Xiaoxi Zhuang - US grants

DESCRIPTION (provided by applicant): This R21 proposal aims to develop a versatile gene-trap approach for insertional mutagenesis in mouse embryonic stem (ES) cells. The specific applications of this approach include: 1) It will generate null mutations in trapped genes by transcriptional termination rather than protein truncation. 2) Expression of the trapped gene can be controlled spatially by tissue-specific repair of the null mutation following recombinase-mediated excision of the trap vector. 3) Expression of the trapped gene can be controlled temporally by a tetracycline-dependent promoter in the trap vector. 4) The expression pattern of the trapped gene can be easily assessed by B-galactosidase expression. 5) Genomic DNA of the trapped gene can be rapidly cloned and sequenced following plasmid rescue. 6) cDNA of the trapped gene with full-length coding sequence can be rapidly cloned and sequenced following 3' RACE. 7) The promoter of the trapped gene can be used to express any genes of interest following recombinase mediated cassette exchange in ES cells and replacement of the trap vector with the gene of interest. Specific aim 1 is to construct the trap vector. Aim 2 is to test all functional units in the trap vector in ES cells. Aim 3 is to test the above applications in mice derived from independent ES cell clones. The long-term goal of this project is to generate a C57BL/6 ES cell library and mutant mice that will permit spatial and temporal control of expression of trapped genes. They will provide valuable resources to the scientific community for the study of gene function in vivo.

DESCRIPTION (provided by applicant): Lack of inhibitory control over apparently voluntary motor acts is a behavioral symptom found in attention deficit hyperactivity disorder, drug addiction, Tourette's Syndrome, and obsessive-compulsive disorder. It is highly debated whether impaired inhibitory control is due to hyper- or hypo-dopaminergic activity. To investigate its neurobiological basis, we have generated "hyperdopaminergic" transgenic mice by reducing the dopamine transporter expression (DAT knockdown). Our preliminary studies suggest that DAT knockdown mice model aspects of impaired inhibitory control and that impaired postsynaptic dopamine D2 receptor function may underlie such behavioral deficits. Aim 1 of the proposed studies is to determine the specific biochemical changes underlying impaired inhibitory control in DAT knockdown mice. We will first identify the behavioral mechanisms underlying impaired inhibitory control in DAT knockdown mice: whether it's delay aversion or response inhibition, two processes implicated as core behavioral deficits in impaired inhibitory control. We will then investigate what signaling molecule is responsible for the impaired postsynaptic D2 receptor function in DAT knockdown mice: receptor, G protein, receptor-G protein coupling or adenytyl cyclase activity. We will take both behavioral measures and biochemical measures from each animal and examine the degree of association between them. Aim 2 is to test the hypothesis that impaired postsynaptic D2 receptor function underlies impaired inhibitory control using a pharmacological rescue approach. Our preliminary studies suggest that chronic D2 antagonist treatment is able to reverse behavioral deficits in DAT knockdown mice. We will analyze D2 signaling and behavioral changes in DAT knockdown mice after chronic treatment with D2 antagonists. We will test the hypothesis that such treatment will reverse D2 signaling and behavioral deficits in DAT knockdown mice. Aim 3 is to test directly the hypothesis that impaired D2 receptor function underlies impaired inhibitory control using mice that lack or have reduced postsynaptic D2 receptors.

[unreadable] DESCRIPTION (provided by applicant): While motor habits are important for the development of motor skills, aberrant motor habits are implicated in drug addiction. Motor habits are uniquely different from other types of memory. They take a long time to form and are hard to be modified. We hypothesize that this unique feature makes it hard to reverse aberrant motor habits in drug addiction. What makes motor habits different from other types of memory? The answer may lie in the unique signaling pathways in striatal medium spiny neurons (MSNs). The converged inputs to the MSNs from glutamatergic neurons in the cortex and from dopamine neurons in the midbrain are implicated in corticostriatal synaptic plasticity and habit learning. Based on published studies and studies done in our lab, we hypothesize that adenylyl cyclase type V (ACS) which is selectively and highly expressed in adult striatum is a key determinant of stability of corticostriatal synapses and stability of motor habits. However, during early postnatal development, adenylyl cyclase type I (AC1) rather than ACS is highly expressed in the striatum. In contrast to ACS which promotes synaptic stability, AC1 promotes synaptic plasticity. Therefore, the silencing of AC1 expression during postnatal development and emergence of ACS as the dominant isoform may be a major event in neural development that has corresponding behavioral consequences. The AC1 promoter sequence is GC rich and contains CpG islands, making it potential targets for epigenetic regulation. The proposed studies aim to 1) examine epigenetic mechanisms in turning off AC1 expression in striatum during development; 2) test the hypothesis that reawaking AC1 expression in adult striatum will make motor habits more susceptible for modifications. These studies will point to potential epigenetic interventions in the future that may be able to treat drug addition by reversing motor habits. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We have generated mutant mice that harbor null alleles of DJ-1 and PINK1. Both genes are linked to recessive form of early-onset familial Parkinson's disease (PD). Our published and unpublished data indicate that DJ-1 deficient mice develop progressive age-dependent hypolocomotion, dopamine system dysregulation, body weight loss and skeletal muscle degeneration. Similarly, muscle degeneration has recently been reported in PINK1 deficient flies. In contrast, PINK1 null mice only display very mild phenotypes (our unpublished data). Recently, PD cases that are heterozygous for DJ-1 and PINK1 mutations have been reported. We reason that DJ-1 and PINK1 might be components of a biochemical pathway and that DJ-1;PINK1 double mutant mice might develop important phenotypes that are not captured by either DJ-1 deficiency or PINK1 deficiency alone. We have successfully obtained DJ-1;PINK1 double null allele (DJ-1 and PINK1 are 11.9 Mb apart). To our surprise, deficiency for both DJ-1 and PINK1 leads to early embryonic lethality, further reinforcing the idea that DJ- 1 and PINK1 might be components of a biochemical pathway that is crucial for cell survival in multiple tissues. We propose to generate a series of mutant mouse lines with intermediate DJ-1 PINK1 gene doses in order to obtain intermediate phenotypes between early embryonic lethality (DJ-1;PINK1 double mutants) and muscle degeneration but no dopamine neuron loss (DJ-1 single mutants). These mice will be important tools to investigate DJ-1 and PINK1 function and their interactions related to the pathogenesis in Parkinson)s disease as well as in other degenerative diseases. PUBLIC HEALTH RELEVANCE: Recently published studies and our own data suggest that two genes, DJ-1 and PINK1, may work together and play important roles in Parkinson's disease and other degenerative diseases. We propose to generate animal models to examine in detail such a biochemical pathway that is important for different degenerative disease. Our results will point to new therapeutic targets and therapeutic approaches. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): L-DOPA-induced dyskinesia (LID) in Parkinson's isease (PD) is due to aberrant corticostriatal plasticity. However, how chronic L-DOPA treatment in advanced stage PD causes such aberrant plasticity remains unknown. We focus on the hypothesis that it is the regulated phasic dopamine release that modifies corticostriatal synaptic strength. In advanced stage PD, L-DOPA is able to restore tonic dopamine but not regulated phasic dopamine release from dopamine neurons due to the loss of dopamine terminals in the striatum. Instead, unregulated fluctuations in dopamine release due to L-DOPA therapy will cause aberrant modification of corticostriatal synaptic strength and LID. cAMP pathway is important for corticostriatal plasticity. The main adenylyl cyclase (AC) isoform in the striatum is the calcium-calmodulin (CaCaM)- insensitive isoform AC5. Accordingly, cAMP production in adult striatum relies on G protein coupled receptors. Such a lack of CaCaM stimulation of cAMP production and its reliance on G protein coupled receptor activation makes corticostriatal neuroplasticity dependent on dopamine input. We have generated transgenic mice that express the CaCaM-dependent AC1 in striatal neurons. We hypothesize that in these mice, cAMP pathway in striatal neurons will be activated through CaCaM and NMDA receptor activation from cortical glutamatergic input independent of dopamine input. In advanced stage PD patients who have already lost most of regulated phasic dopamine release and wide fluctuations of dopamine levels contribute to aberrant plasticity, attenuation of dopamine influence on the aberrant plasticity will blunt the effect of dopamine on dyskinesia. The proposed studies aim to provide proof of principle for its potential therapeutic effects on LID. PUBLIC HEALTH RELEVANCE: L-DOPA-induced dyskinesia is a main problem in arkinson's disease therapy. We will test that adenylyl cyclase is a valid target for modifying corticostriatal plasticity and preventing L-DOPA induced dyskinesia.

DESCRIPTION (provided by applicant): In Parkinson's disease (PD), dopamine replacement therapies work well for the first a few years. However, in late stage PD, such therapies cause dyskinesia that is often more debilitating than PD itself. There is an urgent need to develop alternative approaches to treat motor impairment. We recently found that mice deficient for the striatum enriched adenylyl cyclase (AC) type V (AC5) display dopamine D2 receptor- independent motor control. However they are still dependent on dopamine D1 receptors. We hypothesize that over-expression of adenylyl cyclase type I (AC1) in striatal D1 receptor positive neurons will enable D1 receptor-independent motor control. We further hypothesize that double transgenic mice with both AC1 over-expression in D1 neurons and AC5 deficiency will display dopamine-independent motor control. We propose to generate these transgenic mice and test our hypotheses using dopamine antagonists, dopamine depletion and dopamine neuron lesions. We will examine motor functions using locomotor activity, akinesia test, gait analysis and rotarod. PUBLIC HEALTH RELEVANCE: We will generate transgenic mice with dopamine-independent motor control. Positive results will provide proof of principle and will guide the development of non- dopamine replacement based therapies for Parkinson's disease.

Abstract The rewarding aspects of food is an important driving force mediating the decisions to seek food, including the overeating observed in many cases of obesity. In recent years, studies suggest that a lower functional dopamine system is associated with obesity. Our preliminary data suggest that the dopamine system, the central component of reward pathways, is especially important when food seeking-related energy cost is an important factor in decision to seek food. We hypothesize that individuals with lower dopaminergic function will shift their feeding behavior toward overeating when food is relatively easy to obtain. Our hypothesis would predict that lower dopaminergic function or leptin signaling deficiency in dopamine neurons can in deed cause obesity but only in an environment with abundance of low-cost and energy-rich foods, typical of the modern society. The proposed experiments are designed to test the causal link between hypo-dopaminergic activity and obesity and the hypothesized mechanisms. We will first establish whether hypo-dopaminergic activity and the hypothesized mechanisms contribute to obesity in leptin deficient mice. We will then establish whether hypo-dopaminergic activity and the hypothesized mechanisms contribute to diet-induced obesity. Lastly, we will examine gene-environment interactions in food seeking behavior and obesity. We will test whether dopamine D2 receptor deficient mice will be more susceptible and hyperdopaminergic mice will be more resistant to diet induced obesity in an environment with abundance of energy-rich foods.

DESCRIPTION (provided by applicant): Although energy homeostasis is central in feeding behavior, the decision to eat is not merely a question of hunger. In a natural environment in which food is often scarce, evaluating the benefit of gaining food over the energy cost or risk in seeking and acquiring food, and the decision to either seek food or stay put and conserve energy is essential for survival. However, the genetic and molecular basis of food-seeking decision-making behaviors, and their functional significance in fitness are poorly understood. We propose a bottom-up approach: starting from well-defined behaviors that are essential for food-seeking decision-making in Drosophila, taking advantage of its rich genetic tools and genomic resources. Moreover, flies are ideal for viability studies to examine the relations between genetic variations, differences in decision- making and differences in fitness under different environmental conditions. We have already taken the critical step and designed novel assays. We will optimize the following assays for quantitative analysis in this application. Decision-making in choosing between 1) small free reward or large reward that requires work; 2) small reward or large reward with a relatively low probability in its availability; 3) small reward or large reward that is paired with potential risks of getting noxious food; and 4) immediate small reward or delayed large reward. We will take advantage of natural variation in genetics and behaviors between different D. melanogaster populations living in African. We will establish 200 recombinant inbred strains derived from original African strains with extreme behavioral phenotypes for each decision-making behavior. We will perform QTL mapping and identify QTLs that contribute to the variations in food-seeking decision-making and fitness. We will then use RNAi transgenic lines to identify specific genes and variants responsible for the above behavioral variations. Although the most likely immediate clinical relevance of our study is obesity, it will have a much broader impact on neuroeconomics, the genetic basis of behavior in general, evolution and ecology.

DESCRIPTION (provided by applicant): Epidemiological studies have consistently shown that the incidence of Parkinson's disease (PD) is reduced in smokers. However, the mechanism by which smoking acts to reduce the incidence of PD remains unknown. Our earlier studies have demonstrated that dopamine (DA) blockade promotes increased activity of the inhibitory, indirect pathway leading to learned inhibition of movement, a phenomenon that we term 'aberrant motor learning.' In this application, we aim to test the hypothesis that chronic nicotine by desensitizing ¿2-containing nAChRs, prevents such aberrant learning and lessens PD symptoms. Specific Aim 1 will dissociate whether ¿2 deactivation on DA neurons is sufficient to block aberrant motor learning and whether the specific deletion of this population of ¿2-containing nAChRs prevents the initial acquisition of aberrant learning during DA receptor blockade or facilitates the acquisition of new learning once DA signaling is restored. Specific Aim 2 will delineate the mechanism by which chronic nicotine, acting via ¿2-containing nAChRs, blocks aberrant learning. Given that nicotine administered over a period of time significantly alters DA release, we will test the hypothesis that such adaptive changes have protective effects against aberrant synaptic plasticity induced by DA blockade.

? DESCRIPTION (provided by applicant): Dopamine signaling has been implicated in many aspects of reward. Despite the vast literature on this important subject, it is still highly debate if and how regional and cell-type-specific dopamine signaling plays distinct roles in specific aspects of reward. Both dopamine D1 and D2 receptors are mainly coupled to the cAMP pathway in striatal medium spiny neurons (MSNs). Our studies in recent years indicate that dopamine signaling through cAMP is essential for corticostriatal plasticity. Moreover, the adenylyl cyclase type 5 (AC5) deficient (AC5KO) mice are severely impaired in appetitive associative learning. AC5 is the main adenylyl cyclase in the striatum and nucleus accumbens for dopamine-cAMP signaling. Identification of such a key molecule in appetitive associative learning provides a unique opportunity to perform precise manipulations and to rigorously test the necessity and sufficiency of regional and cell type specific dopamine signaling in specific aspects of appetitive associative learning. One of the most important issues in the field is that the conditioned stimulus (CS) acquires both predictive value and incentive value. Dopamine is implicated in both processes. A second important issue that has not been resolved is that the lack of conditioned approach responses linked to dopamine signaling deficiency could be due to a true lack of CS-reward association or due to the animal's inability to engage motivational and motor systems and promote the approach behavior. Aim 1 of this application will address these issues. For Aim 2, we have generated a condition AC5 allele that will allow us to achieve AC5 gene deletion in specific regions and cell types using the Cre recombinase mediated gene deletion. These studies will allow us to test necessity of AC5 in these cells in supporting specifi behavioral processes. In addition, we will perform virus mediated regional and cell type specific AC5 re-expression on the AC5KO background. This will allow us to test sufficiency, i.e., to test if AC5 in this brain region and this cell type is sufficient in supporting specific behavioral processes.

The therapeutic effects of L-DOPA are remarkable in early stage Parkinson's disease (PD). However, with chronic dopamine replacement therapy, motor side effects such as dyskinesia become a severe problem in advanced PD. Mechanisms underlying PD symptoms and therapy are still poorly understood. Our recent studies in animal models have for the first time demonstrated that experience-dependent aberrant motor learning (learned motor inhibition) under low dopamine conditions may play a major role in PD motor symptoms, which was supported by recent studies on PD patients and by computational models. Moreover, we have demonstrated that glutamatergic inputs in combination with dopamine signaling through the adenylyl cyclase type 5 (AC5) and the cAMP pathway contributes to corticostriatal long-term potentiation and depression (LTP and LTD) in the dopamine D2 receptor-expressing striatal medium spiny neurons (MSNs). More importantly, we have found that aberrant LTP is associated with aberrant motor learning while prevention of such aberrant LTP is associated with prevention of aberrant motor learning. Our behavioral and electrophysiological advances have set the stage for identifying and testing potential PD therapies based on preventing and/or reversing aberrant corticostriatal LTP. In this application, we propose to test the precise conditions/parameters for induction of corticostriatal LTP/LTD in D2-expressing MSNs. We then aim to establish a causal link between aberrant corticostriatal LTP and aberrant motor learning as well as to test treatments that can prevent such aberrant corticostriatal LTP and aberrant motor learning. Finally, we will test the therapeutic effects of preventing and reversing aberrant LTP in PD models.

PROJECT SUMMARY/ABSTRACT The Integrative Training in the Neurobiology of Addictive Behaviors Program at The University of Chicago offers training for both pre- and postdoctoral trainees in drug abuse-related research. We have an outstanding panel of core faculty trainers, who are productive researchers, with successful training credentials of both pre- and postdoctoral trainees. Their expertise and therefore their training areas, range from molecular biology, electrophysiology, pharmacology and animal models of drug addiction, to the social, behavioral and psychopharmacological aspects of human drug use, including etiology, treatment and drug policy. Our goal is to prepare the next generation of scientists to investigate the etiology, prevention and treatment of drug abuse with integrative approaches. We propose a program for 3 predoctoral and 4 postdoctoral trainees. Trainees will be supported by the Training Program for 1-3 years. Trainees obtain specialized intensive training in their `home' laboratories, but they are also exposed to the full breadth of addictions research outside their own area, from molecular to policy. In addition to their doctoral program requirements, predoctoral trainees will participate in a bi-weekly training grant seminar and a didactic course providing a broad overview of addiction (history, epidemiology, pharmacology, treatment and policy). Trainees will also participate in 20-hour rotation experiences in laboratories conducting addictions research distinct from their own expertise. The organizational structure will include an Executive Committee making overall training decisions, a Selection Subcommittee to ensure a good flow and balance of trainees, a Program and Review Subcommittee to monitor trainees' progress through the program, and an External Advisory Committee to provide expert outside consultation. The University of Chicago provides a unique environment with both a long history of interdisciplinary collaboration and recently a significant commitment to neuroscience research. This provides a rich intellectual context for trainees in drug abuse related research. To ensure that there is substantial interaction among the trainees, and between trainees and trainers, we will schedule bi-weekly meetings consisting of journal club presentations, seminars by invited speakers, and didactic classes. We will devote resources to recruiting underrepresented minorities and we will provide comprehensive training in ethical conduct of research.

Abstract Aberrant synaptic plasticity is a key underlying biological mechanism of drug addiction. The long-lasting synaptic changes are dependent on changes in gene expression and evidence support the involvement of epigenetic mechanisms. However, histone modifications, DNA methylation or DNA hydroxymethylation affects gene expression at transcriptional level, which is not only slow in affecting protein synthesis but also lacks synaptic specificity. RNA N6-methyladenosine (m6A) modification has been found to significantly affect RNA splicing, export, localization, translation efficiency and stability. All there are key factors in regulating translation and potentially localized translation in synapses. Both our preliminary data and published studies suggest that deficiency in m6A dependent pathways significantly impairs neuronal function including dopamine signaling and dopamine dependent learning. Therefore we propose to focus on the role of m6A modification of mRNAs in synaptic plasticity and in drug addiction models. We will use mice with the m6A methyltransferase METTL14 deleted in D1 positive striatal neurons in the nucleus accumbens. In Aim 1, we will test the hypothesis that mutant mice have impaired appetitive Pavlovian learning and impaired cue-induced reinstatement of cocaine self-administration. In Aim 2, we will record from brain slices and test if mutant mice have impaired corticostriatal plasticity and diminished changes in corticostriatal synaptic strengths caused by cocaine exposure. In Aim 3, we will determine how m6A RNA methylation regulates synaptic protein translation in responses to drug challenges and other changes on neuronal activity. We will first examine if cocaine exposure in vivo affects level of m6A RNA methylation and identify downstream targets. The rest of the proposed experiments will examine the degree to which m6A RNA methylation affects protein translation in the soma vs. dendrites where local translation is under control of synapses. We will use dissociated neuronal cortical-striatal co-cultures from METTL14 knockout and control mice.

Abstract Local protein synthesis in dendrites plays a significant role in synaptic plasticity. For example, the activity-regulated cytoskeleton-associated protein (ARC/ARG3.1), an immediate early gene, is required for many forms of synaptic plasticity and long-term memory formation. It has been reported that Arc transcription is rapidly induced after novel environmental exposure or stimuli that induce synaptic plasticity. Its messenger RNA (mRNA) is quickly transported to dendrites soon after synthesis for local translation by active synapses. Protein synthesis is a heavily- regulated process. It has been recently discovered that m6A RNA methylation, an epitranscriptomic modification, and m6A reader proteins (RNA binding proteins that recognize m6A and cause downstream functional consequences), play key roles in mRNA nuclear export, transport, localization, translation efficiency and stability. Although the specific significance of m6A RNA methylation in the nervous system is not well understood, using mice deficient in m6A or mice deficient in m6A readers, we have found that m6A RNA methylation plays a significant role in learning and memory. However, the link between m6A regulation of mRNA localization/translation and learning/memory is still unknown. We hypothesize that m6A methylation of mRNAs regulates the rates of protein synthesis from dendritically localized mRNAs and affects synaptic plasticity. As a first step to test our hypothesis, we propose to focus on Arc for the following reasons. We have discovered several m6A sites on Arc mRNA. Arc protein is synthesized locally in dendrites. Arc plays a significant role in synaptic plasticity. We will manipulate m6A levels in the Arc mRNA and determine how Arc localization and local protein synthesis will be affected by m6A and by stimuli that induce synaptic plasticity.

PROJECT SUMMARY Both neurodevelopment and the synaptic plasticity events that underlie learning and memory rely heavily on tightly regulated gene expression programs and rapid, finely-tuned translation of messenger RNA (mRNA) transcripts. The degradation, stability, and translation of mRNA has, in recent years, been found to be regulated by adenosine methylation, which alters both transcript structure and the recruitment of RNA-binding proteins that inform these activities. Recent identification of and experimentation with the methyltransferases (?writers?), demethylases (?erasers?), and specific methyladenosine binding proteins (?readers?) have established that these epitranscriptomic mRNA regulatory processes are both dynamic and tightly regulated. Although the most well-studied of these modifications is N6-methyladenosine (m6A), N1- methyladenosine (m1A) has also recently emerged as a prevalent epitranscriptomic mark. Current methods used to explore these modifications require large sample sizes and are inherently low-resolution. These limitations preclude them from mapping and quantifying the epitranscriptome in specific brain regions, or in clinical biospecimens. Here, we describe preliminary development of innovative technologies to precisely sequence and probe the function of specific m6A and m1A modifications. We propose to leverage these foundations in the service of the following specific aims: 1) Evolve and establish high-resolution, antibody-free m6A and m1A mapping platforms for brain analysis, 2) Design and validate a molecular toolkit to manipulate transcript-specific m6A and m1A modifications in vivo, and 3) Catalog m6A and m1A modifications in the brain across development, neuron populations, activity state, and in synapses, and determine their function in relation to learning and memory. Our findings will illuminate how the epitranscriptomic landscape and specific mRNA transcripts in discrete neuronal populations regulates gene expression to inform complex neuronal processes, such as development, learning and memory, and how perturbations thereof result in abnormal brain function such as learning impairment. Importantly, this translational, functional validation of our new tools, which will be made available to the research community, provides a strong foundation for their usage to specifically interrogate how mRNA modifications are perturbed in other pathological contexts.

Abstract Pyridoxine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting inactive forms of Vitamin B6 (VB6) in diet, including pyridoxine and pyridoxamine, to the only active form, pyridoxal 5'-phosphate (PLP). PLP is a cofactor required for the syntheses of dopamine, serotonin and GABA in the brain. In humans, PNPO deficiency is known to cause neonatal epileptic encephalopathy (NEE). Mutations in PNPO have been increasingly reported in NEE patients. Recent studies also identify PNPO as a contributor to early-onset epilepsies and one of the16 epilepsy genes involved in the common epilepsies. However, due to the lack of animal models, we know little about the developmental or adult functional impact of PNPO deficiency at systems, circuit or cellular level (e.g. involvement of GABA, dopamine or serotonin synthesis) under in vivo conditions. We know little about how mild PNPO deficiency interacts with other genetic defects or environmental factors (e.g. VB6 in diet) to cause seizures or other conditions. We have identified the Drosophila homolog of PNPO and identified a Drosophila mutant (sgll95 flies) with partial PNPO deficiency. Due to low PNPO activity, they are sensitive to dietary VB6 deficiency. We have since generated global knock-down as well as knock-in models in which the endogenous wild-type (WT) fly PNPO was replaced by human mutant PNPO found in patients. Viability during development, lifespan and seizure phenotype of these flies depend on the specific genetic manipulation as well as availability of VB6 in diet. We have also found that PNPO deficiency exacerbated other epileptic mutant alleles in flies with significant synergistic interactions. In Aim 1, we will define specific developmental stages in fly models in which PNPO deficiency leads to lethality and seizures. In Aim 2, we will define brain specific cell types involved in PNPO- deficiency-induced lethality and seizures in fly and mouse models. We will test gene-gene interactions (e.g., PNPO and other known epilepsy genes). In Aim 3, we will generate and characterize fly and mouse models that carry human PNPO mutations. 1