Autism has recently been reported to affect 1 in 150 American children with an estimated cost of 90 billion dollars per year to the US. Autism is a spectrum disorder with wide ranging levels of social deficits and has been difficult to model in animal studies. In a recent animal study using rats, we described a neural circuit that regulates emotional responses to social cues and may be related to deficits in social processing observed with spectrum disorders. A key component of this neural circuit is a discrete population of interneurons containing substance P1 receptors (also known as tachykinin receptor 1;Tacr1) located within the amygdala. These Tacr1 interneurons receive cortical inputs related to social recognition and suppress anxiety-like outputs of BLA projection neurons. Selectively lesioning these interneurons with a targeted toxin, SSP-saporin, a compound that selectively binds and delivers (via receptor internalization) a toxin only to cells with Tacr1, resulted in increased anxiety measures that were not alleviated with anxiolytic social cues such as social familiarity. These Tacr1 interneurons represent a subpopulation of BLA-interneurons that contain the neuropeptides somatostatin (Sst) and cholecystokinin (Cck), and although these cells appear to be pivotal in the regulation of anxiety-like responses, the contribution of these neuropeptides to these responses remains unknown. Furthermore, since the Tacr1-interneurons are only a subpopulation of the Sst- and Cck- containing BLA interneurons, traditional methods like antagonists or gene suppression cannot elucidate the specific contributions of these neuropeptides within the Tacr1-interneuronal circuit. The goal of the proposed research is to develop an in vivo gene silencing technique that will target specific cells and demonstrate the feasibility of use in vivo. The objective of the proposed research is to combine the gene-silencing properties of antisense PNAs with cellular targeting agents of targeted toxins to create a compound capable of targeted gene silencing. Specifically, we will combine the proven Tacr1 targeting agent, SSP with an antisense PNA that blocks translation of Sst mRNA (antiPNASst) resulting in SSP- antiPNAsst. The central hypothesis for the proposed research is that SSP-antiPNAsst will inhibit Sst expression only in cells that have Tacr1 on their plasma membranes. Once this is established this technology can then be used to elucidate the anxiety-modulating role of neuropeptides within the BLA interneurons. Results generated from the study will impact the field of autism by providing a novel method to determine key neural substrates underlying social behaviors. Additionally, these studies potential impact multiple fields of biomedical research by demonstrating the feasibility of targeted, in vivo PNA delivery. This will open the doors to near limitless combinations of targeting agents and PNA-based diagnostic and therapeutic agents, which to date have been limited for in vivo use due to poor membrane permeability of PNAs.
Peptide nucleic acids are powerful molecules for genetic manipulations and represent potential improvement to current methods of gene therapies, including increased specificity, multitude of strategies to regulate gene expression and long-lasting effects on gene expression without viral transfection. However, PNAs are relatively impermeable to membranes, keeping in vivo uses to a minimum. To overcome this problem we plan to develop a novel method of in vivo targeted delivery of PNAs and demonstrate the feasibility of using these compounds in an animal model.
|Lungwitz, Elizabeth A; Stuber, Garret D; Johnson, Philip L et al. (2014) The role of the medial prefrontal cortex in regulating social familiarity-induced anxiolysis. Neuropsychopharmacology 39:1009-19|
|Lungwitz, Elizabeth A; Molosh, Andrei; Johnson, Philip L et al. (2012) Orexin-A induces anxiety-like behavior through interactions with glutamatergic receptors in the bed nucleus of the stria terminalis of rats. Physiol Behav 107:726-32|