Jeffrey D. Rothstein - US grants

The role of diazepam-binding inhibitor (DBI) in the pathogenesis of hepatic encephalopathy will be studied in humans and animals. Recent theories on the neurochemical etiology for hepatic encephalopathy have suggested activation of inhibitory neurotransmitter GABA systems. GABA receptor sites are known to be modulated by benzodiazepines. Furthermore, hepatic encephalopathy can be ameliorated in animals and humans by the administration of benzodiazepine antagonists. The neuropeptide DBI is an endogenous allosteric modulator of BABA systems at the benezodiazepine receptor. These experiments will examine, in animals and humans, whether DBI metabolism is altered in hepatic encephalopathy and how these changes correlate with clinical indices of encephalopathy. In preliminary experiments cerebrospinal fluid DBI was found to be markedly elevated in patients with hepatic encephalopathy and was normal in non-encephalopathic patients with liver disease. A primary goal of the proposed experiments will be to quantify DBI in the CSF of patients with hepatic as well as non- hepatic encephalopathy and to correlate these changes with other clinical abnormalities. Since data acquisition from humans is limited, a major goal of this proposal will be the utilization and analysis of animal models of encephalopathy. Central nervous system DBI will be quantified in various animal models of acute and chronic hepatic encephalopathy as well as animal models of non-hepatic encephalopathy. Experiments are planned to quantify the content of mRNA for DBI in various brain regions to add the understanding of its synthesis. Furthermore, to test the hypothesis that DBI or its metabolites may be responsible for hepatic encephalopathy, the neuropeptide will be administered to the CNS of animals. Finally, the effectiveness and specificity of benzodiazepine antagonists in the reversal of hepatic encephalopathy will be examined in the proposed series of experiments. Understanding of role of DBI in the pathogenesis of hepatic encephalopathy may lead to important pharmacological therapies in this disease.

Glutamate transport is critical for the synaptic inactivation of glutamate, and failure of this process can be neurotoxic, and may participate in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Three transporter subtypes have been identified, GLT-1, EAAC1, and GLAST. We intend to combine immunohistochemical, and physiological approaches to study the L-glutamate sodium-coupled neurotransmitter transporter family. We shall prepare specific antibodies to each cloned transporter, and will then use these antibodies to better understand the defect in glutamate transport in ALS. Finally, under controlled culture conditions, we will selectively study each transporter to better understand their role in excitotoxicity. Aim 1. Localization of Glutamate Transporters - Preparation of selective oligopeptide antibodies to the three rat and human glutamate transporter subtypes. These antibodies will be used as powerful tools to localize glutamate transporter immunoreactive protein in brain and spinal cord at the cellular and ultrastructural level. We hypothesize that GLT-l and GLAST are localized to astroglia, whereas EAAC1 is uniquely located in neurons. Significance: In a selective, yet systematic fashion, we will produce and characterize monospecific antisera to oligopeptides derived from each transporter subtype including EAAC1, GLAST and GLT-1. These reagents will be used to determine the cellular and ultrastructural localization of the various glutamate transporter proteins in rat and human tissue. Aim 2. Glutamate transporters and ALS - To understand the nature of the glutamate transporter defect in ALS. We hypothesize that the loss of glutamate transport in ALS selective for transport subtypes. Significance: We will use monospecific antibodies to each transporter-subtype to identify the specific transporter defect in AL[unreadable] These studies will determine the subtype specificity of the disease, as well as localize the defect regionally and ultrastructurally. Aim 3. Glutamate transporters and chronic neural toxicity - To explore the relationship between individual glutamate transporters and chronic neurotoxicity by selectively blocking the synthesis of individual transporter subtypes, in vitro and in vivo, using antisense oligodeoxynucleotides. In addition, the susceptibility of glutamate transport to oxygen radical mediated insults will be studied in cultured neurons or cells expressing glutamate transporter subtypes. Significance: Chronic neuronal toxicity may involve both direct glutamate injury and oxidative stress. These studies will help dissect the role of neuronal and glia glutamate transporters and determine if a link between oxidative stress and glutamate toxicity could account for neuronal degeneration in ALS.

Endozepine-4 is a newly identified natural chemical that is active at the benzodiazepine receptor (endozepine), and is neither a benzodiazepine nor a peptide. It is a low molecular weight molecule that has a high affinity for the benzodiazepine binding site of the GABAA receptor and is present in pharmacologically relevant concentrations in human and rat brain. It acts selectively at the benzodiazepine receptor. Like the benzodiazepine receptor agonist, diazepam, it can potentiate GABA-mediated chloride fluxes, and can serve as an anticonvulsant. Furthermore, preliminary studies provide evidence that endozepine-4 can cause encephalopathy, and may be the cause of idiopathic recurring stupor and may contribute to hepatic encephalopathy. The proposed studies will examine the role of endozepine-4 in the pathogenesis of idiopathic recurring stupor and hepatic encephalopathy. An animal model will be used to investigate how human and rat endozepine-4 causes encephalopathy and whether these actions occur via the benzodiazepine receptor. (l) We will identify alterations in endozepine-4 in patients with idiopathic recurring stupor and hepatic encephalopathy and determine whether these changes correlate with the clinical level or temporal course of encephalopathy. Significance: Studying endozepine-4 in serum and CSF from patients with these disorders will serve to define its role in idiopathic recurring stupor and hepatic encephalopathy. (2) We propose to test the hypothesis that alterations of endozepine-4 exist in animal models of hepatic encephalopathy and that excess endozepine-4 can cause encephalopathy and therefore contribute to the disease. Significance: Animal models of excess endozepine-4 and animal encephalopathy models will allow us to directly test the hypothesis that endozepine-4 can cause or contribute to encephalopathy. (3) We will test the hypothesis that the behavioral actions of endozepine- 4 are mediated via benzodiazepine receptors by examining its anti-anxiety and anti-convulsant properties. Significance: These studies will determine if naturally occurring endozepine-4 has the functional properties of other synthetic benzodiazepine agonists - anxiolytic and/or anticonvulsant. It is anticipated that the results of these experiments should provide important information on a new neuromodulator that may be an important cause or contributor to certain encephalopathic diseases. Endozepine-4 may also be an important allosteric regulator of GABAergic networks in the nervous system. In addition, the possibility that it could serve as a new class of "natural" anticonvulsants adds additional impetus for its study.

DESCRIPTION: (Applicant's abstract) Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized by selective upper and lower motor neuron degeneration, the pathogenesis of which is unknown. About 60-70 percent of sporadic ALS patients have a 30-95 percent loss of the astroglial glutamate transporter EAAT2 (excitatory amino acid transporter 2) protein in motor cortex and spinal cord. Loss of EAAT2 leads to increased extracellular glutamate and excitotoxic neuronal degeneration. Preliminary studies document multiple abnormal EAAT2mRNAs, including intron-retention and exon-skipping, in the affected tissues of ALS pateints. The aberrant mRNAs were highly abundant and were only found in neuropathologically affected areas of ALS patients, but not in other brain regions. They were found in 65 percent of sporadic ALS patients, but were not found in non-neurologic disease or other diseased controls. They were also detectable in the cerebrospinal fluid of living ALS patients, early in the disease. In vitro expression studies suggest that proteins translated from these aberrant mRNAs may undergo rapid degradation and/or produce a dominant negative effect on normal EAAT2 resulting in loss of protein and activity. We propose a systematic approach to study the biology and relevance of aberrant EAAT2 mRNA and protein in ALS. The specific aims address: 1) Identification and characterization of aberrant EAAT2 mRNA species and proteins in ALS, including disease and tissue specificity, quantification. 2) Using in vitro expression systems we will investigate the biology of the aberrant RNA species to understand how they could account for the loss of EAAT2 in ALS by examining their transporter characteristics-Vmax, Km, conductance properties, stability and cellular trafficking. 3) Develop in vitro model systems to determine the processes that could be responsible for their formation in vivo, including excitotoxicity/oxidative stress, DNA repair, RNA processing proteins, and use astrocytes cultures from ALS patients to study their formation. Our preliminary work suggests that the loss of EAAT2 IN ALS is due to aberrant mRNA, and these aberrant mRNAs could result from RNA processing errors. Aberrant RNA processing could bed an important process in the pathophysiology of neurodegenerative disease and in excitotoxicity. The presence of these mRNA species in also cerebrospinal fluid may have diagnostic utility.

Mutations of the antioxidant enzyme Cu,Zn SOD(SOD1) are responsible for about 20 percent of familial amyotrophic lateral sclerosis (FALS). More than 50 different SOD1 mutations have been identified. The process by which mutant SOD1 causes toxicity is not known, but all the mutants have a common property of binding copper and scavenging free radicals. Aberrant copper regulation has been suggested as one possible toxic property of mutant SOD1. Copper trafficking in mammalian cells is highly regulated. CCS is a newly cloned copper chaperone that functions to deliver copper specifically to SOD1, including mutant SOD1. In this proposal, we will investigate the biology of this new protein and evaluate its possible role in the neurotoxicity of mutant SOD1-mediated motor neuron degeneration. Preliminary studies suggest that CCS is commonly associated with SOD1 and that abnormal intracellular inclusions in transgenic models of ALS may reflect CCS-SOD1 aberrant interactions. We propose a systematic study of CCS, first by generating several oligo peptide antibodies to investigate the cellular and ultrastructural localization of CCS in both human and murine tissues. We hypothesize that CCS distribution will closely follow that of SOD1. Next, we will determine if CCS is associated with cellular abnormalities in transgenic mice expressing mutant SOD1. We will examine and compare neural tissue, obtained over time, from transgenic mice with two different SOD1 mutations (G93A and G85R) which exhibit different time courses of neurodegeneration and neuropathology. Thirdly, we will also examine CNS tissue from sporadic and familial ALS to determine if there aggregates of CCS the disease? Finally, we will test the hypothesis that CCS can contribute to, or cause, the intracellular inclusions seen in ALS models, and that it plays a direct role in mutant SOD1 motor neuron toxicity. Overall, these experiments will help test the hypothesis that 1) CCS is responsible for the cytoplasmic aggregates in astrocytes and neurons, and 2) that CCS participates in mutant SOD1 mediated neurotoxicity.

DESCRIPTION (From the applicant's abstract): Glutamate transport is an essential component of central glutamatergic neurotransmission; studies document an essential role for glutamate transport in the inactivation of synaptically released glutamate and the prevention of excitotoxicity. The understanding of glutamatergic neurotransmission has expanded recently with the identification of new proteins responsible for the regulation and synaptic targeting of these receptors to the synaptic cleft. Studies also suggest that glutamate transporters may be regulated and targeted to the synaptic cleft. We hypothesize that neuronal proteins exist that are responsible for the synaptic targeting and regulation of neuronal glutamate transporter subtypes; we propose to label these proteins GTRAP: glutamate transporter associated protein. In preliminary studies we have identified at least three novel and specific interacting proteins that appear to both positively and negatively modulate neuronal glutamate transporter subtypes. We will identify proteins that interact with the major intracellular domains of the two neuronal glutamate transporter subtypes: EAAT4 and EAACI (EAAT3). The aims of the studies include: 1) Identification and characterization of neuronal glutamate transporter interacting proteins. Using yeast-two hybrid screens, we will identify proteins that interact with both neuronal glutamate transporter subtypes; 2) Determine the specificity and characterization of GTRAP-transporter interaction. 3) Evaluate the cellular and ultrastructural distribution of GTRAPs and 4) to determine the physiological regulation of neuronal glutamate transporters by these novel GTRAPs.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal adult onset neurological disorder that characteristically involve the degeneration of motor neurons. No effective treatment exists to substantially retard the progression of this disease, or to reverse the devastating and fatal disability. The use of stem cells in general and astroglial in particular, appears to represent a new approach to the treatment of neurodegenerative disease: rather than directly combating a pathological process, stem cell-based strategies could reinvoke developmental processes to insert new cells seamlessly into the degenerated environment. Recent studies document the potential of certain stem cells in promoting neuroprotection/recovery in a variety of nervous system injuries. The overall goal of this proposal will be to study the potential of murine astroglial stem cell transplantation as a therapeutic modality for the treatment of animal models ALS. In ALS patients and in animal models, defects in astroglial glutamate transporter GLT-1/EAAT2 lead to excitotoxic neural degeneration. Preliminary studies document that replacement of glutamate transport can effectively and dramatically slow down the disease; while use of selected trophic factors can enhance motor axon growth and also protect motor neurons. Glial-restricted progenitor cells appear to be able to engraft into spinal cord explants (in vitro), express the potentially neuroprotective astroglial GLT-1/EAAT2 glutamate transporter, and potently protect against glutamate-mediated neuronal death. The overall goal of this proposal will be to: 1) Characterize the normal regulation of glutamate transporter subtypes in glial progenitor cells in vitro and after engraftment; 2) Establish the neuroprotection by glial progenitor stem cells using in vitro models. To critically evaluate the role of glutamate transporter versus other properties of GRP (e.g. trophic factor release) we will perform comparisons with GRPs prepared from transporter null mice (GLT-1, GLAST, GLT-1/GLAST); 3) Determine if glial progenitor cells can protect against chronic neuronal injury by examining stem cell differentiation and neuroprotection in vivo in a transgenic animal model of ALS - G93A SOD 1 mutant mice and rats. Over all, these studies will provide data on the utility of astroglial stem cells to alter neurodegeneration in acute and chronic injury models relevant to ALS and hopefully provide important critical pre-clinical information.

DESCRIPTION (provided by applicant): The astroglial transporter GLT-1/EAAT2 is responsible for the largest percentage of glutamate transport in forebrain. Abnormal expression/function of EAAT2 is common to sporadic ALS, to transgenic models of the disease, as well as other neurological injuries. The mechanism that underlies transporter deregulation is not fully understood, but may involve altered promoter activation. We hypothesize that regulating expression of these proteins could provide powerful therapies to retard disease progression. Initial studies provide exciting evidence that increasing EAAT2 protein/activity can retard neurodegeneration in ALS animal models;diminish seizures associated with epilepsy, and retard brain tumor growth. In this proposal we will use recently generated GLT1 and GLAST-promoter reporter mice to evaluate the regulation of transporters. These rodents will be used to study the dysregulation of transporters in neurodegenerative disease. We propose to alter transporter gene activation as a novel means to delay neurodegenerative disease. The completion of these studies will provide a comprehensive understanding of transporter regulation in normal and abnormal CNS and their potential as neuroprotectants. Specifically we propose: 1) To understand the normal regional and temporal regulation of astroglial glutamate transporter gene expression, thru analysis of GLAST-BAC and GLT1-BAC promoter reporter expressing transgenic mice;2) To test the hypothesis that altered glutamate transporter expression in neurological injury is the result of promoter deregulation;3) To determine if altered cellular expression of astroglial glutamate transporters can prevent neuronal degeneration. Using drugs capable of increasing GLT1 and GLAST promoter expression we will determine if increased expression of the astroglial transporter(s) can prevent neural injury in vivo and we will determine if cell specific expression of glutamate transport alters chronic neurodegeneration using an animal model of amyotrophic lateral sclerosis. We hypothesize that over expression of GLT1 or GLAST will be neuroprotective in disease models. Over all Significance: In this proposal, we will develop a basic understanding of expression of GLAST and GLT I, discover reagents that can alter glutamate transporter activity, and determine if these approaches are useful for chronic neurological insults.

DESCRIPTION (provided by applicant): Glutamate transport is critical for the maintenance of low extracellular glutamate and dysregulation of transporters can lead to acute and chronic neurodegeneration. The astroglial transporter EAAT2/GLT1 is the dominant glutamate transporter, accounting for up to 95% of all CNS glutamate transport. Loss of EAAT2 occurs in human diseases and disease models such as ALS, and can contribute to neuronal and astroglial dysfunction and cell death. Recently an alternative splice variant of EAAT2/GLT1, known as EAAT2b/GLT1b, was identified, and early studies suggest it may be present in neurons. Our preliminary data suggest EAAT2b/GLT1b protein expression is regulated and enhanced by neural injury and neurological disease, leading a unique high-level expression in neurons- rather than glia. In this proposal- we will more fully explore the basic biology of this potentially important transport splice variant and its relationship to neuronal/astroglial injury. We hypothesize that EAAT2b/GLT1 is functionally expressed in neural injury as a protective reaction or compensation for the dysregulation of astroglial glutamate transport and may be synaptically active. To test this hypothesis we will: 1) Investigate the altered regional and cellular expression of EAAT2b/GLT1b in ALS and ALS models; 2) Study the function and expression of EAAT2b/GLTb under conditions of neuronal/glial stress; and 3) Analyze the function of GLT1b in neurons vs. astroglia, thru targeted deletion of GLT1/GLT1b from neurons. These studies will help determine if the altered expression of GLT1b in disease is an important physiological response. They will also provide basic physiological properties of the neuronal versus glial pools of GLT1 and GLT1b.

The astroglial transporters EAAT1 and EAAT2 are responsible for the largest percentage of glutamate transport in forebrain. Abnormal expression/function of EAAT2 is common to sporadic ALS and to transgenic models of the disease. In addition, altered function of various molecular subtypes of this transporter family is associated with brain tumor growth, cerebellar ataxia, multiple sclerosis and epilepsy. Regulating expression of these proteins could provide powerful therapies to retard disease progression. No practical pharmacological agents exist that can activate glutamate transporters. Studies using EAAT2 transgenic mice recently provide exciting evidence that increasing EAAT2 protein/activity can retard neurodegeneration in ALS animal models; diminish seizures associated with epilepsy, and retard brain tumor growth. A recent NINDS-organized screen of FDA approved drugs, identified beta-lactam antibiotics as modulators of EAAT2. We have accumulated preliminary data that several screened antibiotics can activate EAAT2 gene expression, leading to increased brain protein up to seven fold. The in vitro and in vivo actions of these drugs have been validated- they can protect against excitotoxic injury to cultured neurons/motor neurons. Finally one beta-lactam, ceftriaxone, can delay loss of muscle strength and increase survival in the ALS transgenic mouse model-even when given at disease onset. In this translational research proposal we will systematically evaluate non-antibiotic beta-lactam compounds and beta-lactam antibiotics as modulators of EAAT1 or EAAT2 expression. We will then go on to identify the most potent agent(s) and thoroughly evaluate the drugs in an in vitro-relevant and animal model of ALS. These sequential phases will set the stage for future practical translation towards rational clinical trial design.

The astroglial transporters EAAT1 and EAAT2 are responsible for the largest percentage of glutamate transport in forebrain. Abnormal expression/function of EAAT2 is common to sporadic ALS and to transgenic models of the disease. In addition, altered function of various molecular subtypes of this transporter family is associated with brain tumor growth, cerebellar ataxia, multiple sclerosis and epilepsy. Regulating expression of these proteins could provide powerful therapies to retard disease progression. No practical pharmacological agents exist that can activate glutamate transporters. Studies using EAAT2 transgenic mice recently provide exciting evidence that increasing EAAT2 protein/activity can retard neurodegeneration in ALS animal models;diminish seizures associated with epilepsy, and retard brain tumor growth. A recent NINDS-organized screen of FDA approved drugs, identified beta-lactam antibiotics as modulators of EAAT2. We have accumulated preliminary data that several screened antibiotics can activate EAAT2 gene expression, leading to increased brain protein up to seven fold. The in vitro and in vivo actions of these drugs have been validated- they can protect against excitotoxic injury to cultured neurons/motor neurons. Finally one beta-lactam, ceftriaxone, can delay loss of muscle strength and increase survival in the ALS transgenic mouse model-even when given at disease onset. In this translational research proposal we will systematically evaluate non-antibiotic beta-lactam compounds and beta-lactam antibiotics as modulators of EAAT1 or EAAT2 expression. We will then go on to identify the most potent agent(s) and thoroughly evaluate the drugs in an in vitro-relevant and animal model of ALS. These sequential phases will set the stage for future practical translation towards rational clinical trial design.

DESCRIPTION (provided by applicant): Although we do not fully know if disease study of cells in Petri dishes can fully emulate the developmental progression that occurs in human adult neurodegenerative disease like ALS, new described technical ability to generate Induced Pluripotent Cells (iPS) from ALS patients provides an exceptional tool by which we can explore these issues. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. But - iPS cell biology is exceptionally new and we do not yet have sufficient information about the reliability of the cells generated, their ability to truly reflect human cell biology, recapitulate the protein, genetic and functional characteristics of native motor neurons and astroglia. Before we can embark on extensive use of these cells for basic/translational research- it would be critical to generate a series of cell lines- all produced under identical conditions, from different fALS mutations, to determine how representative they are for cell type specificity and functional biology. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate familial ALS (fALS) iPS cell lines. Project 1, led by Dr. Eggan will obtain the skin biopsies from FALS and control patients, generate the fibroblast and ultimately the initial iPS lines. We will employ the aid of iZumi, a biotech company to be a central site for uniform protocol iPS cell generation. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab, Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which of the fALS iPS cell lines have the appropriate characteristics of motor neurons and astroglia, through a series of sequential analyses. Only those cell lines that meet final criteria (as compared to human ES cell and prior work on human astroglia) will then go on for final genetic analysis in the Project 4 lab, lead by Tom Maniatis. PUBLIC HEALTH RELEVANCE: Understanding the pathophysiology and development of new therapeutics for ALS has been an enormous challenge. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. )

DESCRIPTION (provided by applicant): Injury and/or dysfunction of astroglia appears to be a component of numerous neurological disorders. Replacement of these cells might be a novel and potent therapy- but engraftment approaches are therapeutically challenging. Adult NG2 cells- the resident adult brain progenitors for oligodendroglia and astroglia are abundant in the rodent and human CNS. We propose to screen rodent and human NG2 cells with a chemical/drug library (singly and in matrix combination) to identify small molecules capable of differentiating these cells to astroglia in vitro and in vivo. We have generated appropriate reporter cells and animals for these assays including rodent NG2-dsRed/GLT1-eGFP, NG2-dsRed-/GLT1 luciferase and human glial progenitor/EAAT2 luciferase cell lines-all suitable for identifying molecules that differentiate progenitors into astroglia. We also have generated the necessary mouse models including NG2/GLT1 BAC reporter mice to evaluate the in vivo efficacy of new astrocyte generation. For future studies, we will test active compounds in our rodent models of neurodegeneration (e.g. G93A SOD1 mouse) to determine the efficacy of the agents in generating new astroglial in diseased mouse models. The aims of the study include: Aim1) Generation and validation of NG2-progenitor/astroglial reporter cell lines for use in drug discovery assays, including rodent NG2 reporter cell lines from NG2-astroglia reporter mice and human progenitor-reporter cell lines. Aim 2) Identification of astroglial transforming small molecules from a diverse chemical compound library and a library of FDA approved drugs in rodent reporter cells, followed by functional validation of new astrogliogenesis from these gliogenic drugs. Finally, the new gliogenic drugs will be validated in the human progenitor cell lines including cell-specific markers and functional activity in vitro. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: This proposal is solely focused on the use of relevant rodent and human progenitor cells as a discovery tool to identify drugs would could transdifferentiate -in vivo-existing adult glial progenitor cells and lead to the generation of new glial cells. The generation of new astroglial cells could be therapeutically valuable for neurological diseases including ALS, Huntington's disease, Alzheimer's disease ,epilepsy, multiple sclerosis and transverse myelitis. Most importantly, it could generate adult stem cell therapy- by activating endogenous adult CNS progenitor cells to differentiate into new astroglia-thereby eliminating need for external cellular based therapy. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

DESCRIPTION (provided by applicant): A growing body of evidence suggests that astrocytes are critical for the support of normal neuronal function, and astrocyte dysfunction may contribute to a number of neurodegenerative diseases. One important function of astrocytes is to transport nutrients from capillaries to neurons. Much of the nutritional support is in the form of glucose;however, it has been theorized that an alternate pathway, involving lactate, may also be an important source of energy for neurons. Astrocytes produce high amounts of lactate through glycolysis, and lactate can be transported from astrocytes to neurons via distinct molecular subspecies- MCT1, MCT4 in astroglia and MCT2 on neurons. Recent in vivo studies suggest that excitatory transmission may be regulated- in part - by networked astrocyte regulation of neuronal metabolism. In vitro, exogenous lactate or co-culturing with astrocytes is able to prevent the neuronal cell death induced by glucose deprivation. This protective function of astrocytes is lost by treatment with inhibitors to astroglial lactate transporters MCT1 and MCT4. In this Proposal we will first determine if astroglial lactate export by MCT1 or MCT4 is necessary for neuronal survival in vitro. These studies will test the hypothesis that astroglial lactate export is a fundamental astroglial support pathway for neurons. We will then determine if astroglial lactate transporters MCT1/MCT4 are A) Participants in normal neuronal function/activity in vivo and B) are necessary for neuronal survival in vivo. We hypothesize that Astroglial MCT transporters are likely to be necessary for normal neuronal function. Finally we will determine if dysregulation of MCT expression contributes to the neurodegeneration in ALS models if and repair of this pathway is neuroprotective. Overall, we hypothesize that astrocytes support neurons via essential lactate transport/export thru astrocyte specific transporters MCT1/MCT4 and that this pathway is a significant part of astroglial dysfunction in neurodegeneration and contributes to motor neuron death in diseases like ALS.

DESCRIPTION (provided by applicant): Induced Pluripotent Cells (IPS) from ALS patients could provide an exceptional tool by which we can disease pathophysiology and druggable targets. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines representing the natural disease in the most relevant cell types- motor neurons and astrocytes- provides unprecedented tools to 1) study cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. We have generated iPS cell lines, under identical conditions, from various familial ALS (fALS) mutations and sporadic ALS (sALS) patients to determine how representative they are for cell type specificity and functional biology. The first set of cells now generated, are ready for use in disease phenotyping paradigms. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate fALS and sALS cell lines and to use them to develop cell specific phenotypic assays. Project 1, lead by Dr. Eggan will generate new critical IPS line- isogenic liens from selected fALS mutations as well as non-integrating IPS lines- to complement our retroviral based IPS collection. In addition Project 1 will fully evaluate the pluripotency of the lines using a novel genetic scorecard system. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab. Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which ofthe fALS IPS cell lines have the appropriate characteristics of motor neurons and astroglia, thru sequential analyses. In addition, both groups will generate Zinc-finger based cell specific reporter cell lines for future use in drug discovery assays. Cell lines that meet final criteria (a compared to human ES cell and prior work on human astroglia) will undergo genetic analysis in the Project 4 lab, lead by Tom Maniatis. Finally, to develop useful tools for therapeutics and pathogenesis, Cores 1 and 2 will generate motor neuron and astroglial disease phenotyping assays. PUBLIC HEALTH RELEVANCE: Understanding the pathophysiology and development of new therapeutics for ALS has been an enormous challenge. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery.

At the time of submission of the orginal ALS iPS Go Grant, there were no well-validated protocols for generating astroglia from human ES or IPS cell lines, although approaches from rodent cells existed. Fortunately, we were able to develop a reliable protocol in both the Henderson lab and the Rothstein labs (Figure 5A). We now have a protocol that works identically in both labs¿with very similar efficiency at differentiation between labs. We have also been able to detect evidence of true astroglial maturation in a number of lines that have advanced sufficiently and these express appropriate astroglial specific proteins and have functional glutamate transport (Figure 5B-D).

DESCRIPTION (provided by applicant): The overall aim of this proposal is to study the pathology of the expanded hexanucleotide repeats in the C9orf72 gene using human iPS differentiated neurons and glia cells. The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene on chromosome 9p21 has been discovered as the cause of approximately 30-50% of familial and up to 10% of sporadic ALS cases as well as 12% of familial FTD cases, making this the most common known genetic cause of ALS/FTD to date. Expanded repeats are highly unstable and potentially yield toxic RNAs that accumulate in the nucleus and are hypothesized to cause cellular dysfunction via aberrant DNA and protein binding. Preliminary data from our laboratory confirm (GGGGCC)n nuclear RNA foci, aberrant gene expression and nuclear retention of RNA binding proteins (RBPs) in C9orf72 patient derived fibroblasts and iPS differentiated neurons. We therefore propose to elaborate on these early findings by generating an extensive genetic profile of ALS/FTD human iPS neurons and glia cells through the use of microarray and validate whether the iPS changes are relevant by comparing to human autopsy C9orf72 brain tissues. Furthermore, we will use C9orf72 iPS cell lines to investigate the RNA toxicity/pathology thru the identification of aberrant accumulation and binding of RNA binding proteins. Finally we will determine if we can abrogate C9orf72 genomic toxicity and pathology with antisense oligonucleotides already designed and validated in our laboratory. The likelihood of success will be greatly enhanced through a collaborative working relationship, the availability and experience of using iPS cells and human tissues. The extensive use of iPS cells to model disease, to cross correlate with human tissues and their use to validate ameliorative antisense therapy provides an important and possibly new direction for understanding disease pathophysiology and therapeutics development. !

Administration Component The Administrative Core is responsible for setting the overall direction of the NeuroLINCS center and for ensuring that the resources and components of the Center are optimally utilized. The successful development and evolution of the NeuroLINCS center requires strong interactions between the leaders and co-leaders of each Component and of the center as a whole. Hence, the NeuroLINCS Administrative Component plays a vital role in facilitating these interactions. Moreover, the Administrative Component and its personnel provide the necessary administrative and fiscal oversight to ensure that the NeuroLINCS center is run efficiently. The NeuroLINCS center involves 5 principal sites with defined responsibilities of growing, differentiating and generating new induced pluripotent stem cell lines (iPSCs), performing data generation assays on human brain cells made from iPSCs in response to perturbagens, performing basic analyses and developing cell signatures through integrated data analysis .methods, and establish community interactions. An integrated and highly collaborative group of investigators with expertise in stem cell biology, IPS cells, quantitative molecular phenotyping (omics and single cell imaging) and bioinformatics will work closely together to generate significant and highly predictive cell signatures. The PIs of the NeuroLINCS center are Steven Finkbeiner (Gladstone), Ernest Frankel (MIT), Jeffrey Rothstein (JHU), Clive Svendsen (Cedars) and Leslie Thompson (UCI), who will serve as leaders and co-leaders of components. Each Component has identified co-investigators/collaborators/consultants appropriate for the planned scientific investigations. Component leaders and co-leaders will also be active participants in NeuroLINCS consortium working groups as they are developed to address specific issues. Results of the genetic, proteomic and other characterization conducted by consortium labs will provide important feedback for further enhancement of induction and differentiation protocols and related methodologies and it is anticipated that this collaborative and iterative approach will lead to the broadest success for the study. An Evaluation Program within the NeuroLINCS is in place to determine if the programs supported are meeting the needs of the research community, are efficiently managed, and demonstrably effective and annual objectives and milestones.

Data Generation Component We propose three broad experimental aims based around the type of assay, perturbagens and technologies being applied that will overlap across the years. The first will use iPSCs from three disease states (non affected, SMA and ALS) in which we have shown specific phenotypes. We will use an iterative approach by first screening for a number of perturbagens of interest to the broad neuroscience community using cost effective assays including simple cell death models and a highly novel imaging analysis system. The second parallel effort will be to use the same iPSC lines but in this case test a set of known cell modifiers (Glutamate, ER stressor and SOD1 ASO) as perturbagens and perform massive parallel quantitative molecular phenotyping (QMP) to generate robust signatures and to define the responses of motor neuron cultures to these perturbagens. We will then perform QMP on neurons, astrocytes and oligodendrocytes from disease and control cells and in response to the same perturbations as above to elucidate signatures across broadly relevant neural cell types. This data will be compared to motor neuron cultures (where expected disease signature will be) with non motor neuron cultures (where no or a more restricted disease signature is expected) to resolve the question of cell type specificity. We will also generate new iPS lines from post mortem human patient tissues to allow clinical pathological signatures to be incorporated into the LINCS data, providing a unique resource to both the SMA and ALS scientific community and to researchers interested in larger questions relating to the CNS. The third is to bring in disease iPS lines from Huntington's and Parkinson's subjects (from the respective NIH consortia and in coordination with various foundations - see letters of support. Overall) and test the specificity of signatures seen in the motor neuron diseases with other neurodegenerative conditions (both disease and response to perturbagens). All of these studies will be done in close association with the data analysis component community section (being responsive to the needs of the community). Given the speed of discovery in iPSC and new molecule generation, we also aim to be flexible in our design to allow incorporation of breakthrough technologies or drugs should they arise.

DESCRIPTION (provided by applicant): There is a critical need to define the state and predict the behavior of human brain cells in health and disease. The number of different cell types in the CNS remains undefined, and despite a demographically ordained wave of neurodegenerative diseases, not a single disease-modifying therapy exists. Our knowledge of the CNS and the foundation for intervening rationally in disease would be dramatically advanced by generating quantitative molecular phenotypes essentially cell signatures of human neurons, astrocytes and oligodendrocytes from healthy people and from patients with motor neuron disease, Huntington's disease, and Parkinson's disease. The CNS is so unique that studying non-neuronal cells does not provide much assistance. Despite this desperate need, the inaccessibility of human brain cells meant studying them would have been impossible until the recent discovery of cellular reprogramming and induced pluripotent stem cell technology. Here we propose to form the NeuroLINCS consortium to accomplish these goals. We have handpicked the team to bring in critical expertise in iPSC technology, disease modeling, transcriptomics, epigenomics, metabolomics, whole genome sequencing, proteomics, high content, high throughput longitudinal single cell analysis, other cell-based assays, bioinformatics, statistics and computational biology. In addition, we are collaborating with Google to bring in special expertise in machine learning and the integration of signatures across platforms into highly predictive models of responses to perturbagens. Together, we expect to develop cell signatures of an array of human brain cell types under different conditions that should be broadly applicable to the LINCs community. We also anticipate generating innovative software tools and approaches that will make the signature generating process cheaper, faster, and more reliable. Besides the unique combination of expertise represented within NeuroLINCS, another distinguishing feature is the long track record that its members have of collaborating with each other. That collaborative spirit will be expressed in NeuroLINCS through its significant and multifaceted community outreach programs. These will involve specific and detailed plans to make the data and tools that NeuroLINCS generates available to the community, to interact with other LINCS sites, and to prepare for DCIC and the prospect of disseminating knowledge and resources at scale.

Community Interactions & Outreach Component The NeuroLINCS Community project will plan to provide resources and tools for a broad user base of basic and clinical scientists. It has a structure to facilitate access to the various genetic and proteomic data sets, the signatures created, and the analysis tools. It is designed to be directed to researchers at the bench, clinicians developing biological disease readouts and those in computational roles. It will incorporate an assessment to demonstrate the utility of the generated resources, methodologies, and analytical tools to LINCS and non-LINCS scientific community. Importantly, it will develop and implement a plan to bring in external collaborators who may have data sets that bear on the development of cell signatures. There is an extensive plan to develop workshops, tutorials, and symposia in conjunction with the use of innovative online technologies for disseminating information to target the major LINCS goals. Finally it will develop bidirectional links with the neuroscience clinical and basic community through a series of collaborations with large National clinical data and tissue-based networks.

DESCRIPTION (provided by applicant): Addiction to cocaine remains a serious unmet medical need for which there is no truly effective treatment. Addiction to cocaine an relapse following withdrawal of the drug are associated with changes in CNS glutamate levels and homeostasis. It has been demonstrated that Ceftriaxone, a cephalosporin antibiotic, increases the expression and function of the major glutamate transporter GLT 1 (Rothstein et al, 2005). Ceftriaxone has also been shown to increase the activity of system xC-, which exchanges extracellular cysteine for intracellular glutamate (Lewerenz et al, 2009). Basal non-synaptic glutamate levels in the nucleus accumbens are largely controlled by system xC-, and a decrease in its activity is the cause of the altered glutamate homeostasis observed in this brain region following cocaine self-administration and extinction training in rats (Baker et al, 2003). The catalytic subunit of xC- is xCT, and we have demonstrated that expression of xCT and GLT-1 are decreased in the nucleus accumbens core following cocaine self- administration (Knackstedt et al, 2010a). We have also shown that Ceftriaxone attenuates cue- and cocaine- primed reinstatement while restoring levels of both xCT and GLT-1 in the nucleus accumbens core (Knackstedt et al, 2010a). Furthermore, the protective effect of Ceftriaxone against relapse lasts for weeks following the last administration of Ceftriaxone (Sondheimer & Knackstedt, 2011). While Ceftriaxone shows preclinical promise as a medication for the treatment of cocaine addiction in humans, it possesses several characteristics that may prevent it from translating from the bench to the clinic. As an antibiotic, it possesses antimicrobial activity and thus chronic use of ceftriaxone can induce resistant strains of bacteria. At the high doses required to achieve therapeutically meaningful CNS concentrations (due to Ceftriaxone's low brain bioavailability), chronic Ceftriaxone will likely produce undesirable side effects such a diarrhea. Additionally, Ceftriaxone requires parenteral infusion and it is unlikely that cocaine dependent-patients would comply with daily intravenous administration of Ceftriaxone. We have recently identified MC-100093, a lead molecule from a series of monocyclic azetadinones, as a potent up-regulator of GLT-1 expression that is orally bioavailable, brain penetrant, and induces GLT-1 up-regulation in an accepted model of cocaine addiction and withdrawal. This proposal aims to further optimize the chemical scaffold represented by MC-100093 using a well-defined screening scheme and a multidimensional approach to arrive at one or more advanced lead molecules that can be advanced to IND-enabling studies.

Data Analysis & Signature Generation Component Our goal is to generate cellular signatures of human neurons in response to perturbagens. Our studies will focus on human neurons, generated from induced pluripotent stem cells (iPSCs) (i-neurons) obtained from both healthy people and patients with neurodegenerative diseases. The cellular signature will be a composite picture of the molecular properties of a neuron that distinguish the state and determine the behavior of the cell. We will generate three classes of cellular signatures. The first will be static signatures based on quantitative molecular phenotyping involving OMIC analysis of the i-neurons. Analysis of the static signatures will highlight critical signaling pathways that distinguish a cellular response to a perturbagen. The second type of signature will be dynamic signatures generated with a novel high throughput, single cell longitudinal analysis system. Robotic Microscopy (RM). RM will be able to pinpoint critical times in the life of i-neurons as their physiology change in response to perturbagens. Analysis of dynamic signatures will guide selection of time points that will be investigated more in depth with methods that generate static signatures. In turn, elements of these static signatures will be perturbed genetically and analyzed by RM to elucidate the epistatic relationship of the components of a signature and to develop explicit multivariate predictive descriptions of cellular responses to perturbations. The third type of signature will emerge from an integration of the individual signatures using clustering methods and machine learning algorithms. The technology to analyze the data of the cellular signatures will be compatible with those produced at other sites in the LINCS network. A major innovation of our program is the implementation of novel data analysis platforms that will produce signatures that will have greater predictive value of a cell's biology than standard technologies. We will integrate Data Analysis and Data Generation, creating feedback loops to allow the cellular signatures that we generate to influence subsequent data generation. In turn, the use of machine learning algorithms in collaboration with Google will allow us to iteratively refine our signatures to make them more predictive in identifying cause and effect relationships from the cellular signatures.

? DESCRIPTION (provided by applicant): Glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excessive activation of glutamate receptors leads to excitotoxicity, which in turn contributes to cell death observed after acute neurologic insults and in chronic neurodegenerative diseases. A family of Na+-dependent transporters controls extracellular Glu and prevents excitotoxic activation of Glu receptors. The GLT1/EAAT2 subtype of transporter mediates the bulk of this activity in the forebrain, and it is almost exclusively expressed in astrocytes. The levels of GLT1 are decreased in several neurologic diseases, including amyotrophic lateral sclerosis (ALS). To study transcriptional regulation of GLT1 we generated a BAC-GLT1-eGFP transgenic mouse that utilizes a large bacterial artificial chromosome (BAC) to express eGFP under the control of the full length GLT1 promoter. To better understand which region of the GLT1 promoter is necessary and/or sufficient for astroglial GLT1 expression, we generated a family of promoter reporter mice that utilize increasing amounts of the 5' non-coding region of the GLT1 gene (2.5, 6.7, 7.9, and 8.3 kilobases) to express tdTomato. When we crossed these mice with the BAC-GLT1-eGFP mice to produce dual reporter mice we made two exciting and unexpected observations. First, the promoter region between 7.9 and 8.3 kb is required for specific expression of tdTomato in astroglia. This region contains a domain that is evolutionarily conserved from rodents to humans, suggesting that this domain is critical for selective in vivo astroglia expression of GLT1. Second, although tdTomato is only found in eGFP-expressing astroglia, not all eGFP-expressing astroglia express tdTomato; the tdTomato/eGFP (double+) astrocytes are enriched in regions where GLT1 selectively decreases in ALS. This suggests that the 8.3 kb portion of the GLT1 promoter is only sufficient to induce expression of GLT1 in a defined subset of astrocytes, providing evidence for the existence of distinct subtypes of astrocytes. Based on these and other preliminary data, we propose two specific aims: 1) We will test the hypothesis that subtypes of astrocytes use different extrinsic stimuli (neurons and endothelia) to activate different intrinsic signals (Pax6 and Notch with associated promoter elements) to control subtype-specific expression of GLT1. We will determine if this differential control of GLT1 generalizes to proteins that are differentially expressed in these subpopulations of astrocytes. We will confirm that these subpopulations of astrocytes are found in humans. 2) We will test the hypothesis that the subtypes of astroglia identified by the 8.3 kb promoter reporter mice are selectively affected in mouse models of ALS. We will confirm pathologic changes using human tissue. Finally, we will test the hypothesis that the subtype of astroglia identified by the 8.3 kb promoter reporter mice selectively contributes to the known non-cell autonomous motor neuron degeneration.

PROJECT SUMMARY A GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), though the underlying disease mechanism is poorly defined. Multiple studies, including our own, support a gain-of-function mechanism of neurotoxicity mediated by the HRE. Expanded repeats may generate toxic RNAs that sequester RNA-binding proteins. They may also be translated via Repeat-Associated Non-ATG Translation (RANT) into toxic dipeptide repeat proteins (DPRs). Both HRE RNA and DPRs are hypothesized to mediate neurotoxicity in C9-ALS/FTD. Multiple recent studies from our lab and others suggest that disruption of the nuclear pore and/or nucleocytoplasmic transport is a primary cause of neurodegeneration in yeast, fly, and induced pluripotent cell (iPS) models of C9- ALS/FTD. In addition, our recent studies, using a C9-ALS/FTD Drosophila model, human iPS neurons derived from C9-ALS patients, and human C9-ALS CNS tissues, suggest that nucleocytoplasmic transport defects may be a fundamental pathway for ALS/FTD pathogenesis amenable to therapy. This proposal will comprehensively investigate the mechanism by which the C9ORf72 HRE disrupts nucleocytoplasmic transport and nuclear pores utilizing several complementary models including C9-ALS fly and mouse models and iPS neurons and brain tissue from C9 ALS/FTD patients, and investigate whether modulation of nucleocytoplasmic transport may be a therapeutic strategy for ALS/FTD. (1) We will determine the morphological and biochemical composition of the nuclear pore complex (NPC) in motor neurons and glia, and characterize NPC pathology in C9-ALS/FTD in fly, iPS, mouse models and human brain. Little is known about CNS NPCs including differences between cell types and ultimately how the NPC constituents, nucleoporins, are dysregulated in C9-ALS/FTD models. Therefore, understanding the basic characteristics of the NPC and nucleocytoplasmic transport in the CNS and in disease models is fundamentally important to dissecting the nature of pathology. (2) We will then investigate the mechanism of nucleocytoplasmic transport disruption in C9-ALS/FTD. We hypothesize that disrupted NPC and/or nucleocytoplasmic transport function causes neurodegeneration due to nuclear loss and/or cytoplasmic accumulation of nuclear export sequence (NES) containing cargo in fly, mouse, and iPS models of C9-ALS. (3) Therefore, we will determine the consequences of nucleocytoplasmic transport disruption in C9-ALS/FTD models. (4) Finally, we will determine if restoration of nucleocytoplasmic transport rescues neurodegeneration in C9-ALS/FTD by employing a series of novel compounds that may have human utility.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized clinically and pathologically by progressive weakness and degeneration of motor neurons. A subset of patients can also have frontotemporal dementia with cortex injury. Though the symptoms of ALS are due to neuron degeneration, extensive research has shown that support cells in the CNS, including microglia, astrocytes, and recently oligodendrocytes, contribute to motor neuron degeneration. Our lab and others have shown that oligodendrocytes degenerate in ALS and that dysfunctional oligodendrocytes contribute to motor neuron degeneration, perhaps through failure of metabolic support to neurons. Oligodendrocyte dysfunction has been found in sporadic ALS, but also familial ALS associated with mutations in superoxide dismutase. Importantly, research from our laboratory has shown that oligodendrocytes play a critical role in neurodegeneration in SOD1 mice, since removing mutant SOD1 specifically from oligodendrocyte precursor cells (OPCs) and oligodendrocytes significantly prolongs lifespan in this mouse model of ALS. In the last several years, many research groups have focused on the recently discovered hexanucleotide repeat expansions (HREs) in C9orf72, which is the most common cause of familial ALS and also a common cause of frontotemporal dementia. These studies have determined that the neurotoxicity is likely due to both RNA- and dipeptide repeats (DPR) protein-mediated events. The exact mechanism by which these events produce toxicity is unknown, but published work by our laboratory and others has demonstrated that nucleocytoplasmic transport and nuclear pore proteins are disrupted in neurons expressing C9orf72HREs and restoration of this critical cell function leads to attenuation of neuronal toxicity. To date, there has been only one study on the role of C9orf72HREs in oligodendrocytes. In this proposal, we will thoroughly investigate the role of C9orf72HREs in OPCs and oligodendrocytes and their contribution to cellular dysfunction and degeneration in cellular and mouse models. We hypothesize that oligodendrocytes are dysfunctional in C9orf72 ALS and that alterations of nucleocytoplasmic transport lead to oligodendrocyte injury and reduced capacity for OPC differentiation. Specifically we propose to determine whether there is oligodendrocyte degeneration and OPC proliferation in ALS patients, and animal models with C9orf72 HREs. Our preliminary studies suggest C9orf72 is highly expressed in oligodendrocytes, which are dysfunctional in C9orf72 patients. We will then determine whether OPCs fail to differentiate and/or oligodendrocytes degenerate in C9orf72 ALS due to direct effect of repeat expansion on oligodendrocytes or an indirect effect from neuronal toxicity. Using oligodendrocyte monocultures and co-cultures with neurons derived from C9orf72 iPS cells and C9BAC mice, along with appropriate controls, we will evaluate OPC and oligodendrocyte proliferation, differentiation, survival, myelination, and support of neurons. We will also evaluate the impact on oligodendrocytes in vivo through viral vectors expressing HREs selectively in oligodendrocytes or neurons. To better understand the mechanism of oligodendroglial injury, we will determine whether OPCs or oligodendrocytes have dysfunctional nucleocytoplasmic transport in ALS patients and C9orf72HREs BAC transgenic mice. Finally, in hopes of using these model systems to mitigate injury, we will determine whether dysfunctional nucleocytoplasmic transport in OPCs and oligodendrocytes can be attenuated through genetic and pharmacologic techniques, including antisense oligonucleotides and nuclear transport modulators.

The intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of Alzheimer's Disease (AD). While tau pathology is correlated with both synaptic loss and neurodegeneration in AD, implicating a major role for tau accumulation in cognitive decline, the mechanisms of tau aggregation and cell toxicity remain unknown. We propose to provide novel insight into the extent to which abnormalities in nuclear pore complexes (NPCs) and the resulting nucleocytoplasmic transport (NCT) defects contribute to AD pathogenesis. NPCs are complex, molecular assemblies consisting of multiple copies of proteins called nucleoporins (Nups) that regulate the macromolecular trafficking of protein and RNA between the nucleus and cytoplasm. Previous reports have identified nuclear membrane irregularities that often associate with NFTs. However, the pathophysiology of tau and its relationship to nuclear function/integrity is not well understood. We have amassed a series of preliminary data demonstrating that disruptions of the NPC and functional nuclear transport are present in cells containing hyperphosphorylated tau in human AD brain, as well as in mouse and cellular models of tauopathy. Given the extent of NPC and NCT dysfunction in ALS and HD, our preliminary studies in AD/FTLD-tau suggest that impaired NPC function may represent a common mechanism of neurodegeneration. Specifically in AD, we find that a major nuclear pore component, Nup98, is mislocalized from the nuclear membrane and co-aggregates with tau in both human brain and transgenic tau mouse models. Tau and Nup98 directly interact and we have discovered that Nup98 can potently promote tau filament formation in vitro. Based on these key findings, we will test the overall hypothesis that tau-Nup98 interactions underlie a pathologically important disruption of nuclear pore function and represent a key mediator of neurotoxicity in AD and related tauopathies. As the ultimate goal of our project is to develop a disease- modifying therapeutic strategy, we will assess whether either clearing tau/Nup98 inclusions or rescuing the NCT defects are necessary and/or sufficient to ameliorate tau toxicity. Our studies specifically will: 1) Investigate alterations in nuclear pore complexes in human AD/FTD and model systems. These studies will establish NPC defects as a novel, pathological feature of tauopathy; 2) Determine the functional alterations in nucleocytoplasmic transport resulting from nuclear pore abnormalities in AD/FTD. These studies will determine if aberrant functional NCT is characteristic of cells containing tau lesions.; and finally, 3) Examine whether disaggregases rescue NPC/NCT pathway defects in vivo. These studies will help to test the hypothesis that NPC defects can contribute to tau toxicity

PROJECT SUMMARY Astroglia are essential for the homeostasis and maintenance of the central nervous system (CNS). They display a vast array of roles such as neurotransmitter metabolism, regulation of synaptic neurotransmitter clearance, extracellular ion buffering, neurotrophic release, immune signaling and blood-brain-barrier maintenance. It is not surprising that astroglia in different regions display diverse functions to maintain their environmental niche. Historically, astroglia were placed into two groups based on their neuroanatomical localization and morphological depictions: protoplasmic astroglia of the grey matter and fibrous astroglia of the white matter. Recent evidence has suggested that astroglia consist of different subpopulations, similar to neuronal functional and molecular heterogeneity. Nevertheless, there still remain large gaps in our understanding of these different subtypes due to a lack of molecular markers to identify and study these populations and how these different astrocytes serve to regulate neurons and their synapses. We recently generated a novel transgenic mouse model that selectively and robustly labels a specific astroglia subpopulation in the adult CNS and thru RNA and protein analyses, have learned that these astroglia are highly and selectively enriched in a secreted protein, norrin. A mutated form of norrin is the cause of a rare neurological degenerative disease, Norrie disease. Our preliminary studies strongly suggest that astroglial norrin plays a significant role in the local formation and/or maintenance of local dendrites and spines. We have early data to suggest that this astrocytic norrin regulates neuronal spine density and dendritic branching in cortical layers. Furthermore, our studies suggest that this astrocyte subpopulation is dramatically affected in amyotrophic lateral sclerosis. In collaboration with Jackson Labs, we recently generated a Norrie disease transgenic mouse which can allow us to explore this protein function in vivo and in disease. We plan several approaches to understand the biology of astroglial norrin and how it may alter dendrites/spines as well its contribution to neurodegeneration in ALS and Norrie disease. We propose to: 1) Evaluate the role of cortical astroglial Norrin in regulating neuronal dendrites and spines in vitro and in vivo. These studies will demonstrate the role that Norrin has in regulating neurons, primarily through dendritic and synaptic development and/or maintenance. 2) Determine whether astroglial mutant Norrin is sufficient to alter synaptic development and/or maintenance in vivo. We have first model of Norrie disease, and will test the hypothesis that norrin can rescue this neurodegenerative disease of astroglia. And 3) Investigate the loss of astroglial norrin in contributing to synaptic loss and neurodegeneration in motor neuron disease models and human ALS. These studies will evaluate the contribution of norrin to synaptic injury in several ALS models and human ALS. Taken together, these findings set the stage to study of this newly identified astrocyte subpopulation in both health and disease, including the potential to generate cell astrocyte-specific therapeutics for several neurological disorders.

PROJECT SUMMARY/ABSTRACT Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), the second most common form of dementia, comprise a spectrum of fatal neurodegenerative diseases. An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene, have been linked to ALS and FTD, although clinically two distinct diseases. The C9orf72 HRE is the most common cause of both familial and sporadic ALS accounting for ~40% and ~8% of patients respectively. Overall, about 10% of ALS cases are familial with the remaining 90% being sporadic. The molecular mechanisms underlying disease pathogenesis remain poorly understood. Defects in nucleocytoplasmic transport (NCT) and the nuclear pore complex (NPC) have recently emerged as a prominent pathomechanism underlying multiple neurodegenerative diseases including C9orf72 ALS/FTD, subsets of sporadic ALS, Alzheimer?s Disease, and Huntington?s Disease. However, little is known about the nature of the injury to the NPC and its individual nucleoporin components themselves. Using induced pluripotent stem cell derived spinal neurons (iPSNs) and postmortem human tissue, we have amassed data that loss of the transmembrane nucleoporin POM121 from NPCs initiates a pathological cascade impacting NPC composition, function and downstream cellular survival. Notably, loss of POM121 is mediated by pathologic G4C2 repeat RNA and not dipeptide repeat poly peptides or loss of C9ORF72 protein. Given that POM121 protein is not mislocalized and POM121 RNA metabolism is unaltered, we hypothesized that POM121 and subsequently altered nucleoporin proteins are aberrantly degraded in the early stages of C9orf72 ALS/FTD pathogenesis. Recent work in yeast and non-neuronal mammalian cells has shown that nuclear CHMP7 ?activates? ESCRT-III mediated degradation of nuclear pore complexes and nuclear envelope components during nuclear pore surveillance and homeostasis. Our new preliminary data suggests that the loss of POM121 from the nucleoplasm and NPCs is initiated by nuclear accumulation of CHMP7. Mechanistically, increased nuclear CHMP7 appears to be the result of G4C2 repeat RNA mediated impaired nuclear export. Thus, our data strongly implicate a CHMP7 degradative pathway in disease pathogenesis. Intriguingly, knockdown of CHMP7 mitigates NPC injury in C9orf72 iPSNs making CHMP7 an attractive therapeutic target in neurodegeneration. In this proposal we will comprehensively investigate this new pathway including studies to 1) Determine the degradative pathway by which CHMP7 mediates nucleoporin removal from NPCs in iPSNs, 2) Investigate the mechanism by which pathologic G4C2 repeat RNA initiates CHMP7 mediated NPC injury. And finally, 3) using a large battery of individual patient iPSN spinal neuron cell lines, evaluate the ability of CHMP7 antisense oligonucleotides to mitigate C9orf72 ALS/FTD and sporadic ALS mediated alterations in the nuclear pore complex and nucleocytoplasmic transport and downstream sensitivity to stressors in iPSNs.