Xiao-Jiang Li - US grants

DESCRIPTION The applicant proposes to study the binding of HAP1 to other cellular proteins. The applicant identified HAP1 in the rat as a protein that binds to huntingtin, the protein that is abnormal in Huntington's disease. Huntington's disease is an autosomal dominant disease characterized by massive neuronal loss that is particularly severe in specific brain regions. The genetic defect is an expansion of a CAG trinucleotide repeat that produces a polyglutamine domain in an expressed protein named huntingtin. The applicant has documented that HAP1 binds to huntingtin in vivo and that there is increased binding of HAP1 to huntingtin that has an expanded polyglutamine domain. He has also found that HAP1 associates with accessory proteins for the microtubular motor proteins kinesin and dynein that participate in the anterograde and retrograde transport in the neuron. These accessory proteins are kinesin light chain and dynactin p150Glued. Also, HAP1 associates with various membranous organelles. Thus the applicant developed the ingenious and powerful hypothesis that HAP1 plays a role in targeting motor protein complexes to intracellular structures; also, that HAP1 may coordinate the two types of transport. The applicant further hypothesizes that mutant huntingtin may interact with HAP1 in such a way that the association of HAP1 with these other molecules is perturbed and thus intracellular trafficking is affected. Thus the applicant proposes three specific aims. The first is to clone and characterize homologues of human HAP1. In this aim, he proposes also to generate antibodies to human HAP1 protein, characterize the binding properties of human HAP1 to huntingtin, determine the regional distribution of human HAP1, and determine the subcellular localization of HAP1 in human and monkey brain and compare this to human huntingtin. In Aim 2, he will investigate the association of HAP1 with microtubular motor binding proteins and intracellular organelles. In this aim, he will also determine which regions of HAP1 that bind to kinesin light chain, dynactin p150Glued, and intracellular organelles. He will also examine the effects of phosphorylation, calcium level, and other cellular factors on the association of HAP1 with huntingtin and to the proteins involved in intracellular transport. He will also identify the potential proteins that link HAP1 to intracellular vesicle membranes. In Aim 3, he will study the effect of huntingtin on the association of HAP1 with motor binding proteins and organelles. In particular, he will study huntingtin with different numbers of glutamine repeats on the self-interaction of HAP1 and on HAP1's interactions with other molecules using a variety of techniques.

Nerve cells, or neurons, have elongated branches called neurites that sometimes reach long distances to make connections with other parts of the brain. Inside the cell are structures called organelles that are important for transport of materials from one place to another. For transport within the cell, important biochemicals often are packaged in small bubble-like containers called vesicles. The actual mechanisms of transport are not fully understood, but some proteins are believed to act like motors, transporting vesicles along 'tracks' of organelles called microtubules. This project investigates a recently discovered neural protein called HAP1, which is associated with microtubules and some kinds of vesicles. Biochemical and microscopic studies will examine how the HAP1 protein binds to form a molecular complex with some of the motor proteins associated with microtubule transport mechanisms. Results from this work will be important in understanding intracellular transport mechanisms, and so will be important beyond neuroscience to cell biology in general, and to developmental biology.

DESCRIPTION (provided by applicant): Intracellular trafficking of organelles and molecules, which is vitally important to the function of neuronal processes and nerve terminals, involves a variety of neuronal proteins that couple motor protein complexes with intracellular cargoes and structures. HAPl, a neural protein that binds to the Huntington's disease protein huntingtin, appears to play roles in intracellular transport and other neuronal functions. First, our studies show that it interacts with dynactin p150 and kinesin light chain, which are involved in microtubule dependent transport. Second, HAP1 is associated with a number of intracellular organelles or structures, especially a unique cytoplasmic inclusion of unknown function. Recently, we also observed that HAP1 is involved in neurite outgrowth and is colocalized with huntingtin in presynaptic terminals. We hypothesize that the interactions of HAP1 with various proteins confer its multiple and diverse functions, which may include the development of neurites and synaptic plasticity. The abnormal interaction of HAP1 with mutant huntingtin may affect some of HAP1's neuronal functions and contribute to the neuropathology of HD. Three aims are proposed to test the above hypothesis. (I) We will identify the regions in HAP1 responsible for binding to KLC or dynactin p150 to examine whether neurite outgrowth is affected by inhibiting the interaction between HAP1 and KLC or dynactin p150. (2) We will identify the nature of the unique cytoplasmic inclusion to understand how HAP1 is associated with this subcellular structure. We will investigate how HAP1 is targeted to synaptic vesicles and whether it is involved in synaptic vesicle recycling. (3) We will examine whether mutant huntingtin affects HAP1's protein interactions and its related functions. These studies aim to advance our understanding of neuronal function of HAP1 and huntingtin. They will also help us to understand the mechanism of the specific neuropathology in Huntington's disease.

Eight inherited neurological disorders have been found to associate with an expansion of a glutamine repeat in the protein products of the disease genes. Increasing evidence has shown that the expanded glutamine repeat causes the disease proteins to aggregate in the nucleus and to abnormally interact with other proteins, Studies of brains from patients with Huntington's disease (HD) and HD animal models have indicated that N- terminal fragments of huntingtin with expanded polyglutamine accumulate in the nucleus and are toxic to neurons. However, the mechanisms of these pathological events remain unknown. The aim of this proposal is to investigate the mechanisms of cellular pathology caused by intranuclear huntingtin with expanded polyglutamine. We will use cellular and animal models to address two important questions: (1) how N-terminal fragments of huntingtin with expanded polyglutamine are accumulated in the nucleus, and (2) how intranuclear mutant huntingtin induces cellular dysfunction. Accordingly, two specific aims will be pursued. Aim 1 is to study how N- terminal huntingtin with expanded polyglutamine is generated and accumulates in the neuronal nucleus and to identify proteins or cellular factors that keep mutant huntingtin in the nucleus. Aim 2 is to investigate how mutant huntingtin may interact with transcription factors and affects gene transcription. These studies will advance our knowledge about the pathogenesis of HD and provide vital information to help develop therapeutic strategies.

DESCRIPTION (provided by applicant): Synaptic toxicity of the Huntington's disease protein Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion in the N- terminal region of huntingtin (htt). It is characterized by progressive neurodegeneration that preferentiall affects the medium spiny neurons in the striatum. Furthermore, polyQ expansion leads to ht becoming misfolded and aggregated in an age-dependent manner. Thus, HD and other aging-related neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, share the major features of late-onset and selective neurodegeneration, as well as age-dependent protein aggregation. Because of its well-defined genetic mutation and neuropathology, HD makes an ideal system for investigating the pathogenesis of age-dependent neurodegenerative diseases. Moreover, numerous studies have shown that mutant htt can impair synaptic function, an early neuropathologic event that is also common in many other age-dependent neurological disorders. Despite the fact that mutant ht can affect a variety of synaptic functions, how exactly synaptic dysfunction contributes to HD neuropathology remains unclear. We also do not know how mutant htt can preferentially accumulate in the axons of medium spiny neurons in HD knock-in (KI) mice that express full-length mutant htt. Since it is medium spiny neurons that are mostly affected in HD, understanding the mechanism for this preferential accumulation is important to unravel the pathogenesis of HD and develop treatments for the early neuropathology of HD. We hypothesize that mutant N-terminal htt fragments may interact abnormally with axonal proteins to preferentially accumulate in axonal terminals of medium spiny neurons and affect synaptic function. To test these hypotheses, we have generated a novel transgenic HD mouse model that selectively expresses N-terminal mutant htt in axonal terminals. Using this HD mouse model, we will explore whether axonal terminal mutant htt can cause neurological symptoms and affect synaptic function, as well as identify the common pathological events that also occur in HD KI mice. We will then examine whether and how N-terminal mutant htt fragments can preferentially accumulate in axons and affect the function of medium spiny neurons by identifying the axonal proteins that may abnormally interact with mutant htt. These studies also have implications for our understanding of synaptic dysfunction in other age-dependent neurodegenerative diseases that are also caused by misfolded proteins.

DESCRIPTION (provided by applicant): Huntington disease (HD) belongs to a neurodegeneration disease family in which selective neuronal loss is caused by misfolded proteins. In HD, a polyglutamine (polyQ) expansion (>37 glutamines) in the N-terminal region of Huntington (htt) causes N-terminal htt fragments to misfold or aggregate, conferring neuropathology. Consistently, N-terminal htt fragments containing an expanded polyQ are able to interact with a number of proteins to mediate multiple pathological pathways. Thus, an important therapy approach is to inhibit the expression of mutant Huntington (htt) or its activity by blocking its abnormal interactions with other important proteins. Intracellular antibody (intrabody) provides a promising approach to achieve this goal, as intrabody is able to interact with an antigen intracellular to block its activity ortoxicity. However, normal htt is required for cell survival, and deletion of htt causes cell degeneration. Thus, a challenge for using the intrabody therapy for HD is to identify an intrabody that can selectively inhibit the toxicity of mutant htt but not interfere with the pivotal function of normal htt. Using the same antigen for an antibody (EM48) that reacts preferentially with misfolded and aggregated htt, we have established a hybridoma cell line that generates a monoclonal antibody (mEM48), which also preferentially reacts with mutant htt. By isolating the gene encoding mEM48 from this cell line, we have generated an intrabody that selectively binds mutant htt. We propose to characterize the protective effect of this intrabody on htt's toxicity in HD cellular models. We will also modify this intrabody to increase its expression level and stability. Finally, we will deliver this intrabody to HD mouse brain via viral vectors and to examine its protection against the neuropathology in HD brain. This proof of principle study will have a broad implication for the treatment of other neurodegeneration diseases that are also caused by misfolded proteins.

DESCRIPTION (provided by applicant): Nuclear accumulation of polyglutamine (polyQ) disease proteins is a common pathological feature of polyQ diseases, which contributes to altered gene expression and neuropathology. In Huntington disease (HD), N- terminal fragments of the disease protein huntingtin (htt) contain an expanded polyQ tract (>38 glutamines) and accumulate in the nucleus in aged neuronal cells, though these fragments do not have known nuclear localization sequences. Prior studies show that expanded polyQ can also cause a small cytoplasmic protein, hypoxanthine-guanine-phosphoribosyltransferase (HPRT), to accumulate in the nucleus even when this protein is tagged with nuclear export sequences. We found that N-terminal htt binds to the nuclear pore protein Tpr, which is involved in nuclear export, and that the binding is decreased by polyQ expansion or aggregation. We hypothesize that small N-terminal htt fragments generated by proteolysis are able to shuttle between the cytoplasm and nucleus. The nuclear export of N-terminal htt is facilitated by its interaction with Tpr. PolyQ misfolding and aggregation reduces the interaction of htt with Tpr and the nuclear export of htt. All these could also lead to abnormal interactions of mutant htt with transcription factors. To test these hypotheses, we propose three aims in this application. In Aim-1, we will identify the binding sites in htt and Tpr for their interaction. We will study whether polyQ misfolding, ubiquitination, or SUMOylation modulates this interaction to alter intranuclear accumulation of htt. In Aim-2, we will study whether the nuclear capacity to remove misfolded htt is decreased in neurons and whether this decrease results from impaired function of the proteasome or chaperones, which then leads to the selective nuclear accumulation of mutant htt in neurons. In Aim-3, we will use immunoprecipitation and mass spectrometry to identify transcription factors whose interactions with mutant htt in HD mouse brains are associated with disease progression and/or selective neurodegeneration. Understanding the molecular mechanism for the nuclear accumulation of mutant htt in HD-affected neurons will help develop a therapeutic strategy to treat HD.

PROJECT SUMMARY Obesity is a major health problem worldwide and also a leading risk factor for various diseases, including type 2 diabetes, stroke, and cardiovascular diseases. Since neuronal activities in the brain are critical for maintaining systemic energy homeostasis, abnormal neuronal functions could lead to the development of obesity; thus, unraveling the complex neuronal mechanisms behind the central control of energy homeostasis is a high priority if we are to understand the biology of obesity and eventually treat or alleviate the health burdens it imposes. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a newly identified neurotrophic factor whose protective efficacy has been confirmed in several neurodegenerative diseases, but its endogenous function in the brain remains largely unknown. Recently, we generated a transgenic mouse model in which MANF is overexpressed in the central nervous system. Surprisingly, MANF transgenic mice become obese and exhibit hyperphagia. Moreover, we found endogenous MANF is highly enriched in the hypothalamus, and its expression is closely linked to the feeding status of the mice. These observations led us to hypothesize that MANF is involved in the hypothalamic control of food intake and energy homeostasis. Specific Aims for testing this hypothesis are: Aim (1) To evaluate the phenotypes of mice by increasing or reducing MANF expression in the hypothalamus. We will use virus transduction and CRISPR/Cas9 technology to modulate MANF levels specifically in the hypothalamus and evaluate the metabolic phenotypes of the mice after such modulations; Aim (2) To identify hypothalamic MANF partners in the regulation of energy homeostasis. We will perform affinity purification chromatography followed by tandem mass spectrometry to comprehensively study the MANF interactome in the hypothalamus and how such interactions shape the function of MANF. The results of this study will broaden our knowledge about the neuronal functions that regulate energy homeostasis. Understanding the molecular mechanisms of MANF signaling will yield valuable insights for developing potential MANF-based therapeutic strategies to treat obesity and related neurodegenerative disorders. !

Huntington's disease (HD) is a devastating neurodegenerative disease caused by expansion of a polyglutamine (polyQ) domain in distinct proteins with different functions. In HD, the polyQ domain is located in the N-terminal region of huntingtin (Htt). This N-terminal region is well conserved in a wide range of species, but polyQ expansion can lead to misfolding and subsequent toxicity of N-terminal fragments of Htt. Since a lack of Htt causes embryonic lethality in mice, Htt is also thought to be essential for animal development and survival. Reducing the expression of mutant Htt is widely accepted as an important strategy for treating HD, so considerable efforts have gone into developing siRNA and anti-sense oligonucleotides to suppress the expression of mutant Htt. These approaches have also raised concerns that markedly suppressing Htt expression could lead to side effects by diminishing the normal function of Htt; however, whether Htt can preserve critical functions without the N-terminal domain that contains the polyQ domain remains unknown. Addressing this issue is important if we are to develop a new strategy to treat HD: if the N-terminal polyQ domain is not required for essential Htt functions and can be removed, complete elimination of the N-terminal region of Htt is now possible since the recent development of the genomic editing tool, CRISPR/Cas9. In this competitive renewal application, we will use CRISPR/Cas9 to investigate the toxicity of N-terminal mutant Htt fragments and therapeutic effects by removing the polyQ-containing N-terminal region. In Aim 1, we will use CRISPR/Cas9 to introduce mutations in the mouse Htt gene in embryos from HD 140Q KI mice to generate truncated mutant Htt genes that express different N-terminal Htt fragments and can be transmitted to offspring via the germline. Using the newly established HD KI mice that express different N-terminal mHtt fragments containing the same polyQ repeat (140Q) at the endogenous level, we will examine the relationship between the length of N-terminal mutant Htt fragments and their nuclear accumulation and toxicity in striatal neurons. In Aim 2, we will use CRISPR/Cas9 to remove the N-terminal polyQ domain as a therapeutic strategy. We will explore whether removing the N-terminal polyQ domain in Htt can eliminate neuropathology without affecting neuronal survival and function in adult mice. These studies will use HD knock-in mice in which mutant Htt is expressed at the same endogenous level as in HD patients. We hope these studies will not only provide new insight into the pathogenesis of N-terminal mutant Htt fragments, but also allow us to develop a novel therapeutic strategy to treat Huntington's disease and other polyQ diseases.

Project Summary Neuronal function of huntingtin-associated protein Huntingtin-associated protein-1 (Hap1) was first identified as a neuronal protein that interacts with the Huntington's disease (HD) protein, huntingtin (Htt). Hap1's binding to Htt is enhanced by expanded polyglutamine repeats in the N-terminal region of Htt. Moreover, unlike Htt, which is ubiquitously expressed, Hap1 is expressed primarily in neuronal cells, suggesting that it is a good candidate for involvement in the selective neurodegeneration in HD. Extensive studies have shown that Hap1 and Htt associate with each other in the intracellular trafficking of various vesicles and proteins. Also, like Htt, Hap1 is essential for early development and neurogenesis. Our recent studies showed that the function of both Hap1 and Htt is cell type and age dependent. We also know that neurogenesis is important for early brain development and the repair of neuronal damage in the adult brain. Although both Htt and Hap1 participate in neurogenesis, and impaired neurogenesis is seen in HD brains, whether Hap1 and Htt work together to regulate neurogenesis and whether mutant Htt affects neurogenesis via its interaction with Hap1 remain unknown. We hypothesize that Hap1 and Htt participate in age- and environmental stress-dependent neurogenesis and that mutant Htt affects this function by its interaction with Hap1. To test this hypothesis, we will use Hap1 KO and Htt KO mice as well as HD140Q knock-in mice to examine the role of the interaction of Hap1 and Htt in postnatal and adult neurogenesis. We will focus on neurogenesis in the hypothalamus and hippocampus, as loss of Hap1 in these two regions is found to cause body weight loss and depression, two well-known phenotypes that also occur in HD patients. We will use CRISPR/Cas9 to selectively deplete Htt expression or to increase the production of N-terminal mutant Htt in HD140Q KI mice to examine whether they have any effects on neurogenesis. Using these approaches, we propose two aims in this application. Aim 1 will investigate whether Hap1 and Htt work together to promote neurogenesis. Aim 2 will explore whether mutant Htt affects neurogenesis via its abnormal interaction with Hap1. The studies seek to provide new insight into the cell type- and age-dependent function of Hap1 and Htt, as well as the selective neuropathology of HD. Findings from these studies will also help us uncover therapeutic strategies for the specific neuropathology and phenotypes in HD. !

A variety of neurological diseases are caused by mutations in the disease genes that result in gain-of-toxicity in the brain. Lowering or blocking the expression of mutant genes is considered an effective therapeutic strategy for the treatment of these neurological disorders. In Huntington?s disease, the CAG repeat expansion in exon1 of the huntingtin gene leads to selective neurodegeneration and progressive neurological symptoms, which are incurable with current therapies. We will use a newly developed technology, CRISPR/Cas9, to eliminate the expression of mutant huntingtin in Huntington disease mice. Our preliminary studies have shown the promising effect of CRISPR/Cas9 to alleviate neurotoxicity and neurological symptoms in Huntington?s disease mice. However, the long-term effects of CRISPR/Cas9 and the safety issue of this new technology remain to be investigated. The current application will examine the gene targeting efficiency of modified Cas9 in adult mouse brains in R21 studies. The R33 studies will rigorously examine the long-term effects of removing mutant huntingtin in Huntington?s disease mice and potential side effects caused by CRISPR/Cas9. Given the increasing demand to use CRISPR/Cas9 to remove mutant genes in the brain to treat a variety of neurological disorders, our studies will have broad implications for the future clinical use of CRISPR/Cas9 to ameliorate neurological symptoms in brain diseases.