Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

Secondhand Smoke Exposure Impairs Ion Channel Function and Contractility of Mesenteric Arteries.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

ß2 Adrenergic Receptor Complexes with the L-Type Ca2+ Channel CaV1.2 and AMPA-Type Glutamate Receptors: Paradigms for Pharmacological Targeting of Protein Interactions.

Formation of signaling complexes is crucial for the orchestration of fast, efficient, and specific signal transduction. Pharmacological disruption of defined signaling complexes has the potential for specific intervention in selected regulatory pathways without affecting organism-wide disruption of parallel pathways. Signaling by epinephrine and norepinephrine through α and ß adrenergic receptors acts on many signaling pathways in many cell types. Here, we initially provide an overview of the signaling complexes formed between the paradigmatic ß2 adrenergic receptor and two of its most important targets, the L-type Ca2+ channel CaV1.2 and the AMPA-type glutamate receptor. Importantly, both complexes contain the trimeric Gs protein, adenylyl cyclase, and the cAMP-dependent protein kinase, PKA. We then discuss the functional implications of the formation of these complexes, how those complexes can be specifically disrupted, and how such disruption could be utilized in the pharmacological treatment of disease.

Tissue-specific adrenergic regulation of the L-type Ca2+ channel CaV1.2.

Ca2+ influx through the L-type Ca2+ channel Cav1.2 triggers each heartbeat. The fight-or-flight response induces the release of the stress response hormone norepinephrine to stimulate ß-adrenergic receptors, cAMP production, and protein kinase A activity to augment Ca2+ influx through Cav1.2 and, consequently, cardiomyocyte contractility. Emerging evidence shows that Cav1.2 is regulated by different mechanisms in cardiomyocytes compared to neurons and vascular smooth muscle cells.

ß-blockers augment L-type Ca2+ channel activity by targeting spatially restricted ß2AR signaling in neurons.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of ß-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate ß2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated ß2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant ß2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant ß2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which ß-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.