Faculty Bibliography

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.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

Acoustic startle response (ASR) assesses hyperarousal, a core symptom of posttraumatic stress disorder (PTSD). Intranasal neuropeptide Y (NPY) administration was shown to prevent hyperarousal in single prolonged stress (SPS) rodent PTSD model. However, it is unclear how ASR itself alters responses to stress. Rats (A-S-A) were exposed to acoustic startle (AS) 1 day before SPS (ASR1) and 2 weeks afterward (ASR2). Other groups were exposed in parallel to either AS (A-A) or SPS or neither. SPS enhanced ASR2. In relevant brain areas, mRNA levels were determined by qRT-PCR. In mediobasal hypothalamus, AS or SPS each increased CRH mRNA levels without an additive effect. Exposure to AS appeared to dampen some responses to SPS. The SPS-triggered reduction of GR and FKBP5 gene expression was not observed in A-S-A group. In locus coeruleus, SPS increased CRHR1 and reduced Y2R mRNAs, but not in A-S-A group. In both regions, AS altered NPY receptor gene expression, which may mediate dampening responses to SPS. In second experiment, intranasal NPY administered 2 weeks after SPS reversed hyperarousal symptoms for at least 7 days. This study reveals important effects of AS on the NPY system and demonstrates that intranasal NPY elicits long-lasting reversal of traumatic stress-triggered hyperarousal.

Late-stage pancreatic cancer harbors a fibrotic and immune-excluded tumor microenvironment that impedes immunotherapy success. A key to unlocking pancreatic cancer immunotherapy may be treating early-stage pancreatic cancer, when peripancreatic inflammation promoted by the microbiome potentiates oncogenic signaling and suppresses innate and adaptive immunity. Hence, understanding the role of microbiota in pancreatic cancer initiation, progression, and immunosuppression is crucial. We propose that not only are microbiota targets for immunomodulation in this disease, but also that microbiome profiling has a potential role in pancreatic cancer screening. Furthermore, combining microbiome profiling with liquid and tissue biopsy may validate the early pancreatic cancer treatment approach of microbiome modulation and immunotherapy.

We previously found GPR133 (ADGRD1), an orphan adhesion GPCR, is De novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells (GSCs). Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic tumor initiation in vivo in mice. Analysis of TCGA data indicates that increased GPR133 transcription inversely correlates with patient survival in GBM. While these findings underscore the importance of GPR133 in GBM and suggest an essential role in tumor growth, its ligand and mechanism of activation remain unknown. Toward identifying GPR133 ligands, we used GPR133's N-terminal ectodomain as bait and performed affinity co-immunoprecipitation (CoIP) followed by mass spectrometry as an unbiased screening approach. We identified 490 extracellular proteins with enriched binding to GPR133 compared to control. Reverse CoIP using the 15 most abundant candidate ligands as bait to purify the receptor confirmed this interaction reproducibly in 4 candidates. Despite this binding, overexpression of these candidate ligands, or addition of purified recombinant protein, is not sufficient to increase receptor signaling as assessed by cAMP levels in HEK293 cells. This suggests that ligand binding to the GPR133 ectodomain may not be sufficient by itself to induce receptor activation. We hypothesize receptor activation requires mechanical forces in addition to ligand binding. Consistent with this hypothesis, the GPR133 binding proteins we have identified may be anchored to the extracellular matrix, mediating such mechanical force. To test whether mechanical shearing of the extracellular domain is sufficient for receptor activation, we used Dynabeads coupled to antibody against GPR133's N-terminal ectodomain, and indeed observed receptor activation leading to elevated cAMP levels. No activation was observed when Dynabeads devoid of antibody were used. This mode of GPR133 activation might indicate a role in sensing mechanical/viscoelastic properties of GBM extracellular matrix, which may be relevant to tumor cell migration and invasion