions among faculty and students from different departments and colleges at the University of Iowa that share an interest in Pharmacological Sciences. The program was initially funded in July 2004 for a period of three years and with three predoctoral slots to provide the initial resources necessary for further development of an interdisciplinary curriculum and training program that is at the core of the pharmacological sciences. As evidence of the interdisciplinary nature of this program, our trainees come from six different departments (Chemistry, Medicinal & Natural Products Chemistry [MNPC], Pharmacology, Physiology, Biochemistry, Anatomy & Cell Biology) in three colleges (Liberal Arts and Sciences, Pharmacy, and Medicine). The recruitment of 19 trainees with strong-to-outstanding credentials from outside the Department of Pharmacology (of a total of 27) is tangible evidence that this TG functions as a highly effective mechanism to attract the interest and promote the interdisciplinary training of graduate students in the Pharmacological Sciences regardless of departmental affiliation. This TG has led to a significant increase in the teaching, mentoring and research interactions among faculty and students in the two core departments, Pharmacology in the Carver College of Medicine (CCOM), and Medicinal and Natural Products Chemistry (MNPC) in the College of Pharmacy (COP) during the last funding period. Moreover, with a pool of 51 excellent faculty trainers from across the University, the program is strongly positioned to qualify for an additional five years of funding. A modest expansion of our TG from currently 6 to 8 slots is justified by 1) the quality and size of our applicant pool, 2) the diversity of our trainee pool (15% URMs, 41% women), and most importantly, 3) the success of our graduates. An expansion of this TG will aid our ability to attract nationwide students with excellent credentials to the Pharmacological Sciences and foster interdepartmental and intercollegiate collaboration at the U of Iowa. The curriculum established for this TG provides both basic and advanced instruction in Pharmacological Sciences, and has undergone continual review and revision to ensure that it fulfills the needs of the program and the students. The two 5-week, 1 semester hour (sh) modules Principles in Pharmacology (71:135) and Pharmacogenetics and Pharmacogenomics (71:136) serve as a concise and highly effective introductory course sequence for all trainees of this TG. Modularized in 2010 from a 3 sh course, this course sequence attracts a sizable number of graduate students (5-year average: 15 per year, of which 2/3rd are from outside the Pharmacology Ph.D. program). These students come from various departments and Ph.D. programs including Pharmacology, MNPC, Molecular & Cell Biology, Neuroscience, Physiology & Biophysics, Biochemistry, Anatomy & Cell Biology, and Chemistry. The increased enrollment reflects the successful reorganization of the course, enhanced emphasis on modern (genetics and genomics) in addition to core (dynamics and kinetics) subdisciplines, as well as enhanced interactions between trainers and trainees with common interests. We also host a unique course specifically for trainees of this program: Advanced Problem Solving in Pharmacological Sciences (71:250). Each month it features an in-depth lecture by a trainer on a research problem related to Pharmacological Sciences. Closely mentored by the trainer, students then work as a group to solve a problem and write a substantial NIH style research proposal. Finally, all trainees along with trainers attend the semi-weekly Pharmacology Seminar (71:204), which consists of research presentations by students in the graduate program in Pharmacology, the trainees of this program, as well as postdoctoral fellows and on- and off campus faculty.

DESCRIPTION (provided by applicant): Mitochondria are essential for ATP production, calcium homeostasis, and programmed cell death, and mitochondrial dysfunction is a hallmark of neuronal injury and degeneration. Mitochondria are highly dynamic organelles shaped by frequent fission and fusions events. While mutations in broadly expressed mitochondrial fusion enzymes are known to cause common neurodegenerative diseases (Charcot-Marie-Tooth disease and dominant optic atrophy), physiological regulatory mechanisms impacting the mitochondrial fission/fusion equilibrium are poorly defined. We previously identified an outer mitochondrial protein kinase and phosphatase pair, PKA/AKAP1 and PP2A/Bbeta2, which regulate mitochondrial shape via reversible phosphorylation of a highly conserved, inhibitory phosphorylation site (S656) in the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). In addition to controlling neuronal survival, PKA/PP2A-mediated phospho-regulation of Drp1 has profound consequences for dendrite and synapse development in cultured hippocampal neurons. This proposal builds on these in vitro results by exploring the role of PP2A/Bbeta2 and its antagonist PKA/AKAP1 in postnatal brain development and function in vivo. It addresses the overarching hypothesis that OMM- localized PKA and PP2A bidirectionally regulate synaptic connectivity and plasticity and ultimately cognition via Drp1 phosphorylation and mitochondrial dynamics. To this end, aim 1 will analyze brains of AKAP1 and Bbeta2 KO, as well as AKAP1/Bbeta2 double KO mice for altered development of dendrites, dendritic spines, and synapses. Aim 2 examines the impact of these structural changes on synaptic plasticity and learning and memory. Aim 2 also addresses the mechanism by which Bbeta2 and AKAP1 influence postnatal brain development and function by rescuing deficits with wild-type and mutant B?2 and AKAP1 expression, and by replacing endogenous with phospho-mutant Drp1 via stereotaxic delivery of lentivirus. These studies are meant to elucidate regulatory mechanisms controlling neuronal wiring and plasticity via mitochondrial dynamics. Knowledge gained with this proposal may spur the development of better treatments for CNS disorders involving abnormal synaptic connectivity and transmission.

Project Summary / Abstract Neurodevelopmental disorders including intellectual disability, autism, juvenile intractable seizures, and schizophrenia have high socioeconomic impact, yet poorly understood etiologies. Recent large scale sequencing efforts identified de novo mutations as a major cause of neurodevelopmental disorders. Most de novo mutations arise in the paternal germline to confer a growth advantage to mutant spermatogonia in what has been termed ?selfish spermatogonial selection?. Protein phosphatase 2 (PP2A), one of the major Ser/Thr phosphatases is a known regulator of growth and differentiation and a suspected tumor suppressor. A trimeric enzyme of catalytic (C), scaffolding (A), and variable regulatory subunits (B,B',B''), PP2A can exist in >50 subunit combinations in mammalian cells, presumably with distinct localization, substrates, and regulatory mechanisms. A surge of de novo mutations in PP2A uncovered since 2015 defined two new classes of autosomal-dominant mental retardation. The most common class is caused by recurrent missense mutations in one of the 12 PP2A regulatory subunit genes, PPP2R5D, the product of which, B'? predominates in human testes and brain. The same de novo PPP2R5D mutations cause human overgrowth, a syndrome commonly associated with intellectual disability and autism. This exploratory proposal seeks to identify molecular mechanisms by which recurrent de novo mutations in PPP2R5D (B'?) cause neurodevelopmental disorders. Because some neurodevelopmental disorders are reversible, our results may lead to new pharmacological interventions. Predicated by our published work predating the discovery of PP2A mutations in mental retardation, we hypothesize that de novo mutations in PPP2R5D cause neurodevelopmental disorders by a novel change-of-function mechanism. Specifically, we suggest that basic amino acids introduced into an acidic substrate-binding surface alter PP2A substrate specifity to impair some and favor other dephosphorylation events. This in turn may enhance growth/proliferative signaling pathways over those that mediate cell cycle exit, differentiation, and morphogenesis. To address this hypothesis, the two aims of this proposal will delineate consensus sequences for dephosphorylation by wild-type and mutant PP2A enzymes, identify their cellular substrates by quantitative phosphoproeomics, and uncover phenotypes in cell models of neuronal development. Fundamental insights from this proposal are expected to pave the way for new patient-derived cell models, animal models, diagnostic tests, as well as ultimately for PP2A-targeted therapies of neurodevelopmental disorders.  

Project Summary / Abstract Presenting with chronic pain or loss of sensation, peripheral diabetic neuropathy (PDN) is a debilitating comorbidity of diabetes that affects at least half the diabetic patient population. Since only palliative treatments are available, there is an urgent need for therapies that prevent or reverse the ?dying back? degeneration of peripheral axons in PDN. Recent evidence suggests that diabetes compromises mitochondrial structure and function in sensory neurons. However, the underlying mechanisms are unknown. Mitochondrial shape is controlled by opposing fission and fusion events. Mutations in mitochondrial fusion enzymes cause neurological disorders that present similarly to neurological complications in diabetic patients. Specifically, mitofusin-2 mutations result in Charcot-Marie-Tooth disease type 2A, a peripheral neuropathy characterized by primary axon degeneration, while mutations in Opa1 cause dominant optic atrophy, the most common form of hereditary blindness. The mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is activated by dephosphorylation of a highly conserved inhibitory PKA phosphorylation site. Two phosphatases target this site to promote mitochondrial fission, the Ca2+-dependent phosphatase calcineurin and a neuron-specific and mitochondria- localized isoform of protein phosphatase 2A containing the B?2 regulatory subunit (PP2A/B?2). We generated a mouse knock-out (KO) of B?2 and found elongated mitochondria in neurons, consistent with deletion of a Drp1 activator. B?2 KO results in a striking reduction in infarct volume following ischemic stroke, indicating that mitochondrial elongation is neuroprotective. Conversely, knocking out A Kinase Anchoring Protein 1 (AKAP1), the protein that recruits PKA to the outer mitochondrial membrane to maintain Drp1 in a phosphorylated and inhibited state, causes mitochondrial fragmentation and exacerbates stroke injury. Supported by preliminary evidence that B?2 KO mice are resistant to peripheral neuropathy in both type-1 and type-2 diabetes models, the present proposal seeks proof-of-concept evidence for B?2 (and other, as yet undiscovered, neuron-specific Drp1 activators) as a drug target for the treatment of PDN. We further propose to investigate how diabetes causes mitochondrial fragmentation in sensory neurons and how inhibiting mitochondrial fragmentation protects peripheral axons in diabetes. Using new mouse models and innovative in vivo imaging approaches, we will test the overarching hypothesis that dysregulation of the mitochondrial fission/fusion equilibrium contributes to the pathogenesis of diabetic neuropathy, and that inhibition of Drp1- dependent mitochondrial fission provides neuroprotection via improvement of mitochondrial metabolism, reduction of ROS, modulation of mitochondrial Ca2+ transport and enhanced regeneration of sensory axons. We anticipate that these studies will shed light on PDN etiology, suggest new therapeutic strategies, and thus help improve quality of life for a rapidly growing diabetic population.

Project Summary Optimal functioning of the medial prefrontal cortex (mPFC) relies on synaptic connections made onto dendritic spines in pyramidal neurons. Prefrontal dysfunction resulting from chronic stress and stress-related psychiatric illnesses are each linked to decreases in dendritic spine number and shape alterations in this cortical region. Work from our laboratory and others has shown that chronic stress and elevated glucocorticoids, the end products of the hypothalamo-pituitary-adrenal stress axis activation, induce structural deficits marked by dendritic spine loss in mPFC and impaired prefrontal cognitive functions. While some progress has been made in elucidating the biochemical and genetic components underlying disrupted prefrontal plasticity in animal models of psychiatric illnesses, more work is needed to identify the cellular mechanisms accounting for these changes to help develop targets for therapeutic intervention. In this regard, recent consideration has been given to the idea that mitochondrial deficiencies may contribute to chronic stress-induced cognitive impairments and the pathogenesis of human psychiatric disorders. A-kinase anchoring protein 1 (AKAP1) is a mitochondrial scaffolding protein that recruits protein kinase A (PKA) to the outer mitochondrial membrane leading to phosphorylation and inactivation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1). We have previously shown that AKAP1 increases mitochondrial membrane potential, an indicator of the cell's ability to generate ATP by oxidative phosphorylation, whereas deletion of AKAP1 leads to mitochondrial fission and dendritic spine loss in cortical neurons. These observations have culminated in the novel hypothesis that chronic stress-induced alterations in prefrontal structural and functional plasticity are mediated by diminished AKAP1 signaling in mPFC neurons. Therefore, the goal of this exploratory/developmental R21 is to examine the role of AKAP1 in chronic stress-induced dendritic spine loss and functional compromise in mPFC neurons, thus developing a data set for a future R01. In aim 1, we will interrogate whether CVS's adverse effects on prefrontal dendritic spine structure are accompanied by AKAP1 loss, Drp1 dephosphorylation/activation, and mitochondrial fragmentation. Aim 2 will use AKAP1 KO mice and lentiviral delivery of wild type and PKA-binding deficient AKAP1 to identify the signaling mechanisms accounting for disruption of prefrontal mitochondrial, dendritic spine, and behavioral alterations. The long-term goal of this line of research is to elucidate the key mechanisms linking mitochondrial dynamics to stress-related prefrontal dysfunction, as this is a common underlying feature of stress-related psychiatric disorders such as major depressive illness.