Concise review: Spinal cord injuries: how could adult mesenchymal and neural crest stem cells take up the challenge?

Since several years, adult/perinatal mesenchymal and neural crest stem cells have been widely used to help experimental animal to recover from spinal cord injury. More interestingly, recent clinical trials confirmed the beneficial effect of those stem cells, which improve functional score of patients suffering from such lesions. However, a complete understanding of the mechanisms of stem cell-induced recovery is seriously lacking. Indeed, spinal cord injuries gathered a wide range of biochemical and physiopathological events (such as inflammation, oxidative stress, axonal damage, demyelination, etc.) and the genuine healing process after cell transplantation is not sufficiently defined. This review aims to sum up recent data about cell therapy in spinal cord lesions using mesenchymal or recently identified neural crest stem cells, by describing precisely which physiopathological parameter is affected and the exact processes underlying the observed changes. Overall, although significant advances are acknowledged, it seems that further deep mechanistic investigation is needed for the development of optimized and efficient cell-based therapy protocols.

α-Synuclein membrane association is regulated by the Rab3a recycling machinery and presynaptic activity.

α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity.

Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.

Effect of Ser-129 phosphorylation on interaction of α-synuclein with synaptic and cellular membranes.

In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.

Adult bone marrow: which stem cells for cellular therapy protocols in neurodegenerative disorders?

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crests (NCSCs) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSCs). In this paper, we will review all information available concerning NCSC from adult tissues and their possible use in regenerative medicine. Moreover, as multiple recent studies showed the beneficial effect of bone marrow stromal cells in neurodegenerative diseases, we will discuss which stem cells isolated from adult bone marrow should be more suitable for cell replacement therapy.

Correction: In Vivo Tumorigenesis Was Observed after Injection of In Vitro Expanded Neural Crest Stem Cells Isolated from Adult Bone Marrow.

[This corrects the article DOI: 10.1371/journal.pone.0046425.].

Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington's disease.

Neural and mesenchymal stem cells have been proposed as alternative sources of cells for transplantation into the brain in neurodegenerative disorders. However, the endogenous factors controlling their engraftment within the injured parenchyma remain ill-defined. Here, we demonstrate significant engraftment of undifferentiated exogenous mesenchymal or neural stem cells throughout the lesioned area in a rat model for Huntington's disease, as late as 8 weeks post-transplantation. We show that stem cell factor (SCF), strongly up-regulated within host cells in the damaged striatum, is able to activate the SCF receptor c-kit and its signaling pathway and to promote the migration and proliferation of mesenchymal and neural stem cells in vitro. Furthermore, c-kit receptor blockade alters neural stem cell distribution within the lesioned striatum. Host SCF expression is observed in atypical cells expressing glial fibrillary acidic protein and doublecortin in the lesioned striatum and in migrating doublecortin-positive progenitors. Taken together, these data demonstrate that SCF produced in situ in the lesioned striatum is an important factor in promoting the engraftment of stem cells within the lesioned brain.

Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

BACKGROUND: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood. RESULTS: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol. CONCLUSION: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells.

BACKGROUND: Bone marrow stromal cells and radial glia are two stem cell types with neural phenotypic plasticity. Bone marrow mesenchymal stem cells can differentiate into osteocytes, chondrocytes and adipocytes, but can also differentiate into non-mesenchymal cell, i.e. neural cells in appropriate in vivo and in vitro experimental conditions. Likewise, radial glial cells are the progenitors of many neurons in the developing cortex, but can also generate astrocytes. Both cell types express nestin, an intermediate filament protein which is the hallmark of neural precursors. RESULTS: In this study, we demonstrate that thrombin, a multifunctional serine protease, stimulates the growth of radial glial cells (RG) and mesenchymal stem cells (MSCs) in a dose-dependent manner. In RG, the mitogenic effect of thrombin is correlated with increased expression of nestin but in MSCs, this mitogenic effect is associated with nestin down-regulation. Both cell types express the PAR-1 type receptor for Thrombin and the effect of Thrombin on both cell types can be mimicked by its analogue TRAP-6 activating specifically this receptor subtype or by serum which contains various amount of thrombin. Moreover, we also demonstrate that serum deprivation-induced expression of nestin in MSCs is inhibited by high cell density (> 50,000 cells/cm2). CONCLUSION: This work shows that thrombin stimulates the growth of both RG and MSCs and that nestin expression by MSCs and RG is regulated in opposite manner by thrombin in vitro. Thrombin effect is thus associated in both cell types with a proliferating, undifferentiated state but in RG this involves the induction of nestin expression, a marker of immaturity for neural progenitors. In MSCs however, nestin expression, as it corresponds to a progression from the mesenchymal "undifferentiated", proliferating phenotype toward acquisition of a neural fate, is inhibited by the mitogenic signal.

Beta-carbolines induce apoptosis in cultured cerebellar granule neurons via the mitochondrial pathway.

N-butyl-beta-carboline-3-carboxylate (betaCCB) is, together with 2-methyl-norharmanium and 2,9-dimethylnorharmanium ions, an endogenously occurring beta-carboline. Due to their structural similarities with the synthetic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), harman and norharman compounds have been proposed to be involved in the pathogenesis of Parkinson's disease. While also structurally related, betaCCB has received much less interest in that respect although we had previously demonstrated that it induces the apoptotic cell death of cultured cerebellar granule neurons (CGNs). Herein, we have investigated the molecular events leading to CGN apoptosis upon betaCCB treatment. We first demonstrated that betaCCB-induced apoptosis occurs in neurons only, most likely as a consequence of a specific neuronal uptake as shown using binding/uptake experiments. Then we observed that, in betaCCB-treated CGNs, caspases 9, 3 and 8 were successively activated, suggesting an activation of the mitochondrial pathway. Consistently, betaCCB also induced the release from the mitochondrial intermembrane space of two pro-apoptotic factors, i.e. cytochrome c and apotptosis inducing factor (AIF). Interestingly, no mitochondrial membrane depolarisation was associated with this release, suggesting a mitochondrial permeability transition pore-independent mechanism. The absence of any neuroprotective effect provided by two mPTP inhibitors, i.e. cyclosporine A and bongkrekic acid, further supported this hypothesis. Together, these results show that betaCCB is specifically taken up by neuronal cells where it triggers a specific permeabilization of the outer mitochondrial membrane and a subsequent apoptotic cell death.

Peripheral benzodiazepine receptor (PBR) ligand cytotoxicity unrelated to PBR expression.

Some synthetic ligands of the peripheral-type benzodiazepine receptor (PBR), an 18 kDa protein of the outer mitochondrial membrane, are cytotoxic for several tumor cell lines and arise as promising chemotherapeutic candidates. However, conflicting results were reported regarding the actual effect of these drugs on cellular survival ranging from protection to toxicity. Moreover, the concentrations needed to observe such a toxicity were usually high, far above the affinity range for their receptor, hence questioning its specificity. In the present study, we have shown that micromolar concentrations of FGIN-1-27 and Ro 5-4864, two chemically unrelated PBR ligands are toxic for both PBR-expressing SK-N-BE neuroblastoma cells and PBR-deficient Jurkat lymphoma cells. We have thereby demonstrated that the cytotoxicity of these drugs is unrelated to their PBR-binding activity. Moreover, Ro 5-4864-induced cell death differed strikingly between both cell types, being apoptotic in Jurkat cells while necrotic in SK-N-BE cells. Again, this did not seem to be related to PBR expression since Ro 5-4864-induced death of PBR-transfected Jurkat cells remained apoptotic. Taken together, our results show that PBR is unlikely to mediate all the effects of these PBR ligands. They however confirm that some of these ligands are very effective cytotoxic drugs towards various cancer cells, even for reputed chemoresistant tumors such as neuroblastoma, and, surprisingly, also for PBR-lacking tumor cells.

Astrocytic and neuronal fate of mesenchymal stem cells expressing nestin.

Classically, bone marrow mesenchymal stem cells (MSC) differentiate in vivo or in vitro into osteocytes, chondrocytes, fibroblasts and adipocytes. Recently, it was reported by several groups that MSC can also adopt a neural fate in appropriate in vivo or in vitro experimental conditions. However, it is unclear if those cells are really able to differentiate into functional neural cells and in particular into functional neurons. Some observations suggest that a cell fusion process underlies the neural fate adoption by MSC in vivo and first attempts to reproduce in vitro this neural fate decision in MSC cultures were unsuccessful. More recently, however, in several laboratories including ours, differentiation of MSC cultivated from adult rat bone marrow into astrocytes and neuron-like cells was demonstrated. More precisely, we stressed the importance of the expression by MSC of nestin, an intermediate filament protein associated with immaturity in the nervous system, as a pre-requisite to adopting an astrocytic or a neuronal fate in a co-culture paradigm. Using this approach, we have also demonstrated that the MSC-derived neuron-like cells exhibit several electrophysiological key properties classically devoted to neurons, including firing of action potentials. In this review, we will discuss the neurogenic potential of MSC, the factor(s) required for such plasticity, the molecular mechanism(s) underlying this neural plasticity, the importance of the environment of MSC to adopt this neural fate and the therapeutic potential of these observations.

Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4.

BACKGROUND: Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies. RESULTS: In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings) and we report here that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1) this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2) anti-BMP4 antibodies inhibit the nestin-positive mesenchymal stem cells conditioned medium inducing effect on astrogliogenesis. CONCLUSIONS: When thinking carefully about mesenchymal stem cells as candidates for cellular therapy in neurological diseases, their effects on resident neural cell fate have to be considered.

Bone marrow stromal stem cells transplantation in mice with acute spinal cord injury.

Spinal cord injured experimental animals are widely used for studying pathophysiological processes after central nervous system acute traumatic lesion and elaborating therapeutic solutions, some of them based on stem cell transplantation. Here, we describe a protocol of spinal cord contusion in C57BL/6J mice, directly followed by bone marrow stromal stem cells transplantation. This model allows for the characterization of neuroprotective and neurorestorative abilities of these stem cells in a context of spinal cord trauma.

Concise review: adult mesenchymal stem cells, adult neural crest stem cells, and therapy of neurological pathologies: a state of play.

Adult stem cells are endowed with in vitro multilineage differentiation abilities and constitute an attractive autologous source of material for cell therapy in neurological disorders. With regard to lately published results, the ability of adult mesenchymal stem cells (MSCs) and neural crest stem cells (NCSCs) to integrate and differentiate into neurons once inside the central nervous system (CNS) is currently questioned. For this review, we collected exhaustive data on MSC/NCSC neural differentiation in vitro. We then analyzed preclinical cell therapy experiments in different models for neurological diseases and concluded that neural differentiation is probably not the leading property of adult MSCs and NCSCs concerning neurological pathology management. A fine analysis of the molecules that are secreted by MSCs and NCSCs would definitely be of significant interest regarding their important contribution to the clinical and pathological recovery after CNS lesions.