Anxiety disorders, such as posttraumatic stress disorder (PTSD), are characterized by a high prevalence and debilitating symptoms. However, the current first-line treatment for these conditions, which consists of selective serotonin reuptake inhibitors (SSRIs) and cognitive behavioral therapy, alongside symptomatic treatment with benzodiazepines, does not represent by far a functional solution for all affected patients. Therefore, identifying and characterizing novel candidates for alternative anxiolytic therapies are a crucial focus of psychiatric and neurobiological research. This study focuses on Neuropeptide S (NPS), a newly identified endogenous neuropeptide that has been shown to exert strong anxiolytic effects upon intracerebral injection in rodents. In an approach that combines basic research with incipient clinically relevant application, novel mechanisms and brain targets of NPS-mediated anxiolytic effects were identified, and a noninvasive application procedure also applicable in patients, namely the intranasal administration, was established for the first time for NPS in mouse models. In a first step, the feasibility of intranasal NPS delivery was established in mice using fluorophore-coupled NPS to allow intracerebral tracking. This method permitted for the first time tracking of intranasally applied substances within the brain at a single-cell resolution. These results not only proved the applicability of intranasal NPS administration in the mouse, but also allowed identification and characterization of hitherto undescribed cerebral NPS target cells, which were shown to be most likely exclusively neurons. Moreover, specific uptake of fluorescently labeled NPS in the hippocampus provided the first direct evidence linking this brain region, a well-known major player in the regulation of fear expression, to the NPS circuitry. Further investigation into the functional role of the hippocampus in NPS-elicited anxiolytic effects revealed that local microinjections of NPS into the ventral CA1 (vCA1) region are sufficient to elicit anxiolysis in C57BL6/N mice on the elevated plus maze (EPM). In a second step, behavioral and molecular effects of intranasal NPS treatment were characterized in C57BL/6N mice. Intranasal application of NPS was shown here to produce anxiolytic effects similar to those described by others after intracerebral injection. This finding represents the basis for the implementation of a future NPS-based therapy via nasal sprays in patients suffering from anxiety disorders. Furthermore, the molecular effects of NPS treatment on cerebral protein expression were examined here for the first time. This research led to identification of novel downstream targets of NPS-mediated regulation in the hippocampus and the prefrontal cortex. These new targets include proteins involved in the glutamatergic system and in synaptic plasticity, both of which are known to be dysregulated in anxiety disorders. Finally, the effects of intranasal NPS treatment, hitherto described only in non-pathological animal models, were examined for the first time in mouse models of anxiety disorders, namely the high anxiety behavior (HAB) mice and a mouse model of PTSD. In HAB mice, NPS treatment elicited anxiolytic effects similar to those observed in C57BL/6N mice. In the mouse model of PTSD, NPS counteracted disease-related changes in expression levels of hippocampal synaptic proteins. To sum up, this work expands the current state-of-knowledge concerning the molecular and mechanistic background of NPS-mediated anxiolysis by characterizing the role of the hippocampus in the NPS circuitry and by identifying novel downstream targets of NPS. The data on anxiolytic effects of intranasal NPS treatment especially in mouse models of anxiety disorders furthermore establishes the therapeutic potential of NPS as a novel anxiolytic treatment.

1s

Jul 30, 2012

Plastid transformation is a valuable technique for both basic and applied science. In basic science the technique is used to study chloroplast function. Applied approaches deal with the potential of the plastid for the production of medicinal therapeutics, in most cases vaccine antigens coupled to adjuvants. Adjuvants are used for the trans-mucosal delivery of attached cargoes. Cell penetrating peptides (CPPs) emerged as valuable tools for the delivery of cell-impermeable cargoes across cell barriers more than twenty years ago. Although the exact mechanism of CPP penetration of cells is still discussed, the applied value of CPPs is documented in a number of clinical studies. Recently, scientists working in the CPP field launched a call for an alternative expression platform for CPP fusion peptides / proteins. Only a short time before, CPPs were introduced into plant science and some impressive first results, manipulating plant cells from the “outside”, were achieved. The present study aimed at combining the fields of plastid transformation and CPPs from the “inside”. We report the first expression of CPP fusion proteins in a plant, more precisely in the plastid. The approach focused on three aspects of CPP fusion protein expression in the organelle: (A) the principal feasibility of CPP-fusion protein expression in the plastid, (B) the location of CPP fusion proteins in the plant cell upon plastid-based expression and (C) the use of plastids for the manufacture of CPP fusions to provide an alternative to the bacterial expression system. Nine prominent CPPs were employed in three vector series to investigate these aspects. In vector series I the selected CPPs were fused to the fluorescent protein eGFP to provide an optical read-out; in vector series II, the CPPs were fused to Arabidopsis MYB transcription factor PAP1 to provide a biological read-out and in vector series III, two CPPs were fused to the human enzyme PAH to introduce plant-based CPP fusion protein expression. Taken together, the expression of CPP fusion in the plastid turned out to be feasible. Transplastomic plants reached homoplasmy, produced viable seeds and stably inherited the desired trait to their progenies in a maternal fashion. Only low protein accumulation levels were detected. Pleiotropic effects occured at the low protein accumulation levels observed. Localisation of CPP fusion proteins was shown to be restricted to the plastid. An inability of CPP fusion proteins isolated from vector series I to penetrate protoplasts, young plant tissue and human cell lines was revealed. The value of a plastid-based manufacture of CPP fusion proteins for clinical approaches failed to be demonstrated due to low fusion protein accumulation levels. Bottlenecks of the current study are discussed and suggestions are made to provide a framework for future efforts.

1s

Jun 21, 2012