Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.     

Bone marrow transplantation for constitutional pure red cell aplasia

Constitutional pure red cell aplasia (CPRCA) is a syndrome of failed erythropoiesis usually diagnosed within the first year of life. Four patients with CPRCA received transplants with marrow from their HLA- identical, mixed lymphocyte culture-nonreactive siblings. All patients were resistant to corticosteroid therapy and were dependent on regular red cell transfusions for at least 5 years. Three patients were conditioned with procarbazine, antithymocyte globulin, cyclophosphamide, and busulfan, and one was conditioned with antithymocyte serum, cyclophosphamide, and busulfan. Three patients promptly had successful engraftments with establishment of donor hematopoiesis. One patient initially rejected his graft but received a successful retransplant. All patients are currently alive with Karnofsky performance scores of 100 and normal erythropoiesis of donor origin. Despite a history of multiple transfusions, bone marrow transplantation is a potentially curative therapy for patients with CPRCA.     

Minimally invasive coronary artery bypass grafting versus percutaneous transluminal coronary angioplasty with stenting in isolated high-grade stenosis of the proximal left anterior descending coronary artery: six months' angiographic and clinical follow-up of a prospective randomized study

OBJECTIVE: We sought to compare minimally invasive coronary artery bypass grafting (surgical intervention) with percutaneous transluminal coronary angioplasty with primary stenting (stenting) in patients having an isolated high-grade stenosis (American College of Cardiology/American Heart Association classification type B2 or C) of the proximal left anterior descending coronary artery. At 6 months, both procedures were compared on the basis of quantitative angiography and clinical outcome. METHODS: Both treatments were compared in a single-center, prospective, randomized study. The primary end point of this study was quantitative angiographic outcome at 6 months. The secondary end point was 6-month clinical outcome. Statistical analysis was performed in accordance with the intention-to-treat principle. RESULTS: From March 1997 to September 1999, patients with angina pectoris caused by an isolated high-grade stenosis of the proximal left anterior descending coronary artery were randomly assigned to surgical intervention (n = 51) or stenting (n = 51). At 6 months, quantitative coronary angiography showed an anastomotic stenosis rate of 4% after surgical intervention and a restenosis rate of 29% after stenting (P <.001). Periprocedural events did not significantly differ between surgical intervention and stenting. After surgical intervention, 2 patients died; no patients died after stenting. After 6 months, no significant difference was found for major adverse cardiac or cerebral events and need for repeat target vessel revascularization. After 6 months, return of angina pectoris, physical work capacity, and use of antianginal drugs did not significantly differ between treatments. CONCLUSIONS: After 6 months, surgical intervention had a significantly better angiographic outcome than stenting in patients with an isolated high-grade stenosis of the proximal left anterior descending coronary artery. Clinical outcome did not significantly differ between treatments.     

Sympathetic Neural Adaptation to Hypocaloric Diet With or Without Exercise Training in Obese Metabolic Syndrome Subjects

OBJECTIVE

Sympathetic nervous system (SNS) overactivity contributes to the pathogenesis and target organ complications of obesity. This study was conducted to examine the effects of lifestyle interventions (weight loss alone or together with exercise) on SNS function.

RESEARCH DESIGN AND METHODS

Untreated men and women (mean age 55 ± 1 year; BMI 32.3 ± 0.5 kg/m²) who fulfilled Adult Treatment Panel III metabolic syndrome criteria were randomly allocated to either dietary weight loss (WL, n = 20), dietary weight loss and moderate-intensity aerobic exercise (WL+EX, n = 20), or no treatment (control, n = 19). Whole-body norepinephrine kinetics, muscle sympathetic nerve activity by microneurography, baroreflex sensitivity, fitness (maximal oxygen consumption), metabolic, and anthropometric measurements were made at baseline and 12 weeks.

RESULTS

Body weight decreased by −7.1 ± 0.6 and −8.4 ± 1.0 kg in the WL and WL+EX groups, respectively (both P < 0.001). Fitness increased by 19 ± 4% (P < 0.001) in the WL+EX group only. Resting SNS activity decreased similarly in the WL and WL+EX groups: norepinephrine spillover by −96 ± 30 and −101 ± 34 ng/min (both P < 0.01) and muscle sympathetic nerve activity by −12 ± 6 and −19 ± 4 bursts/100 heart beats, respectively (both P < 0.01), but remained unchanged in control subjects. Blood pressure, baroreflex sensitivity, and metabolic parameters improved significantly and similarly in the two lifestyle intervention groups.

CONCLUSIONS

The addition of moderate-intensity aerobic exercise training to a weight loss program does not confer additional benefits on resting SNS activity. This suggests that weight loss is the prime mover in sympathetic neural adaptation to a hypocaloric diet.The metabolic syndrome (MetS) is an increasingly prevalent multidimensional risk factor for cardiovascular disease and type 2 diabetes (1). Its etiology is complex and incompletely understood, but thought to involve the interplay between metabolic susceptibility, lifestyle factors, and the acquisition of excess visceral adiposity (2). Scientific studies performed over the last 2 decades strongly support the relevance of the sympathetic nervous system (SNS) in both the pathogenesis and target organ complications of MetS obesity (3).Several indexes of SNS activity, such as urinary norepinephrine excretion, norepinephrine spillover from sympathetic nerves, and postganglionic muscle sympathetic nerve activity (MSNA) are increased in subjects with MetS, even in the absence of hypertension (4–7). Among the adiposity indexes, abdominal visceral fat is most strongly associated with elevated MSNA (8). Because of the bidirectional relationship between sympathetic activation and insulin resistance, much debate has focused on their chronology. Prospective studies with 10–20 years follow-up indicate that elevated plasma norepinephrine concentration (9) and sympathetic reactivity (10) precede and predict future rise in BMI and development of insulin resistance. Although seemingly counterintuitive, sympathetic activation may be causally linked to obesity via β-adrenoceptor desensitization (11) and insulin resistance (12,13). In established obesity, metabolic, cardiovascular (baroreflex impairment), and medical conditions (obstructive sleep apnea) contribute significantly to sympathetic neural drive and further aggravate insulin resistance, hence establishing a vicious cycle (3,7). Chronic sympathetic activation is associated with an increased prevalence of preclinical cardiovascular and renal changes that are recognized predictors of adverse clinical prognosis (3,14,15).Weight loss and exercise are recommended as first-line treatments for MetS. The Diabetes Prevention Program and the Oslo Diet and Exercise Study have shown the marked clinical benefits of intensive lifestyle intervention on the resolution of the MetS (16,17). Individually, both weight loss (5) and exercise training (18,19) cause sympathoinhibition and improvement in MetS components. We have previously reported that moderate weight loss (7% of body weight) by diet alone is accompanied by reductions in whole-body norepinephrine spillover and MSNA and improvement in spontaneous cardiac baroreflex function in middle-aged MetS subjects (5). Because exercise is often added to energy restriction in the treatment of obesity, it is pertinent to clarify its additive benefits. Augmented improvements in metabolic, anthropometric, and cardiovascular parameters have been observed after combined exercise training and dietary weight loss in some (17,20,21), but not other studies (22), and there are limited data regarding their combined effect on sympathetic activity (23). Exercise training may potentially augment weight loss induced sympathoinhibition by promoting a greater loss of fat relative to lean mass (20,21), by further improvement in insulin sensitivity (24) and reduction in plasma leptin concentration (21), and by potentiation of baroreceptor sensitivity (18).The present study was conducted to 1) test the hypothesis that weight loss by combined hypocaloric diet and aerobic exercise training would be associated with greater sympathoinhibition and improvement in MetS components than hypocaloric diet alone and 2) to examine the interrelationships between reduction in sympathetic tone and concurrent changes in anthropometric, metabolic (insulin sensitivity, plasma leptin concentration), and cardiovascular parameters. A moderate-intensity bicycle riding protocol was chosen as the exercise intervention, based on an earlier study that demonstrated attenuation in whole-body and renal norepinephrine spillover rates with this regimen in healthy men (19).     

Clinical and neuropsychological follow up at 12 months in patients with complicated Parkinson's disease treated with subcutaneous apomorphine infusion or deep brain stimulation of the subthalamic nucleus

BACKGROUND: The clinical condition of advanced Parkinson's disease (PD) patients is often complicated by motor fluctuations and dyskinesias which are difficult to control with available oral medications. OBJECTIVE: To compare clinical and neuropsychological 12 month outcome following subcutaneous apomorphine infusion (APO) and chronic deep brain stimulation of the subthalamic nucleus (STN-DBS) in advanced PD patients. METHODS: Patients with advanced PD and medically untreatable fluctuations underwent either APO (13 patients) or STN-DBS (12 patients). All patients were clinically (UPDRS-III, AIMS, 12 h on-off daily) and neuropsychologically (MMSE, Hamilton-17 depression, NPI) evaluated at baseline and at 12 months. APO was discontinued at night. RESULTS: At 12 months APO treatment (74.78+/-24.42 mg/day) resulted in significant reduction in off time (-51%) and no change in AIMS. Levodopa equivalent medication doses were reduced from 665.98+/-215 mg/day at baseline to 470+/-229 mg/day. MMSE, NPI, and Hamilton depression scores were unchanged. At 12 months STN-DBS resulted in significant clinical improvement in terms of reduction in daily off time (-76%) and AIMS (-81%) as well as levodopa equivalent medication doses (980+/-835 to 374+/-284 mg/day). Four out of 12 patients had stopped oral medications. MMSE was unchanged (from 28.6+/-0.3 to 28.4+/-0.6). Hamilton depression was also unchanged, but NPI showed significant worsening (from 6.58+/-9.8 to 18.16+/-10.2; p<0.02). Category fluency also declined. CONCLUSIONS: Both APO and STN-DBS resulted in significant clinical improvement in complicated PD. STN-DBS resulted in greater reduction in dopaminergic medications and provided 24 h motor benefit. However, STN-DBS, unlike APO, appears to be associated with significant worsening on NPI resulting from long term behavioral problems in some patients.     

Glial activation in white matter following ischemia in the neonatal P7 rat brain

This study examines cell death and proliferation in the white matter after neonatal stroke. In postnatal day 7 injured rat, there was a marked reduction in myelin basic protein (MBP) immunostaining mainly corresponding to numerous pyknotic immature oligodendrocytes and TUNEL-positive astrocytes in the ipsilateral external capsule. In contrast, a substantial restoration of MBP, as indicated by the MBP ratio of left-to-right, occurred in the cingulum at 48 (1.27 +/- 0.12) and 72 (1.30 +/- 0.18, P < 0.05) h of recovery as compared to age-matched controls (1.03 +/- 0.14). Ki-67 immunostaining revealed a first peak of newly generated cells in the dorsolateral hippocampal subventricular zone and cingulum at 72 h after reperfusion. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 h and NG2 pre-oligodendrocytes at 72 h of recovery. Microglia infiltration occurs over several days in the cingulum, and a huge quantity of macrophages reached the subcortical white matter where they engulfed immature oligodendrocytes. The overall results suggest that the persistent activation of microglia involves a chronic component of immunoinflammation, which overwhelms repair processes and contributes to cystic growth in the developing brain.