Zhen Zhao - US grants

SUMMARY Alzheimer's disease (AD) is the most common form of dementia in the elderly, manifesting progressive neurodegenerative conditions including amyloid plaque and neurofibrillary tangle formation, and cognitive impairment. Genetic inheritance is estimated to determine nearly 80% of the AD cases. Besides the well-known familial mutations in APP, PSEN1 and PSEN2 genes found in early-onset AD cases, over 30 loci or genes are associated sporadic late-onset AD (LOAD) as indicated by recent genome-wide association studies and whole exosome/genome sequencing projects. APOE and PICALM are among the top of the list. APOE encodes the lipid carrier apolipoprotein E protein. Among its three major isoforms (?2, ?3, and ?4), ?3 is the most common isoform, ?4 is unarguably the strongest genetic risk factor for LOAD, and ?2 is the less frequent but is protective for AD. These isoforms also differentially affect molecular and cellular events that are important for amyloid ? (A?) metabolism and neurodegeneration. On the other hand, PICALM encodes the phosphatidylinositol binding clathrin assembly protein, and is confirmed by nearly all GWAS studies as a major AD-associated gene. PICALM controls receptor internalization and subsequent intracellular trafficking of clathrin-coated vesicles. It plays key roles in mediating brain clearance of A?, regulating activities of ?- and ?-secretases for A? production, mitigating A? toxicity in neurons, and promoting Tau clerance via autophagy. More interestingly, the unique genetic interaction between APOE and PICALM in AD has been demonstrated based on population studies, as PICALM genotypes at multiple AD-associated confer risk predominantly in ?4 carriers, and AD risk PICALM rs3851179G allele and APOE ?4 allele synergistically affect cortex volume and working memory function in AD patients. However, the mechanism underpinning this interaction in AD is still unknown. Based on the PICALM's interactome and functions in maintaining cell surface protein functions, as well as our preliminary findings showing impaired APOE lipidation and reduced level of surface ABCA1 cholesterol and phospholipid transporter in PICALM deficient mice, we hypothesize that PICALM may facilitate APOE lipidation and A? metabolism by controlling the function of ABCA1 transporter, and therefore risk PICALM rs3851179G and APOE ?4 alleles adversely affect AD pathogenesis. To test this hypothesis, we propose to: i) determine the cellular and molecular mechanisms of PICALM in facilitating APOE lipidation and characterize PICALM- dependent internalization and trafficking of ABCA1 transporter (AIM 1); ii) explore the functional impact of PICALM and APOE's synergistic interaction in vivo on neurodegenerative phenotypes (AIM 2). We expect to gather first-hand evidence that the risk alleles of two genes synergistic influence AD pathogenesis, and establish the molecular and cellular mechanisms of interaction between APOE and PICALM both in vitro and in vivo. The outcomes of the studies will provide new insights into the inheritability, etiology and pathogenesis of AD, and serve as a foundation for future studies to therapeutically target this interaction for AD diagnosis and treatment.

SUMMARY Neuronal functions and brain connectivity require a highly coordinated neurovascular unit (NVU). Neurons and vascular cells are not just adjacently located; they communicate with each other vigorously via different signaling modules. Pericytes are vascular mural cells of the endothelium and vital integrators of NVU functions, including maintaining the blood-brain barrier (BBB) and vascular integrity, regulating blood flow and tissue oxygenation, modulating neuroinflammation and supporting neuronal health. Pericyte injury and loss occur commonly in CNS diseases including Alzheimer?s disease and dementia. Our current knowledge implicates a critical role of pericytes for neuronal functions, which calls for a thorough investigation of pericyte?neuronal communication for different neuronal functions in health and particularly in Alzheimer?s disease. Using new 3D co-culture systems and novel transgenic models, we found that pericytes can directly regulate neurogenesis and neuronal functions, which can be attributed to pericyte-derived insulin-like growth factor 2. IGF2 is a peptide hormone with multiple roles in regulating metabolic functions and developmental processes. Human with IGF2 mutation and mice lacking IGF2 exhibited strong growth defects with abnormal neural development. IGF2 is produced locally in the brain; however, the roles of brain IGF2 in neurogenesis and neuronal dysfunction in CNS diseases are poorly understood. Our preliminary studies additionally indicated that IGF2 mediates pericyte-neuronal communication by activating a noncanonical IGF2R-G?i-PLC pathway to enhance neuronal functions, as well as stimulating a canonical PI3K/Akt pathway to promote neurogenesis or suppressing Tau-phosphorylation. Here, we propose to study the functional crosstalk between pericytes and neurons, and examine the influence of IGF2-mediated paracrine signaling on neurogenesis during development (AIM1), on neuronal maturation and functions in adult (AIM2), and on AD-like pathogenesis (AIM3). Follow the Rigor and Reproducibility guidelines, we plan to: i) explore pericyte?neuronal crosstalk using 3D co-culture systems; ii) pinpoint the receptor mediated signaling by manipulating gene expressions and key kinase activities; iii) to determine the role of pericyte-specific IGF2 on neurogenesis and neuronal functions in new pericyte ablation and Igf2 conditional knockout mouse models; iv) examine the role of IGF2- mediated pericyte?neuronal crosstalk during AD-like pathogenies in mice using complex behavioral tests and histological analysis. We hope to generate first evidence of functional pericyte-neuron crosstalk for brain function in health and diseases, and pinpoint the mechanism of this signaling at molecular level for IGF2-mediated pericyte-neuron crosstalk. The outcomes may provide new insights to the IGF system and neurovascular interaction in brain, and close an important gap between metabolic diseases and CNS neurodegenerative diseases such as AD.

ABSTRACT Dysregulation of endocytosis and endosomal trafficking pathways contributes to the pathogenesis of Alzheimer?s disease and related dementia (ADRD). Beside controlling amyloid precursor processing and ?-amyloid (A?) production in neuronal cells, endocytosis and endosomal trafficking pathways also exist in brain endothelial cells and govern the A? clearance cross Blood-brain barrier (BBB) through receptor mediated transport (RMT). A properly functioning RMT is highly selective due to the spatial distribution of the receptors and specific interaction with their ligands, which ensures the exclusive entry of essential peptides and proteins into the brain and effective clearance of toxic waste from brain to blood in maintaining CNS health and functions. However, our understanding of this unique RMT system within the BBB remains very limited. RMT is tightly regulated by products of AD risk genes, such as Apolipoprotein E and phosphatidylinositol-binding clathrin assembly protein (PICALM). PICALM, a highly validated risk gene for Alzheimer?s disease, is also an endosomal protein and a key component of the RMT machinery at the BBB. PICALM controls the RMT transcytosis across the BBB by facilitating the clathrin-mediated endocytosis and intracellular trafficking of cell surface receptors and their ligands. PICALM deficiency in mice results in defected transferrin trafficking and diminished brain clearance of Alzheimer?s amyloid-? peptides (A?) across the BBB. Therefore, delineate the molecular mechanism of PICALM-mediated transcytosis events offers new opportunities in advancing our understanding of the BBB RMT system, as well as its role in AD pathogenesis. Through in-depth analysis of PICALM?s interactome and functional target validations using an in vitro BBB model, we found that PICALM interacting mitotic regulator (PIMREG) is a novel functional partner for PICALM in the brain endothelial cells and is required for the later steps of RMT transcytosis. Therefore, we hypothesize that PIMREG is an integral component of the RMT machinery, and teams up with PICALM and other proteins in controlling the intracellular trafficking and transcytosis of cargo vesicles, which is essential for brain homeostasis and A? clearance. To test our hypothesis, we propose to determine the function of PICALM-PIMREG complex in controlling endosomal trafficking events in primary brain endothelial cells (AIM 1), understand the role PIMREG in regulating the PICALM-mediated RMT transcytosis of different ligands across the in vitro BBB model (AIM 2), and probe PIMREG?s function in vivo for PICALM-dependent A? clearance across the BBB using antisense oligonucleotides (ASOs) (AIM 3). We expect to generate unique new insights into the biology and molecular mechanism of RMT transcytosis at the BBB, and provide first-hand evidence of a novel component of PICALM- dependent A? clearance across the BBB, which will expand our understanding of RMT and its roles in BBB dysfunction and consequent neurodegeneration in ADRD.

SUMMARY Zika virus (ZIKV) is a single-stranded RNA virus of the Flaviviridae family. It rapidly spread worldwide during 2015-2016 and is causally associated with fetal microcephaly, intrauterine growth retardation, and other congenital malformations. ZIKV is reported to infect placenta and fetal brain during pregnancy, particularly targeting human neural stem and progenitor cells (NSCs). Among the flavivirus family, only ZIKV is linked to microcephaly, suggesting uniqueness of ZIKV infection compared to other members, which calls for a better understanding of the molecular drivers of ZIKV immune evasion and pathogenesis in fetal brain. In addition, host molecular targets of ZIKV proteins remain elusive, which not only limits our understanding of ZIKV infection and pathogenesis, but also impedes anti-ZIKV drug development. Since the ZIKV outbreak in 2015, we have focused on understanding the complexity of ZIKV infection and pathogenesis of microcephaly. To fully understand the roles of viral proteins during ZIKV life cycle, we established the ZIKV-host interactome in human iPSC-derived NSCs. By analyzing this ZIKV-host interactome, we found that the key microRNA processing protein DICER was the top target of ZIKV capsid protein, and DICER deficiency facilitated ZIKV infection in mouse embryonic NSCs. Dysregulation of microRNAs has been associated with many human disease diseases, including developmental neurological disorders such as microcephaly. More importantly, DICER-dependent microRNA production is commonly used by plants, fungi and invertebrates, and remains active in mammalian stem cells to produce antiviral small RNAs from the viral genomes, which inhibits viral replication via RISC-mediated RNA interference. Mechanistically, we further identified that ZIKV capsid directly interacts with DICER and blocks its ribonuclease activity, dampening the production of both viral interfering RNAs and host microRNAs that are essential for neurogenesis. Therefore, we hypothesize that ZIKV can efficiently suppress the DICER-mediated antiviral viRNA pathway in host cells with its capsid protein; and by antagonizing host microRNA machinery, ZIKV capsid also intervenes neural development and causes microcephaly and other birth defects. Under the current application, we propose to further investigate capsid-dependent suppression of DICER function as a unique determinant of ZIKV immune evasion and pathogenesis, using different ZIKV strains and capsid variants in both human fetal NSCs and a mouse model of prenatal infection. By understanding the unique role of DICER in ZIKV infection and its associated microcephaly, we hope to define a capsid-dependent difference between the Brazilian and African strains (AIMs 1-2), and provide a proof-of-concept whether boosting this viRNA-dependent innate immune system is applicable as a novel approach to reverse the pathogenesis of ZIKV in fetal brain (AIMs 2-3). The outcomes of this application will also provide broader insight for other CNS infectious diseases.