Faculty Bibliography

To understand the molecular anatomy of myelin membranes, we performed a large-scale, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based lipidome and proteome screen on freshly purified human and murine myelin fractions. We identified more than 700 lipid moieties and above 1,000 proteins in the two species, including 284 common lipids and 257 common proteins. This study establishes the first comprehensive map of myelin membrane components in human and mice. Although this study demonstrates many similarities between human and murine myelin, several components have been identified exclusively in each species. Future quantitative validation studies focused on interspecies differences will authenticate the myelin membrane anatomy. The combined lipidome and proteome map presented here can nevertheless be used as a reference library for myelin health and disease.

BACKGROUND: Normal development and the response to injury both require cell growth, migration and morphological remodeling, guided by a complex local landscape of permissive and inhibitory cues. A standard approach for studying by such cues is to culture cells on uniform substrates containing known concentrations of these molecules, however this method fails to represent the molecular complexity of the natural growth environment. RESULTS: To mimic the local complexity of environmental conditions in vitro, we used a contact micropatterning technique to examine cell growth and differentiation on patterned substrates printed with the commonly studied growth permissive and inhibitory substrates, poly-L-lysine (PLL) and myelin, respectively. We show that micropatterning of PLL can be used to direct adherence and axonal outgrowth of hippocampal and cortical neurons as well as other cells with diverse morphologies like Oli-neu oligodendrocyte progenitor cell lines and fibroblast-like COS7 cells in culture. Surprisingly, COS7 cells exhibited a preference for low concentration (1 pg/mL) PLL zones over adjacent zones printed with high concentrations (1 mg/mL). We demonstrate that micropatterning is also useful for studying factors that inhibit growth as it can direct cells to grow along straight lines that are easy to quantify. Furthermore, we provide the first demonstration of microcontact printing of myelin-associated proteins and show that they impair process outgrowth from Oli-neu oligodendrocyte precursor cells. CONCLUSION: We conclude that microcontact printing is an efficient and reproducible method for patterning proteins and brain-derived myelin on glass surfaces in order to study the effects of the microenvironment on cell growth and morphogenesis.

Bradykinin, acting via the bradykinin B2 receptor (B2R), is a potent stimulator of adrenomedullary catecholamine biosynthesis and release and likely plays an important role in the adrenomedullary stress response. However, the effects of stress on the expression of this receptor in the adrenal medulla are currently unclear. Here, we examined the changes in adrenomedullary B2R gene expression in male rats in response to single (1 time) and repeated (6 times) exposure to 2 hours immobilization stress (IMO). Immediately after 1 or 6 times IMO, B2R mRNA levels were increased by 9-fold and 7-fold, respectively, and returned to unstressed control levels 3 hours later. This large, but transient, increase in mRNA elicited a doubling of protein levels 3 hours after the stress exposure. Next, the role of the hypothalamic-pituitary-adrenocortical axis in the stress-induced upregulation of B2R gene expression was examined. Treatment with endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids dose-dependently increased B2R mRNA levels in adrenomedullary-derived PC12 cells. Furthermore, cortisol supplementation at levels mimicking stress exposure elevated B2R mRNA levels in the adrenal medulla of hypophysectomized rats. In response to 1 exposure to IMO, the stress-triggered rise in plasma corticosterone and adrenomedullary B2R mRNA levels was attenuated in CRH-knockout mice and absent in pharmacologically adrenalectomized rats, indicating a requirement for glucocorticoids in the upregulation of B2R gene expression with stress. Overall, the increase in B2R gene expression in response to the stress-triggered rise in glucocorticoids likely enhances catecholamine biosynthesis and release and may serve as an adaptive response of the adrenomedullary catecholaminergic system to stress.

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Since its identification 20 years ago, the biological basis for the high breast cancer risk in women who have germline BRCA1 mutations has been an area of intense study for three reasons. First, BRCA1 was the first gene shown to associate with breast cancer risk, and therefore serves as model for understanding genetic susceptibility. Second, the type of breast cancer that occurs in these women has specific features that have engendered new hypotheses about the cancer biology. Third, it is hoped that understanding the origins of this disease may provide the means to prevent disease. Resolving this question has proven extremely challenging because the biology controlled by BRCA1 is complex. Our working model is that the high frequency of basal-like breast cancer in BRCA1 mutation carriers is the result of a self-perpetuating triad of cellular phenotypes consisting of: (i) intrinsic defects in DNA repair and centrosome regulation that lead to genomic instability and increases spontaneous transformation; (ii) aberrant lineage commitment; and (iii) increased proliferation due to in large part to increased IGF-1 activity. We propose that the last is key and is a potential entree for preventing breast cancer in BRCA1 mutation carriers.

Autophagy is a lysosomal degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and protein aggregates. These substrates reach lysosomes by several distinct mechanisms, including delivery within endosomes as well as autophagosomes. Completion of digestion involves dynamic interactions among compartments of the autophagic and endocytic pathways. Neurons are particularly vulnerable to disruptions of these interactions, especially as the brain ages. Not surprisingly, mutations of genes regulating autophagy cause neurodegenerative diseases across the age spectrum with exceptional frequency. In late-onset disorders such as Alzheimer's disease, amyotrophic lateral sclerosis and familial Parkinson's disease, defects arise at different stages of the autophagy pathway and have different implications for pathogenesis and therapy. This Review provides an overview of the role of autophagy in neurodegenerative disease, focusing particularly on less frequently considered lysosomal clearance mechanisms and their considerable impact on disease. Various therapeutic strategies for modulating specific stages of autophagy and the current state of drug development for this purpose are also evaluated.

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2alpha, in Oca2-null melanocytes, eIF2alpha was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1alpha phosphatase complex. Gadd34-complex inhibition blocked eIF2alpha dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2alpha dephosphorylation as an adaptive mechanism to ER stress.

RATIONALE: To date, there has been no specific marker of the first heart field to facilitate understanding of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages are essential steps toward modeling diseases of the CCS and for development of biological pacemakers. OBJECTIVE: To investigate HCN4 as a marker for the first heart field and for precursors of distinct components of the CCS, and to gain insight into contributions of first and second heart lineages to the CCS. METHODS AND RESULTS: HCN4CreERT2, -nuclear LacZ, and -H2BGFP mouse lines were generated. HCN4 expression was examined by means of immunostaining with HCN4 antibody and reporter gene expression. Lineage studies were performed using HCN4CreERT2, Isl1Cre, Nkx2.5Cre, and Tbx18Cre, coupled to coimmunostaining with CCS markers. Results demonstrated that, at cardiac crescent stages, HCN4 marks the first heart field, with HCN4CreERT2 allowing assessment of cell fates adopted by first heart field myocytes. Throughout embryonic development, HCN4 expression marked distinct CCS precursors at distinct stages, marking the entire CCS by late fetal stages. We also noted expression of HCN4 in distinct subsets of endothelium at specific developmental stages. CONCLUSIONS: This study provides insight into contributions of first and second heart lineages to the CCS and highlights the potential use of HCN4 in conjunction with other markers for optimization of protocols for generation and isolation of specific conduction system precursors.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1(+) progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1(+) progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1(+) progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.