Athar N. Malik

IN VITRO CHONDROGENESIS OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN A PHOTOPOLYMERIZING HYDROGEL

Mesenchymal stem cells (MSCs) from skeletally mature goats were encapsulated in a photopolymerizing poly(ethylene glycol)-based hydrogel and cultured with or without transforming growth factor beta1 (TGF) to study the potential for chondrogenesis in a hydrogel scaffold system amenable to minimally invasive implantation. Chondrogenic differentiation was evaluated by histological, biochemical, and RNA analyses for the expression of cartilage extracellular matrix components. The two control groups studied were MSCs cultured in monolayer and MSCs encapsulated in the hydrogel and cultured for 6 weeks in chondrogenic medium without TGF-beta1 (6wk-TGF). The three experimental time points for encapsulated cells studied were 0 days (0d), 3 weeks, and 6 weeks in chondrogenic medium with TGF-beta1 at 10 ng/ml (3wk+TGF and 6wk+TGF). MSCs proliferated in the hydrogels with TGF-beta1. Glycosaminoglycan (GAG) and total collagen content of the hydrogels increased to 3.5% dry weight and 5.0% dry weight, respectively, in 6wk+TGF constructs. Immunohistochemistry revealed the presence of aggrecan, link protein, and type II collagen. Upregulation of aggrecan and type II collagen gene expression compared with monolayer MSCs was demonstrated. Type I collagen gene expression decreased from 3 to 6 weeks in the presence of TGF-beta1. 6wk-TGF hydrogels produced no GAG and only moderate amounts of collagen. However, immunohistochemistry and RT-PCR demonstrated a small amount of spontaneous differentiation in this control group. This study demonstrates the ability to encapsulate MSCs to form cartilage-like tissue in vitro in a photopolymerizing hydrogel. This system may be useful for minimally invasive implantation, MSC differentiation, and engineering of composite tissue structures with multiple cellular phenotypes.

Activity-dependent regulation of inhibitory synapse development by Npas4

Neuronal activity regulates the development and maturation of excitatory and inhibitory synapses in the mammalian brain. Several recent studies have identified signalling networks within neurons that control excitatory synapse development. However, less is known about the molecular mechanisms that regulate the activity-dependent development of GABA (γ-aminobutyric acid)-releasing inhibitory synapses. Here we report the identification of a transcription factor, Npas4, that plays a role in the development of inhibitory synapses by regulating the expression of activity-dependent genes, which in turn control the number of GABA-releasing synapses that form on excitatory neurons. These findings demonstrate that the activity-dependent gene program regulates inhibitory synapse development, and suggest a new role for this program in controlling the homeostatic balance between synaptic excitation and inhibition.

Using Whole-Exome Sequencing to Identify Inherited Causes of Autism

Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole-exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1). At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.

Experimental Model for Cartilage Tissue Engineering to Regenerate the Zonal Organization of Articular Cartilage

OBJECTIVE Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Genome-wide identification and characterization of functional neuronal activity-dependent enhancers

Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) and binding of the transcriptional coactivator CREBBP (also called CBP) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

MEF2D Drives Photoreceptor Development through a Genome-wide Competition for Tissue-Specific Enhancers

Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors (TFs) play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood. Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites and redirects it to retina-specific enhancers that lack the consensus MEF2-binding sequence. Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs and through selective activation of these enhancers to regulate tissue-specific genes.

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase–independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease.

Evolution of Osteocrin as an activity-regulated factor in the primate brain

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.

Neurogenic Stress Cardiomyopathy After Aneurysmal Subarachnoid Hemorrhage

BACKGROUND Neurogenic stress cardiomyopathy (NSC) is a known complication of aneurysmal subarachnoid hemorrhage (SAH). Detailed analyses of risk factors for its occurrence across large cohorts are relatively sparse. METHODS A consecutive group of 300 patients with aneurysmal SAH was reviewed for the presence of markers of myocardial injury, including electrocardiogram changes (long QT, T-wave inversion), elevated plasma troponin levels (≥0.1), and echocardiogram findings (decreased ejection fraction and wall motion abnormalities). NSC was defined as the presence of at least 1 marker of myocardial injury. Univariate and multivariate analyses were conducted to assess the correlation of NSC and individual markers of myocardial injury with age, gender, medical comorbidities, medications, current smoking status, Hunt-Hess grade, and Fisher grade. Medical comorbidities were assessed based on reported medical history or reported use of comorbidity-specific medications at the time of presentation. RESULTS Across the cohort, 27% of patients had a plasma troponin elevation of at least 0.1; 13%, a prolonged QT interval; 16%, new T-wave inversions; 18%, a depressed ejection fraction (<55%); and 15%, echocardiographic wall motion abnormalities. After a multivariate analysis, significant risk factors for NSC included higher Hunt-Hess grade on presentation (odds ratio [OR] = 2.33, P = 4.52 × 10(-6)), current smoking status (OR = 2.00, P = 0.030), and older age (OR = 1.03, P = 0.048). Hypertension was protective against NSC (OR = 0.48, P = 0.031). Patient gender, hyperlipidemia, diabetes, coronary artery disease, statin use, beta blocker use, angiotensin-converting enzyme inhibitor use, aspirin use, and thicker SAH (Fisher grade 3) were not significant risk factors for NSC. CONCLUSIONS Higher Hunt-Hess grade, current smoking status, lack of hypertension, and older age were the strongest predictors of NSC.