Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease.

CRISPR/Cas9 technology has transformed our ability to manipulate the genome and epigenome, from efficient genomic editing to targeted localization of effectors to specific loci. Through the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators, the role of gene-regulatory and epigenetic pathways in basic biology and disease processes can be directly queried. Here, we discuss emerging CRISPR-based methodologies, with specific consideration of neurobiological applications of human induced pluripotent stem cell (hiPSC)-based models.

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Nociceptin receptor activation does not alter acquisition, expression, extinction and reinstatement of conditioned cocaine preference in mice.

Growing evidence indicates that targeting nociceptin receptor (NOP) signaling may have therapeutic efficacy in treating alcohol and opioid addiction. However, little is known about the therapeutic value of selective NOP agonists for the treatment of cocaine dependence. Recently, we identified a highly selective, brain-penetrant NOP small molecule agonist (SR-8993), and using this compound, we previously showed that nociceptin receptor activation attenuated consolidation of fear-related memories. Here, we sought to determine whether SR-8993 also affects the rewarding properties of cocaine. Using a conditioned place preference (CPP) procedure, we show that SR-8993 (3 or 10 mg/kg) failed to disrupt acquisition or expression of cocaine CPP (7.5 or 15 mg/kg) in C57BL/6 mice. Additionally, SR-8993 did not affect rate of extinction or reinstatement (yohimbine- and cocaine-induced) of cocaine CPP. These studies indicate that selective activation of NOP may not be sufficient in reducing behavioral responses to cocaine.

Diffusion tensor imaging properties and neurobehavioral outcomes in children with hydrocephalus.

BACKGROUND AND PURPOSE: White matter structural alterations and the correlation with neuropsychological deficits in children with hydrocephalus have not been well investigated. In this prospective study, the objectives were the following: 1) to apply DTI to detect in vivo white matter alterations based on diffusion properties in children with acute hydrocephalus, 2) to quantify early neuropsychological deficits, and 3) to explore the correlation between potential neuropsychological deficits and abnormalities in functionally related white matter. MATERIALS AND METHODS: A total of 44 children, 24 with hydrocephalus and 20 controls, were enrolled in the study. DTI indices, FA, MD, AD, and RD, were evaluated in the gCC, sCC, PLIC, and ALIC. The ABAS-II was used as a broad screener of development, including conceptual, social, practical, and motor skills. The correlation between the Motor Scale and DTI indices in the PLIC was analyzed. RESULTS: DTI analyses showed that the gCC and sCC in children with hydrocephalus had lower FA and higher MD, driven by the increased RD with statistical significance (P < .05) or trend-level significance (P = .06). The PLIC and ALIC had significantly higher AD in children with hydrocephalus (P < .05). On the ABAS-II, parent ratings of general adaptive skills, conceptual skills, and motor skills were significantly lower in children with hydrocephalus (all at P < .05). The MD and RD values in the PLIC were found to have trend-level or significant correlation with the Motor Scale (P = .057, .041, respectively). CONCLUSIONS: DTI reveals alterations in the white matter structure in children with hydrocephalus with preliminary findings suggesting correlation with clinical motor deficits.

Enhanced gene transfer to rabbit jugular veins by an adenovirus containing a cyclic RGD motif in the HI loop of the fiber knob.

Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting. However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR). Designing vectors specifically targeted to alpha(v) integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC. RGD, a receptor-binding motif that can interact with alpha(v) integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a beta-galactosidase reporter gene. Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture. Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg. Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins. Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg. Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations. A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD. These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting.

Breeding of retroviruses by DNA shuffling for improved stability and processing yields.

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.

Antiretroviral agents inhibit infection of human cells by porcine endogenous retroviruses.

The efficacy of antiretroviral drugs against porcine endogenous retroviruses (PERV) that may be harbored in pig organs intended for transplantation was examined in human cells in vitro. The nucleoside analogs zidovudine and dideoxyinosine were found to effectively inhibit PERV replication.

Neural cell response to multiple novel sites on laminin-1.

The basement membrane protein laminin-1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the laminin-1 molecule. In this study, 545 overlapping 12- to 14-mer synthetic peptides, corresponding to most of the amino acid sequence of the alpha1, beta1, and gamma1 chains of laminin-1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the laminin-1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike-like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell-type specificity in their activity. These studies show that (1) there are multiple and distinct sites on laminin-1 for cell adhesion and neurite-like outgrowth and (2) that there are neural cell-type-specific active domains. The multiple active sites found explains, in part, the potent activity of laminin-1 on neurite outgrowth.

Cell adhesive sequences in mouse laminin beta1 chain.

Laminin-1, a major component of the basement membrane, consists of three different chains, alpha1, beta1, and gamma1. We sought to identify cell adhesive sequences from the mouse laminin beta1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin beta1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin beta1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.

In vitro analysis of transformation potential associated with retroviral vector insertions.

While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, GlnBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that GlnBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro.

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI-anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI-anchored proteins have been found on both the axonal and somatodendritic cell-surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication-incompetent retroviral vector to express a model GPI-anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high-density monolayers, and maintained for 1-9 days under serum-free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP-expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP-expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well-polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI-anchored protein expressed in a well-polarized primary neuronal cell type in vitro; other signals, such as those present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon-specific GPI-anchored proteins.

Cell binding sequences in mouse laminin alpha1 chain.

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.

Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro.

The basement membrane glycoprotein laminin-1 is a potent stimulator of neurite outgrowth. Although a variety of laminin isoforms have been described in recent years, the role of alternative laminin isoforms in neural development remains largely uncharacterized. We found that a polyclonal antibody raised against the alpha1, beta1, and gamma1 chains of laminin-1 and a monoclonal antibody raised against the alpha2 chain of laminin-2 detect immunoreactive material in neuronal cell bodies in the developing mouse cerebellum. In addition, laminin-1-like immunoreactivity was found in cell types throughout the cerebellum, but laminin-alpha2-like immunoreactivity was restricted to the Purkinje cells. Purified laminin-1 and laminin-2 stimulated neurite outgrowth in primary cultures of mouse cerebellar granule neurons to a similar extent, whereas the synthetic peptides tested appeared to be active only for cell adhesion and not for stimulation of neurite outgrowth. The E8 proteolytic fragment of laminin-1 contained full neurite outgrowth activity. The identity of laminins expressed in granule neurons was also examined by Western blotting; laminin-like complexes were associated with the cell and appeared to have novel compositions. These results suggest that laminin-like complexes play important roles in cerebellar development.

Neuronal laminins and their cellular receptors.

The laminins are a family of extracellular matrix glycoproteins expressed throughout developing neural tissues. The laminins are potent stimulators of neurite outgrowth in vitro for a variety of cell types, presumably reflecting an in vivo role in stimulating axon outgrowth. In recent years, the laminins have been shown to occur in several distinct isoforms; currently, the precise functional differences between the laminin variants are not well understood. A variety of neuronal surface receptors have been identified for one laminin isoform, laminin-1. These receptors include several members of the integrin family, as well as non-integrin laminin-binding proteins such as LBP-110, the 67 kDa laminin-receptor, alpha-dystroglycan, and beta 1,4 galactosyltransferase. Little is currently known about receptors for other laminin isoforms.

The neural cell adhesion molecule expresses a tyrosine-independent basolateral sorting signal.

Transmembrane isoforms of the neural cell adhesion molecule, N-CAM (N-CAM-140 and N-CAM-180), are vectorially targeted from the trans-Golgi network to the basolateral domain upon expression in transfected Madin-Darby canine kidney cells (Powell, S. K., Cunningham, B. A., Edelman, G. M., and Rodriguez-Boulan, E. (1991) Nature 353, 76-77). To localize basolateral targeting information, mutant forms of N-CAM-140 were constructed and their surface distribution analyzed in Madin-Darby canine kidney cells. N-CAM-140 deleted of its cytoplasmic domain shows a non-polar steady state distribution, resulting from delivery from the trans-Golgi network to both the apical and basolateral surfaces. This result suggests that entrance into the basolateral pathway may occur without cytoplasmic signals, implying that apical targeting from the trans-Golgi network is not a default mechanism but, rather, requires positive sorting information. Subsequent construction and analysis of a nested set of C-terminal deletion mutants identified a region of 40 amino acids (amino acids 749-788) lacking tyrosine residues required for basolateral targeting. Addition of these 40 amino acids is sufficient to restore basolateral targeting to both the non-polar cytoplasmic deletion mutant of N-CAM as well as to the apically expressed cytoplasmic deletion mutant of the p75 low affinity neurotrophin receptor (p75(NTR)), indicating that this tyrosine-free sequence is capable of functioning independently as a basolateral sorting signal. Deletion of both cytoplasmic and transmembrane domains resulted in apical secretion of N-CAM, demonstrating that the ectodomain of this molecule carries recessive apical sorting information.

Development of polarity in cerebellar granule neurons.

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex.

Identification of cell binding sequences in mouse laminin gamma1 chain by systematic peptide screening.

Laminin-1, a major component of basement membranes, consists of three different chains designated alpha1, beta1, and gamma1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin gamma1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the gamma1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-alpha2beta1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin gamma2 chain. These results suggest that the specific sequences on the laminin gamma1 chain are biologically active and interact with distinct cell surface receptors.

Job stress versus success factors for case management.

Job stress is all too common in the case management profession. Recognizing the signs and symptoms of stress and understanding its causes will help the nurse case manager minimize the deleterious effects of "burnout." The following article is an excerpt from the book. Nursing Case Management: A Practical Guide to Success in Managed Care. The author explores eight techniques to reduce stress and improve performance. The information can be used by the individual case manager or by supervisors and coworkers trying to help fellow case managers.

Incidence of retinal detachment after cataract surgery and neodymium: YAG laser capsulotomy.

The objective of this retrospective study was to determine the incidence of retinal detachment (RD) in patients following cataract extraction with intraocular lens placement and after neodymium:YAG (Nd:YAG) laser capsulotomy. This study comprised 1092 patients (1168 eyes) who had cataract extraction and related procedures between January 1986 and December 1992 identified from the coding and billing database. Of the 1092 patients, 215 (244 eyes) had had Nd:YAG laser capsulotomy. Their charts were reviewed for incidence of RD, and these data were correlated with age, sex, axial length, surgical complications, and other surgical procedures done at the time of cataract extraction. The incidence of RD following phacoemulsification alone was 0.75% (6/799), with a mean time between cataract extraction and RD of 11.6 months. The cases of RD after extracapsular cataract extraction, combined phacoemulsification and trabeculectomy, combined extracapsular cataract extraction and penetrating keratoplasty, and combined phacoemulsification and anterior vitrectomy were too few to draw any conclusions. The incidence of RD following Nd:YAG laser capsulotomy was 0.82% (2/244), with a mean time of 32 months between cataract surgery and capsulotomy and 13.5 months between capsulotomy and RD. There was a statistically significant higher incidence of RD after posterior capsule rupture and anterior vitrectomy than after uncomplicated phacoemulsification (2/12 versus 6/799). In conclusion, the rate of RD after uncomplicated phacoemulsification was less than or similar to the rate found in other recent studies. It was not statistically different from the rate following phacoemulsification and Nd:YAG laser capsulotomy (0.82%). This study confirms the increased risk of RD following posterior capsule rupture and anterior vitrectomy.