MAB324 Sigma-AldrichAnti-Neuron Specific Enolase Antibody, clone 5E2

Castration-resistant prostate cancers (CRPCs) that relapse after androgen deprivation therapies (ADTs) are responsible for the majority of mortalities from prostate cancer (PCa). While mechanisms enabling recurrent activity of androgen receptor (AR) are certainly involved in the development of CRPC, there may be factors that contribute to the process including acquired neuroendocrine (NE) cell-like behaviors working through alternate (non-AR) cell signaling systems or AR-dependent mechanisms. In this study, we explore the potential relationship between the AR axis and a novel putative marker of NE differentiation, the human male protocadherin-PC (PCDH-PC), in vitro and in human situations. We found evidence for an NE transdifferentiation process and PCDH-PC expression as an early-onset adaptive mechanism following ADT and elucidate AR as a key regulator of PCDH-PC expression. PCDH-PC overexpression, in turn, attenuates the ligand-dependent activity of the AR, enabling certain prostate tumor clones to assume a more NE phenotype and promoting their survival under diverse stress conditions. Acquisition of an NE phenotype by PCa cells positively correlated with resistance to cytotoxic agents including docetaxel, a taxane chemotherapy approved for the treatment of patients with metastatic CRPC. Furthermore, knockdown of PCDH-PC in cells that have undergone an NE transdifferentiation partially sensitized cells to docetaxel. Together, these results reveal a reciprocal regulation between the AR axis and PCDH-PC signals, observed both in vitro and in vivo, with potential implications in coordinating NE transdifferentiation processes and progression of PCa toward hormonal and chemoresistance.

When the axons of primary sensory neurons project into the embryonic mammalian spinal cord, they bifurcate and extend rostrocaudally before sending collaterals to specific laminae according to neuronal subclass. The specificity of this innervation has been suggested to be the result both of differential sensitivity to chemorepellants expressed in the ventral spinal cord and of the function of Ig-like neural cell adhesion molecules in the dorsal horn. The relationship between these mechanisms has not been addressed. Focussing on the pathfinding of TrkA+ NGF-dependent axons, we demonstrate for the first time that their axons project prematurely into the dorsal horn of both L1 and TAG-1 knockout mice. We show that axons lacking TAG-1, similar to those lacking L1, are insensitive to wild-type ventral spinal cord (VSC)-derived chemorepellants, indicating that adhesion molecule function is required in the axons, and that this loss of response is explained in part by loss of response to Sema3A. We present evidence that TAG-1 affects sensitivity to Sema3A by binding to L1 and modulating the endocytosis of the L1/neuropilin 1 Sema3A receptor complex. However, TAG-1 appears to affect sensitivity to other VSC-derived chemorepellants via an L1-independent mechanism. We suggest that this dependence of chemorepellant sensitivity on the functions of combinations of adhesion molecules is important to ensure that axons project via specific pathways before extending to their final targets.

Developmental neurotoxicity is a major issue in human health and may have lasting neurological implications. In this preliminary study we exposed differentiating Ntera2/clone D1 (NT2/D1) cell neurospheres to known human teratogens classed as non-embryotoxic (acrylamide), weakly embryotoxic (lithium, valproic acid) and strongly embryotoxic (hydroxyurea) as listed by European Centre for the Validation of Alternative Methods (ECVAM) and examined endpoints of cell viability and neuronal protein marker expression specific to the central nervous system, to identify developmental neurotoxins. Following induction of neuronal differentiation, valproic acid had the most significant effect on neurogenesis, in terms of reduced viability and decreased neuronal markers. Lithium had least effect on viability and did not significantly alter the expression of neuronal markers. Hydroxyurea significantly reduced cell viability but did not affect neuronal protein marker expression. Acrylamide reduced neurosphere viability but did not affect neuronal protein marker expression. Overall, this NT2/D1-based neurosphere model of neurogenesis, may provide the basis for a model of developmental neurotoxicity in vitro.

OBJECTIVE: Multiple genes contribute to autism spectrum disorder (ASD) susceptibility. One particularly promising candidate is the MET gene, which encodes a receptor tyrosine kinase that mediates hepatocyte growth factor (HGF) signaling in brain circuit formation, immune function, and gastrointestinal repair. The MET promoter variant rs1858830 allele C is strongly associated with ASD and results in reduced gene transcription. Here we examined expression levels of MET and members of the MET signaling pathway in postmortem cerebral cortex from ASD cases and healthy control subjects. METHODS: Protein, total RNA, and DNA were extracted from postmortem temporal cortex gray matter samples (BA 41/42, 52, or 22) belonging to eight pairs of ASD cases and matched control subjects. MET protein expression was determined by Western blotting; messenger RNA expression of MET and other related transcripts was assayed by microarray and quantitative reverse transcriptase polymerase chain reaction. RESULTS: MET protein levels were significantly decreased in ASD cases compared with control subjects. This was accompanied in ASD brains by increased messenger RNA expression for proteins involved in regulating MET signaling activity. Analyses of coexpression of MET and HGF demonstrated a positive correlation in control subjects that was disrupted in ASD cases. INTERPRETATION: Altered expression of MET and related molecules suggests dysregulation of signaling that may contribute to altered circuit formation and function in ASD. The complement of genes that encode proteins involved in MET activation appears to undergo long-term compensatory changes in expression that may be a hallmark contribution to the pathophysiology of ASD.

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.

Most clinical protocols involving adenovirus (Ad) vectors for gene therapy use a vector based on serotype 5 (Ad5). We believe that this serotype is not suitable for all gene therapy applications and that alternative vectors based on other serotypes should be developed. We have compared the ability of Ad5, Ad11p, Ad16p, and a chimpanzee Ad (CV23) to infect human low-passage brain tumor cells as well as primary glioma cells sorted into a CD133(+) and CD133(-) population. Cancer stem cells have been shown to reside in the CD133(+) population of cells in human glioma tumors and they are of considerable interest in glioma therapy. Ad16p and CV23 infected the low-passage brain tumor cell lines and also the CD133(+) and CD133(-) primary tumor cells most efficiently. Interestingly, as the passage number of the cells increased, the infection capacity of Ad5 increased significantly, whereas this was not seen for CV23. To ensure the therapeutic effect of Ad vectors on brain tumors, the vector must be capable of addressing both the CD133(+) cancer stem cells and the CD133(-) cells of the tumor. In particular, Ad16 and CV23 are meeting this challenge.

Extracellular matrix proteins (ECMs) play a significant role in the transfer of mechanical strain to monocyte-derived macrophages (MDMs) affecting morphological changes in a foreign body reaction. This study investigated how the functional responses of U937 macrophage-like cells differed when subjected to 2 dynamic strain types (nonuniform biaxial or uniform uniaxial strain) while cultured on siloxane membranes coated with either collagen type I or RGD peptide repeats (ProNectin). Biaxial strain caused an increase in intracellular esterase and acid phosphatase (AP) activities, as well as monocyte-specific esterase (MSE) protein levels in cells that were seeded on either uncoated surfaces (shown previously) or collagen, but not ProNectin. Released AP activity, but not released esterase activity, was increased on all surfaces. Biaxial strain increased IL-6, but not IL-8 on all surfaces. When cells were subjected to uniaxial strain, intracellular esterase increased on coated surfaces only, whereas intracellular AP activity was unaffected. Both esterase and AP released activities increased on all surfaces. Uniaxial strain increased the release of IL-6 on all surfaces, but IL-8 on coated surfaces only. This study demonstrated for the first time that ECM proteins could specifically modulate cellular responses to different types of strain. Using this approach with an in vitro cell system may help to unravel the complex function of MDMs in the foreign-body reaction.

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.

Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.

Progress in stem cell biology research is enhancing our ability to generate specific neuron types for basic and applied studies and to design new treatments for neurodegenerative diseases. In the case of Parkinson's disease (PD), alternative human dopaminergic (DAergic) neurons other than primary fetal tissue do not yet exist. One possible source could be human neural stem cells (hNSCs), although the yield in DAergic neurons and their survival are very limited. [see figure]. In this study, we found that Bcl-X(L) enhances (one-to-two orders of magnitude) the capacity for spontaneous dopaminergic differentiation of hNSCs, which then exceeds that of cultured human ventral mesencephalic tissue. Bcl-X(L) also enhanced total neuron generation by hNSCs, but to a lower extent. Neuronal phenotypes other than DA were not affected by Bcl-X(L), indicating an exquisitely specific effect on DAergic neurons. In vivo, grafts of Bcl-X(L)-overexpressing hNSCs do generate surviving human TH+ neurons in the adult rat 6-OH-dopamine lesioned striatum, something never seen when naive hNSCs were transplanted. Most of the data obtained here in terms of the effects of Bcl-X(L) are consistent with an enhanced survival type of mechanism and not supportive of induction, specification, or proliferation of DAergic precursors. From this in vitro and in vivo evidence, we conclude that enhancing Bcl-X(L) expression is important to obtain human DAergic neurons from hNSCs. These findings may facilitate the development of drug-screening and cell-replacement activities to discover new therapeutic strategies for PD.