A prospective short-term study to evaluate methodologies for the assessment of disease extent, impact, and wound evolution in patients with dystrophic epidermolysis bullosa.

BACKGROUND: Standardized assessments for dystrophic epidermolysis bullosa (DEB) are needed. This prospective, multicenter, 4-week, observational study was designed to evaluate DEB assessments for suitability as clinical trial endpoints. METHODS: Patients with confirmed DEB diagnosis and ≥ 5 measurable wounds were included. The primary outcome was change from baseline in wound surface area (WSA) of 5 selected wounds by 3-dimensional imaging. Secondary endpoints were change from baseline in clinician global assessment (CGA) of WSA, wound characteristics, disease-related questionnaires and instruments (disease severity, quality of life [QoL], pain and disability, and itch), and tolerability of procedures. RESULTS: Of 30 enrolled patients, 29 completed the study (of whom, 28 had recessive DEB). Median age was 17.8 years (range, 3.8-58.7). All patients developed new or recurrent wounds during the 4-week study. Of the wounds selected at baseline, 45/150 (30.0%) healed by week 2; an additional 38 healed by week 4, while 8 of those healed at week 2 had recurred by week 4 for a total of 75/150 (50.0%) healed wounds at week 4. Mean values for WSA, CGA, and disease-related questionnaire and instrument scores remained steady during this 4-week observational study. Of the 10 disease-related questionnaires and instruments assessed, the scores for the Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) and the Instrument for Scoring Clinical Outcomes for Research of Epidermolysis Bullosa (iscorEB) did not substantially overlap between moderate and severe disease. Between mild and moderate disease, only the EBDASI scores did not substantially overlap. CONCLUSIONS: These results stress the dynamic nature of wounds, even during a 4-week period of observation, and suggest that a combination of clinician-assessed outcomes and patient-/caregiver-reported outcomes is needed to provide a comprehensive assessment of DEB severity and impact. In addition, these results support the use of EBDASI and iscorEB to monitor disease severity as both produced scores that did not substantially overlap between disease severity strata. Clinical trial registration ClinicalTrials.gov, NCT02178969 . Registered 4 June 2014, https://clinicaltrials.gov/ct2/show/NCT02178969 .

PI3K signaling effects in hypothalamic neurons mediated by estrogen.

Multiple mechanisms mediate the effects of estrogen in the central nervous system, including signal transduction pathways such as protein kinase A, protein kinase C, and phosphatidylinositol 3-kinase (PI3K) pathways. Previously we demonstrated that estrogen regulates a number of PI3K-related genes in the hypothalamus, including the PI3K p55gamma regulatory subunit. We hypothesized that PI3K activation is critical for the effects of estrogen and that the p55gamma subunit may be more prevalent than the p85alpha regulatory subunit in the hypothalamus. Therefore, in the present study, we compared the mRNA distribution of the p55gamma and p85alpha regulatory subunits by using in situ hybridization in guinea pig. Expression level of p55gamma mRNA was greater than p85alpha in most hypothalamic nuclei. Twenty-four hours of estrogen treatment increased p55gamma mRNA expression in the paraventricular, suprachiasmatic, arcuate, and ventromedial nuclei, and little or no change was observed for p85alpha mRNA. Quantitative real-time PCR confirmed the in situ hybridization results. Next, we investigated the general role of PI3K signaling in the estrogen-mediated changes of arcuate proopiomelanocortin (POMC) neuronal excitability by using whole-cell recording. One cellular mechanism by which estrogen increases neuronal excitability is to desensitize (uncouple) gamma-aminobutyric acid type B (GABA(B)) receptors from their G-protein-gated inwardly rectifying K(+) channels in hypothalamic neurons. We found that the PI3K inhibitors wortmannin and LY294002 significantly reduced the estrogen-mediated GABA(B) receptor desensitization in POMC arcuate neurons, suggesting that PI3K signaling is a critical downstream mediator of the estrogen-mediated rapid effects. Collectively, these data suggest that the interplay between estrogen and PI3K occurs at multiple levels, including transcriptional and membrane-initiated signaling events that ultimately lead to changes in homeostatic function.

Estrogen regulation of genes important for K+ channel signaling in the arcuate nucleus.

Estrogen affects the electrophysiological properties of a number of hypothalamic neurons by modulating K(+) channels via rapid membrane actions and/or changes in gene expression. The interaction between these pathways (membrane vs. transcription) ultimately determines the effects of estrogen on hypothalamic functions. Using suppression subtractive hybridization, we produced a cDNA library of estrogen-regulated, brain-specific guinea pig genes, which included subunits from three prominent K+ channels (KCNQ5, Kir2.4, Kv4.1, and Kvbeta(1)) and signaling molecules that impact channel function including phosphatidylinositol 3-kinase (PI3K), protein kinase Cepsilon (PKCepsilon), cAMP-dependent protein kinase (PKA), A-kinase anchor protein (AKAP), phospholipase C (PLC), and calmodulin. Based on these findings, we dissected the arcuate nucleus from ovariectomized guinea pigs treated with estradiol benzoate (EB) or vehicle and analyzed mRNA expression using quantitative real-time PCR. We found that EB significantly increased the expression of KCNQ5 and Kv4.1 and decreased expression of KCNQ3 and AKAP in the rostral arcuate. In the caudal arcuate, EB increased KCNQ5, Kir2.4, Kv4.1, calmodulin, PKCepsilon, PLCbeta(4), and PI3Kp55gamma expression and decreased Kvbeta(1). The effects of estrogen could be mediated by estrogen receptor-alpha, which we found to be highly expressed in the guinea pig arcuate nucleus and, in particular, proopiomelanocortin neurons. In addition, single-cell RT-PCR analysis revealed that about 50% of proopiomelanocortin and neuropeptide Y neurons expressed KCNQ5, about 40% expressed Kir2.4, and about 60% expressed Kv4.1. Therefore, it is evident that the diverse effects of estrogen on arcuate neurons are mediated in part by regulation of K(+) channel expression, which has the potential to affect profoundly neuronal excitability and homeostatic functions, especially when coupled with the rapid effects of estrogen on K(+) channel function.

Membrane-initiated signaling of estrogen in the brain.

It is well known that many of the actions of estrogen in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there now exists compelling evidence for membrane steroid receptors for estrogen in hypothalamic and other brain neurons. It is not well understood how estrogen signals via membrane receptors, and how these signals influence not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for some time that estrogen can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane-delimited events. In addition, estrogen can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review considers our current knowledge of rapid membrane-initiated and intracellular signaling by estrogen in the brain, and the nature of receptors involved and how they contribute to homeostatic functions.

Estrogen modulation of hypothalamic neurons: activation of multiple signaling pathways and gene expression changes.

Hypothalamic target neurons of estrogen include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and gamma-aminobutyric acid (GABA) neurons. These and other hypothalamic neurons are involved in regulating numerous homeostatic functions including reproduction, thermoregulation, stress responses, feeding and motivated behaviors. Using a combination of techniques to examine the molecular mechanisms leading to physiological changes induced by estrogen, we find that both rapid effects and transcriptional changes alter excitability of hypothalamic neurons. We have identified membrane-initiated, rapid signaling pathways through which 17beta-estradiol (E2) alters synaptic responses in these neurons using whole-cell patch recording in hypothalamic slices from ovariectomized female guinea pigs. E2 rapidly uncouples mu-opioid and GABA(B) receptors from G protein-gated inwardly rectifying K+ (GIRK) channels in POMC and dopamine neurons as manifested by a reduction in the potency of mu-opioid and GABA(B) receptor agonists to activate these channels. Inhibitors of phospholipase C, protein kinase C and protein kinase A block the actions of E2, indicative that the E2 receptor is G protein-coupled to activation of this cascade. Taking advantage of an animal model we developed to investigate estrogen's feedback actions on secretion of gonadotropin-releasing hormone (GnRH), we studied the transcriptional changes induced by estrogen using suppression subtractive hybridization (SSH) and microarray analysis. Many of the observed mRNA expression changes include transcripts encoding proteins critical for neurotransmitter release and receptor dynamics. Some of these include gec-1, PI3-kinase p55gamma, rab11a GTPase, synaptobrevin2, synaptogyrin, taxilin, Ca2+-dependent activator protein for secretion (CAPS) and a number of proteins containing pleckstrin homology domains-domains that are involved in plasma membrane targeting of their host protein. In situ hybridization and quantitative film autoradiography analysis on selected transcripts show differential distribution and expression in hypothalamic nuclei. Furthermore, single-cell PCR analysis reveals these genes to be expressed in neurons such as POMC (and GnRH). Whether these expression changes are mediated by the classical or membrane estrogen receptors has yet to be delineated. More detailed investigations of transcript spatial localization within neurons and their temporal expression, i.e., within minutes or hours, will provide more insight regarding how estrogen alters neuronal excitability and synaptic efficacy that ultimately lead to changes in complex behavior.

Suppression subtractive hybridization and microarray identification of estrogen-regulated hypothalamic genes.

The gonadal steroid estrogen is a pleiotropic hormone that has multiple effects on numerous cellular functions. One of estrogen's major targets is the brain, where the steroid not only affects growth, differentiation, and survival of neurons, but also regulates cell excitability. Because estrogen modulates multiple, overlapping signaling pathways, it has been difficult to scrutinize the transcriptional activity of the steroid. Therefore, we still lack a global picture of how different genes interact and are regulated by estrogen. Herein we report the use of suppression subtractive hybridization followed by custom microarray analysis of thousands of genes that are differentially expressed during the negative feedback phase of the female reproductive cycle. We have found a number of key transcripts that are regulated by estrogen and contribute to the alteration in synaptic transmission and hence excitability of hypothalamic neurons (e.g., GABA neurons). These include gec-1, GABA(B)R2, PI3 kinase subunit p55gamma, and a number of proteins containing pleckstrin homology domains that are critical for plasma membrane targeting. Studies are underway to refine our analysis to individual nuclei and individual cells. However, what has emerged from this highly sensitive microarray analysis is that estrogen affects neuronal plasticity in hypothalamic neurons not only by transcription of new membrane proteins (e.g., receptors and channels), but also by altering expression of downstream signaling molecules and proteins involved in neurosecretory pathways.