Biogenic synthesis of dopamine/carboxymethyl cellulose/TiO2 nanoparticles using Psidium guajava leaf extract with enhanced antimicrobial and anticancer activities.

The green synthesis of metal oxide nanoparticles (NPs) has garnered considerable attention from researchers due to its utilization of eco-friendly solvents during synthesis and cost-effective approaches. This study focuses on the synthesis of titanium oxide (TiO2) and dopamine (DA) carboxymethyl cellulose (CMC)-doped TiO2 (DA/CMC/TiO2) NP using Psidium guajava leaf extract, while also investigating the structural, optical, and morphological and biocidal potential of the prepared NPs. Significantly larger zones of inhibition were observed for DA/CMC/TiO2 NPs compared to TiO2 against various pathogens. Moreover, the MTT assay was carried out to evaluate the anticancer activity of the prepared samples against MG-63 cells, and the results revealed that DA/CMC/TiO2 NPs exhibited significantly higher level of anticancer activity compared to TiO2. The experimental results demonstrated that DA/CMC/TiO2 NPs exhibited enhanced anticancer activity in a dose-dependent manner when compared to TiO2 NPs.

Influence of 6-Hydroxydopamine Toxicity on α-Synuclein Phosphorylation, Resting Vesicle Expression, and Vesicular Dopamine Release.

Post mortem studies on familial and sporadic Parkinson's disease patient striatal tissue have shown that nearly 90% of α-synuclein deposited in Lewy-bodies is phosphorylated at serine-129 (pSyn-129) as opposed to only 4% in normal human brain. We aimed to find the influence of endogenous neurotoxin 6-hydroxydopamine (6-OHDA) on α-synuclein phosphorylation, resting vesicles, and vesicular dopamine release. The relative distribution of pSyn-129+ cells in apoptotic and non-apoptotic populations at different 6-OHDA concentrations was assessed along with changes in oxidant-antioxidant system, mitochondrial membrane-potential, and intracellular-Ca2+ . Exposing SH-SY5Y cells to different concentrations of 6-OHDA for 48 h showed cell-death and apoptosis. Immunocytochemical analysis indicated an increase in pSyn-129 with increasing 6-OHDA concentration, and ELISA-estimation showed a significant increase in the pSyn-129 to α-synuclein ratio. FACS analysis also showed a significant increase in pSyn-129; and at sub-lethal 6-OHDA concentrations, pSyn-129+ cells were primarily distributed in the non-apoptotic population, suggesting that phosphorylation of α-synuclein precedes apoptosis. At higher 6-OHDA concentrations, the pSyn-129+ cell count significantly increased in the apoptotic population and decreased in the non-apoptotic population. Cytosolic co-localization of α-synuclein and ubiquitin was noticed at higher doses of 6-OHDA. FACS analysis showed decrease in vesicular monoamine transporter-2 (VMAT2) expression in 6-OHDA-treated cells, confirmed by reduction in functional dopamine-release on KCl and ATP stimulation. Significant decrease in VMAT2 expression and vesicular dopamine-release were observed with the lower 6-OHDA concentration, together with mild occurrence of apoptosis and significant increase in phosphorylated α-synuclein. This suggests that at sub-lethal 6-OHDA concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-synuclein. J. Cell. Biochem. 117: 2719-2736, 2016. © 2016 Wiley Periodicals, Inc.

By Changing Dimensionality, Sequential Culturing of Midbrain Cells, rather than Two-Dimensional Culture, Generates a Neuron-Glia Ratio Closer to in vivo Adult Midbrain.

The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-L-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker ß-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of ß-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres.

Directed differentiation of human embryonic stem cell-line HUES9 to dopaminergic neurons in a serum-free defined culture niche.

Although there are several reports on differentiation of human embryonic stem cells to dopaminergic neurons, notable heterogeneity exists in the reported yields of tyrosine hydroxylase (TH)-positive cells. For benchmarking performance and efficiency standards in future applications of hESC-derived dopaminergic neurons, there is thus a dire need of well-defined directed differentiation protocols. Pal et al. [Pal et al. 2009 Exp Biol Med (Maywood) 234:1230-3] demonstrated predisposition of HUES9 towards ectodermal lineage, but the directed differentiation of HUES9 to dopaminergic neurons has not yet been reported. Therefore, we report here a simple two-step protocol using suitable ECM and serum-free induction medium for generating dopaminergic cells from HUES9-derived embryoid bodies. Flow cytometry analysis of the neural progenitors obtained after the first step gave an enriched yield of cells immune-positive for nestin (99.6 ± 0.1%), musashi12 (98.1 ± 1.5%) and Sox2 (95.4 ± 2.6%). Most of these cells also expressed the proliferation marker Ki67 (83.8 ± 1.5%), whereas the presence of the undifferentiated stem cell marker Oct4 was negligible. In the second step, when these neural progenitors were exposed to midbrain cues sonic hedgehog and fibroblast growth factor 8 along with bFGF, the differentiated cells showed an upregulation of dopaminergic-related transcription factors Nurr1 and Engrailed1. Immunocytochemistry and flow cytometry analysis showed that these differentiated cells were positive for the mature neuronal marker Map2ab (96.2 ± 1.5%) and dopaminergic neuronal marker TH (71.9 ± 4.4%). Thus, the data demonstrate novel findings of the directed differentiation of HUES9 to dopaminergic neurons using well-defined serum-free nutrient supplements.

A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe syndrome.

Human brain development is a complex process where multiple cellular and developmental events are coordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS) is an X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1, which encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a 'disease-in-a-dish' model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development. This article has an associated First Person interview with the first author of the paper.

Influence of intranasal exposure of MPTP in multiple doses on liver functions and transition from non-motor to motor symptoms in a rat PD model.

Besides the effects on the striatum, the impairment of visceral organs including liver functions has been reported in Parkinson's disease (PD) patients. However, it is yet unclear if liver functions are affected in the early stage of the disease before the motor phase has appeared. The aim of our present study was thus to assess the effect of intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in different doses on striatum and liver functions. Deterioration of non-motor activities appeared on single exposure to MPTP along with rise in striatum oxidative stress and decline in antioxidant levels. Decreases in dopamine, noradrenaline, and GABA and increase in serotonin were detected in striatum. Motor coordination was impaired with a single dose of MPTP, and with repeated MPTP exposure, there was further significant impairment. Locomotor activity was affected from second exposure of MPTP, and the impairment increased with third MPTP exposure. Impairment of liver function through increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels was observed after first MPTP insult, and it worsened with second and third administrations. First administration of MPTP triggered systemic inflammation showing significant increase in inflammatory markers in the liver. Our data shows for the first time that an intranasal route of entry of MPTP affects liver from the non-motor phase of PD itself, occurring concomitantly with the reduction of striatal dopamine. It also suggests that a single dose is not enough to bring about progression of the disease from non-motor to locomotor deficiency, and a repeated dose is needed to establish the motor severity phase in the rat intranasal MPTP model.

Astrocyte-Like Cells Differentiated from Dental Pulp Stem Cells Protect Dopaminergic Neurons Against 6-Hydroxydopamine Toxicity.

Dental pulp stem cells (DPSCs) are promising for use in neurodegenerative-diseases because of their neural crest origin. While neuronal differentiation of DPSCs has been shown, their plasticity towards astrocyte-like cells remains to be studied. We aimed to examine differentiation potential of DPSCs to astrocytes and their consequent neuroprotective role towards dopaminergic (DA) neurons under 6-hydroxydopamine (6-OHDA) toxicity. Induction of DPSCs to astrocytes with differentiation factors showed definitive increase in astrocyte-specific markers glial fibrillary acidic protein (GFAP), and excitatory amino acid transporter 2 along with glial calcium-binding protein S100ß through FACS and immunofluorescence assays. RT-PCR and ELISA showed significant increase in BDNF and GDNF expression and secretion in astrocyte-differentiated DPSCs over naïve DPSCs. Neuroprotective role of these cells on DA neurons under 6-OHDA stress was evaluated by both contact and non-contact methods. FACS analysis of PKH26-stained SH-SY5Y homogenous cells in contact method and of TH immunopositive cells in primary midbrain culture in non-contact method both indicated higher survival of DA neurons in astrocyte-differentiated DPSCs over naïve DPSCs. Recovery of ß-tubulin III and TH immunopositive cells was reduced in the presence of TrkB inhibitor, suggesting a key neuroprotective role of BDNF secretion by DPSCs. When nitric oxide (NO) release was inhibited by L-NAME in primary midbrain culture, BDNF release in co-culture under 6-OHDA stress reduced further in naïve DPSCs than in astrocyte-differentiated DPSCs, suggesting that BDNF release in naïve DPSCs is primarily regulated by paracrine signaling while for differentiated DPSCs, it is equally through autocrine and paracrine signaling with NO being the mediator. In conclusion, we suggest that DPSCs exposed to glial commitment cues exhibit substantial differentiation towards astrocyte-like cells with better neuroprotective activity against 6-OHDA toxicity than naïve DPSCs.

Location and Number of Astrocytes Determine Dopaminergic Neuron Survival and Function Under 6-OHDA Stress Mediated Through Differential BDNF Release.

While astrocytes throughout the CNS share many common traits, they exhibit significant differences in function and number among brain regions. The aim of the present study is to assess the effect of region-specificity and number of astrocytes on the survival of dopaminergic neurons under stress, and to understand the possible mechanism by which these astrocytes extend neuroprotection to dopaminergic neurons. Purified astrocytes obtained from forebrain, midbrain, and hindbrain region were characterized through FACS and immunofluorescence. Co-culture experiments (using trans-wells) were then performed to measure the effect of region-specificities and numbers of astrocytes on primary midbrain culture under 6-OHDA stress. Cell survival augmented with an increase in astrocyte seeding number and total cell survival was comparable among the different region-specific astrocytes for all numbers. However, striking differences were observed in dopaminergic neuronal (TH) cell survival in the presence of midbrain astrocytes in comparison to forebrain and hindbrain astrocytes at all seeding numbers. At 75 µM 6-OHDA insult, while cell survival was comparable in purified astrocytes from the different brain regions, a distinct increase in BDNF secretion (significantly higher than its constitutive release) was noted for midbrain astrocytes compared to forebrain and hindbrain astrocytes. The TH immunopositive population decreased when TrkB inhibitor was added to the co-culture under 6-OHDA toxicity, suggesting that BDNF released by co-cultured astrocytes plays a key role in the survival of dopaminergic neurons. This BDNF release decreased in presence of NO inhibitor and increased in the presence of NO donor (DETA/NO). We conclude that the BDNF released from astrocytes under 6-OHDA toxicity is mediated through NO release through both autocrine and paracrine signaling, and this BDNF release is primarily responsible for the differential effect of region-specific astrocytes on TH neuron survival under these conditions.