Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid β (Aβ). Tight interactions with substrates occur at an exosite located ~30Å away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9Å crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite, and not to the catalytic site. In agreement with observed high Km values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all thirteen cysteines is insensitive to the inhibition by S-nitroso-glutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing towards an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.

Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1 Ϫ/Ϫ mouse cells exhibited significantly reduced induction of HIF-1␣ protein, HIF-1␣ transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1␣ pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.

The impact of traumatic brain injury during the perinatal period, which coincides with glial cell (astrocyte and oligodendrocyte) maturation was assessed to determine whether a second insult, e.g., increased inflammation due to remote bacterial exposure, exacerbates the initial injury's effects, possibly eliciting longer-term brain damage. Thus, a murine multifactorial injury model incorporating both mechanisms consisting of perinatal penetrating traumatic brain injury, with or without intraperitoneal injection of lipopolysaccharide (LPS), an analog of remote pathogen exposure has been developed. Four days after injury, gene expression changes for different cell markers were assessed using mRNA in situ hybridization (ISH) and qPCR. Astrocytic marker mRNA levels increased in the stab-alone and stab-plus-LPS treated animals indicating reactive gliosis. Activated microglial/macrophage marker levels, increased in the ipsilateral sides of stab and stab-plus LPS animals by P10, but the differences resolved by P15. Ectopic expression of glial precursor and neural stem cell markers within the cortical injury site was observed by ISH, suggesting that existing precursors and neural stem cells migrate into the injured areas to replace the cells lost in the injury process. Furthermore, single exposure to LPS concomitant with acute stab injury affected the oligodendrocyte population in both the injured and contralateral uninjured side, indicating that after compromise of the blood-brain barrier integrity, oligodendrocytes become even more susceptible to inflammatory injury. This multifactorial approach should lead to a better understanding of the pathogenic sequelae observed as a consequence of perinatal brain insult/injury, caused by combinations of trauma, intrauterine infection, hypoxia and/or ischemia in humans.

Introduction: Male hypogonadism results from insufficient secretion of testosterone (T) and is characterized by low serum T concentrations. Common symptoms of hypogonadism include decreased libido, impotence, weakness, low energy, depression and/or loss of motivation, memory and concentrating issues, and sleep disturbances. Several forms of T replacement are available. Testosterone undecanoate (TU) is a testosterone prodrug available in oral formulations. A novel TU formulation, SOV2012-F1, has been submitted for FDA consideration under the name KYZATREX®. While TU efficacy is measured by serum total T, patientfocused endpoints such as Patient Reported Outcomes (PROs) are valuable indicators of well-being and psychosexual symptom abatement. Methods: A Phase 3, randomized, multicenter, open-label, active-controlled trial, comparing SOV2012F1 (testosterone undecanoate capsules) (n=214) with AndroGel® (1.62% topical testosterone gel) (n=100) enrolled males aged 18 to 65 years with hypogonadism (serum total T levels ≤281 ng/dL). A key exploratory endpoint was change from Baseline (ΔBL) after 52 weeks of treatment in the following PROs: International Prostate Symptom Score (IPSS), Psychosexual Daily Questionnaire (PDQ), Short Form Health Survey 36 item (SF-36), and the International Index of Erectile Function (IIEF). Results: Total or overall scores for all PROs (IPSS, PDQ, SF-36 and IIEF) showed increased improvement in the SOV2012-F1 group relative to the Androgel group, and all but IPSS demonstrated improvement relative to BL. For IPSS, due to the potential that T could worsen urinary symptoms, the ΔBL would ideally be small to reflect minimal impact. Change for the SOV2012-F1 and AndroGel groups was, respectively, 0.6 and 1.0. Further, the IPSS total score was not significantly different from BL in the patients receiving SOV20212-F1 (p = 0.5659). For PDQ, a clinically meaningful improvement of sexual desire in hypogonadal men age ≥65 years is ≥0.7; mean ΔBL was 1.6 in the SOV2012-F1 group versus 1.4 in the AndroGel group. In the SF-36, the mean ΔBL total score was 83.7 in the SOV2012-F1 group and 70.2 in the AndroGel group. Further, post hoc analysis of the Health Change category found a significant (p ≤ 0.05) improvement in patient perspectives on health over the course of the study. The overall satisfaction score of the IIEF trended towards significance for the SOV2012-F1 group with a mean ΔBL score of 2.3 versus and 1.6 in the AndroGel group. The ΔBL for the 4 domains of male sexual function were small and consistent between the SOV2012-F1 and AndroGel groups. Comparable results were noted for Early Withdrawals and All Subjects across all PROs. Conclusion: Treatment with SOV2012-F1 for 52 weeks exceeded AndroGel patient satisfaction as measured by PROs including IPSS, PDQ, SF-36 and IIEF, demonstrating clinical distinction. Further analysis of SOV2012-F1 will be forthcoming.