Neuroimmune pathways involvement in neurodegeneration of R6/2 mouse model of Huntington's disease.

Mechanisms of tissue damage in Huntington's disease (HD) involve excitotoxicity, mitochondrial damage, and neuroinflammation, including microglia activation. CD47 is a membrane protein that interacts with the inhibitory immunoreceptor SIRPα. Engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis, promoting a "don't-eat-me" signal. These proteins are involved in the immune response and are downmodulated in inflammatory diseases. The involvement of inflammation and of the inflammasome in HD has already been described. In this study, we focused on other factors that can be involved in the unregulated inflammatory response that accelerates and exacerbate the neurodegenerative process in HD. Our results show that CD47 on striatal neurons decreased in HD mice, while it increased in wild type mice with age. SIRPα, on the other hand, was present in neurons in the wild type and increases in the R6/2 mice at all stages. Recruitment of SIRPα and binding to CD47 promotes the activation through phosphorylating events of non-receptor protein tyrosine phosphatase SHP-1 and SHP-2 in neurons and microglia. SHP phosphatases are able to curb the activity of NLRP3 inflammasome thereby reducing the detrimental effect of neuroinflammation. Such activity is mediated by the inhibition (dephosphorylation) of the proteins signal transducer and activator of transcription (STAT). We found that activated SHP-1 was present in microglia and neurons of WT mice at 5 and 13 weeks, increasing with time; while in R6/2 it was not localized in neurons but only in microglia, where it decreases with time. Consequently, STAT1 was overexpressed in neurons of R6/2 mice, as an effect of lack of modulation by SHP-1. Thus, our results shed light on the pathophysiology of neuronal damage, on one hand, paving the way toward a modulation of signal transducer proteins by specific inhibitors to achieve neuroprotection in HD, on the other.

Morpho-Functional Changes of Nigral Dopamine Neurons in an α-Synuclein Model of Parkinson's Disease.

BACKGROUND: The accumulation of α-synuclein (α-syn) fibrils in intraneuronal inclusions called Lewy bodies and Lewy neurites is a pathological signature of Parkinson's disease (PD). Although several aspects linked to α-syn-dependent pathology (concerning its spreading, aggregation, and activation of inflammatory and neurodegenerative processes) have been under intense investigation, less attention has been devoted to the real impact of α-syn overexpression on structural and functional properties of substantia nigra pars compacta (SNpc) dopamine (DA) neurons, particularly at tardive stages of α-syn buildup, despite this has obvious relevance to comprehending mechanisms beyond PD progression. OBJECTIVES: We aimed to determine the consequences of a prolonged α-syn overexpression on somatodendritic morphology and functions of SNpc DA neurons. METHODS: We performed immunohistochemistry, stereological DA cell counts, analyses of dendritic arborization, ex vivo patch-clamp recordings, and in vivo DA microdialysis measurements in a 12- to 13-month-old transgenic rat model overexpressing the full-length human α-syn (Snca+/+ ) and age-matched wild-type rats. RESULTS: Aged Snca+/+ rats have mild loss of SNpc DA neurons and decreased basal DA levels in the SN. Residual nigral DA neurons display smaller soma and compromised dendritic arborization and, in parallel, increased firing activity, switch in firing mode, and hyperexcitability associated with hypofunction of fast activating/inactivating voltage-gated K+ channels and Ca2+ - and voltage-activated large conductance K+ channels. These intrinsic currents underlie the repolarization/afterhyperpolarization phase of action potentials, thus affecting neuronal excitability. CONCLUSIONS: Besides clarifying α-syn-induced pathological landmarks, such evidence reveals compensatory functional mechanisms that nigral DA neurons could adopt during PD progression to counteract neurodegeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Neuropathology of the Basal Ganglia in SNCA Transgenic Rat Model of Parkinson's Disease: Involvement of Parvalbuminergic Interneurons and Glial-Derived Neurotropic Factor.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the accumulation of alpha-synuclein, encoded by the SNCA gene. The main neuropathological hallmark of PD is the degeneration of dopaminergic neurons leading to striatal dopamine depletion. Trophic support by a neurotrophin called glial-derived neurotrophic factor (GDNF) is also lacking in PD. We performed immunohistochemical studies to investigate neuropathological changes in the basal ganglia of a rat transgenic model of PD overexpressing alfa-synuclein. We observed that neuronal loss also occurs in the dorsolateral part of the striatum in the advanced stages of the disease. Moreover, along with the degeneration of the medium spiny projection neurons, we found a dramatic loss of parvalbumin interneurons. A marked decrease in GDNF, which is produced by parvalbumin interneurons, was observed in the striatum and in the substantia nigra of these animals. This confirmed the involvement of the striatum in the pathophysiology of PD and the importance of GDNF in maintaining the health of the substantia nigra.

Emerging Role of NLRP3 Inflammasome/Pyroptosis in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease characterized by several symptoms encompassing movement, cognition, and behavior. The mutation of the IT15 gene encoding for the huntingtin protein is the cause of HD. Mutant huntingtin interacts with and impairs the function of several transcription factors involved in neuronal survival. Although many mechanisms determining neuronal death have been described over the years, the significant role of inflammation has gained momentum in the last decade. Drugs targeting the elements that orchestrate inflammation have been considered powerful tools to treat HD. In this review, we will describe the data supporting inflammasome and NLRP3 as a target of therapeutics to fight HD, deepening the possible mechanisms of action underlying these effects.

A2A Receptor Dysregulation in Dystonia DYT1 Knock-Out Mice.

We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/- knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3-0.4 µm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/-, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/-, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/-, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/-, opposite changes of A2A receptors' expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia.

Modulation of Inflammasome and Pyroptosis by Olaparib, a PARP-1 Inhibitor, in the R6/2 Mouse Model of Huntington's Disease.

Pyroptosis is a type of cell death that is caspase-1 (Casp-1) dependent, which leads to a rapid cell lysis, and it is linked to the inflammasome. We recently showed that pyroptotic cell death occurs in Huntington's disease (HD). Moreover, we previously described the beneficial effects of a PARP-1 inhibitor in HD. In this study, we investigated the neuroprotective effect of Olaparib, an inhibitor of PARP-1, in the mouse model of Huntington's disease. R6/2 mice were administered Olaparib or vehicle from pre-symptomatic to late stages. Behavioral studies were performed to investigate clinical effects of the compound. Immunohistochemical and Western blotting studies were performed to evaluate neuroprotection and the impact of the compound on the pathway of neuronal death in the HD mice. Our results indicate that Olaparib administration starting from the pre-symptomatic stage of the neurodegenerative disease increased survival, ameliorated the neurological deficits, and improved clinical outcomes in neurobehavioral tests mainly by modulating the inflammasome activation. These results suggest that Olaparib, a commercially available drug already in use as an anti-neoplastic compound, exerts a neuroprotective effect and could be a useful pharmaceutical agent for Huntington's disease therapy.

Pyroptotic cell death in the R6/2 mouse model of Huntington's disease: new insight on the inflammasome.

Mechanisms of tissue damage in Huntington's disease involve excitotoxicity, mitochondrial damage, and neuroinflammation, including microglia activation. In the present study, we investigate the role of pyroptosis process in the striatal neurons of the R6/2 mouse model of Huntington's disease. Transgenic mice were sacrificed at 4 and 13 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that NLRP3 and Caspase-1 were intensely expressed in 13-week-old R6/2 mice. Moreover, NLRP3 expression levels were higher in striatal spiny projection neurons and in parvalbumin interneurons, which are prone to degenerate in HD.

Dystonia: Sparse Synapses for D2 Receptors in Striatum of a DYT1 Knock-out Mouse Model.

Dystonia pathophysiology has been partly linked to downregulation and dysfunction ofdopamine D2 receptors in striatum. We aimed to investigate the possible morpho-structuralcorrelates of D2 receptor downregulation in the striatum of a DYT1 Tor1a mouse model. Adultcontrol Tor1a+/+ and mutant Tor1a+/- mice were used. The brains were perfused and free-floatingsections of basal ganglia were incubated with polyclonal anti-D2 antibody, followed by secondaryimmune-fluorescent antibody. Confocal microscopy was used to detect immune-fluorescent signals.The same primary antibody was used to evaluate D2 receptor expression by western blot. The D2receptor immune-fluorescence appeared circumscribed in small disks (0.3-0.5 µm diameter), likelyrepresenting D2 synapse aggregates, densely distributed in the striatum of Tor1a+/+ mice. In theTor1a+/- mice the D2 aggregates were significantly smaller (µm2 2.4 ± SE 0.16, compared to µm2 6.73± SE 3.41 in Tor1a+/+) and sparse, with ~30% less number per microscopic field, value correspondentto the amount of reduced D2 expression in western blotting analysis. In DYT1 mutant mice thesparse and small D2 synapses in the striatum may be insufficient to "gate" the amount ofpresynaptic dopamine release diffusing in peri-synaptic space, and this consequently may result ina timing and spatially larger nonselective sphere of influence of dopamine action.

Neuroprotective Effects of Doxycycline in the R6/2 Mouse Model of Huntington's Disease.

Mechanisms of tissue damage in Huntington's disease involve excitotoxicity, mitochondrial damage, and inflammation, including microglia activation. Immunomodulatory and anti-protein aggregation properties of tetracyclines were demonstrated in several disease models. In the present study, the neuroprotective and anti-inflammatory effects of the tetracycline doxycycline were investigated in the mouse model of HD disease R6/2. Transgenic mice were daily treated with doxycycline 20 mg/kg, starting from 4 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that doxycycline-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the saline-treated ones. Primary outcome measures such as striatal atrophy, neuronal intranuclear inclusions, and the negative modulation of microglial reaction revealed a neuroprotective effect of the compound. Doxycycline provided a significantly increase of activated CREB and BDNF in the striatal neurons, along with a down modulation of neuroinflammation, which, combined, might explain the beneficial effects observed in this model. Our findings show that doxycycline treatment could be considered as a valid therapeutic approach for HD.

Modulation of Phospho-CREB by Systemically Administered Recombinant BDNF in the Hippocampus of the R6/2 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease due to an expansion of a trinucleotide repeats in IT15 gene encoding for the protein huntingtin. Motor dysfunction, cognitive decline, and psychiatric disorder are typical clinical signs of HD. In HD, mutated huntingtin causes a major loss of brain derived neurotrophic factor (BDNF), causing striatal atrophy. Moreover, a key involvement of BDNF was observed in the synaptic plasticity that controls the acquisition and/or consolidation of certain forms of memory. We studied changes in hippocampal BDNF and in CREB in the R6/2 mouse model of HD. Moreover, we investigated if the beneficial effects of systemically administered recombinant BDNF observed in the striatum and cortex had an effect also on the hippocampus. Osmotic minipumps that chronically released recombinant BDNF or saline solution from 4 weeks of age until euthanasia were implanted into R6/2 and wild type mice. Our data show that BDNF is severely decreased in the hippocampus of R6/2 mice, while BDNF treatment restored its physiological levels. Moreover, the chronic administration of recombinant BDNF promoted the increment of phosphorylated CREB protein. Our study demonstrates the involvement of hippocampus in the pathology of R6/2 model of HD and correlates the beneficial effects of BDNF administration with increased hippocampal levels of BDNF and pCREB.

Conditioned medium from amniotic cells protects striatal degeneration and ameliorates motor deficits in the R6/2 mouse model of Huntington's disease.

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro-inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM-hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune-based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM-hAMSC display less severe signs of neurological dysfunction than saline-treated ones. CM-hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM-hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM-hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM-hAMSC could act by modulating inflammatory cells, and more specifically microglia.

Role of Phosphodiesterases in Huntington's Disease.

Huntington's disease (HD) is an autosomal-dominant rare inherited neurodegenerative disease characterized by a wide variety of symptoms encompassing movement, cognition and behaviour. The cause of the disease is a genetic mutation in the huntingtin protein. The mutation leads to an unstable CAG expansion, translated into a polyglutamine domain within the disease protein. Indeed, huntingtin has a CAG/polyglutamine expansion in the range of 6-39 units in normal individuals, whereas it reaches 39-180 units in HD patients. Mutant huntingtin interacts with and impairs the function of a number of transcription factors. Indeed, the expression and function of cAMP response element-binding protein (CREB) and the brain-derived neurotrophic factor (BDNF) are severely affected in HD. Drugs targeting CREB loss of function and BDNF decrease have been considered as powerful tools to treat HD. Recently, cyclic nucleotide phosphodiesterase (PDE) inhibitors have been shown to reduce striatal and cortical degeneration in transgenic mouse model of HD. The neuroprotective effect is due to the competency of PDE4, 5 and 10 inhibitors to positively modulate CREB and BDNF protein levels, both in striatum and cortex in HD models. In this chapter, we will summarize the data supporting the use of PDE inhibitors as a therapeutic approach to fight HD, deepening the possible mechanisms of action underlying these effects.

Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington's Disease.

Poly (ADP-ribose) polymerases (PARPs) are enzymes that catalyze ADP-ribose units transfer from NAD to their substrate proteins. It has been observed that PARP-1 is able to increase both post-ischemic and excitotoxic neuronal death. In fact, we have previously shown that, INO-1001, a PARP-1 inhibitor, displays a neuroprotective effect in the R6/2 model of Huntington's disease (HD). In this study, we investigated the effects of PARP-1-inhibition on modulation of phosphorylated c-AMP response element binding protein (pCREB) and CREB-binding protein (CBP) localization in the different striatal neuronal subsets. Moreover, we studied the neurodegeneration of those interneurons that are particularly vulnerable to HD such as parvalbuminergic and calretininergic, and of other subclasses of interneurons that are known to be resistant, such as cholinergic and somatostatinergic interneurons. Transgenic mice were treated with INO-1001 (10 mg/Kg daily) starting from 4 weeks of age. Double-label immunofluorescence was performed to value the distribution of CBP in ubiquitinated Neuronal intranuclear inclusions (NIIs) in the striatum. INO-1001-treated and saline-treated brain sections were incubated with: goat anti-choline acetyl transferase; goat anti-nitric oxide synthase; mouse anti-parvalbumin and mouse anti-calretinin. Morphometric evaluation and cell counts were performed. Our study showed that the PARP inhibitor has a positive effect in sparing parvalbumin and calretinin-containing interneurons of the striatum, where CREB was upregulated. Moreover, INO-1001 promoted CBP localization into the nuclei of the R6/2 mouse. The sum of our data corroborates the previous observations indicating PARP inhibition as a possible therapeutic tool to fight HD.

PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington's Disease.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington's disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.

PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment.

BACKGROUND: Cancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM). METHODS: MTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFRα/ß and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors. RESULTS: In this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFRα and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFRα significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized. CONCLUSIONS: We have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.

Cord blood CD34+ cells expanded on Wharton's jelly multipotent mesenchymal stromal cells improve the hematopoietic engraftment in NOD/SCID mice.

OBJECTIVE: This study aims to investigate the capability of Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSC) to support the in vitro expansion of hematopoietic stem/progenitor cells (HSPC) derived from cord blood (CB) in the absence of exogenous cytokines, and the effect on engraftment of the expanded cells in a mouse model. METHODS: CB-CD34+ cells were seeded on WJ-MSC layer and cultured in HP01 serum-free medium. Day-7 and day-13 expanded cells were transplanted in NOD/SCID mice. After 8 wk, engraftment was evaluated in mouse bone marrow as percentage of human CD45+ cells. RESULTS: CD34+ population was expanded without increasing the differentiation rate. Co-culture increased the expansion of the CD34+ cells by 2.0 and 7.3 times after 7 and 13 d, respectively, and maintained the CD34+ cells up to day 20. In particular, earlier CD34+/CD90+ and CD34+/CD33- subtypes were increased. An advantage of the day-7 co-cultured HSPC in respect of HSPC at day 0 in the engraftment of NOD/SCID mice was obtained both as percentage of mice engrafted (100% vs. 75%) and as percentage of chimerism. CONCLUSIONS: Although the increase in hematopoietic progenitors is not dramatic as in the presence of added cytokines, this study demonstrates the effectiveness of the WJ-MSC not only to preserve the CD34+ population but also to improve the repopulating efficacy of the amplified HSPC, also in the absence of added cytokines and growth factors.

Tumor initiating cells and chemoresistance: which is the best strategy to target colon cancer stem cells?

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called "cancer stem cells" (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.

The purpose of this study was to investigate the Wharton's jelly mesenchymal stem cells differentiation ability toward neuronal fate. Human Wharton's jelly mesenchymal stem cells (hWJMSC) have been isolated from human umbilical cord of full-term births and characterized by flow cytometry analysis for their stem mesenchymal properties through specific surface markers expression (CD73, CD90, and CD105). hWJMSC mesodermal lineage differentiation ability and karyotype analysis were assessed. The trans-differentiation of hWJMSC into neural lineage was investigated in presence of forskolin, an agent known to increase the intracellular levels of cAMP. A molecular profile of differentiated hWJMSC was performed by microarray technology which revealed 1,532 statistically significant modulated genes respect to control cells. Most of these genes are mainly involved in functional neuronal signaling pathways and part of them are specifically required for the neuronal dopaminergic induction. The acquisition of the dopaminergic phenotype was evaluated via immunocytochemistry and Western blot analysis revealed the significant induction of Nurr1, NeuroD1, and TH proteins expression in forskolin-induced hWJMSC. Moreover, the treatment with forskolin promoted, in hWJMSC, a strong upregulation of the neurotrophin Trk receptors related to the high release of brain-derived neurotrophic factor. Taken together these findings show that hWJMSC may be represent an optimal therapeutic strategy for neurological diseases.

Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation.

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood-brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.