Identification of a highly neurotoxic α-synuclein species inducing mitochondrial damage and mitophagy in Parkinson's disease.

Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.

Blocking c-Jun N-terminal kinase (JNK) translocation to the mitochondria prevents 6-hydroxydopamine-induced toxicity in vitro and in vivo.

Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-Sab(KIM1) protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-Sab(KIM1) via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-Sab(KIM1) decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-Sab(KIM1) at day 7 were 750 nm, which was ∼3.4-fold higher than the IC(50) for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.

Inhibition of JNK mitochondrial localization and signaling is protective against ischemia/reperfusion injury in rats.

To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.

What Should the Surgeons Do at the Family Meeting: A Multi-Disciplinary Qualitative Description of Surgeon Participation in Palliative Care Discussions.

OBJECTIVE: National guidelines have suggested that quality surgical care should incorporate effective palliative care (PC). Numerous barriers to surgeon participation remain and the domains of optimal surgeon participation are unclear. DESIGN: Eight semi-structured and multi-professional focus groups with 34 total participants. Discussion was transcribed, and qualitative approaches were used to encode, identify, and categorize emergent themes. SETTING: Oregon Health & Science University, Portland Oregon. A tertiary care teaching hospital. PARTICIPANTS: 34 multi-disciplinary participants in eight focus groups, identified on a volunteer basis. RESULTS: Key themes defining domains of optimal surgeon/palliative practice include: (1) "primary/secondary PC" which detailed conflict between the surgeon's desire to be part of palliative discussions and competing clinical/time demands. (2) "role/responsibility" described the tension surgeons feel around a desire to provide honest and goal concordant care (3) "teamwork/conflict" detailed the approach to disagreement among multidisciplinary teams. CONCLUSIONS: In this qualitative analysis, emergent themes suggest that surgeons want to be involved in the PC of their patients but are limited by available time and competing for ethical obligations. Tension between competing communication and care obligations and PC goals is common, and discord around patient goals remains an issue. This work highlights the need for a standardized curriculum to improve the PC of surgical patients.

Failure to repair endogenous DNA damage in ß-cells causes adult-onset diabetes in mice.

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in ß-cells can drive ß-cell dysfunction and diabetes, via deletion of Ercc1, a key DNA repair gene, in ß-cells. Mice harboring Ercc1-deficient ß-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in ß-cells and a significant loss of ß-cell mass. Using electron microscopy, we identified ß-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some ß-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1-deficiency in ß-cells also resulted in loss of ß-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1-deficient ß-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive ß-cell dysfunction and provide a mechanism by which age increases the risk of T2DM.

Teaching primary palliative care to general surgical residents: A novel pedagogical approach drawn from multi-disciplinary focus group data.

BACKGROUND: Primary palliative care (PPC) is provided by the primary team and is essential for high-quality surgical care. There is a recognized PPC clinical and research need but little work on the optimal way to teach PPC to general surgery residents. We sought to define important factors of PPC pedagogy (i.e. nature and practice of teaching). METHODS: Eight semi-structured and multi-professional focus groups (n = 34) were performed. Discussion was transcribed, and de-identified. Qualitative approaches were used to encode, identify, and categorize emergent themes. RESULTS: Emergent themes included: establishing a baseline knowledge, use of existing resources, simulation and debriefings, and emphasis on authentic clinical opportunities with graduated responsibility. A tension between resident entrustability and hesitancy of faculty to entrust was identified. CONCLUSIONS: PPC must be taught in surgical residency and the themes identified here will inform development and implementation of a PPC curriculum.

Supporting surgical residents learning clinical palliative care: Insights from multi-disciplinary focus groups.

BACKGROUND: A shortage of palliative care (PC) sub-specialists highlights the need for quality PC provided by treating surgeons, although no established curriculum exists to teach surgical residents PC skills. To guide curriculum development, we sought to determine what modifiable factors contribute to surgical residents successfully providing PC. METHODS: Eight focus groups with 34 participants were conducted. Semi-structured interviews were recorded, transcribed, and de-identified. Inductive thematic analysis was utilized to encode, identify, and categorize emergent themes. RESULTS: Barriers to resident involvement in PC included: Limited Knowledge/Inexperience, Communication Difficulties, Time Constraints, and Burnout. Factors supporting resident involvement included: Patient Relationship/Rapport, Expertise Guiding PC Discussions, and Institutional Support. Communication skills that support successful PC delivery include establishing rapport, managing conflicts, avoiding bias, and acknowledging personal/scientific limitations. DISCUSSION: This work identifies modifiable factors that support surgical residents providing PC. Faculty and institutional support, resident education on PC principles, and expanding clinical experience with PC may be the most modifiable from a programmatic perspective. Curriculum and process development focused on these areas will help optimize surgical resident's success delivering PC.

Adnexal Torsion Predictive Tool Maintains Applicability in a Heterogeneous Pediatric Population.

STUDY OBJECTIVE: A previously published scoring system showed promise in identifying adnexal torsion in adolescents. However, published patients were homogeneously Caucasian/White. We sought to assess whether this scoring system was generalizable to a more diverse population that is predominantly African American and overweight. DESIGN: Retrospective chart review. SETTING: Tertiary academic hospital. PARTICIPANTS: Female patients 0-21 years of age undergoing surgery for suspected ovarian torsion from 2010 to 2019. INTERVENTIONS: Records were reviewed for patients, including laboratory studies, imaging, surgery, and pathology. Significance was determined for clinical and imaging findings, and retrospective composite scores were calculated for each participant as suggested by the scoring system. MAIN OUTCOME MEASURES: Determination of whether the previously published composite score was predictive of pediatric adnexal torsion in our population. RESULTS: A total of 57 cases of suspected torsion were included. Approximately 60% of patients were African American, 10% Hispanic, and 30% Caucasian/White. The average body mass index (BMI) across ethnicities was 29. Our findings significantly correlated with previously published predictors for all components. The presence of nausea/vomiting, leukocytosis, ovarian volume and ratio were found to be significant. The post hoc calculated composite score was applied to our cohort, and more than 90% of confirmed torsion would have been identified. CONCLUSION: Our study suggests that a previously published composite score assessing torsion can successfully be used to predict torsion in a more diverse setting than the original study population, potentially accelerating surgical management of patients with the condition.

Critical decisions in the trauma intensive care unit: Are we practicing primary palliative care?

BACKGROUND: Devastating injuries require both urgent assessment by a trauma service and early attention to patients' goals of care (GOC). American College of Surgeons Trauma Quality Improvement Program (TQIP) guidelines recommend an initial palliative assessment within 24 hours of admission and family meeting, if needed, within 72 hours. We hypothesize that a primary palliative care-based practice improves adherence to TQIP guidelines in trauma patients. METHODS: All adult trauma patients who died while inpatient from January 2014 to December 2018 were reviewed. Timing of GOC discussions, transition to comfort measures only (CMO), and the utilization of specialty palliative services were analyzed with univariate analysis. RESULTS: During the study period, 415 inpatients died. Median Injury Severity Score was 26 (interquartile range [IQR], 17-34), median age was 67 years (IQR, 51-81 years), and 72% (n = 299) transitioned to CMO before death. Documented GOC discussions increased from 77% of patients in 2014 to 95% of patients in 2018 (p < 0.001), and in 2018, the median time to the first GOC discussion was 15 hours (IQR, 7- 24 hours). Specialty palliative care was consulted in 7% of all patients. Of patients who had at least one GOC discussion, 98% were led by the trauma intensive care unit (TICU) team. Median time from admission to first GOC discussion was 27 hours (IQR, 6-91 hours). Median number of GOC discussions was 1 (IQR, 1-2). Median time to CMO after the final GOC discussion was 0 hours (IQR, 0-3). Median time to death after transition to CMO was 4 hours (IQR, 1-18 hours). CONCLUSION: Of those who died during index admission, we demonstrated significant improvement in adherence to American College of Surgeons TQIP palliative guidelines across the 5-year study period, with the TICU team guiding the majority of GOC discussions. Our TICU team has developed an effective primary palliative care approach, selectively consulting specialty palliative care only when needed. LEVEL OF EVIDENCE: Therapeutic/care management, level III.

Microbial symbionts regulate the primary Ig repertoire.

The ability of immunoglobulin (Ig) to recognize pathogens is critical for optimal immune fitness. Early events that shape preimmune Ig repertoires, expressed on IgM+ IgD+ B cells as B cell receptors (BCRs), are poorly defined. Here, we studied germ-free mice and conventionalized littermates to explore the hypothesis that symbiotic microbes help shape the preimmune Ig repertoire. Ig-binding assays showed that exposure to conventional microbial symbionts enriched frequencies of antibacterial IgM+ IgD+ B cells in intestine and spleen. This enrichment affected follicular B cells, involving a diverse set of Ig-variable region gene segments, and was T cell-independent. Functionally, enrichment of microbe reactivity primed basal levels of small intestinal T cell-independent, symbiont-reactive IgA and enhanced systemic IgG responses to bacterial immunization. These results demonstrate that microbial symbionts influence host immunity by enriching frequencies of antibacterial specificities within preimmune B cell repertoires and that this may have consequences for mucosal and systemic immunity.

Pharmacological Inhibition of c-Jun N-terminal Kinase Reduces Food Intake and Sensitizes Leptin's Anorectic Signaling Actions.

The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin's anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance.

Serum- and Glucocorticoid-Inducible Kinase 1 Confers Protection in Cell-Based and in In Vivo Neurotoxin Models via the c-Jun N-Terminal Kinase Signaling Pathway.

Serum glucocorticoid kinase 1 (SGK1) has been shown to be protective in models of Parkinson's disease, but the details by which it confers benefit is unknown. The current study was designed to investigate the details by which SGK1 confers neuroprotection. To do this we employed a cellular neurodegeneration model to investigate c-Jun N-terminal kinase (JNK) signaling and endoplasmic reticulum (ER) stress induced by 6-hydroxydopamine. SGK1-expressing adenovirus was created and used to overexpress SGK1 in SH-SY5Y cells, and dexamethasone was used to increase endogenous expression of SGK1. Oxidative stress, mitochondrial dysfunction, and cell death were monitored to test the protective effect of SGK1. To investigate the effect of SGK1 overexpression in vivo, SGK1-expressing adenovirus was injected into the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and protection of dopaminergic neurons was quantitatively assessed by tyrosine hydroxylase immunohistochemistry. SGK1 overexpression was found to decrease reactive oxygen species generation, alleviate mitochondrial dysfunction, and rescue cell death in vitro and in vivo by inactivating mitogen-activated protein kinase kinase 4 (MKK4), JNK, and glycogen synthase kinase 3ß (GSK3ß) and thereby decreasing ER and oxidative stress. These results suggest that therapeutic strategies for activation of SGK1 may have the potential to be neuroprotective by deactivating the JNK and GSK3ß pathways.

Bis-aryl urea derivatives as potent and selective LIM kinase (Limk) inhibitors.

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (>400-fold), potent inhibition of cofilin phosphorylation in A7r5, PC-3, and CEM-SS T cells (IC50 < 1 µM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 µM inhibited only Limk1 and STK16 with ≥80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors.

Small Molecule c-jun-N-terminal Kinase (JNK) Inhibitors Protect Dopaminergic Neurons in a Model of Parkinson's Disease.

There are currently no drugs to treat neurodegeneration in Parkinson's disease (PD) and all existing medications only treat symptoms, lose efficacy over time, and produce untoward side effects. In the current work, we report the first highly selective, orally bioavailable, c-jun-N-terminal kinase (JNK) inhibitor for protection of dopaminergic neurons in vitro and in vivo. At 300 nM this compound showed statistically significant protection of primary dopaminergic neurons exposed to 1-methyl-4-phenylpyridinium (MPP(+)), had pharmacokinetic properties in rodents consistent with twice daily (b.i.d.) dosing, and was orally efficacious at 30 mg/kg in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Moreover, a dose-dependent target modulation of c-jun phosphorylation served as a biomarker for demonstrating on-target inhibition of JNK as the mechanism of action for this compound. Collectively these results suggest that this JNK inhibitor could be a promising therapeutic neuroprotective agent in the treatment of Parkinson's disease.