Lily Y. Jan - US grants

We propose to study embryogenesis of the nervous system in Drosophila. Techniques employed include genetics and the use of antibodies. Monoclonal antibodies that recognize all, or part of, nervous tissues have been generated and may be useful in two aspects: On the one hand, they serve as markers in studying neural development in normal and mutant animals. On the other hand, they provide a chance for me to get at molecules that are potentially important for development. A major difficulty of the latter approach concerns the question of causality. Even if the antigen recongized by a monoclonal antibody shows an interesting distribution and developmental profile, it is difficult to know whether the antigen itself is developmentally important or whether it is merely a secondary byproduct. This may be resolved if one can show that the monoclonal antibody functionally blocks specific processes during development. Alternatively, if mutations of the structural gene coding for an antigen block specific processes during development, that antigen is probably important developmentally. For genetic studies Drosophila has been a favorable animal. A variety of developmental mutations have been isolated and analyzed. It is possible to use methods such as segmental aneuploidy to localize structrual genes, and to isolate mutations of these genes once they are localized on the salivary chromosome. The wealth of Drosophila genetics may be used to test critically whether a particular gene product is important developmentally. The monoclonal antibodies that we have generated so far subdivide neurons and fiber pathways in various ways and show different developmental profiles. Further, immunohistochemical experiments revealed that the pattern of fiber pathways in the central nervous system of Drosophila embryos appeared very similar to that in grasshopper embryos. To study neurla development in Drosophila embryos, we hope to draw from the rich source of information obtained from studies on grasshoppers. A number of monoclonal antibodies generated so far recognize embryonic nervous tissue in both insects and may serve as a bridge, allowing one to combine Drosophila genetics with advantages of larger insects such as the grasshopper.

One of our approaches to neurobiology is to study perturbations in synaptic function or development caused by single gene mutations. In much the same way as previously done for biosynthetic pathways in microorganisms, specific components of neural activity or steps in neural development may be studied by altering specific genes. Neuromuscular junctions in Drosophila larvae are very accessible to neurophysiology, because the preparation is thin and visible with Normarski optics, and the nerve terminals are readily accessible to experimental manipulations. The quantal nature of transmitter release, the ionic basis of the membrane resting potential and the excitatory junctional potential, as well as postsynaptic action of L-glutamate, have been worked out in detail. Since single-gene mutations affecting the synapse can be isolated, one can combine genetics, electrophysiology and biochemistry in studying the synapse. So far three mutations affecting neuromuscular transmission have been found, mapped genetically and studied electrophysiologically. In addition, several mutations in the bithorax gene complex, studied extensively by Dr. E. B. Lewis at Caltech, were found to produce specific changes in the larval muscle pattern. More mutations affecting the synapse will be studied with the hope of revealing hitherto unsuspected processes underlying synapse formation or changes of synaptic efficacy.

The hippocampus has been implicated in memory acquisition in man and other mammals. Slice preparations of the hippocampus allow cellular studies of synaptic transmission and its long term potentiation (LTP), processes reminiscent of associative learning. Mechanisms underlying LTP are likely to involve presynaptic changes in the amount of transmitters released as well as postsynaptic changes in the receptors or the intracellular second messengers involved in transducing the signal. LTP may last for hours and its maintenance requires protein synthesis, indicating that the long term changes in synaptic efficacy may involve gene regulation and structural alterations of the synapse. Transcriptional regulators such as c-fos and zif 268 are found to be up regulated at the transcriptional level by electrical activities in different regions of the brain including the hippocampus. The transient increase of these transcriptional regulators are likely to be the first in a chain of events that lead to long term changes in the expression of genes coding for specific functional elements and structural components of the synapse. From studies of cultured PC12 neurons, it is apparent that some of the genes coding for transcriptional regulators (e.g. c-fos) are themselves regulated at the transcriptional level not only by electrical activities but also by nerve growth factor. These findings suggest that the trophic factors present in the adult brain are likely to control not only the survival of neurons and their processes but also the expression of some of the genes involved in synaptic plasticity. Conversely, receptors of neurotransmitters such as the serotonin receptor have been found to play a potential role in neurite outgrowth, and to stimulate cell growth and division. These new findings illustrate the convergence of "functional" and "developmental" studies. This convergence is particularly important in studies of learning and memory which may involve both functional and structural changes of neurons and synapses. The proposed study includes, on the one hand, identification and characterizations of molecules potentially involved in these processes, including transmitter receptors, channels, trophic factors and their receptors, and transcriptional regulators, and on the other hand, functional studies at the cellular level on LTP, and possible involvement of these molecules in LTP. This concerted effort, combining the different expertise of the five investigators in this group, allow the approach to the general questions concerning the basis of learning and memory in a manner that is beyond the reach of any one of these researchers.

The hippocampus has been implicated in memory acquisition in man and other mammals. Slice preparations of the hippocampus allow cellular studies of synaptic transmission and its long term potentiation (LTP), processes reminiscent of associative learning. Mechanisms underlying LTP are likely to involve presynaptic changes in the amount of transmitters released as well as postsynaptic changes in the receptors or the intracellular second messengers involved in transducing the signal. LTP may last for hours and its maintenance requires protein synthesis, indicating that the long term changes in synaptic efficacy may involve gene regulation and structural alterations of the synapse. Transcriptional regulators such as c-fos and zif 268 are found to be up regulated at the transcriptional level by electrical activities in different regions of the brain including the hippocampus. The transient increase of these transcriptional regulators are likely to be the first in a chain of events that lead to long term changes in the expression of genes coding for specific functional elements and structural components of the synapse. From studies of cultured PC12 neurons, it is apparent that some of the genes coding for transcriptional regulators (e.g. c-fos) are themselves regulated at the transcriptional level not only by electrical activities but also by nerve growth factor. These findings suggest that the trophic factors present in the adult brain are likely to control not only the survival of neurons and their processes but also the expression of some of the genes involved in synaptic plasticity. Conversely, receptors of neurotransmitters such as the serotonin receptor have been found to play a potential role in neurite outgrowth, and to stimulate cell growth and division. These new findings illustrate the convergence of "functional" and "developmental" studies. This convergence is particularly important in studies of learning and memory which may involve both functional and structural changes of neurons and synapses. The proposed study includes, on the one hand, identification and characterizations of molecules potentially involved in these processes, including transmitter receptors, channels, trophic factors and their receptors, and transcriptional regulators, and on the other hand, functional studies at the cellular level on LTP, and possible involvement of these molecules in LTP. This concerted effort, combining the different expertise of the five investigators in this group, allow the approach to the general questions concerning the basis of learning and memory in a manner that is beyond the reach of any one of these researchers.

To better understand mechanisms that underlie the formation of the mammalian central nervous system and its functional and anatomical adjustments in response to experience, processes central to mental health, the Silvio Conte Center for Neuroscience Research at UCSF plans to study synaptic plasticity and its modulation by transmitters such as serotonin, neuronal survival and organization regulated by neurotrophins, and specification of cell fate and identity of neurons during their formation. The strategy is to employ organisms amenable to genetics for the characterization of genes important for neural development, to use molecular technologies for the study of ion channels, neurotrophins and transmitter receptors, and to pursue mechanistic analysis of synaptic plasticity in the hippocampus of the mammalian brain. These studies are relevant for the development of antidepressant, antiemetic and antipsychotic drugs, the design of regimen to reduce or remedy neuronal damage during ischemia or anoxia, or in the course of neurodegenerative diseases such as amyotrophic lateral sclerosis and parkinson's disease. Recent prospective studies reveal that schizophrenia manifests itself in behavioral abnormalities early in life, indicating that it is a developmental disorder. Thus, therapy for such mental disorders may also benefit from better mechanistic understanding of neural development and function.

embryonic stem cell; tissue /cell preparation; biomedical facility; genetically modified animals; laboratory mouse;

The long-term objectives are to molecularly characterize potassium channels and to understand their functional roles in the mammalian brain. Specifically, the plan over the next five years is (1) to study the function and regulation of the Kv1.4 voltage-gated [potassium channels prevent in the axons and nerve terminals of many central neurons, (2) to study the possible role of a G protein-activated potassium channel as an effector that mediates the action of a wide range of neurotransmitter including serotonin, acetylcholine and GABA, and (3) to test the possibility that neuronal excitability is regulated by the energy level of the neuron partly via inhibition of a potassium channel by ATP. In addition to contributing to our understanding of the basic mechanisms underlying the function and plasticity of the mammalian brain, these studies may be relevant to current efforts tin the development of drugs that affect potassium channel function, used for treatment of diseases such as diabetes, arrhythmia, multiple sclerosis, or incontinence. Better understanding of the function and molecular diversity of potassium channels in the mammalian brain may also improve the likelihood of developing regimen to minimize neuronal damage due to ischemia or anoxia or treatments for behavioral disorders. One such disorder, episodic ataxia, has been found to arise from mutations of the Kv1.1 voltage-gated potassium channel. The research design is to first clone potassium channel genes that are expressed in the mammalian brain, and then study these potassium channels using two complimentary approaches. First, expression of cloned potassium channels in cell lines or Xenopus oocytes makes it possible to carry out mechanistic studies on the assembly, function and regulation of individual channel types. Second, expression of wildtype or mutated potassium channels in transgenic mice or removal of the endogenous potassium channels in transgenic mice or removal of the endogenous potassium channel gene function via homologous recombination will be carried out to analyze the function of these potassium channels at the level of central neuronal signaling and behavior.

DESCRIPTION (provided by applicant): Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of a variety of central and peripheral neurons. For example, CaCC contributes to signal amplification of sensory inputs and regulation of excitability of both sensory and central neurons. The long- term objectives are to understand how these channels work, and how they regulate neuronal activity. Reflecting an intense interest in CaCCs as potential therapeutic targets for hypertension, cystic fibrosis and other diseases, there have been extensive efforts to determine the molecular identity of CaCCs. Because the channel properties and expression patterns of several reported molecular candidates do not match those for native CaCCs, several years ago we began the undertaking for expression cloning, leading to the identification of Xenopus and mouse TMEM16A, as well as mouse TMEM16B as CaCC subunits. In 2008, two concurrent studies were published around the same time as ours, and all three reached the same conclusion that mammalian TMEM16A corresponds to CaCC. By now, several studies of TMEM16A knockout mice have shown that TMEM16A is required for CaCC in exocrine glands and airway epithelia. With the TMEM16 family of "transmembrane proteins with unknown function" emerging as a novel family of ion channels, even the most basic questions are open and now amenable to molecular and genetic studies: How does calcium activate CaCC? How many TMEM16A subunits are present in a CaCC channel? Does TMEM16A correspond to the CaCC in sensory neurons of the dorsal root ganglion (DRG)? Is TMEM16A up regulated following denervation and, if so, does it influence nerve regeneration and/or neuropathic pain? Denervation causes up regulation of CaCC of DRG neurons - one of the best examples of neuronal CaCC, hence one specific aim of this proposal is to examine the involvement of TMEM16A in CaCC of DRG neurons with or without sciatic nerve lesion, and to explore potential roles of TMEM16A in pain sensitivity and neuropathic pain, which develops after nerve injury or in diseases like diabetes, herpes, and cancer. To better understand how CaCC channel traffic and activity may be controlled by cytosolic calcium, we will carry out biochemical and mutagenesis studies of TMEM16A, which can be heterogeneously expressed to generate CaCC. We will also use a combination of approaches to determine the CaCC stoichiometry - an important question for better appreciation of CaCC function and regulation, and the diversity of CaCCs. PUBLIC HEALTH RELEVANCE: Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of neurons in the central and peripheral nervous system. Having recently established the TMEM16 family of "transmembrane proteins of unknown function" as a novel ion channel family that includes TMEM16A and TMEM16B as CaCC subunits, we propose to use heterologous expression systems to study how CaCC channels work. We will also characterize CaCC endogenous to the dorsal root ganglion (DRG) and examine the role of TMEM16A in pain sensitivity and neuropathic pain. Bearing in mind that CaCC of DRG sensory neurons is up regulated after denervation, we have designed our experiments to lay the groundwork for future studies of the potential roles of CaCC in nerve regeneration and/or neuropathic pain, which develops after nerve injury, in diseases like diabetes, herpes zoster injection and cancer, and may also be induced by chemotherapy.

DESCRIPTION (provided by applicant): Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of a variety of central and peripheral neurons. For example, CaCC contributes to signal amplification of sensory inputs and regulation of excitability of both sensory and central neurons. The long- term objectives are to understand how these channels work, and how they regulate neuronal activity. Reflecting an intense interest in CaCCs as potential therapeutic targets for hypertension, cystic fibrosis and other diseases, there have been extensive efforts to determine the molecular identity of CaCCs. Because the channel properties and expression patterns of several reported molecular candidates do not match those for native CaCCs, several years ago we began the undertaking for expression cloning, leading to the identification of Xenopus and mouse TMEM16A, as well as mouse TMEM16B as CaCC subunits. In 2008, two concurrent studies were published around the same time as ours, and all three reached the same conclusion that mammalian TMEM16A corresponds to CaCC. By now, several studies of TMEM16A knockout mice have shown that TMEM16A is required for CaCC in exocrine glands and airway epithelia. With the TMEM16 family of transmembrane proteins with unknown function emerging as a novel family of ion channels, even the most basic questions are open and now amenable to molecular and genetic studies: How does calcium activate CaCC? How many TMEM16A subunits are present in a CaCC channel? Does TMEM16A correspond to the CaCC in sensory neurons of the dorsal root ganglion (DRG)? Is TMEM16A up regulated following denervation and, if so, does it influence nerve regeneration and/or neuropathic pain? Denervation causes up regulation of CaCC of DRG neurons - one of the best examples of neuronal CaCC, hence one specific aim of this proposal is to examine the involvement of TMEM16A in CaCC of DRG neurons with or without sciatic nerve lesion, and to explore potential roles of TMEM16A in pain sensitivity and neuropathic pain, which develops after nerve injury or in diseases like diabetes, herpes, and cancer. To better understand how CaCC channel traffic and activity may be controlled by cytosolic calcium, we will carry out biochemical and mutagenesis studies of TMEM16A, which can be heterogeneously expressed to generate CaCC. We will also use a combination of approaches to determine the CaCC stoichiometry - an important question for better appreciation of CaCC function and regulation, and the diversity of CaCCs.

DESCRIPTION (provided by applicant): The phenothiazine group of drugs including thioridazine has been widely prescribed for the treatment of psychiatric disorders since the 1940s. The anti-cancer activities of these antipsychotics have intrigued clinicians and researchers since the early 1970s. Whereas thioridazine and several other phenothiazines can block the cardiac hERG voltage-gated potassium channels, they remain on the market because their risk for prolonging the cardiac QTc interval is outweighed by their beneficial effects. The possibility that potassium channel block is one molecular mechanism by which these antipsychotics protect against cancer has never been considered before. We propose to test the original hypothesis that thioridazine protects against medulloblastoma (MB) growth and metastasis by blocking the EAG2 voltage-gated potassium channels that are upregulated in a subset of MBs of human patients, particularly in metastatic MBs. Specifically, we hypothesize that (1) thioridazine block of EAG2 channels prevents MB cell volume reduction for premitotic condensation (PMC) so as to cause cell cycle arrest - those MB cells that venture beyond the G2 phase encounter mitotic catastrophe and perish via apoptosis, and (2) thioridazine block of EAG2 channels reduces water efflux from the trailing edge of migrating MB cells, thereby interfering with MB cell migration by preventing the trailing edge of the cell from shrinking - a local volume regulation essential for cell movement. To test our hypothesis that thioridazine block of EAG2 potassium channels that appear on the surface of mitotic MB cells and on the trailing edge of migrating MB cells to protect against MB growth and metastasis, we will conduct in vitro and in vivo studies to experimentally validate the prediction that the anti-cancer activites of thioridazine can be mimicked by pharmacological treatment with astemizole, a structurally unrelated EAG2 channel blocker. We will further test whether the anti-cancer activities of these two EAG2 channel blockers resemble the effects of reducing EAG2 expression of human MB cells in vitro and in vivo - for mice bearing human MB xenograft, as well as the effects of knocking out Eag2 in mouse MB models. Additional controls will be conducted to confirm that the anti-cancer activities of thioridazine that arise from its block of EAG2 channels are occluded by shRNA knockdown of EAG2 or genetic deletion of Eag2 in mouse models.

Project Summary The goal of this project is to generate data and reagents that help uncover critical functions of the poorly characterized members of ion channels. We prioritize mouse models, given the high value investigators place on phenotypes observed for genetic perturbation. However, we focus on co-perturbation of ion channel genes and their interacting genetic components as opposed to singly altering ion channel genes in mouse models. This approach will validate our proteomics approaches in the most definitive manner- in vivo. We see in vivo exploration as an essential step to evaluate ion channel function. Our major aims include mapping ion channel interactions and complexes using a high-throughput proteomics platform at UCSF. These data will be interrogated using integrative approaches established by the Monarch Initiative, where biochemical interactions will be validated and prioritized for further study. Another major aim is function-centric: we use mouse models to elucidation of human disease mechanisms, where we embrace a genetic interaction scheme to uncover ion channel redundancy and polygenic effects. Together these integrative approaches complement each other, specifically the in vivo genetic interaction platform interrogates those genes identified from proteomics and bioinformatics analysis. We generate critical reagents and data including antibodies and gene expression patterns. Our broad goal is to identify ion channel phenotypes relevant to fundamental human disease. We bring on board a number of ion channel experts and mouse phenotyping specialists who can accomplish this major programs goals in an efficient manner.

We made the surprise finding that loss of TMEM16C function led to the elimination of the majority of POA neurons that increase their firing rate with rising temperature of the preoptic area of the anterior hypothalamus (POA) ? warm-sensitive neurons that are on top of a command chain for thermoregulation. To test for the involvement of TMEM16C in thermoregulation and febrile seizure, we examined TMEM16C conditional knockout (cKO) mice and found that mutant pups with TMEM16C removed from their central neurons cannot maintain their body temperature and are prone to develop hyperthermia-induced seizure. To look for molecular markers of temperature sensitive POA neurons, we conducted single-cell RNAseq of 68 POA neurons following recording from these neurons in brain slices subjected to temperature changes, and validated a molecular and genetic marker for temperature sensitive POA neurons. By generating cKO mice with TMEM16C removed from temperature sensitive POA neurons, we aim to test the function of TMEM16C in the specification of warm-sensitive neurons, thermoregulation, and hyperthermia-induced seizure. Given the GWAS association of TMEM16C with febrile seizure and our finding that rodent pups without neuronal TMEM16C are prone to exhibit hyperthermia-induced seizures, TMEM16C cKO mice provide animal models for febrile seizure, which affects 2-8% of young children. Because complex febrile seizures are associated with hippocampal sclerosis as the epileptogenic pathology, we will test the hypothesis that TMEM16C regulates hippocampal neuronal excitability by recording action potentials and synaptic potentials in hippocampal neurons from mice with TMEM16C removed either via nestin-Cre that is expressed in all neurons or via Drd3-Cre that targets ~50% of hippocampal pyramidal neurons. We will also record from temperature sensitive POA neurons in TMEM16C cKO mice and sibling controls to determine how TMEM16C modulates temperature sensitive POA neuronal activity. We will further test the hypothesis that removing TMEM16C from central neurons causes alteration of sodium-activated potassium current in these neurons. Finally, we will adopt the recently developed split GFP approach involving the use of mass spectrometry to identify proteins that are associated with TMEM16C in specific neuronal types.

Project Summary/Abstract Maintenance of body temperature at the optimal level is crucial for survival, and it requires homeostatic feedback regulation based on monitoring the temperature of internal organs as well as the environment. Homeostatic thermoregulation in response to changes of brain temperature relies on the temperature- sensitive neurons in the preoptic area of the anterior hypothalamus (POA). These neurons constitute about one third of the medial and lateral POA neurons and are intermingled with temperature-insensitive neurons that control drinking, feeding, sleep, and parental behaviors. For eight decades since the discovery of temperature-sensitive neurons in the brain, electrophysiology has been the only way to identify these central neurons. Having identified the first molecular marker for temperature-sensitive POA neurons by combining single-cell RNA-seq with whole-cell patch-clamp recording, we will identify central neurons that are upstream or downstream of these temperature-sensitive POA neurons, to elucidate the central neuronal circuitry for thermoregulation. To identify central neurons that receive input from temperature-sensitive POA neurons, we will use trans-synaptic tracers, and further verify these synaptic connections by using the PGDS Cre-line to drive channelrhodopsin expression in temperature-sensitive POA neurons for optogenetic activation in brain slices. To test whether specific neuronal types in the suprachiasmatic nucleus (SCN) innervate temperature- sensitive POA neurons to modulate the circadian variation of body temperature, we will use Cre-lines for these SCN neuronal types to drive trans-synaptic tracer expression. We will also use these Cre-lines to drive channelrhodopsin expression in SCN neurons, and record from POA neurons to determine whether they receive SCN input and whether their firing rate changes when the temperature of the brain slice is altered. In addition to identifying central neurons that are upstream or downstream of temperature-sensitive POA neurons, this proposed project includes mechanistic studies on the nature of the signals used by temperature-sensitive POA neurons to alter the activity of their downstream neurons so as to modulate body temperature, to test the hypothesis that, besides classical transmitters, endogenous PGD2 mediates thermoregulation. These studies will generate predictive models at a conceptual level of understanding thermoregulation.

Project Summary/Abstract Not applicable. No change from Parent Award.

Project Summary/Abstract No changes from the awarded grant.

Project Summary/Abstract To initiate molecular characterization of the CaCC calcium-activated chloride channels that have been found in multiple neuronal types since 1980s, we first showed that CaCC is formed by TMEM16A or TMEM16B in 2008. The mammalian TMEM16 family with ten members turns out to be surprisingly diverse, with family members acting as calcium-activated ion channels and/or calcium-activated lipid scramblase. The TMEM16 family members provide a variety of activities in central neurons to serve important functions such as modulation of neuronal excitability and thermoregulation. Indeed, some of the mammalian TMEM16 family members have been associated with human diseases such as febrile seizure and neurodegeneration as well as Scott syndrome, a bleeding disorder. Therefore, it will be important to conduct molecular and cell biological investigations to learn about the mechanisms that underlie the functions of these TMEM16 family members. To ask how the calcium-activated chloride channel works, we solved cryo-EM structures of calcium-free and calcium-bound TMEM16A and carried out structure-inspired site-directed mutagenesis to identify 10 pore- lining residues important for anion selectivity and 7 pore-lining residues near pore constrictions important for channel gating. We then showed that TMEM16B modulates action potential waveform and firing patterns in multiple brain regions. To ask how TMEM16F fulfils the dual functions of calcium-activated ion channel and calcium-activated lipid scramblase, we examined cryo-EM structures of TMEM16F and conducted structure- inspired site-directed mutagenesis to provide evidence for separate pathways in TMEM16F for ion permeation and lipid scrambling. To establish the physiological importance of TMEM16 family members, we generated knockout (KO) mice to show that they provide mouse models for human diseases, such as the bleeding disorder Scott syndrome (TMEM16F), febrile seizure (TMEM16C), and the progressive neurodegenerative disease spinocerebellar ataxia (TMEM16K). To monitor endogenous TMEM16C in various cell types in the brain, we modified the split GFP approach by using CRISPR mediated knock-in to fuse the FLAG tag along with the 11th beta strand of GFP to the C-terminus of TMEM16C, and expressing GFP1-10 (the rest of GFP) in a Cre-dependent manner in specific cell types. This approach will allow visualization of fluorescently tagged endogenous proteins as well as identification of their associated proteins for better understanding of their physiological functions.