Transcriptome and Metabolite Changes during Hydrogen Cyanamide-Induced Floral Bud Break in Sweet Cherry.

Release of bud dormancy in perennial woody plants is a temperature-dependent process and thus flowering in these species is heavily affected by climate change. The lack of cold winters in temperate growing regions often results in reduced flowering and low fruit yields. This is likely to decrease the availability of fruits and nuts of the Prunus spp. in the near future. In order to maintain high yields, it is crucial to gain detailed knowledge on the molecular mechanisms controlling the release of bud dormancy. Here, we studied these mechanisms using sweet cherry (Prunus avium L.), a crop where the agrochemical hydrogen cyanamide (HC) is routinely used to compensate for the lack of cold winter temperatures and to induce flower opening. In this work, dormant flower buds were sprayed with hydrogen cyanamide followed by deep RNA sequencing, identifying three main expression patterns in response to HC. These transcript level results were validated by quantitative real time polymerase chain reaction and supported further by phytohormone profiling (ABA, SA, IAA, CK, ethylene, JA). Using these approaches, we identified the most up-regulated pathways: the cytokinin pathway, as well as the jasmonate and the hydrogen cyanide pathway. Our results strongly suggest an inductive effect of these metabolites in bud dormancy release and provide a stepping stone for the characterization of key genes in bud dormancy release.

Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering.

Almond and sweet cherry are two economically important species of the Prunus genus. They both produce the cyanogenic glucosides prunasin and amygdalin. As part of a two-component defense system, prunasin and amygdalin release toxic hydrogen cyanide upon cell disruption. In this study, we investigated the potential role within prunasin and amygdalin and some of its derivatives in endodormancy release of these two Prunus species. The content of prunasin and of endogenous prunasin turnover products in the course of flower development was examined in five almond cultivars - differing from very early to extra-late in flowering time - and in one sweet early cherry cultivar. In all cultivars, prunasin began to accumulate in the flower buds shortly after dormancy release and the levels dropped again just before flowering time. In almond and sweet cherry, the turnover of prunasin coincided with increased levels of prunasin amide whereas prunasin anitrile pentoside and ß-D-glucose-1-benzoate were abundant in almond and cherry flower buds at certain developmental stages. These findings indicate a role for the turnover of cyanogenic glucosides in controlling flower development in Prunus species.

Intranasally applied neuropeptide S shifts a high-anxiety electrophysiological endophenotype in the ventral hippocampus towards a "normal"-anxiety one.

The neurobiological basis of pathological anxiety and the improvement of its pharmacological treatment are a matter of intensive investigation. Here, using electrophysiological techniques in brain slices from animals of the high anxiety-related behavior (HAB) and normal anxiety-related behavior (NAB) mouse model, we show that basal neurotransmission at ventral hippocampal CA3-CA1 synapses is weaker in HAB compared to NAB mice. We further demonstrate that paired-pulse facilitation (PPF) and long-term potentiation (LTP) at these synapses are more pronounced in slices from HAB animals. Based on previous findings, we also examined whether intranasal delivery of neuropeptide S (NPS), which increasingly emerges as a potential novel treatment option for anxiety symptoms occurring in a variety of diseases like anxiety disorders, posttraumatic stress disorder, and major depression, impacts on the high-anxiety electrophysiological endophenotype in HAB mice. Strikingly, we detected enhanced basal neurotransmission and reduced PPF and LTP in slices from NPS-treated HAB animals. Collectively, our study uncovers a multifaceted high-anxiety neurophysiological endophenotype in the murine ventral hippocampus and provides the first evidence that an intranasally applied neuropeptide can shift such an endophenotype in an anxiety-regulating brain structure towards a "normal"-anxiety one.

Identification and characterization of HPA-axis reactivity endophenotypes in a cohort of female PTSD patients.

Analysis of the function of the hypothalamic-pituitary-adrenal (HPA)-axis in patients suffering from posttraumatic stress disorder (PTSD) has hitherto produced inconsistent findings, inter alia in the Trier Social Stress Test (TSST). To address these inconsistencies, we compared a sample of 23 female PTSD patients with either early life trauma (ELT) or adult trauma (AT) or combined ELT and AT to 18 age-matched non-traumatized female healthy controls in the TSST which was preceded by intensive baseline assessments. During the TSST, we determined a variety of clinical, psychological, endocrine and cardiovascular parameters as well as expression levels of four HPA-axis related genes. Using a previously reported definition of HPA-axis responsive versus non-responsive phenotypes, we identified for the first time two clinically and biologically distinct HPA-axis reactivity subgroups of PTSD. One subgroup ("non-responders") showed a blunted HPA-axis response and distinct clinical and biological characteristics such as a higher prevalence of trauma-related dissociative symptoms and of combined AT and ELT as well as alterations in the expression kinetics of the genes encoding for the mineralocorticoid receptor (MR) and for FK506 binding protein 51 (FKBP51). Interestingly, this non-responder subgroup largely drove the relatively diminished HPA axis response of the total cohort of PTSD patients. These findings are limited by the facts that the majority of patients was medicated, by the lack of traumatized controls and by the relatively small sample size. The here for the first time identified and characterized HPA-axis reactivity endophenotypes offer an explanation for the inconsistent reports on HPA-axis function in PTSD and, moreover, suggest that most likely other factors than HPA-axis reactivity play a decisive role in determination of PTSD core symptom severity.

A role for synapsin in FKBP51 modulation of stress responsiveness: Convergent evidence from animal and human studies.

Both the molecular co-chaperone FKBP51 and the presynaptic vesicle protein synapsin (alternatively spliced from SYN1-3) are intensively discussed players in the still insufficiently explored pathobiology of psychiatric disorders such as major depression, schizophrenia and posttraumatic stress disorder (PTSD). To address their still unknown interaction, we compared the expression levels of synapsin and five other neurostructural and HPA axis related marker proteins in the prefrontal cortex (PFC) and the hippocampus of restrained-stressed and unstressed Fkbp5 knockout mice and corresponding wild-type littermates. In addition, we compared and correlated the gene expression levels of SYN1, SYN2 and FKBP5 in three different online datasets comprising expression data of human healthy subjects as well as of predominantly medicated patients with different psychiatric disorders. In summary, we found that Fkbp5 deletion, which we previously demonstrated to improve stress-coping behavior in mice, prevents the stress-induced decline in prefrontal cortical (pc), but not in hippocampal synapsin expression. Accordingly, pc, but not hippocampal, synapsin protein levels correlated positively with a more active mouse stress coping behavior. Searching for an underlying mechanism, we found evidence that deletion of Fkbp5 might prevent stress-induced pc synapsin loss, at least in part, through improvement of pc Akt kinase activity. These results, together with our finding that FKBP5 and SYN1 mRNA levels were regulated in opposite directions in the PFC of schizophrenic patients, who are known for exhibiting an altered stress-coping behavior, provide the first evidence of a role for pc synapsin in FKBP51 modulation of stress responsiveness. This role might extend to other tissues, as we found FKBP5 and SYN1 levels to correlate inversely not only in human PFC samples but also in other expression sites. The main limitation of this study is the small number of individuals included in the correlation analyses. Future studies will have to verify the here-postulated role of the FKBP51-Akt kinase-synapsin pathway in stress responsiveness.

Identification of a role for the ventral hippocampus in neuropeptide S-elicited anxiolysis.

Neuropeptide S (NPS) increasingly emerges as a potential novel treatment option for anxiety diseases like panic and posttraumatic stress disorder. However, the neural underpinnings of its anxiolytic action are still not clearly understood. Recently, we reported that neurons of the ventral hippocampus (VH) take up intranasally administered fluorophore-conjugated NPS and, moreover, that application of NPS to mouse brain slices affects neurotransmission and plasticity at hippocampal CA3-CA1 synapses. Although these previous findings define the VH as a novel NPS target structure, they leave open whether this brain region is directly involved in NPS-mediated anxiolysis and how NPS impacts on neuronal activity propagation in the VH. Here, we fill this knowledge gap by demonstrating, first, that microinjections of NPS into the ventral CA1 region are sufficient to reduce anxiety-like behavior of C57BL/6N mice and, second, that NPS, via the NPS receptor, rapidly weakens evoked neuronal activity flow from the dentate gyrus to area CA1 in vitro. Additionally, we show that intranasally applied NPS alters neurotransmission and plasticity at CA3-CA1 synapses in the same way as NPS administered to hippocampal slices. Thus, our study provides, for the first time, strong experimental evidence for a direct involvement of the VH in NPS-induced anxiolysis and furthermore presents a novel mechanism of NPS action.

Long-lasting hippocampal synaptic protein loss in a mouse model of posttraumatic stress disorder.

Despite intensive research efforts, the molecular pathogenesis of posttraumatic stress disorder (PTSD) and especially of the hippocampal volume loss found in the majority of patients suffering from this anxiety disease still remains elusive. We demonstrated before that trauma-induced hippocampal shrinkage can also be observed in mice exhibiting a PTSD-like syndrome. Aiming to decipher the molecular correlates of these trans-species posttraumatic hippocampal alterations, we compared the expression levels of a set of neurostructural marker proteins between traumatized and control mice at different time points after their subjection to either an electric footshock or mock treatment which was followed by stressful re-exposure in several experimental groups. To our knowledge, this is the first systematic in vivo study analyzing the long-term neuromolecular sequelae of acute traumatic stress combined with re-exposure. We show here that a PTSD-like syndrome in mice is accompanied by a long-lasting reduction of hippocampal synaptic proteins which interestingly correlates with the strength of the generalized and conditioned fear response but not with the intensity of hyperarousal symptoms. Furthermore, we demonstrate that treatment with the serotonin reuptake inhibitor (SSRI) fluoxetine is able to counteract both the PTSD-like syndrome and the posttraumatic synaptic protein loss. Taken together, this study demonstrates for the first time that a loss of hippocampal synaptic proteins is associated with a PTSD-like syndrome in mice. Further studies will have to reveal whether these findings are transferable to PTSD patients.

Intranasally administered neuropeptide S (NPS) exerts anxiolytic effects following internalization into NPS receptor-expressing neurons.

Experiments in rodents revealed neuropeptide S (NPS) to constitute a potential novel treatment option for anxiety diseases such as panic and post-traumatic stress disorder. However, both its cerebral target sites and the molecular underpinnings of NPS-mediated effects still remain elusive. By administration of fluorophore-conjugated NPS, we pinpointed NPS target neurons in distinct regions throughout the entire brain. We demonstrated their functional relevance in the hippocampus. In the CA1 region, NPS modulates synaptic transmission and plasticity. NPS is taken up into NPS receptor-expressing neurons by internalization of the receptor-ligand complex as we confirmed by subsequent cell culture studies. Furthermore, we tracked internalization of intranasally applied NPS at the single-neuron level and additionally demonstrate that it is delivered into the mouse brain without losing its anxiolytic properties. Finally, we show that NPS differentially modulates the expression of proteins of the glutamatergic system involved inter alia in synaptic plasticity. These results not only enlighten the path of NPS in the brain, but also establish a non-invasive method for NPS administration in mice, thus strongly encouraging translation into a novel therapeutic approach for pathological anxiety in humans.

FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior.

BACKGROUND: The Hsp90 cochaperone FK506 binding protein 5 (FKBP5) is an established regulator of the glucocorticoid receptor (GR), and numerous genetic studies have linked it to stress-related diseases such as major depression or posttraumatic stress disorder. However, translational studies including genetic animal models are lacking. METHODS: Mice deficient of FKBP5 were generated and analyzed in comparison with wildtype littermates. They were subjected to several test paradigms characterizing their emotionality, stress reactivity, and coping behavior as well as hypothalamus-pituitary-adrenal axis function and regulation. Moreover, protein expression of GR and FKBP5 was determined in different brain structures 8 days after stress exposure. The combined dexamethasone/corticotropin-releasing hormone test was performed both in mice and healthy human subjects of different FKBP5 genotypes. The GR function was evaluated by reporter gene assays. RESULTS: Under basal conditions, deletion of FKBP5 did not change exploratory drive, locomotor activity, anxiety-related behavior, stress-coping, or depression-like behavior. After exposure to different acute stressors of sufficient intensity, however, it led to a more active coping behavior. Moreover, loss of FKBP5 decreased hypothalamus-pituitary-adrenal axis reactivity and GR expression changes in response to stressors. In mice and humans, the FKBP5 genotype also determined the outcome of the dexamethasone/corticotropin-releasing hormone test. CONCLUSIONS: This study in mice and humans presents FKBP5 as a decisive factor for the physiological stress response, shaping neuroendocrine reactivity as well as coping behavior. This lends strong support to the concept emerging from human studies of FKBP5 as important factor governing gene-environment interactions relevant for the etiology of affective disorders.