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.

Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina.

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.

Efficient targeting to storage granules of human proinsulins with altered propeptide domain.

In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide.

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.

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.

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.

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.

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.

Autopsy services for dementia patients.

Chiefs of Pathology of every nonfederal hospital in Maryland were surveyed to determine their hospitals' experience with autopsies of dementia patients. Few pathology services had extensive, ongoing experience with these postmortem examinations, and many hospitals lacked special histochemical techniques or a referral system to evaluate them. Problems exist in obtaining postmortem examinations for dementia patients, especially those who die outside facilities with Autopsy Services.

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.

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.

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.

New techniques lead to advances in epithelial cell polarity.

We have utilized cell surface biotinylation assays to study protein targeting signals and pathways in polarized epithelial cells. These studies have revealed that in MDCK cells, most proteins are sorted intracellularly and are targeted directly to the surface; in other cell types, protein targeting may be mediated by a selective retrieval event. Studies on both intact and permeabilized cells demonstrate that microtubules facilitate apical but not basolateral delivery. Recent transfection studies in MDCK cells have identified glycosyl phosphatidyl inositol (GPI) as an apical targeting signal; interaction of the GPI moiety with glycolipids preferentially expressed on the apical surface may mediate this process. Several proteinaceous basolateral targeting signals have also been recently described.

Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.

The calcium-independent neural cell adhesion molecule N-CAM is expressed transiently during development in many tissues, including epithelia. The three naturally occurring principal isoforms of N-CAM differ in the way in which they associate with the membrane and in their cytoplasmic domains. These isoforms are generated by developmentally regulated alternative splicing of a single gene: the large cytoplasmic domain (ld) form (relative molecular mass 180,000 (Mr 180K] is specific for post-mitotic neurons; the 120K small cytoplasmic domain (ssd) and 140K small surface domain (sd) forms also occur on other cell types. One function of the different isoforms could be to specify cellular localization; for example, glycosyl phosphatidyl inositol (GPI)-membrane anchoring acts as a targeting signal for expression on the apical surface of polarized epithelial cells. Neurons and epithelial cells may use similar mechanisms for polarizing their plasma membrane proteins. We have therefore investigated the targeting of GPI-anchored (ssd N-CAM, 120K) and transmembrane forms of N-CAM (sd N-CAM, 140K; ld N-CAM, 180K) by comparing the expression of each after transfection of the appropriate complementary DNAs into polarized epithelial cells. We find that isoforms with alternative modes of membrane association are targeted to different surfaces of polarized epithelial cells: ssd N-CAM is expressed on the apical surface, whereas sd and ld N-CAM are expressed on the basolateral surface. These results suggest that the different isoforms of N-CAM determine their own diverse cellular destinations. They also support the hypothesis that the GPI anchor acts as an apical targeting signal in epithelia.

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.

Genetics of exopolysaccharide production by mucoid Pseudomonas aeruginosa.

The secretion of the exopolysaccharide, alginate, is believed to contribute to the predilection for persistence of P. aeruginosa in respiratory tract infections of cystic fibrosis patients. To understand more about the pathway of alginate biosynthesis, we have cloned a gene, alg-50, which is involved in alginate biosynthesis. The alg-50 gene was physically mapped on a DNA fragment from P. aeruginosa by deletion analysis and transposition mutagenesis. The alginate trait is unstable, and another clone was found which may contain genes involved in this phenomenon. The two uronic acid components in alginate can vary, and a gene was cloned which increases the L-guluronate concentration of alginate produced by P. aeruginosa.

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.