Visualization of sympathetic neural innervation in human white adipose tissue

Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, to date, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabelling of sympathetic nerve terminals in whole WAT and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize sympathetic innervation of human WAT from obese individuals in a three-dimensional manner. We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.     

Withdrawal of stimulation may initiate the transition of effector to memory CD4 cells.

The initial steps that determine development of memory in CD4 cells are unknown. To distinguish an intrinsic capacity of effectors to become memory cells from contributions of as yet undefined survival factors, we analyzed the effects of withdrawal of signals via TCR, costimulation, and cytokines from Th1 or Th2 primary effectors induced in vitro from TCR-transgenic CD4 cells. Withdrawal of stimulation caused the transition of effectors to resting populations with a memory phenotype that did not undergo division following transfer to normal syngeneic recipients. The return of effectors to rest was accompanied by acquisition of the capacity to function as memory cells in vivo as defined by extended persistence and a more rapid response to Ag in vivo than naive cells in adoptive hosts. Upon challenge with Ag, these in vitro-rested Th1 and Th2 cells were similar to long-term in vivo-rested memory cells, but distinct from in vitro-generated primary effectors and in vivo-restimulated memory effectors by their ability to resist apoptosis. Cessation of stimulation may occur when activated CD4 cells exit lymphoid tissues after priming and transition to memory may be initiated if effectors either fail to gain access to Ag in peripheral tissues where restimulation can lead to activation-induced cell death or do not receive sufficient stimuli to continue a response. Our results suggest that the first stage leading to stable CD4 memory could occur stochastically and independently of instructional processes and as such, the development of memory may be a default pathway when signals that direct responses are not received.     

iPSC-derived fibroblasts demonstrate augmented production and assembly of extracellular matrix proteins

Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of l-ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.     

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.     

Intrinsic linear heterogeneity of amyloid β protein fibrils revealed by higher resolution mass-per-length determinations

Amyloid β proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by dark-field and transmission electron microscopy in a preparation of seeded amyloid β protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when mass-per-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.     

Regulation of splicing: the importance of being translatable

RNA sequences that conform to the consensus sequence of 5' splice sites but are not used for splicing occur frequently in protein coding genes. Mutational analyses have shown that suppression of splicing at such latent sites may be dictated by the necessity to maintain an open reading frame in the mRNA. Here we show that stop codon frequency in introns having latent 5' splice sites is significantly greater than that of introns lacking such sites and significantly greater than the expected occurrence by chance alone. Both observations suggest the occurrence of a general mechanism that recognizes the mRNA reading frame in the context of pre-mRNA.     

The Effects of Chronic Amitriptyline on Zebrafish Behavior and Monoamine Neurochemistry

Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50 μg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at 50 μg/L evoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-β-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.