Faculty Bibliography

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either White or African American (AA) skin. Two dimensional gel electrophoresis (2-DGE) and mass spectrometry demonstrated that Heat Shock Protein 70-1A (Hsp70-1A) protein levels were significantly higher in AA melanocytes compared to White melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70-family members in tyrosinase post-translational modification. Additionally, pharmacologic inhibition and siRNA-mediated down-regulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.

Hypoxia in tumors signifies resistance to therapy. Despite a wealth of tumor histology data, including anti-pimonidazole staining, no current methods use these data to induce a quantitative characterization of chronic tumor hypoxia in time and space. We use image-processing algorithms to develop a set of candidate image features that can formulate just such a quantitative description of xenographed colorectal chronic tumor hypoxia. Two features in particular give low-variance measures of chronic hypoxia near a vessel: intensity sampling that extends radially away from approximated blood vessel centroids, and multithresholding to segment tumor tissue into normal, hypoxic, and necrotic regions. From these features we derive a spatiotemporal logical expression whose truth value depends on its predicate clauses that are grounded in this histological evidence. As an alternative to the spatiotemporal logical formulation, we also propose a way to formulate a linear regression function that uses all of the image features to learn what chronic hypoxia looks like, and then gives a quantitative similarity score once it is trained on a set of histology images.

The signaling pathways that regulate myelination in the PNS remain poorly understood. Phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, activated in Schwann cells by neuregulin and the extracellular matrix, has an essential role in the early events of myelination. Akt/PKB, a key effector of phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, was previously implicated in CNS, but not PNS myelination. Here we demonstrate that Akt plays a crucial role in axon ensheathment and in the regulation of myelin sheath thickness in the PNS. Pharmacological inhibition of Akt in DRG neuron-Schwann cell cocultures dramatically decreased MBP and P0 levels and myelin sheath formation without affecting expression of Krox20/Egr2, a key transcriptional regulator of myelination. Conversely, expression of an activated form of Akt in purified Schwann cells increased expression of myelin proteins, but not Krox20/Egr2, and the levels of activated Rac1. Transgenic mice expressing a membrane-targeted, activated form of Akt under control of the 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter, exhibited thicker PNS and CNS myelin sheaths, and PNS myelin abnormalities, such as tomacula and myelin infoldings/outfoldings, centered around the paranodes and Schmidt Lanterman incisures. These effects were corrected by rapamycin treatmentin vivo Importantly, Akt activity in the transgenic mice did not induce myelination of nonmyelinating Schwann cells in the sympathetic trunk or Remak fibers of the dorsal roots, although, in those structures, they wrapped membranes redundantly around axons. Together, our data indicate that Akt is crucial for PNS myelination driving axonal wrapping by unmyelinated and myelinated Schwann cells and enhancing myelin protein synthesis in myelinating Schwann cells. SIGNIFICANCE STATEMENT: Although the role of the key serine/threonine kinase Akt in promoting CNS myelination has been demonstrated, its role in the PNS has not been established and remains uncertain. This work reveals that Akt controls several key steps of the PNS myelination. First, its activity promotes membrane production and axonal wrapping independent of a transcriptional effect. In myelinated axons, it also enhances myelin thickness through the mTOR pathway. Finally, sustained Akt activation in Schwann cells leads to hypermyelination/dysmyelination, mimicking some features present in neuropathies, such as hereditary neuropathy with liability to pressure palsies or demyelinating forms of Charcot-Marie-Tooth disease. Together, these data demonstrate the role of Akt in regulatory mechanisms underlying axonal wrapping and myelination in the PNS.

Although counteracting innate defenses allows oncolytic viruses (OVs) to better replicate and spread within tumors, CD8(+) T-cells restrict their capacity to trigger systemic anti-tumor immune responses. Herpes simplex virus-1 (HSV-1) evades CD8(+) T-cells by producing ICP47, which limits immune recognition of infected cells by inhibiting the transporter associated with antigen processing (TAP). Surprisingly, removing ICP47 was assumed to benefit OV immuno-therapy, but the impact of inhibiting TAP remains unknown because human HSV-1 ICP47 is not effective in rodents. Here, we engineer an HSV-1 OV to produce bovine herpesvirus UL49.5, which unlike ICP47, antagonizes rodent and human TAP. Significantly, UL49.5-expressing OVs showed superior efficacy treating bladder and breast cancer in murine models that was dependent upon CD8(+) T-cells. Besides injected subcutaneous tumors, UL49.5-OV reduced untreated, contralateral tumor size and metastases. These findings establish TAP inhibitor-armed OVs that evade CD8(+) T-cells as an immunotherapy strategy to elicit potent local and systemic anti-tumor responses.

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophyin vitroandex vivo In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.-Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossificationviaassociating with progranulin growth factor.

Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet beta cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8(+) T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8(+) T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158-166 and 282-290) and one in a non-beta cell protein, dopamine beta-hydroxylase (aa 233-241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DbetaH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DbetaH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

One of the grand challenges faced by neuroscience is to delineate the determinants of interindividual variation in the comprehensive structural and functional connection matrices that comprise the human connectome. At present, this endeavor appears most tractable at the macroanatomic scale, where intrinsic brain activity exhibits robust patterns of synchrony that recapitulate core functional circuits at the individual level. Here, we use a classical twin study design to examine the heritability of intrinsic functional network properties in 101 twin pairs, including network activity (i.e., variance of a network's specific temporal fluctuations) and internetwork coherence (i.e., correlation between networks' specific temporal fluctuations). Five of 7 networks exhibited significantly heritable (23.3-65.2%) network activity, 6 of the 21 internetwork coherences were significantly heritable (25.6-42.0%), and 11 of the 21 internetwork coherences were significantly influenced by common environmental factors (18.0-47.1%). These results suggest that the source of interindividual variation in functional connectome has a modular architecture: individual modules represented by intrinsic connectivity networks are genetic controlled, while environmental factors influence the interplays between the modules. This work further provides network-specific hypotheses for discovery of the specific genetic and environmental factors influencing functional specialization and integration of the human brain.

INTRODUCTION: PI3K/AKT/mTOR up-regulation results in accelerated activation of the primordial follicle (PMF) pool and has been implicated in cyclophosphamide (CY)-induced 'follicular exhaustion'. We compared follicular dynamics in murine models of CY-induced POI and age-related DOR, hypothesizing that 'follicular exhaustion' may occur similarly in both groups. METHODS: C57BL/6 female mice aged 8 wks (n=25) housed in identical conditions were assigned to 5 groups: 150mg/kg CY intraperitoneal (IP) x1, 75mg/kg CY IP weekly x3, control (8 wks and 11 wks), and DOR (13 months). Blood was extracted by terminal cardiac puncture for anti-mullerian hormone (AMH). Ovaries were paraffin-embedded, sectioned, and H&E stained; blinded follicle counts were confirmed by two reviewers. Follicle counts are presented as follicles/section area (mm2); data presented as mean+/-SEM. One-way ANOVA and student's t-test were used for statistical analysis (p<0.05). 'Follicular exhaustion' was calculated as the ratio of total growing follicles (primary, secondary, antral) to PMFs. RESULTS: A DOR murine model was confirmed by low AMH in 13-mth-old mice compared to controls (7.2+/-0.1 vs. 12.1+/-0.5, p<0.05) and fewer PMFs/mm2 (0.6+/-0.2) compared to 8-wk (7.7+/-1.2) and 11 wk controls (6.0+/-1) (p<0.05). A CY-induced POI model was confirmed by lower AMH in 150mg/kg CY-exposed mice compared to age-matched controls (9.6+/-0.9 vs. 12.1+/-0.5) and fewer PMFs/mm2 (2.5+/-0.4 vs. 7.7+/-1.2, p<0.05). CY-treated mice had more primary & secondary follicles/mm2 compared to controls (p<0.05), suggesting increased follicle activation. PMF counts were similar in POI [75mg/kg CY (2.4 +/- 0.5) and 150mg/ kg CY (1.9+/-0.3)] compared to DOR (0.6+/-0.2) despite an 11-mth age difference. Importantly, similar rates of 'follicular exhaustion' were seen in CY-exposed POI mice (75mg/kg CY ratio 1.9; 150mg/kg CY ratio 2.6) and DOR mice (ratio 2.4). In contrast, low 'follicular exhaustion' ratios were seen in both control groups (8-wks ratio 0.64; 11-wks ratio 0.89) signifying maintenance of follicles as PMFs. CONCLUSIONS: Accelerated PMF activation and subsequent follicular exhaustion occur similarly in murine models of CY-induced POI and age-related DOR. Future studies should investigate if PI3K/AKT/mTOR up-regulation is also involved in DOR, suggesting possible implications for prevention