Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Schizophrenia is a devastating brain disorder that affects millions of people worldwide and exhibits a strong genetic component. In a key discovery, deletions or duplications of larger stretches of chromosomal DNA that lead to copy number variations (CNVs) were identified two decades ago (1, 2). CNVs occur unexpectedly frequently, are often de novo, and usually affect multiple genes depending on the size of the deleted or duplicated stretch of DNA. Strikingly, the biggest genetic risk for schizophrenia was identified in three unrelated CNVs: a duplication of region 16p11.2 and deletions of 22q11.2 and of 2p16.3 (3–9). Of these CNVs, 16p11.2 and 22q11.2 CNVs affect more than 20 genes, whereas 2p16.3 CNVs impact only one or more exons of a single gene, NRXN1, which encodes the presynaptic cell-adhesion molecule neurexin-1 (4, 7, 9–12). NRXN1 CNVs confer an approximately 10-fold increase in risk of schizophrenia, and additionally strongly predispose to other neuropsychiatric disorders, especially autism and Tourette syndrome (13, 14). Moreover, genome-wide association studies using DNA microarrays identified common changes in many other genes that predispose to schizophrenia with smaller effect sizes (15–21). Viewed together, these studies indicate that variations in a large number of genes are linked to schizophrenia. Among these genetic variations, heterozygous exonic CNVs of NRXN1 are rare events, but nevertheless constitute the most prevalent high-risk single-gene association at present.Neurexins are central regulators of neural circuits that control diverse synapse properties, such as the presynaptic release probability, the postsynaptic receptor composition, and synaptic plasticity (22–28). To test whether heterozygous NRXN1 mutations might cause functional impairments in human neurons, we previously generated conditionally mutant human embryonic stem (ES) cells that enabled induction of heterozygous NRXN1 deletions using Cre-recombinase (29). We then analyzed the effects of the deletion on the properties of neurons induced from the conditionally mutant ES cells using forced expression of Ngn2, a method that generates a relatively homogeneous population of excitatory neurons that are also referred to as induced neuronal (iN) cells (30). These experiments thus examined isogenic neurons without or with a heterozygous NRXN1 loss-of-function mutation that mimicked the schizophrenia-associated 2p16.3 CNVs, enabling precise control of the genetic background. The heterozygous NRXN1 deletion produced a robust but discrete impairment in neurotransmitter release without major changes in neuronal development or morphology (29). These results were exciting because they suggested that a discrete impairment in neurotransmitter release could underlie the predisposition to schizophrenia conferred by the 2p16.3 CNV, but these experiments did not reveal whether the NRXN1 mutation induces the same synaptic impairment in schizophrenia patients (31).The present project was initiated to achieve multiple overlapping aims emerging from the initial study on human NRXN1 mutations (29). First, we aimed to validate or refute the results obtained with neurons generated from engineered conditionally mutant ES cells with neurons generated from patient-derived induced pluripotent stem (iPS) cells containing NRXN1 mutations (Fig. 1A). This goal was pursued in order to gain confidence in the disease-relevance of the observed phenotypes. Second, we wanted to test whether the observed phenotype is independent of the laboratory of analysis (i.e., whether it is sufficiently robust to be replicated at multiple sites) (Fig. 1A). This goal was motivated by the observation of limited reproducibility in some studies of the phenotypes of patient-derived neurons. We hypothesized that this lack of reproducibility is due to variations in experimental conditions rather than an experimental failure, and designed our studies to demonstrate robustness of the findings through replication. Third, we aimed to generate reagents that could be broadly used by the scientific community for investigating the cellular basis of neuropsychiatric disorders (32). This goal was prompted by the challenges posed by the finding that many different genes appear to be linked to schizophrenia. Fourth, we aimed to definitively establish or exclude the possibility that human neurons are uniquely sensitive to a heterozygous loss of NRXN1 compared with mouse neurons (Fig. 1B). The goal here was to test whether at least as regards to NRXN1, mouse and human neurons exhibit fundamental differences. Fifth and finally, we hoped to gain further insights into the mechanisms by which NRXN1 mutations predispose to schizophrenia, an obviously needed objective given our lack of understanding of this severe disorder. As described in detail below, our data provide advances toward meeting these goals, establishing unequivocally that heterozygous NRXN1 deletions in human but not in mouse neurons cause a robust impairment in neurotransmitter release that is replicable in multiple laboratories.Open in a separate windowFig. 1.Overall study design illustrating the experimental approach to analyze human heterozygous NRXN1 loss-of-function mutations, to achieve cross-laboratory and cross-platform validation of observed phenotypes, and to perform cross-paradigm evaluations of these phenotypes in human and mouse neurons. (A) Experimental strategy for analyzing the functional effects of heterozygous NRXN1 loss-of-function mutations in human patient-derived neurons and for validating the observed phenotypes in a cross-laboratory and cross-platform comparison. PBMCs from schizophrenia patients with NRXN1 deletions and from control individuals were reprogrammed into iPS cells by Rutgers University (RUCDR Infinite Biologics). iPS cells that passed QC were shipped to Stanford and to FCDI for expansion, banking, and transdifferentiation into induced neurons. The indicated subsequent analyses were carried out at Stanford University and at Rutgers University. FCDI manufactured industry-scale human induced neurons that were shipped to Rutgers for analysis, whereas Stanford generated induced neurons at an academic single-laboratory scale for analysis. (B) Experimental strategy to evaluate the conservation of NRXN1-deletion phenotypes observed in human neurons in mouse neurons (cross-paradigm evaluation). Human and mouse stem cells that carried heterozygous engineered conditional NRXN1/Nrxn1 deletions were transdifferentiated into neurons by Ngn2 expression and analyzed using similar approaches to ensure comparability. In this approach, isogenic human and mouse neurons without or with NRXN1/Nrxn1 deletions were compared to test whether side-by-side analysis of human and mouse neurons prepared by indistinguishable approaches yields similar phenotypes.     

Risk in elderly patients after stentless versus stented aortic valve surgery

Recent studies suggest that the hemodynamic advantage of stentless bioprostheses over the stented type improves long-term survival after aortic valve replacement, but the more complex and time-consuming implantation technique may increase the risks of operative death and postoperative complications. Between April 1996 and June 2001, 519 patients with a mean age of 76 +/- 5 years underwent aortic valve replacement using a stentless (Medtronic Freestyle, n = 277) or stented bioprosthesis (Medtronic Mosaic, n = 242). Multiple logistic regression analysis considering different patient populations revealed no increased risk of operative death, postoperative complications, or neurological impairment after implantation of a stentless bioprosthesis. Survival curves in respect of 367 patients who underwent aortic valve replacement up to September 2000 and were followed up for 3 years were not different (p = 0.98). As the patients were elderly, improved survival due to implantation of a stentless valve could not be demonstrated within this time span.     

Evaluation of a computed-tomography-based assessment scheme in treatment decision-making for isolated orbital floor fractures

Introduction

Treatment decisions for fractures of the orbital floor are based on clinical appearance, ophthalmological examination, and computed tomography (CT) scans. In extensive fractures, decisions are easily made between conservative and surgical treatment. However, objective parameters are rare in inconclusive cases.

The relative contributions of the prosthetic and sound limb to balance control in unilateral transtibial amputees

In unilateral transtibial amputees maintenance of standing balance is compromised due to the lack of active ankle control in the prosthetic limb. The purpose of this study is to disentangle the contribution of the prosthetic and sound limb to balance control following waist-pull perturbations. We compared the contribution of the hip and ankle joints to balance control of 15 unilateral transtibial amputees and 13 able-bodied controls after been externally perturbed through release of a pulling force. Perturbations were applied in four different directions. Outcome measure was the proportion of joint moment integrated over time generated by the hip and ankle joints in order to restore static stability after perturbation. Analyses revealed that perturbations in backward/forward direction were recovered mainly by the ankle strategy. The amputees compensated for the absence of active ankle control in the prosthetic limb by increasing the ankle moment in the sound limb. Interestingly, the passive properties of the prosthetic foot contributed to balance control, which has important implications for prosthetic fitting and standing stability in lower limb amputees. Amputees and controls resisted perturbations in medio-lateral direction by generating the necessary hip moments. Finally, these findings are discussed with respect to prosthetic design and rehabilitation processes.     

Role of Breast Surgery in BRCA Mutation Carriers

BRCA mutation carriers have a life-long breast cancer risk between 55 and 85% and a high risk of developing breast cancer at a very young age, depending on the type of mutation. The risk of developing contralateral breast cancer after a first breast cancer is elevated up to 65%, especially in case of BRCA1 mutation and young age at the first breast cancer. Since bilateral prophylactic mastectomy is associated with a risk reduction of 90–95% of developing primary or contralateral breast cancer, this option is a key point within the counseling process for patient information and shared decision-making of mutation carriers. Although the local control after breast-conserving therapy in mutation carriers seems to be comparable to that of sporadic breast cancer patients, individual patient information and counseling should include all alternative procedures of oncologically adequate mastectomy techniques and immediate reconstruction. Excellent cosmetic results, high levels of life quality, and good patient acceptance can be achieved with the recent developments in reconstructive surgery of the breast.