No abstract

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni 2ϩ , inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around Ϫ20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/ calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 and is characterized by insulin-dependent diabetes mellitus, optic atrophy, and deafness. To investigate the cause of β-cell failure, we used induced pluripotent stem cells to create insulin-producing cells from individuals with Wolfram syndrome. WFS1-deficient β-cells showed increased levels of endoplasmic reticulum (ER) stress molecules and decreased insulin content. Upon exposure to experimental ER stress, Wolfram β-cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. Importantly, 4-phenyl butyric acid, a chemical protein folding and trafficking chaperone, restored normal insulin synthesis and the ability to upregulate insulin secretion. These studies show that ER stress plays a central role in β-cell failure in Wolfram syndrome and indicate that chemical chaperones might have therapeutic relevance under conditions of ER stress in Wolfram syndrome and other forms of diabetes.

The protein nucleobindin-2 (NUCB2, also known as nesfatin) was recently implicated as a mediator of anorexia and catabolism in the central nervous system, and has been suggested to act as a cleaved and secreted messenger. Given the overlap of signalling molecules between the brain and pancreas, we have explored the presence of NUCB2 in the islets of Langerhans. We also performed an investigation of the dynamic regulation of pancreatic NUCB2 in different metabolic states. NUCB2-like immunoreactivity was detected by immunofluorescence in all human and rat islet b-cells (as detected by co-localization with insulin), but not in other islet cells or in the exocrine pancreas. Islet NUCB2 content, as measured by enzyme immunoassay, did not change significantly following an overnight fast, but was substantially lower in islets isolated from an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rats (48% of non-diabetic Wistar rat control). Serum levels, however, were not different between Wistar and GK rats. The release of NUCB2 from isolated rat islets was significantly elevated following glucose challenge (123%), but this effect was substantially lower than that observed for insulin (816%). In contrast, serum levels of NUCB2 showed a reversible decrease in an i.p. glucose tolerance test. These data suggest a role for NUCB2 in b-cell function and a potential involvement in diabetic pathology. However, our findings, together with previous reports, appear more compatible with intracellular actions rather than with endocrine/paracrine communication, and suggest that NUCB2 in serum derives primarily from non-islet sources.

z and enhanced catalysis to the ribosomal complex [32][33][34][35][36] . Optimization of the poly(A) tail length (100-300 nucleotides) has proven critical in balancing the synthetic capability of a given mRNA 34,36 . Similarly, improved 5′ cap analogs not only increase translational capacity but also enhance capping efficiency, from 70% to 95%, greatly improving the in vitro transcription process 32,37 . The composition of the 3′ and 5′ UTRs can also be customized for the target cell of interest, increasing the efficiency and tissue specificity of translation 35,38,39 .At present, most mRNA products contain a synthetic UTR sequence from α-globin or β-globin [38][39][40] , but UTR optimization can further improve protein expression by a few fold 41,42 . Careful screening and customization to the target of interest could conceivably offer a wide range of improvements in future UTR sequences, allowing each mRNA to be tailored to the targeted cell and disease-induced microenvironment to maximize protein synthesis per mRNA transcript [41][42][43][44] .Perhaps the most critical advances in mRNA vaccines and therapeutics lie in the discovery that the inclusion of chemically modified nucleosides, particularly in uridine moieties, can markedly increase protein expression after in vitro or in vivo transfection. The chemical modifications of most new RNA formulations to date have been central in intellectual property claims 45,46 . Thus far, over 130 different naturally occurring chemical modifications of RNA have been reported 47,48 . The interest in methylpseudouridine and other modified nucleosides centers on their capacity to greatly reduce (up to 100-fold) detection by the Toll-like receptors of the innate immune system, resulting in an increase in protein expression in vivo compared to unmodified mRNA 40,[49][50][51][52][53] . Combinations of different types of chemical modifications, carriers, for the treatment of chronic conditions. The fifth section provides a comprehensive table and summary of current clinical trends in mRNA therapeutics. Finally, the sixth section considers the scope of mRNA therapeutics and guiding principles for near-term and longer-term clinical development of this novel therapeutic modality.