Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont.

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Experience With Unfavorable Iliac Access When Performing Fenestrated/Branched Endovascular Aneurysm Repair.

PURPOSE: To review a single-center experience with fenestrated and branched endovascular aneurysm repair (f/bEVAR) in patients with challenging iliac anatomies. MATERIALS AND METHODS: A retrospective review of the department's database identified 398 consecutive patients who underwent complex endovascular repair f/bEVAR between January 2010 and June 2018; of these, 67 had challenging accesses. The strategies implemented to overcome access issues were reviewed, using a dedicated scoring system to evaluate the access (integrating diameter, tortuosity, calcification, and previous open or endovascular repair). RESULTS: In this subgroup of patients, the most common graft design was a 4-vessel fenestrated endograft (27, 40.3%). Hostile access was due to small diameter (<7 mm) in 25 patients (37.3%) and/or concentric calcifications in 19 patients (26.9%). Mean iliac diameter was 5.5±2.6 mm on the right side and 6.0±2.5 mm on the left side. Previous open or endovascular aortoiliac repair had been performed in 15 patients (22.4%), and 20 patients (29.9%) had a stent previously implanted in at least 1 iliac artery, resulting in the inability to perform standard fenestrated repair with access from both sides. Five patients (7.5%) had a single patent iliac access. Eight distinctive strategies were identified to overcome these access issues, including the use of preloaded renal catheters in the endograft delivery system, angioplasty, graft modification (branches instead of fenestrations or 4 preloaded fenestrations), a conduit via a retroperitoneal approach, iliac artery recanalization, and/or the multiple puncture technique. Technical success was achieved in 62 cases (92.5%). Four patients had access complications and 1 died in the early postoperative period of multiorgan failure. Median follow-up was 24.6 months (IQR 7.2, 41.3). Clinical success at the end of follow-up was achieved in 57 patients (85.1%). During follow-up, 14 patients died, including 4 from an aorta-related cause. CONCLUSION: Dedicated strategies can be implemented to overcome hostile iliac access in patients with complex aneurysms when f/bEVAR is required. Typically, these maneuvers are associated with favorable outcomes.

Editor's Choice - Durability of Open Repair of Juxtarenal Abdominal Aortic Aneurysms: A Multicentre Retrospective Study in Five French Academic Centres.

OBJECTIVES: With a focus on renal function, the goal of this multicentre study was to assess peri-operative complications and late mortality of open surgical repair (OSR) of juxtarenal abdominal aortic aneurysms (JRAAA). METHODS: From February 2005 to December 2015, 315 consecutive patients undergoing elective OSR of a JRAAA in five French academic centres were evaluated retrospectively. The definition of JRAAA was an aortic aneurysm extending up to but not involving the renal arteries, i.e., a short neck <10 mm. End points included post-operative death; acute kidney injury (AKI) defined by the RIFLE (Risk, Injury, Failure, Loss of function, End stage renal disease) criteria; and long term follow-up with freedom from chronic renal decline (CRD) and any graft related complications. Factors predictive of renal insufficiency were determined by multivariable analysis. RESULTS: Of 315 patients, 292 (92.6%) were men (mean age 68 ± 8 years), and 73 (23.2%) had baseline chronic kidney disease (CKD) with an estimated glomerular filtration rate of <60 mL/min/1.73 m2. The level of aortic clamping was supracoeliac (n = 11), suprarenal (n = 235), or inter-renal above one renal artery (n = 69). The mean duration of renal artery clamping was 24 ± 7 min (range 10-55 min). Eleven patients (3.5%) presented with a renal artery stenosis that was treated conservatively. Perfusion of the renal arteries with a chilled Ringer's solution was used selectively in seven patients (2.2%). The overall 30 day mortality was 0.9% (three patients). AKI occurred in 53 patients (16.8%). Nine patients (2.9%) required temporary dialysis and one patient required chronic dialysis. Predictors of AKI were pre-existing CKD (odds ratio [OR] 2.25, 95% confidence interval [CI] 1.13-4.48; p = .021], diabetes (OR 3.15, 95% CI 1.48-6.71; p = .003), hypertension (OR 3.38, 95% CI 1.33-8.57; p = .01), and age (OR 1.05, 95% CI 1.01-1.10; p = .014). The level of aortic clamping and duration of renal artery clamping were not associated with an increased risk of AKI. The Kaplan-Meier survival estimate was 71% ± 5% at five years. Predictors of CRD during follow up were AKI (hazard ratio [HR] 15.81, 95% CI 5.26-47.54; p = .001), diabetes (HR 4.56, 95% CI 1.57-13.17; p = .005), and pre-existing CKD (HR 2.93, 95% CI 1.19-7.20; p = .019), with freedom from CRD of 89% ± 3% at five years. Surveillance imaging was obtained by computed tomography angiography in 290 patients (92.6%) at a mean follow up of 4.3 ± 2.4 years. Renal artery occlusion occurred in two patients (0.7% of imaged renal arteries). One patient (1.9%) had an aneurysm of the visceral aorta and eight patients had a descending thoracic aneurysm. CONCLUSIONS: This multicentre study suggests that in fit patients, open JRAAA repair can be performed with acceptable operative risk with durable results in terms of both graft integrity and preservation of renal function.

Cryopreserved arterial allografts for in situ reconstruction of abdominal aortic native or secondary graft infection.

OBJECTIVE: The objective of this study was to evaluate the early and long-term outcome of cryopreserved arterial allografts (CAAs) used for in situ reconstruction of abdominal aortic native or secondary graft infection and to identify predictors of mortality. METHODS: We retrospectively included 71 patients (mean age, 65.2 years [range, 41-84 years]; men, 91.5%) treated for abdominal aortic native or secondary graft infection (65 prosthetic graft infections; 16 of them had secondary aortoenteric fistula, 2 venous graft infections, and 4 mycotic aneurysms) by in situ reconstruction with CAA in the university hospitals of Clermont-Ferrand and Saint-Etienne from 2000 to 2016. The cryopreservation protocol was identical in both centers (-140°C). Early (<30 days) and late (>30 days) mortality and morbidity, reinfection, and CAA patency were assessed. Computed tomography was performed in all survivors. Survival was analyzed with the Kaplan-Meier method. Univariate analyses were performed with the log-rank test and multivariate analysis with the Cox regression model. RESULTS: Mean follow-up was 45 months (0-196 months). Early postoperative mortality rate was 16.9% (11/71). Early postoperative CAA-related mortality rate was 2.8% (2/71); both patients died of proximal anastomotic rupture on postoperative days 4 and 15. Early CAA-related reintervention rate was 5.6% (4/71); all had an anastomotic rupture, and two were lethal. Early postoperative reintervention rate was 15.5% (11/71). Intraoperative bacteriologic samples were positive in 56.3%, and 31% had a sole microorganism. Escherichia coli was more frequently identified in the secondary aortoenteric fistula and Staphylococcus epidermidis in the infected prosthesis. Late CAA-related mortality rate was 2.8%: septic shock at 2 months in one patient and proximal anastomosis rupture at 1 year in one patient. Survival at 1 year, 3 years, and 5 years was 75%, 64%, and 54%, respectively. Multivariate analysis identified type 1 diabetes (hazard ratio, 2.49; 95% confidence interval, 1.05-5.88; P = .04) and American Society of Anesthesiologists class 4 (hazard ratio, 2.65; 95% confidence interval, 1.07-6.53; P = .035) as predictors of mortality after in situ CAA reconstruction. Reinfection rate was 4% (3/71). Late CAA-related reintervention rate was 12.7% (9/71): proximal anastomotic rupture in one, CAA branch stenosis/thrombosis in five, ureteral-CAA branch fistula in one, and distal anastomosis false aneurysm in two. Primary patency at 1 year, 3 years, and 5 years was 100%, 93%, and 93%, respectively. Assisted primary patency at 1 year, 3 years, and 5 years was 100%, 96%, and 96%, respectively. No aneurysm or dilation was observed. CONCLUSIONS: The prognosis of native or secondary aortic graft infections is poor. Aortic in situ reconstruction with CAA offers acceptable early and late results. Patients with type 1 diabetes and American Society of Anesthesiologists class 4 are at higher risk of mortality.

G2019S LRRK2 enhances the neuronal transmission of tau in the mouse brain.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 mutations typically give rise to Lewy pathology in the brains of PD subjects yet can induce tau-positive neuropathology in some cases. The pathological interaction between LRRK2 and tau remains poorly defined. To explore this interaction in vivo, we crossed a well-characterized human P301S-tau transgenic mouse model of tauopathy with human G2019S-LRRK2 transgenic mice or LRRK2 knockout (KO) mice. We find that endogenous or pathogenic LRRK2 expression has minimal effects on the steady-state levels, solubility and abnormal phosphorylation of human P301S-tau throughout the mouse brain. We next developed a new model of tauopathy by delivering AAV2/6 vectors expressing human P301S-tau to the hippocampal CA1 region of G2019S-LRRK2 transgenic or LRRK2 KO mice. P301S-tau expression induces hippocampal tau pathology and marked degeneration of CA1 pyramidal neurons in mice, however, this occurs independently of endogenous or pathogenic LRRK2 expression. We further developed new AAV2/6 vectors co-expressing human WT-tau and GFP to monitor the neuron-to-neuron transmission of tau within defined hippocampal neuronal circuits. While endogenous LRRK2 is not required for tau transmission, we find that G2019S-LRRK2 markedly enhances the neuron-to-neuron transmission of tau in mice. Our data suggest that mutant tau-induced neuropathology occurs independently of LRRK2 expression in two mouse models of tauopathy but identifies a novel pathogenic role for G2019S-LRRK2 in promoting the neuronal transmission of WT-tau protein. These findings may have important implications for understanding the development of tau neuropathology in LRRK2-linked PD brains.

Eversion carotid endarterectomy without shunt: concerning 1385 consecutive cases.

BACKGROUND: The aim of this study was to evaluate the perioperative results of eversion carotid endarterectomy (e-CEA) without shunt at 30 days. METHODS: From January 2004 to December 2013, 1385 e-CEAs were performed in 981 men and 404 women, for 268 hemispheric, 55 ocular and 12 oculopyramidal symptoms of carotid stenosis. The average age was 71.1 years. The contralateral internal carotid artery (ICA) was occluded in 77 cases. All e-CEAs were performed using Vanmaele technique, with blood pressure monitoring and under general anesthesia except in two cases (locoregional anesthesia alone). The need for application of an intra-arterial shunt was evaluated using visual quantification of adequate retrograde ICA pressure based on the quality of back-bleeding from the ICA. If well pulsatile, a shunt was not required. Otherwise, the systolic blood pressure was increased until a good quality ICA back-flow was obtained. RESULTS: Freedom from intra-arterial shunt placement was 100% as a result of estimation and augmentation of arterial perfusion to demonstrate pulsatile perfusion by retrograde ICA filling. A peroperative angiography was performed in 910 cases. All surgical sites were evaluated postoperatively by Duplex imaging. The overall stroke and death rate was 1.3%. Nine (0.7%) patients died perioperatively. The 24 (1.7%) non-fatal neurologic events were ipsilateral: 6 (0.4%) disabling and 9 (0.6%) regressive stroke, 3 (0.2%) permanent and 1 (0.1%) transient ocular ischemia, and 5 (0.4%) transient ischemic attacks. Three (0.2%) patients had a perioperative myocardial infarction. Eleven compressive neck hematomas (0.8%) were reoperated in emergency. CONCLUSIONS: E-CEA can be performed safely, as a routine technique, based on the surgeon's evaluation of arterial back-bleeding and an increase in ipsilateral arterial perfusion with standard anesthetic procedures. Also e-CEA may be considered a cost effective method of reducing the frequency of intra-arterial shunt placement and adjuncts used to assess adequate cerebral perfusion of the ipsilateral carotid artery during e-CEA.

Note: Localization based on estimated source energy homogeneity.

Acoustic signal localization is a complex problem with a wide range of industrial and academic applications. Herein, we propose a localization method based on energy attenuation and inverted source amplitude comparison (termed estimated source energy homogeneity, or ESEH). This inversion is tested on both synthetic (numerical) data using a Lamb wave propagation model and experimental 2D plate data (recorded with 4 accelerometers sensitive up to 26 kHz). We compare the performance of this technique with classic source localization algorithms: arrival time localization, time reversal localization, and localization based on energy amplitude. Our technique is highly versatile and out-performs the conventional techniques in terms of error minimization and cost (both computational and financial).

Outcomes of the Endovascular Treatment for the Supra-Aortic Trunks Occlusive Disease: A 14-Year Monocentric Experience.

BACKGROUND: Endovascular treatment of proximal supra-aortic trunks (SAT) has become a safe and reliable alternative to conventional open surgery, with a lower morbimortality rate and good short- and middle-term patency rates. The aim of our study was to assess the long-term results of endovascular treatment of proximal lesions of the SAT (brachiocephalic trunk, common carotid artery, and subclavian artery) and identify predictive risk factors of restenosis. METHODS: From 1999 to 2013, 67 consecutive stenotic lesions of the proximal SAT were treated by angioplasty (13.4%) or stenting (86.6%) in 63 patients with a mean age of 65.5 years (40-87). Procedures were performed under general (69%), local (24%), or locoregional (7%) anesthesia, with percutaneous puncture (47.8%) or open access (52.2%). Patients were followed up for 3, 6, and 12 months, and then every year with clinical examination, Doppler ultrasound and if required an angio-CT scan. RESULTS: The technical success rate was 98.5%. There was no postoperative death or strokes. One myocardial infarction occurred at day 2. There were 2 access complications: a nonsurgical hematoma after brachial access and a brachial thrombosis postpuncture. The mean follow-up was 4.5 years (2-163 months). The primary- and assisted-patency rates were 90.1%, 86.4%, 77.9% and 93.3%, 91.4%, 82.9% at 1, 2, and 5 years, respectively. Eleven restenosis (16.4%) occurred at 28.5 months (3, 0-112, 0) of follow-up. Four of them required an endovascular repair and 3 required a surgical one. The restenosis rate was 17.5% in the stented group on average at 30.2 months of follow-up (range, 3.0-112.0) and 10% in the group of patients with angioplasty alone at 8 months of follow-up, without significant statistically difference (P = 0.9). No predictive risk factor of restenosis was statistically identified. CONCLUSIONS: The endovascular treatment of proximal stenosis of SAT is a safe, reliable, and efficient technique with a low morbidity and mortality. The long-term results are good, but restenosis can occur. Long-term follow-up should be performed to detect and treat restenosis.

Type B Aortic Dissection with Abdominal Aortic Aneurysm Rupture 1 Year after Endovascular Repair of Abdominal Aortic Aneurysm.

We report a patient who developed a type B aortic dissection and ruptured his aneurysmal sac 1 year after endovascular abdominal aortic aneurysm repair (EVAR), despite standard follow-up. This 79-year-old man was presented to emergency room with acute abdominal pain and an acute lower limb ischemia. Computed tomography scan showed an acute type B aortic dissection feeding the aneurysmal sac of the EVAR. The aneurysm rupture occurred during imaging. Type B aortic dissection is a rare cause of aneurysmal rupture after EVAR. The first postoperative computed tomography scan should maybe include the arch and the descending thoracic aorta to rule out an iatrogenic dissection after EVAR.

Does the technique of carotid endarterectomy determine postoperative hypertension?

BACKGROUND: Hypertension (HT) after carotid endarterectomy (CEA) is a risk factor for postoperative myocardial infarction, stroke, and neck hematoma. We compared the incidence of postoperative HT within the week after eversion CEA (e-CEA) and patch closure CEA (p-CEA). Postoperative HT was defined as a systolic blood pressure (sBP) ≥ 160 mm Hg and/or the need for postoperative vasodilatators. The aim of our study was to determine if the technique of CEA had an effect on postoperative HT. METHODS: Between January 2010 and June 2011, we prospectively reviewed 560 consecutive endarterectomies (340 p-CEAs and 220 e-CEAs) performed in 443 patients under general anesthesia. All had >70% stenoses, 119 were symptomatic, and 441 asymptomatic. We compared preoperative, peroperative, and postoperative sBP and diastolic blood pressure, carotid sinus nerve block, postoperative intravenous and oral antihypertensive medications, neurologic and cardiac complications, and mortality. RESULTS: The e-CEA group had a higher incidence of women (36.4% vs. 21.8%, P = 0.0002) and HT (85.0% vs. 78.2%, P = 0.04). The e-CEAs had a significantly higher incidence of carotid sinus nerve block (93.6% vs. 15.6%, P < 0.0001). The incidence of postoperative HT was not significantly different between the 2 groups (75.9% in the e-CEA group versus 68.5% in the p-CEA group, P = 0.06). The average postoperative sBP between postoperative hour (H) 2 and H12 was significantly higher in the e-CEA group but <160 mm Hg. The sBP dropped between H2 and H6, and this decrease was greater in the p-CEA group (30% vs. 15% in the e-CEA group). The need for postoperative antihypertensive medication was not different between the 2 groups. One independent risk factor of postoperative HT was identified: history of HT. The rate of postoperative complications was not significantly different between the 2 groups. CONCLUSIONS: The e-CEA technique is not a risk factor and does not have an effect on postoperative HT. The postoperative sBP was more stable in this group. Eversion carotid endarterectomy has been considered, in the literature, as a risk factor of postoperative hypertension. We conducted a large prospective and comparative study of the endarterectomy technique by eversion and with conventional patch closure. The primary end point was the blood pressure value and the administration of antihypertensive treatment. Our study shows that postoperative hypertension after carotid endarterectomy is not related to the surgical technique. Changes in blood pressure after carotid endarterectomy by eversion are lower than those observed after conventional endarterectomy with patch closure. This technique prevents the occurrence of possible hypotension occurrence, which can be the cause of perioperative complications.

Imprinted Zac1 in neural stem cells.

Neural stem cells (NSCs) and imprinted genes play an important role in brain development. On historical grounds, these two determinants have been largely studied independently of each other. Recent evidence suggests, however, that NSCs can reset select genomic imprints to prevent precocious depletion of the stem cell reservoir. Moreover, imprinted genes like the transcriptional regulator Zac1 can fine tune neuronal vs astroglial differentiation of NSCs. Zac1 binds in a sequence-specific manner to pro-neuronal and imprinted genes to confer transcriptional regulation and furthermore coregulates members of the p53-family in NSCs. At the genome scale, Zac1 is a central hub of an imprinted gene network comprising genes with an important role for NSC quiescence, proliferation and differentiation. Overall, transcriptional, epigenomic, and genomic mechanisms seem to coordinate the functional relationships of NSCs and imprinted genes from development to maturation, and possibly aging.

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD.

Modeling LRRK2 Pathobiology in Parkinson's Disease: From Yeast to Rodents.

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene represent the most common cause of familial Parkinson's disease (PD) with autosomal dominant inheritance, whereas common variation at the LRRK2 genomic locus influences the risk of developing idiopathic PD. LRRK2 is a member of the ROCO protein family and contains multiple domains, including Ras-of-Complex (ROC) GTPase, kinase, and protein-protein interaction domains. In the last decade, the biochemical characterization of LRRK2 and the development of animal model s have provided important insight into the pathobiology of LRRK2. In this review, we comprehensively describe the different models employed to understand LRRK2-associated PD, including yeast, invertebrates, transgenic and viral-based rodents, and patient-derived induced pluripotent stem cells. We discuss how these models have contributed to understanding LRRK2 pathobiology and the advantages and limitations of each model for exploring aspects of LRRK2-associated PD.

Roles of imprinted genes in neural stem cells.

Imprinted genes and neural stem cells (NSC) play an important role in the developing and mature brain. A central theme of imprinted gene function in NSCs is cell survival and G1 arrest to control cell division, cell-cycle exit, migration and differentiation. Moreover, genomic imprinting can be epigenetically switched off at some genes to ensure stem cell quiescence and differentiation. At the genome scale, imprinted genes are organized in dynamic networks formed by interchromosomal interactions and transcriptional coregulation of imprinted and nonimprinted genes. Such multilayered networks may synchronize NSC activity with the demand from the niche resembling their roles in adjusting fetal size.

Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.

Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

Early-life stress reduces DNA methylation of the Pomc gene in male mice.

Early-life stress (ELS) increases the vulnerability thresholds for stress-related diseases such as major depression and anxiety by inducing alterations in the structure and function of neural circuits and endocrine pathways. We previously demonstrated the contribution of epigenetic mechanisms to the long-term programming of the hypothalamo-pituitary-adrenal axis activity following ELS exposure in male mice. Here, ELS comprising daily separation of pups from their dams on postnatal days 1-10 was observed to up-regulate the expression of the pituitary proopiomelanocortin (Pomc) gene; POMC serves as a prohormone for ACTH, a key mediator of the adrenocortical response to stress. Detailed analysis revealed that the increase in Pomc mRNA levels results from a reduction in DNA methylation at a critical regulatory region of the Pomc gene; interestingly, this change occurs with some delay after ELS and persists for up to 1 year. Using a Pomc-expressing pituitary cell line (AtT20), we confirmed a role for DNA methylation in restraining Pomc expression under resting conditions: specifically, we show that CpG site-specific methylation of the Pomc promoter represses Pomc mRNA transcription. Further, we show high-affinity binding of methyl-CpG binding protein-2 to the distal promoter of Pomc, suggesting that methyl-CpG binding protein-2 acts in association with the chromatin modifiers histone deacetylase 2 and DNA methyltransferase 1 to repress Pomc gene expression. Collectively, these experiments contribute to our understanding of the mechanisms through which environmental cues are translated into stable changes ("cellular memory") in neuroendocrine cells.

Zac1 regulates cell cycle arrest in neuronal progenitors via Tcf4.

Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57(Kip2), a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57(Kip2) via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.

Zac1 regulates astroglial differentiation of neural stem cells through Socs3.

Cell-fate decisions and differentiation of embryonic and adult neural stem cells (NSC) are tightly controlled by lineage-restricted and temporal factors that interact with cell-intrinsic programs and extracellular signals through multiple regulatory loops. Imprinted genes are important players in neurodevelopment and mental health although their molecular and cellular functions remain poorly understood. Here, we show that the paternally expressed transcriptional regulator Zac1 (zinc finger protein regulating apoptosis and cell cycle arrest) is transiently induced during astroglial and neuronal differentiation of embryonic and adult NSC lines. Thereby, Zac1 transactivates Socs3 (suppressor of cytokine signaling 3), a potent inhibitor of prodifferentiative Jak/Stat3 signaling, in a lineage-specific manner to prevent precocious astroglial differentiation. In vivo, Zac1 and Socs3 colocalize in the neocortical ventricular zone during incipient astrogliogenesis. Zac1 overexpression in primary NSCs delays astroglial differentiation whereas knockdown of Zac1 or Socs3 facilitates formation of astroglial cells. This negative feedback loop is unrelated to Zac1's cell cycle arrest function and specific to the Jak/Stat3 pathway. Hence, reinstating Jak/Stat3 signaling in the presence of increased Zac1 expression allows for timely astroglial differentiation. Overall, we suggest that the imprinted gene Zac1 curtails astroglial differentiation of NSCs in the developing and adult brain.

Performance of mouse neural stem cells as a screening reagent: characterization of PAC1 activity in medium-throughput functional assays.

The self-renewal and phenotypic properties of neural stem cells make them an abundant and more physiologically relevant alternative to recombinant cell lines for drug screens to identify ligands acting at neural targets. Here, the authors use high-throughput phenotypic and signaling assays to test the ability of neural stem cells isolated from postnatal mouse hippocampus (mNSCs) to deliver high-content and physiologically relevant data on native peptide receptor activity. The authors find that mNSCs express PAC1 but not the related VPAC1 and VPAC2 receptors. PAC1 promotes both the proliferation of mNSCs and their differentiation into neuronal-like cells. In addition, the authors show that PAC1 stimulates markedly different extracellular signal-regulated kinase signals in mNSCs than in recombinant CHO-PAC1 cells and is able to couple to Ca(2+) elevation only in CHO-PAC1 cells. These data suggest that G-protein coupling in CHO-PAC1 cells is nonphysiological, which may affect the ligand binding properties of the receptor and thus distort the results of a screen by increasing numbers of false positives/negatives. This work reinforces the emerging pharmacological theory that recombinant cell lines are often inappropriate models of natively expressing primary cells, and the authors conclude that mNSCs are a viable and relevant physiological alternative for use in high-throughput drug screens.