AB3080 Sigma-AldrichAnti-Green Fluorescent Protein Antibody

The pathology of spinal cord injury (SCI) makes it appropriate for cell-based therapies. Treatments using neural stem cells (NSCs) in animal models of SCI have shown positive outcomes, although uncertainty remains regarding the optimal cell source. Pluripotent cell sources such as embryonic stem cells (ESCs) provide a limitless supply of therapeutic cells. NSCs derived using embryoid bodies (EB) from ESCs have shown tumorigenic potential. Clonal neurosphere generation is an alternative method to generate safer and more clinically relevant NSCs without the use of an EB stage for use in cell-based therapies. We generated clonally derived definitive NSCs (dNSCs) from ESC. These cells were transplanted into a mouse thoracic SCI model. Embryonic stem cell-derived definitive neural stem cell (ES-dNSC)-transplanted mice were compared with controls using behavioral measures and histopathological analysis of tissue. In addition, the role of remyelination in injury recovery was investigated using transmission electron microscopy. The SCI group that received ES-dNSC transplantation showed significant improvements in locomotor function compared with controls in open field and gait analysis. The cell treatment group had a significant enhancement of spared neural tissue. Immunohistological assessments showed that dNSCs differentiated primarily to oligodendrocytes. These cells were shown to express myelin basic protein, associate with axons, and support nodal architecture as well as display proper compact, multilayer myelination in electron microscopic analysis. This study provides strong evidence that dNSCs clonally derived from pluripotent cells using the default pathway of neuralization improve motor function after SCI and enhance sparing of neural tissue, while remaining safe and clinically relevant.

The presubiculum, located between hippocampus and entorhinal cortex, plays a fundamental role in representing spatial information, notably head direction. Little is known about GABAergic interneurons of this region. Here, we used three transgenic mouse lines, Pvalb-Cre, Sst-Cre, and X98, to examine distinct interneurons labeled with tdTomato or green fluorescent protein. The distribution of interneurons in presubicular lamina for each animal line was compared to that in the GAD67-GFP knock-in animal line. Labeling was specific in the Pvalb-Cre line with 87% of labeled interneurons immunopositive for parvalbumin (PV). Immunostaining for somatostatin (SOM) revealed good specificity in the X98 line with 89% of fluorescent cells, but a lesser specificity in Sst-Cre animals where only 71% of labeled cells were immunopositive. A minority of ∼6% of interneurons co-expressed PV and SOM in the presubiculum of Sst-Cre animals. The electrophysiological and morphological properties of fluorescent interneurons from Pvalb-Cre, Sst-Cre, and X98 mice differed. Distinct physiological groups of presubicular interneurons were resolved by unsupervised cluster analysis of parameters describing passive properties, firing patterns and AP shapes. One group consisted of SOM-positive, Martinotti type neurons with a low firing threshold (cluster 1). Fast spiking basket cells, mainly from the Pvalb-Cre line, formed a distinct group (cluster 3). Another group (cluster 2) contained interneurons of intermediate electrical properties and basket-cell like morphologies. These labeled neurons were recorded from both Sst-Cre and Pvalb-Cre animals. Thus, our results reveal a wide variation in anatomical and physiological properties for these interneurons, a real overlap of interneurons immuno-positive for both PV and SOM as well as an off-target recombination in the Sst-Cre line, possibly linked to maternal cre inheritance.

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

Erythropoietin (EPO) has potent neuroprotective effects. The short-term delivery of high-dose EPO seemed to improve patients' neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future in vivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level.A mouse EPO cDNA was subcloned into the pCMS-EGFP vector and transfected into NIH/3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO-overexpressing cell line, EPO-3T3-EGFP, was established. To further confirm the neuroprotective abilities of secreted EPO from EPO-3T3-EGFP cells, a cell model of neurodegeneration, PC12-INT-EGFP, was applied.The expression level of EPO was highly elevated in EPO-3T3-EGFP cells, and an abundant amount of EPO secreted from EPO-3T3-EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, the survival rate of PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-INT-EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF-M, phosphorylated-NF-M, and the α-INT-EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment.The established EPO-overexpressing NIH/3T3 cell line, EPO-3T3-EGFP, may provide a material for future studies of cell-based therapies for neurodegenerative diseases via the secretion of EPO on a short-term, high-dose, regional basis.

Midbrain dopamine neurons fire in bursts conveying salient information. Bursts are associated with pauses in tonic firing of striatal cholinergic interneurons. Although the reciprocal balance of dopamine and acetylcholine in the striatum is well known, how dopamine neurons control cholinergic neurons has not been elucidated. Here, we show that dopamine neurons make direct fast dopaminergic and glutamatergic connections with cholinergic interneurons, with regional heterogeneity. Dopamine neurons drive a burst-pause firing sequence in cholinergic interneurons in the medial shell of the nucleus accumbens, mixed actions in the accumbens core, and a pause in the dorsal striatum. This heterogeneity is due mainly to regional variation in dopamine-neuron glutamate cotransmission. A single dose of amphetamine attenuates dopamine neuron connections to cholinergic interneurons with dose-dependent regional specificity. Overall, the present data indicate that dopamine neurons control striatal circuit function via discrete, plastic connections with cholinergic interneurons.

Delivery of proteins has been regarded as the safest and most useful application in therapeutic application of stem cells, because proteins can regulate gene expression transiently without any genomic alteration. However, it is difficult to accurately measure efficiency or quantity of intracellular protein uptake. Here, we performed a comparison study of cell-penetrating peptide (CPP)-conjugated protein delivery system using seven arginine and Streptolysin O (SLO)-mediated system. To compare CPP- and SLO-mediated protein delivery systems, we used GFP and ESRRB protein, which is known to regulate pluripotency-related genes, for delivery into human bone marrow stromal cells (hBMSCs) and human testicular stromal cells (hTSCs). We found that CPP-conjugated protein delivery was more efficient, lower cytotoxicity, and higher biological activity than SLO-mediated protein delivery system. These results suggest that delivery of CPP-conjugated proteins is an efficient tool for introducing biologically active proteins into cells and may have important implications in clinical cell-based therapy.

The kidney is a homeostatic organ required for waste excretion and reabsorption of water, salts and other macromolecules. To this end, a complex series of developmental steps ensures the formation of a correctly patterned and properly proportioned organ. While previous studies have mainly focused on the individual signaling pathways, the formation of higher order receptor complexes in lipid rafts is an equally important aspect. These membrane platforms are characterized by differences in local lipid and protein compositions. Indeed, the cells in the Xenopus pronephric kidney were positive for the lipid raft markers ganglioside GM1 and Caveolin-1. To specifically interfere with lipid raft function in vivo, we focused on the Sterol Carrier Protein 2 (scp2), a multifunctional protein that is an important player in remodeling lipid raft composition. In Xenopus, scp2 mRNA was strongly expressed in differentiated epithelial structures of the pronephric kidney. Knockdown of scp2 did not interfere with the patterning of the kidney along its proximo-distal axis, but dramatically decreased the size of the kidney, in particular the proximal tubules. This phenotype was accompanied by a reduction of lipid rafts, but was independent of the peroxisomal or transcriptional activities of scp2. Finally, disrupting lipid micro-domains by inhibiting cholesterol synthesis using Mevinolin phenocopied the defects seen in scp2 morphants. Together these data underscore the importance for localized signaling platforms in the proper formation of the Xenopus kidney.

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. Here we show that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor DIMM, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form DIMM-dependent fluorescent puncta that are coassociated by super-resolution microscopy. DIMM is required to maintain SYT-α and SYT-β protein levels in DIMM-expressing cells in vivo. In neurons normally lacking all three proteins (DIMM(-)/SYT-α(-)/SYT-β(-)), DIMM misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if DIMM is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by DIMM overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by DIMM at a post-transcriptional level.