Generation of transplantable striatal projection neurons from human ESCs

In this issue of Cell Stem Cell, Ma et?al. (2012) report a differentiation protocol for generating striatal projection neurons from human embryonic stem cells with high efficiency. The cells survive transplantation, reconnect striatal circuitry, and restore motor function in a mouse model of striatal neurodegeneration that mimics the central pathohology of Huntington's disease.     

Ligatin from embryonic chick neural retina

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.     

Expression of glutamate transporters in human and rat retina and rat optic nerve

l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).     

Activating transcription factor 3 (ATF3) expression in the neural retina and optic nerve of zebrafish during optic nerve regeneration

Fish, unlike mammals, can regenerate axons in the optic nerve following optic nerve injury. We hypothesized that using microarray analysis to compare gene expression in fish which had experienced optic nerve lesion to fish which had undergone a similar operation but without optic nerve injury would reveal genes specifically involved in responding to optic nerve injury (including repair), reducing detection of genes involved in the general stress and inflammatory responses. We discovered 120 genes were significantly (minimally two-fold with a P-value ≤ 0.05) differentially expressed (up or down) at one or more time point. Among these was ATF3, a member of the cAMP-response element binding protein family. Work by others has indicated that elevated cAMP could be important in axon regeneration. We investigated ATF3 expression further by qRT-PCR, in situ hybridization and immunohistochemistry and found ATF3 expression is significantly upregulated in the ganglion cell layer of the retina, the nerve fiber layer and the optic nerve of the injured eye. The upregulation in retina is detectable by qRT-PCR by 24 h after injury, at which time ATF-3 mRNA levels are 8-fold higher than in retinas from sham-operated fish. We conclude ATF3 may be an important mediator of optic nerve regeneration-promoting gene expression in fish, a role which merits further investigation.     

Vertebrate neural cell-fate determination: lessons from the retina

Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion during development. Studies of cell-fate determination in the vertebrate retina have uncovered several fundamental principles by which this is achieved. Most notably, a model for vertebrate cell-fate determination has been proposed that combines findings on the relative roles of extrinsic and intrinsic regulators in controlling cell-fate choices. At the heart of the model is the proposal that progenitors pass through intrinsically determined competence states, during which they are capable of giving rise to a limited subset of cell types under the influence of extrinsic signals.     

A lens fibre differentiation factor from calf neural retina

During growth of the eye lens, epithelial cells differentiate into fibre cells under the influence of neural retina. The fibre differentiation factor (FDF) was partially characterized from calf retina-conditioned medium, using lens epithelial explants from young rats, to provide a bioassay for differentiation. FDF was associated with large-protein aggregates, the smallest of which eluted at approximately 500-600 kD on Sephacryl S-300 columns and migrated as a single protein band near 600 kD on gradient gels. This protein resolved into nine major peptides on SDS-polyacrylamide gels, ranging between 23 and 27 kD. Eight of these peptides were present oa four doublets, but did not appear to contain specific carbohydrate residues. The approximately 500-600 kD complex could be slightly disrupted by trypsin or heat treatment to release a less stable 90 kD component. Fractionation of FDF invariably led to loss of activity, possibly due to gradual dissociation into less active and/or less stable components. A working hypothesis suggested by these findings is that FDF is associated with a small group of peptides, each contributing an essential function to the process of fibre differentiation.     

Sharpening of directional selectivity from neural output of rabbit retina

The estimation of motion direction from time varying retinal images is a fundamental task of visual systems. Neurons that selectively respond to directional visual motion are found in almost all species. In many of them already in the retina direction selective neurons signal their preferred direction of movement. Scientific evidences suggest that direction selectivity is carried from the retina to higher brain areas. Here we adopt a simple integrate-and-fire neuron model, inspired by recent work of Casti et al. (2008), to investigate how directional selectivity changes in cells postsynaptic to directional selective retinal ganglion cells (DSRGC). Our model analysis shows that directional selectivity in the postsynaptic cells increases over a wide parameter range. The degree of directional selectivity positively correlates with the probability of burst-like firing of presynaptic DSRGCs. Postsynaptic potentials summation and spike threshold act together as a temporal filter upon the input spike train. Prior to the intricacy of neural circuitry between retina and higher brain areas, we suggest that sharpening is a straightforward result of the intrinsic spiking pattern of the DSRGCs combined with the summation of excitatory postsynaptic potentials and the spike threshold in postsynaptic neurons.     

Isolation of an adhesion-mediating protein from chick neural retina adherons

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.     

Ligatin from embryonic chick neural retina inhibits retinal cell adhesion

Ligatin, a filamentous cell-surface protein purified from embryonic chick neural retina, has been found to inhibit the reassociation of dissociated retinal cells. This inhibition was demonstrated using two methods, a single cell disappearance assay and an improved monolayer collection assay utilizing microtiter plates. Monomeric ligatin at approximately 20 μg/ml inhibited rates of adhesion, but polymeric ligatin and tryptic fragments of ligatin were ineffective. Ligatin's inhibitory effect is suggested to be mediated through binding to retinal cell surfaces since preincubation of dissociated retinal cells with monomeric ligatin inhibited the cells' adhesiveness and removed the inhibitory activity from the culture media. Ligatin homologues prepared from mammalian tissues were ineffective in inhibiting retinal cell adhesion, suggesting a tissue and/or species specificity. Similarities in physicochemical and biological properties suggest that ligatin may be the inhibitor of adhesion previously described by Merrell et al.[Merrell, R., Gottlieb, D. I., and Glaser, L. (1975). J. Biol. Chem., 250, 4825].     

Mass spectrometry quantification of PICALM and AP180 in human frontal cortex and neural retina

Recent genome-wide association studies have suggested that endocytic factors, such as phosphatidylinositol-binding clathrin assembly protein (PICALM), may be implicated in the development of Alzheimer disease (AD). The cellular functions of PICALM are in line with this possibility: (i) PICALM is involved in regulation of amyloid-β levels and (ii) PICALM is important for a presynaptic function, which is diminished in AD. To facilitate the analysis of PICALM, we developed a quantitative method to assess the expression level of PICALM in various biological samples. For this purpose, a stable isotope-labeled quantification concatamer (QconCAT) of PICALM was designed, expressed, purified, and characterized. The PICALM QconCAT was first used as an internal standard in a multiple reaction monitoring assay to measure PICALM concentrations in the human frontal cortex, a tissue strongly affected by AD. A second endocytic factor that is highly homologous to PICALM and also functions in clathrin-mediated endocytosis, clathrin coat assembly protein AP180, was quantified as well. Because age-related macular degeneration shares several clinical and pathological features with AD, the measurements were then extended to human normal neural retina. Overall, the developed method is suitable for PICALM and AP180 quantitative analysis in various biological samples of interest.     

Purification and comparison of glutamine synthetase from chicken liver, brain and neural retina

Glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) was isolated from chicken liver, brain and neural retina. The specific activities of the purified enzyme preparations from the three different sources were similar. They were composed of subunits of the same molecular weight (43 K) and were immunologically indistinguishable. Slight differences were detectable among them in relation to the amino acid compositions and regulation of their activities by the several effectors tested.