Joachim Herz, MD - US grants

low density lipoprotein receptor; protein structure function; blood lipoprotein metabolism; very low density lipoprotein; gene targeting; molecular chaperones; gene expression; adipose tissue; genetic polymorphism; intracellular transport; vascular endothelium; liver metabolism; recombinase; receptor binding; membrane transport proteins; genetic mapping; X ray crystallography; laboratory mouse; laboratory rabbit; genetically modified animals; polymerase chain reaction;

DESCRIPTION (provided by applicant): In this application we are using multidisciplinary approaches to investigate the common molecular mechanisms by which lipoprotein receptors of the low-density lipoprotein (LDL) receptor gene family regulate on one hand the integrity of the vascular wall, and on the other control development and function of the brain in the embryo as well as in the adult. During the first funding period we showed that the very-low-density lipoprotein receptor (VLDLR) and the Apolipoprotein E receptor-2 (ApoER2) function not only as transporters for cholesterol and other lipids, but also as developmentally essential signaling receptors that convey an important positional cue to migrating neurons that controls the formation of the neocortex, the hippocampus and the cerebellum. We showed that transmission of this signal involves the activation of a series of intracellular kinases. We also demonstrated that the same pathway remains active in the adult brain, where it no longer controls cell migration, but instead participates directly in neurotransmission, learning and memory. This novel role in the mature nervous system is of particular and immediate biomedical importance, since ApoE, a cholesterol transport molecule that binds to this class of lipoprotein receptors and can interfere with their signaling functions, is genetically strongly associated with Alzheimer's disease (AD). One focus during the next funding period thus will be the conceptual implications for the molecular pathogenesis of AD as well as atherosclerosis that arise from these findings. Specifically, we will investigate how VLDLR and ApoER2 interact with ApoE, signaling molecules and neurotransmitter receptors in the synapses of neurons in the adult brain and in the vascular wall, and how lipoprotein receptors integrate such diverse functions in vivo. This may provide novel targets for rational drug design and treatment of atherosclerosis as well as of AD, both disorders of increasing medical and socio-economic importance.

DESCRIPTION (provided by applicant): Members of the low-density-lipoprotein (LDL) receptor gene family have recently been found to participate in signal transduction pathways during the development of the brain. Reelin, a large secreted protein that bears no resemblance to lipoproteins and is not involved in lipid transport, binds to the extracellular domains of two members of this gene family, termed VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2). Binding of this ligand activates a cytoplasmic signaling cascade that apparently involves tyrosine kinases as well as the serine/threonine kinases Cdk5 and GSK-3beta. Disruption of this pathway results in abnormal phosphorylation of the microtubule-associated protein tau and therefore is likely to impair axonal transport processes, which are dependent on the normal function of microtubules. ApoER2 and VLDLR both are expressed abundantly on the surface of embryonic neurons as well as on neurons in the mature brain, where they can also function as receptors for Apolipoprotein E (ApoE). ApoE exists in three major isoforms. One of these isoforms, ApoE4 is genetically associated with late-onset Alzheimer disease. The biochemical basis by which ApoE4 predisposes its carriers to late-onset Alzheimer disease is poorly understood. We hypothesize that ApoE4 acts as a competitor for signaling molecules such as Reelin and reduces their binding to the receptors on the neuronal surface, thereby impairing physiological signaling pathways that regulate neuronal transport and synaptic protein transport. This model would provide a mechanistic basis on which the loss of synapses, which correlates with dementia and precedes the formation of plaques and neurofibrillary tangles, could be explained. The goal of the current proposal is to elucidate the cytoplasmic signaling pathways that are activated by the binding of Reelin to its receptors on the neuronal cell surface and to study the effect of Reelin on axonal transport, the activity of molecular motors such as dynein and kinesin, and the transport of synaptic proteins to their target sites.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area 15 "Translational Science" and the specific Challenge Topic 15-NS-103: "Demonstration of 'proof-of-concept'for a new therapeutic approach in a neurological disease." Frontotemporal dementia (FTD) is a devastating and progressive neurodegenerative disease characterized by profound behavioral and personality changes. FTD has been under-diagnosed and is now recognized as the most common form of dementia in people under age 65. There are no therapies for FTD, and affected individuals usually die within 5-8 years. In our aging population, FTD presents a huge burden for patients, caregivers, and the health care system. However, thanks to the recent identification of disease-causing genetic mutations, there is now new hope for major insights into FTD pathogenesis and for new therapies. Loss-offunction mutations in the human progranulin gene (GRN) account for ~20% of cases of familial FTD via a disease mechanism of GRN haploinsufficiency (loss of 50% of the protein expression);more than sixty pathogenic GRN mutations have been reported in the past two years. Many of these mutations are nonsense mutations (that generate a premature stop codon and degradation of the mutant GRN mRNA). Thus, emerging data suggest that FTD may be, at least in part, a disease of deficiency of progranulin. We hypothesize that increasing progranulin levels may provide an important therapeutic strategy for FTD patients with progranulin deficiency and possibly for all FTD patients. The goal of this study therefore is to identify and provide proof-ofconcept studies for drugs that can increase GRN expression. In this proposal, we focus on two key mechanisms to increase GRN expression: 1) increasing expression from mutant GRN alleles in those subjects with nonsense mutations, and 2) increasing expression from the normal GRN allele. Aim 1 focuses on increasing expression from GRN nonsense alleles. We will determine the efficacy of nonsense mutation- suppressing drugs to elevate progranulin levels in a murine GRN nonsense mutation model. Aim 2 focuses on increasing expression from normal GRN alleles. For this aim, we will perform high-throughput screening assays to identify FDA-approved compounds that increase normal GRN expression. We will then determine if candidate compound(s) increase endogenous Grn expression in murine models both in vitro and in vivo. For subjects with GRN haploinsufficiency, treatment simply by increasing GRN expression by as little as 10-15% may be hugely beneficial. Moreover, agents that increase GRN expression in general may be useful for all causes of FTD and even for a variety of neurodegenerative diseases. Aim 3 is to test the compounds identified as successful in Aims 1 and 2 in human cell lines derived from FTD patients with GRN haploinsufficiency. The goal will be to identify compounds that are efficacious in human FTD cell lines and to lay the groundwork for human clinical trials. Using progranulin as the focus, the studies we propose will help to rapidly identify and test new therapies for this devastating neurodegenerative disease. PUBLIC HEALTH RELEVANCE: One form of frontotemporal dementia (FTD) is frequently caused by nonsense mutations in a presumed signaling protein, Progranulin (GRN). This project aims to develop and perform preclinical testing of a combinatorial drug treatment approach that aims at restoring normal GRN levels and function, as an approach to prevent or delay FTD onset.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid plaques and neurofibrillary tangles. Overexpression or mutations of the amyloid precursor protein (APP) gene create soluble amyloid-beta (A?) oligomers, which are the toxic particles that alter synaptic plasticity y decreasing long-term potentiation (LTP, a paradigm for learning and memory) and enhancing long-term depression (LTD, a paradigm for forgetfulness). This suggests that the synaptic dysfunction found early in AD is A?-driven. Interestingly, the most important genetic risk factor n the pathogenesis of AD is Apolipoprotein E (ApoE) ?4 genotype. Carriers of the ?4 allele of ApoE (ApoE4) are at increased risk for AD compared with those carrying the more common ?3 allele (ApoE3), whereas the ?2 (ApoE2) allele decreases the risk. One mechanism by which ApoE4 promotes AD involves a propensity for impaired clearance and increased aggregation of ?-amyloid; however, that alone may not fully explain the origins of the synaptic dysfunction, which begins long before the amyloid plaques become prominent indicators of blooming AD. Work from our laboratory has revealed how ApoE4 dampens postsynaptic efficacy by impairing Reelin signaling and vesicular trafficking of ApoE and glutamate receptors. Reelin, a ligand for the ApoE receptors Apoer2 and Vldlr, physiologically strengthens the synapse and counteracts A?-induced synaptic suppression by promoting the phosphorylation, and thus reducing the endocytosis, of glutamate receptors. ApoE4 impairs ApoE receptor recycling along with the associated glutamate receptors, thereby diminishing synaptic strength. A? oligomers cause endocytosis of AMPA receptors and synaptic suppression at least in part through Class 1 mGluR-dependent LTD-inducing mechanisms. A? oligomers and the drug DHPG (Dihydroxyphenylglycine) activate mGluRs and increase the tyrosine phosphatase activity of striatal-enriched tyrosine phosphatase (STEP), which dephosphorylates and thereby induces AMPA and NMDA receptor endocytosis. By contrast, Reelin activates Src Family kinases (SFKs), which phosphorylate ionotropic glutamate receptors and block their endocytosis. What remains unclear is how Reelin, ApoE isoforms and ApoE receptors interact with the LTD-inducing postsynaptic machinery to balance synaptic strength through local protein synthesis and in particular GluA1-4 and STEP expression. In this proposal we will investigate whether Reelin alters mGluR- LTD, and whether reduction of Reelin signaling by the AD-promoting ApoE4 isoform enhances mGluR-LTD and the expression of Ca2+-permeable, excitotoxicity-promoting GluA2-lacking AMPAR during aging. Our specific aims will address the roles of Reelin and ApoE isoforms on 1) regulation of mGluR-LTD and AMPA receptor trafficking, 2) induction of LTD and postsynaptic protein translation of FMRP (Fragile X Mental Retardation Protein), Arc and STEPa, and 3) interaction with APP and ?-amyloid at the synapse. Our results will define the roles of Reelin and ApoE isoforms as modulators of A?-induced synaptic suppression.

PROJECT SUMMARY According to the most recent Alzheimer's Association report, 2015 Alzheimer's Disease Facts and Figures, one out of nine Americans over the age of 65 has Alzheimer's Disease (AD) and an estimated 40-65% of them carry at least one copy of the ?4 allele of gene for a cholesterol transport protein, apolipoprotein E (ApoE). Despite being one of the highest risk factors for AD (second only to age), the mechanism by which ApoE4 increases AD occurrence is unknown. ApoE transports cholesterol to neurons via ApoE receptors, which are members of the low density lipoprotein (LDL) receptor gene family. Some of these ApoE receptors, i.e. LRP1, Apoer2, VLDL receptor (Vldlr), and Lrp4, are intrinsic components of central and peripheral synapses, where they serve as essential regulators of neurotransmission through cytoplasmic signaling and neurotransmitter trafficking. The progressive neurodegeneration in AD first presents as memory loss brought on by synaptic dysfunction. Amyloid-? (A?), the central component in the trademark plaques that build up in the brains of people with AD, are a product of the amyloid precursor protein, APP, and a likely source of this early dysfunction. We have shown previously that A?-induced synaptic suppression can be prevented through ApoE receptor activation and ApoE4 selectively impairs this synaptoprotective function by sequestering the ApoE receptor, Apoer2. Apoer2, an essential CNS ApoE receptor, is endogenously expressed in multiple alternatively spliced forms, indicating a physiological need for functionally diverse forms of the receptor. We have found that differential splicing of an extracellular O-glycosylation domain dramatically alters Apoer2 abundance, synaptic function and fear memory. Apoer2 can also modify the formation of A? through multiple interactions with APP that effect APP processing. Therefore, understanding the regulation and function of Apoer2 is central to understanding the mechanisms by which ApoE4 causes synaptic dysfunction in AD. Accumulating evidence has identified Apoer2 as a key regulator of synaptic homeostasis. In this application, we propose to investigate the consequences of the two main physiological splicing events of Apoer2 on gene expression, protein interactions, behavior and cognition. In Aim 1 we will employ Apoer2- deficient mice and mice expressing various splice forms of Apoer2 to explore how Apoer2 regulates gene expression. In Aim 2, we will explore the protein interactome of these Apoer2 isoforms and probe how the various ApoE isoforms affect their trafficking and signaling, as well as their ability to regulate APP processing. In Aim 3, we will explore how endogenous Apoer2 splice variants modify behavior and cognition and how they affect cognitive deficits in mice with human ApoE isoforms or A?-overproduction.

Haploinsufficiency of the GRN gene encoding Progranulin (PGRN) is the genetic cause for a common form of frontotemporal lobar degeneration (FTLD) giving rise to a distinctive frontotemporal dementia syndrome. It is the second most common form of dementia after Alzheimer's disease and currently no effective cure exists for either form of neurodegeneration. Complete lack of PGRN causes a form of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous form of lysosomal storage disease in which the digestion of cellular membranes and glycosphingolipids is impaired, resulting in the accumulation of large misshaped lysosomes especially in neurons. This discovery has shaped our current understanding of GRN haploinsufficiency as a genetically distinct latent form of lysosomal dysfunction that accelerates the `normal' progressively diminishing lysosomal capacity during aging. Lysosomal dysfunction syndromes are thought to induce the production of inflammatory cytokines in part through the reduced generation of physiological lipid ligands for inflammation suppressing nuclear hormone receptors, which further promotes neurodegeneration by increasing microglial activation and synaptophagy. FTLD caused by GRN haploinsufficiency offers a unique therapeutic avenue by increasing gene expression from the remaining functional allele. In theory, doubling of baseline GRN expression should completely negate the risk for this form of FTLD in affected individuals. Our team has developed a comprehensive small molecule discovery strategy that has led to the identification of several chemically and mechanistically distinct classes of GRN transcriptional enhancers. The purpose of this project is to investigate the biochemical and cell biological mechanisms through which these small molecules act on the GRN gene and optimize their specificity and preclinical efficacy. This will be achieved by pursuing three major aims: Aim 1 Prioritize and optimize PGRN enhancers for preclinical development based upon efficacy and functional signatures from integrated ex vivo and in vivo studies; Aim 2 Determine the translational potential of PGRN enhancing compounds by evaluating their ability to normalize the cellular and brain transcriptome and lipidome; and Aim 3 Determine the translational potential of PGRN enhancing compounds by investigating their effects on cellular membrane lipid composition and the lysosomal lipidome and proteome.