Modeling the functional genomics of autism using human neurons.

Human neural progenitors from a variety of sources present new opportunities to model aspects of human neuropsychiatric disease in vitro. Such in vitro models provide the advantages of a human genetic background combined with rapid and easy manipulation, making them highly useful adjuncts to animal models. Here, we examined whether a human neuronal culture system could be utilized to assess the transcriptional program involved in human neural differentiation and to model some of the molecular features of a neurodevelopmental disorder, such as autism. Primary normal human neuronal progenitors (NHNPs) were differentiated into a post-mitotic neuronal state through addition of specific growth factors and whole-genome gene expression was examined throughout a time course of neuronal differentiation. After 4 weeks of differentiation, a significant number of genes associated with autism spectrum disorders (ASDs) are either induced or repressed. This includes the ASD susceptibility gene neurexin 1, which showed a distinct pattern from neurexin 3 in vitro, and which we validated in vivo in fetal human brain. Using weighted gene co-expression network analysis, we visualized the network structure of transcriptional regulation, demonstrating via this unbiased analysis that a significant number of ASD candidate genes are coordinately regulated during the differentiation process. As NHNPs are genetically tractable and manipulable, they can be used to study both the effects of mutations in multiple ASD candidate genes on neuronal differentiation and gene expression in combination with the effects of potential therapeutic molecules. These data also provide a step towards better understanding of the signaling pathways disrupted in ASD.

Lithium regulates adult hippocampal progenitor development through canonical Wnt pathway activation.

Neural stem cells give rise to new hippocampal neurons throughout adulthood, and defects in neurogenesis may predispose an individual to mood disorders, such as major depression. Our understanding of the signals controlling this process is limited, so we explored potential pathways regulating adult hippocampal progenitor (AHP) proliferation and neuronal differentiation. We demonstrate that the mood stabilizer lithium directly expands pools of AHPs in vitro, and induces them to become neurons at therapeutically relevant concentrations. We show that these effects are independent of inositol monophosphatase, but dependent on Wnt pathway components. Both downregulation of glycogen synthase kinase-3beta, a lithium-sensitive component of the canonical Wnt signaling pathway, and elevated beta-catenin, a downstream component of the same pathway produce effects similar to lithium. In contrast, RNAi-mediated inhibition of beta-catenin abolishes the proliferative effects of lithium, suggesting that Wnt signal transduction may underlie lithium's therapeutic effect. Together, these data strengthen the connection between psychopharmacologic treatment and the process of adult neurogenesis, while also suggesting the pursuit of modulators of Wnt signaling as a new class of more effective mood stabilizers/antidepressants.

PBK/TOPK, a proliferating neural progenitor-specific mitogen-activated protein kinase kinase.

We performed genomic subtraction coupled to microarray-based gene expression profiling and identified the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-binding kinase/T-LAK (lymphokine-activated killer T cell) cell originating protein kinase (PBK/TOPK) as a gene highly enriched in neural stem cell cultures. Previous studies have identified PBK/TOPK as a mitogen-activated protein kinase (MAPK) kinase that phosphorylated P38 MAPK but with no known expression or function in the nervous system. First, using a novel, bioinformatics-based approach to assess cross-correlation in large microarray datasets, we generated the hypothesis of a cell-cycle-related role for PBK/TOPK in neural cells. We then demonstrated that both PBK/TOPK and P38 are activated in a cell-cycle-dependent manner in neuronal progenitor cells in vitro, and inhibition of this pathway disrupts progenitor proliferation and self-renewal, a core feature of progenitors. In vivo, PBK/TOPK is expressed in rapidly proliferating cells in the adult subependymal zone (SEZ) and early postnatal cerebellar external granular layer. Using an approach based on transgenically targeted ablation and lineage tracing in mice, we show that PBK/TOPK-positive cells in the SEZ are GFAP negative but arise from GFAP-positive neural stem cells during adult neurogenesis. Furthermore, ablation of the adult stem cell population leads to concomitant loss of PBK/TOPK-positive cells in the SEZ. Together, these studies demonstrate that PBK/TOPK is a marker for transiently amplifying neural progenitors in the SEZ. Additionally, they suggest that PBK/TOPK plays an important role in these progenitors, and further implicates the P38 MAPK pathway in general, as an important regulator of progenitor proliferation and self-renewal.

Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

A family perspective of family/staff interaction in long-term care facilities.

This article explores the ways that families perceive their relationships with the staff in long-term care facilities. Qualitative interviews were conducted with 27 families who had elder members in two facilities in Ontario, Canada. The families identified the staff characteristics that they valued and the ways they developed relationships with staff members.

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Protection by eliprodil against excitotoxicity in cultured rat retinal ganglion cells.

PURPOSE: To test whether eliprodil (SL 82.0715), a unique antagonist for the N-methyl-D-aspartate (NMDA) receptor, is protective in the glutamate-induced cytotoxicity model in cultured rat retinal ganglion cells (RGCs). METHODS: Two to four days after a fluorescent dye, Di-I, was injected near the superior colliculi, neonatal rats were killed, and retinal cells were dissociated and cultured. Survival of RGCs after drug treatment was assayed by counting Di-I fluorescent cells. RESULTS: In rat RGCs, glutamate-induced toxicity with a mean EC50 of 10.7 microM. Only 47% of RGCs survived after a 3-day treatment with 100 microM glutamate. Studies using selective agonists and antagonists indicated that the glutamate-induced toxicity was mediated largely by the NMDA receptor. Pretreatment with eliprodil protected against such toxicity. Eliprodil exhibited a mean IC50 of 1.0 nM (log [IC50] = -9.00 +/- 0.01, mean +/- SEM, n = 3; against cell death produced by 100 microM glutamate). At 1 microM, eliprodil was maximally protective; cell survival in the presence of 100 microM glutamate challenge was 100% +/- 5% (n = 3). This protective effect of eliprodil may be related to its reduction (by 78%) of NMDA-induced currents recorded under patch-clamp recording in these cells. CONCLUSIONS: Eliprodil is protective against glutamate cytotoxicity in retinal neurons. It may be a useful novel compound for the treatment of retinopathies including glaucoma in which excitotoxicity has been implicated.

Role of the low-affinity NGF receptor (p75) in survival of retinal bipolar cells.

We have examined the role of neurotrophins in promoting survival of mammalian rod bipolar cells (RBC) in culture. Retinas taken from 8- to 10-day-old Long-Evans rats were dissociated and cultured in media supplemented with either nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF-2). Survival was measured by the number of cells that were immunoreactive for alpha-, beta-, gamma-PKC, a bipolar cell-specific marker. Compared to untreated cultures, CNTF had no effect on RBC survival, while NGF and NT-3 increased survival only slightly. BDNF, however, increased survival by approximately 300%. Similar results were obtained with FGF-2. Both nerve growth factor (NGF) and an antibody (anti-REX) which interferes with binding to the 75-kD low-affinity neurotrophin receptor (p75NTR) eliminated BDNF-promoted survival, but had no effect on FGF-2-mediated survival. Interestingly, p75NTR was expressed by retinal glia (Müller cells), but not by the bipolar cells themselves, providing for the possibility that BDNF might induce Müller cells to produce a secondary factor, perhaps FGF-2, which directly rescues RBCs. In support of this hypothesis, an antibody that neutralizes FGF-2 attenuated the trophic effects of BDNF, and dramatically reduced survival in cultures with no added growth factors, indicating that there may be an endogenous source of FGF-2 that promotes survival of RBCs in culture. We suggest that BDNF increases production or release of FGF-2 by binding to p75NTR on Müller cells.

Nitric oxide depresses GABAA receptor function via coactivation of cGMP-dependent kinase and phosphodiesterase.

Nitric oxide (NO) is thought to play an essential role in neuronal processing, but the downstream mechanisms of its action remain unclear. We report here that NO analogs reduce GABA-gated currents in cultured retinal amacrine cells via two distinct, but convergent, cGMP-dependent pathways. Either extracellular application of the NO-mimetic S-nitroso-N-acetyl-penicillamine (SNAP) or intracellular perfusion with cGMP depressed GABA currents. This depression was partially blocked by a pseudosubstrate peptide inhibitor of cGMP-dependent protein kinase (PKG), suggesting both PKG-dependent and independent actions of cGMP. cAMP-dependent protein kinase (PKA) is known to enhance retinal GABA responses. 8-Bromoinosine 3',5'-cyclic monophosphate (8Br-cIMP), which activates a type of cGMP-stimulated phosphodiesterase that hydrolyzes cAMP, also significantly reduced GABA currents. 1-Methyl-3-isobutylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, blocked both the action of 8Br-cIMP and the portion of SNAP-induced depression that was not blocked by PKG inhibition. Our results suggest that NO depresses retinal GABAA receptor function by simultaneously upregulating PKG and downregulating PKA.

A fluorescent cellular adhesion assay using insect cell produced human VCAM1.

Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counterreceptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.

Priming of homosynaptic long-term depression in hippocampus by previous synaptic activity.

Mechanisms regulating long-lasting, activity-dependent decreases in synaptic strength are poorly understood. Theoretical studies have suggested that the critical level (threshold) of synaptic activity needed to induce long-term depression (LTD) versus potentiation (LTP) of synaptic transmission should vary as a function of previous synaptic activity. In order to determine whether such a 'sliding threshold' is indeed a physiological phenomenon, we stimulated Schaffer collateral afferents in area CA1 of rat hippocampal slices at frequencies from 1 to 5 Hz before or after inducing LTP.

Fat injection.

The results of grafts of fat obtained through liposuction are presented. They are the outcome of the current revitalization of the old technique of fat extraction and transplant. Both doctor and patient should be aware that a successful treatment is achieved through continuity. Fat grafts evolve with respect to time and permanence with reabsorption a part of the normal evolutionary process. Fat reinjection is a consequence of tissue reabsorption or a complement of the preceding injection. There is no complication associated with repeated reinjection. All cases reported here include patients with two or more reinjections, depending on their pathology and evolution.

A new model of liposuction cannula.

A new design for the suction cannula used in suction curettage is presented. Based on several years' experience in patients from 17 to 63 years of age, the new device allows softer movements intraoperatively, a correct grasp, and excellent suction.

Suction curettage lipectomy.

In 1981, we started performing minor suction curettage with the elements available at that time. In the course of surgical practice, we have modified the suction system and devised a new pattern of suction cannulas adapted for this kind of operation, with all possible variants. During this period, we have treated 150 patients, ranging in age from 17 to 63 years. In the light of present knowledge, we consider it time to provide a systematization of this technique.