Neuroprotective effects of human mesenchymal stem cells on neural cultures exposed to 6-hydroxydopamine: implications for reparative therapy in Parkinson's disease.

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.

TDP-43 is recruited to stress granules in conditions of oxidative insult.

Transactive response DNA-binding protein 43 (TDP-43) forms abnormal ubiquitinated and phosphorylated inclusions in brain tissues from patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. TDP-43 is a DNA/RNA-binding protein involved in RNA processing, such as transcription, pre-mRNA splicing, mRNA stabilization and transport to dendrites. We found that in response to oxidative stress and to environmental insults of different types TDP-43 is capable to assemble into stress granules (SGs), ribonucleoprotein complexes where protein synthesis is temporarily arrested. We demonstrated that a specific aminoacidic interval (216-315) in the C-terminal region and the RNA-recognition motif 1 domain are both implicated in TDP-43 participation in SGs as their deletion prevented the recruitment of TDP-43 into SGs. Our data show that TDP-43 is a specific component of SGs and not of processing bodies, although we proved that TDP-43 is not necessary for SG formation, and its gene silencing does not impair cell survival during stress. The analysis of spinal cord tissue from ALS patients showed that SG markers are not entrapped in TDP-43 pathological inclusions. Although SGs were not evident in ALS brains, we speculate that an altered control of mRNA translation in stressful conditions may trigger motor neuron degeneration at early stages of the disease.

Liquid chromatographic assay for riluzole in mouse plasma and central nervous system tissues.

An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100 ng ml(-1) or g(-1) using 0.1 ml of plasma and about 100mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).

Multiple neurogenic and neurorescue effects of human mesenchymal stem cell after transplantation in an experimental model of Parkinson's disease.

Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.

Transplantation of undifferentiated human mesenchymal stem cells protects against 6-hydroxydopamine neurotoxicity in the rat.

Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.

Erythropoietin does not preserve motor neurons in a mouse model of familial ALS.

Recombinant human EPO (r-Hu-EPO) protects cultured motor neurons from kainate- and serum deprivation-induced toxicity and readily enters the CNS after systemic injection. We examined the effect of rHuEPO in transgenic mice expressing the human Cu/Zn dependent-superoxide dismutase with G93A mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis. rHuEPO (4 unit/g BW s.c. three times/week), increased the haematocrit and induced a slight delay in impairment of motor function as measured by the rotating bar test. However, it did not prolong life span or reduce motor neuron loss in lumbar spinal cord. The effect on motor function may be due to the improvement of skeletal muscle oxygenation induced by chronic EPO administration.

A role for the ELAV RNA-binding proteins in neural stem cells: stabilization of Msi1 mRNA.

Post-transcriptional regulation exerted by neural-specific RNA-binding proteins plays a pivotal role in the development and maintenance of the nervous system. Neural ELAV proteins are key inducers of neuronal differentiation through the stabilization and/or translational enhancement of target transcripts bearing the AU-rich elements (AREs), whereas Musashi-1 maintains the stem cell proliferation state by acting as a translational repressor. Since the gene encoding Musashi-1 (Msi1) contains a conserved ARE in its 3' untranslated region, we focused on the possibility of a mechanistic relationship between ELAV proteins and Musashi-1 in cell fate commitment. Colocalization of neural ELAV proteins with Musashi-1 clearly shows that ELAV proteins are expressed at early stages of neural commitment, whereas interaction studies demonstrate that neural ELAV proteins exert an ARE-dependent binding activity on the Msi1 mRNA. This binding activity has functional effects, since the ELAV protein family member HuD is able to stabilize the Msi1 ARE-containing mRNA in a sequence-dependent way in a deadenylation/degradation assay. Furthermore activation of the neural ELAV proteins by phorbol esters in human SH-SY5Y cells is associated with an increase of Musashi-1 protein content in the cytoskeleton. We propose that ELAV RNA-binding proteins exert an important post-transcriptional control on Musashi-1 expression in the transition from proliferation to neural differentiation of stem/progenitor cells.

Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder involving the selective degeneration of motor neurons. In a small proportion of patients, ALS is caused by mutations in copper/zinc superoxide dismutase (SOD1), and mice overexpressing SOD1(G93A) mutant develop a syndrome that closely resembles the human disease. Excitotoxicity mediated by glutamate AMPA receptors has been suggested to be implicated in the selective susceptibility of motor neurons occurring in ALS. In SOD1(G93A) mice, we found that levels of GluR2 AMPA subunit, which plays a pivotal role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal motor neurons before symptom onset in concomitance with a modest increase of GluR3 expression, a calcium-permeable AMPA subunit. This effect can result in a higher number of calcium-permeable AMPA receptors on motor neurons of SOD1(G93A) mice, predisposing these cells to be injured by AMPA-mediated glutamate firing. In support of this, we showed that treatment with a new noncompetitive AMPA antagonist, ZK 187638, partially protected motor neurons, improved motor function, and prolonged the survival of SOD1(G93A) mice.

A single high dose of cocaine induces behavioural sensitization and modifies mRNA encoding GluR1 and GAP-43 in rats.

Neuroadaptive changes underlying repeated exposure to cocaine-induced behavioural sensitization have been related to modification in the pattern of synaptic connectivity and excitatory transmission. Remarkably, even a single exposure to abused drugs is sufficient to elicit lasting behavioural sensitization. The present study investigated whether in Sprague-Dawley rats a single, behavioural sensitizing dose of cocaine is sufficient to induce changes in the mRNA levels of growth-associated protein 43 (GAP-43), an important protein in mediating experience-dependent plasticity and synaptic reorganization, and of glutamate receptor 1 (GluR1), a subunit of AMPA glutamate receptors, a protein that is up-regulated with repeated cocaine. Single exposure to 20, but not 10 mg/kg cocaine induced locomotor sensitization to a second injection of 10 mg/kg cocaine, observed at 24 h, 48 h and 7 days. Single dose of 20 but not 10 mg/kg cocaine 48 h before scheduled death significantly enhanced GluR1 and GAP-43 mRNA expression in the nucleus accumbens (NAc), both shell and core subregions, and ventral tegmental area (VTA). No changes were found in the levels of mRNA for GluR1 and GAP-43 in the frontal cortex, caudate putamen, dentate gyrus of hippocampus and basolateral nucleus of the amygdala after the single dose of 20 mg/kg cocaine. These results further strengthen the involvement of NAc and VTA in the behavioural sensitization and suggest a role of GAP-43 in the synaptic reorganization associated to drug abuse.