Faculty Bibliography

Nervous system development is instructed by genetic programs and refined by distinct mechanisms that couple neural activity to gene expression. How these processes are integrated remains poorly understood. Here, we report that the regulated release of insulin-like peptides (ILPs) during development of the C. elegans nervous system accomplishes such an integration. We find that the p38 MAP kinase PMK-3, which is required for the differentiation of chemosensory BAG neurons, limits an ILP signal that represses expression of a BAG neuron fate. ILPs are released from BAGs themselves in an activity-dependent manner during development, indicating that ILPs constitute an autocrine signal that regulates the differentiation of BAG neurons. Expression of a specialized neuronal fate is, therefore, coordinately regulated by a genetic program that sets levels of ILP expression during development and by neural activity, which regulates ILP release. Autocrine signals of this kind might have general and conserved functions as integrators of deterministic genetic programs with activity-dependent mechanisms during neurodevelopment.

Brain insulin resistance is a key pathological feature contributing to obesity, diabetes and neurodegenerative disorders, including Alzheimer's Disease (AD). Besides to classic transcriptional mechanism mediated by hormones, posttranscriptional regulation has recently been shown to regulate a number of signaling pathways that could lead to metabolic diseases. Here, we show that miR-7, an abundant miRNA in the brain, targets Insulin Receptor (INSR), Insulin Receptor Substrate-2 (IRS-2) and Insulin Degrading Enzyme (IDE), key regulators of insulin homeostatic functions in the Central Nervous System (CNS) and the pathology of AD. In this study, we found that insulin and Liver X receptor (LXR) activators promote the expression of the intronic miR-7-1 in vitro and in vivo, along with its host gene HNRNPK, an RNA binding protein (RBP) that is involved in insulin action at the posttranscriptional level. Our data show that miR-7 expression is altered in the brains of diet-induced obese mice. Moreover, we found that miR-7 levels are also elevated in brain of AD patients, which inversely correlates with the expression of its target genes IRS-2 and IDE. Furthermore, overexpression of miR-7 increased the levels of extracellular Aβ in neuronal cells and impaired the clearance of extracellular Aβ by microglial cells. Taken together, these results represent a novel branch of insulin action through the HNRNPK-miR-7 axis and highlight the possible implication of these posttranscriptional regulators in a range of diseases underlying metabolic dysregulation in the brain, from diabetes to Alzheimer's disease.

The cerebellum undergoes major rapid growth during the third trimester and early neonatal stage in humans, making it vulnerable to injuries in pre-term babies. Experiments in mice have revealed a remarkable ability of the neonatal cerebellum to recover from injuries around birth. In particular, recovery following irradiation-induced ablation of granule cell precursors (GCPs) involves adaptive reprogramming of Nestin-expressing glial progenitors (NEPs). Sonic hedgehog signaling is required for the initial step in NEP reprogramming; however, the full spectrum of developmental signaling pathways that promote NEP-driven regeneration is not known. Since the growth regulatory Hippo pathway has been implicated in the repair of several tissue types, we tested whether Hippo signaling is involved in regeneration of the cerebellum. Using mouse models, we found that the Hippo pathway transcriptional co-activator YAP1 (Yes-associated protein 1) but not TAZ (transcriptional coactivator with PDZ binding motif, or WWTR1) is required in NEPs for full recovery of cerebellar growth following irradiation one day after birth. Although Yap1 plays only a minor role during normal development in differentiation of NEPs or GCPs, the size of the cerebellum, and in particular the internal granule cell layer produced by GCPs, is significantly reduced in Yap1 mutants after irradiation, and the organization of Purkinje cells and Bergmann glial fibers is disrupted. The initial proliferative response of Yap1 mutant NEPs to irradiation is normal and the cells migrate to the GCP niche, but subsequently there is increased cell death of GCPs and altered migration of granule cells, possibly due to defects in Bergmann glia. Moreover, loss of Taz along with Yap1 in NEPs does not abrogate regeneration or alter development of the cerebellum. Our study provides new insights into the molecular signaling underlying postnatal cerebellar development and regeneration.

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g. stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membrane-less metastable (shape-changing) condensates in the cytoplasm. In our studies we used the same cell lines and methods as used by previous investigators but concluded that wt MxA formed variably-sized spherical or irregular bodies, filaments and even a reticulum distinct from ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of GFP-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24±0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase respectively in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature, and provide evidence for membrane-less MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation, and further report that MxA forms membrane-less metastable (shape-changing) condensates in the cytoplasm consisting of variably-sized spherical or irregular bodies, filaments and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.

Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in two different mouse models of kidney disease. These effects were not related to changes in circulating leukocytes, as bone marrow transplant from miR-33 deficient animals did not have a similar impact on disease progression. Most importantly, targeted delivery of miR-33 peptide nucleic acid (PNA) inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides (pHLIP) as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.

The transport and translation of dendritic mRNAs by RNA-binding proteins (RBPs) allows for spatially restricted gene expression in neuronal processes. Although local translation in neuronal dendrites is now well documented, there is little evidence for corresponding effects on local synaptic function. Here, we report that the RBP Sam68 promotes the localization and translation of Arc mRNA preferentially in distal dendrites of rodent hippocampal CA1 pyramidal neurons. Consistent with Arc function in translation-dependent synaptic plasticity, we find that Sam68 knockout (KO) mice display impaired metabotropic glutamate-receptor-dependent long-term depression (mGluR-LTD) and impaired structural plasticity exclusively at distal Schaffer-collateral synapses. Moreover, by using quantitative proteomics, we find that the Sam68 interactome contains numerous regulators of mRNA translation and synaptic function. This work identifies an important player in Arc expression, provides a general framework for Sam68 regulation of protein synthesis, and uncovers a mechanism that enables the precise spatiotemporal expression of long-term plasticity throughout neurons.

Recurrent Respiratory Papillomatosis (RRP) is a rare disease of the aerodigestive tract caused by the Human Papilloma Virus (HPV) that manifests as profoundly altered phonatory and upper respiratory anatomy. Current therapies are primarily symptomatic; enhanced insight regarding disease-specific biology of RRP is critical to improved therapeutics for this challenging population. Multiplex PCR was performed on oral rinses collected from twenty-three patients with adult-onset RRP every three months for one year. Twenty-two (95.6%) subjects had an initial HPV positive oral rinse. Of those subjects, 77.2% had an additional positive oral rinse over 12 months. A subset of rinses were then compared to tissue samples in the same patient employing HPViewer to determine HPV subtype concordance. Multiple HPV copies (60-787 per human cell) were detected in RRP tissue in each patient, but a single dominant HPV was found in individual samples. These data confirm persistent oral HPV infection in the majority of patients with RRP. In addition, three novel HPV6 isolates were found and identical HPV strains, at very low levels, were identified in oral rinses in two patients suggesting potential HPV subtype concordance. Finally, somatic heteroplasmic mtDNA mutations were observed in RRP tissue with 1.8 mutations per sample and two nonsynonymous variants. These data provide foundational insight into both the underlying pathophysiology of RRP, but also potential targets for intervention in this challenging patient cohort.

Platelets are best known as mediators of hemostasis and thrombosis; however, their inflammatory effector properties are increasingly recognized. Atherosclerosis, a chronic vascular inflammatory disease, represents the interplay between lipid deposition in the artery wall and unresolved inflammation. Here, we reveal that platelets induce monocyte migration and recruitment into atherosclerotic plaques, resulting in plaque platelet-macrophage aggregates. In Ldlr^-/- mice fed a Western diet, platelet depletion decreased plaque size and necrotic area and attenuated macrophage accumulation. Platelets drive atherogenesis by skewing plaque macrophages to an inflammatory phenotype, increasing myeloid suppressor of cytokine signaling 3 (SOCS3) expression and reducing the Socs1:Socs3 ratio. Platelet-induced Socs3 expression regulates plaque macrophage reprogramming by promoting inflammatory cytokine production (Il6, Il1b, and Tnfa) and impairing phagocytic capacity, dysfunctions that contribute to unresolved inflammation and sustained plaque growth. Translating our data to humans with cardiovascular disease, we found that women with, versus without, myocardial infarction have up-regulation of SOCS3, lower SOCS1:SOCS3, and increased monocyte-platelet aggregate. A second cohort of patients with lower extremity atherosclerosis demonstrated that SOCS3 and the SOCS1:SOCS3 ratio correlated with platelet activity and inflammation. Collectively, these data provide a causative link between platelet-mediated myeloid inflammation and dysfunction, SOCS3, and cardiovascular disease. Our findings define an atherogenic role of platelets and highlight how, in the absence of thrombosis, platelets contribute to inflammation.

The three interacting components of the outer blood-retinal barrier are the retinal pigment epithelium (RPE), choriocapillaris, and Bruch's membrane, the extracellular matrix that lies between them. Although previously reviewed independently, this review integrates these components into a more wholistic view of the barrier and discusses reconstitution models to explore the interactions among them. After updating our understanding of each component's contribution to barrier function, we discuss recent efforts to examine how the components interact. Recent studies demonstrate that claudin-19 regulates multiple aspects of RPE's barrier function and identifies a barrier function whereby mutations of claudin-19 affect retinal development. Co-culture approaches to reconstitute components of the outer blood-retinal barrier are beginning to reveal two-way interactions between the RPE and choriocapillaris. These interactions affect barrier function and the composition of the intervening Bruch's membrane. Normal or disease models of Bruch's membrane, reconstituted with healthy or diseased RPE, demonstrate adverse effects of diseased matrix on RPE metabolism. A stumbling block for reconstitution studies is the substrates typically used to culture cells are inadequate substitutes for Bruch's membrane. Together with human stem cells, the alternative substrates that have been designed offer an opportunity to engineer second-generation culture models of the outer blood-retinal barrier.