Dopaminergic Neuronal Imaging in Genetic Parkinson's Disease: Insights into Pathogenesis

Objectives

To compare the dopaminergic neuronal imaging features of different subtypes of genetic Parkinson''s Disease.

Methods

A retrospective study of genetic Parkinson''s diseases cases in which DaTSCAN (123I-FP-CIT) had been performed. Specific non-displaceable binding was calculated for bilateral caudate and putamen for each case. The right:left asymmetry index and striatal asymmetry index was calculated.

Results

Scans were available from 37 cases of monogenetic Parkinson''s disease (7 glucocerebrosidase (GBA) mutations, 8 alpha-synuclein, 3 LRRK2, 7 PINK1, 12 Parkin). The asymmetry of radioligand uptake for Parkinson''s disease with GBA or LRRK2 mutations was greater than that for Parkinson''s disease with alpha synuclein, PINK1 or Parkin mutations.

Conclusions

The asymmetry of radioligand uptake in Parkinsons disease associated with GBA or LRRK2 mutations suggests that interactions with additional genetic or environmental factors may be associated with dopaminergic neuronal loss.     

ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2

Background

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson''s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro.

Methodology/Principal Findings

Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7.

Conclusions/Significance

Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson''s disease pathogenesis.     

Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity

Background

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation.

Methodology/Principal Findings

We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo.

Conclusions/Significance

Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.     

Metabolomic Profiling in LRRK2-Related Parkinson's Disease

Background

Mutations in LRRK2 gene represent the most common known genetic cause of Parkinson''s disease (PD).

Methodology/Principal Findings

We used metabolomic profiling to identify biomarkers that are associated with idiopathic and LRRK2 PD. We compared plasma metabolomic profiles of patients with PD due to the G2019S LRRK2 mutation, to asymptomatic family members of these patients either with or without G2019S LRRK2 mutations, and to patients with idiopathic PD, as well as non-related control subjects. We found that metabolomic profiles of both idiopathic PD and LRRK2 PD subjects were clearly separated from controls. LRRK2 PD patients had metabolomic profiles distinguishable from those with idiopathic PD, and the profiles could predict whether the PD was secondary to LRRK2 mutations or idiopathic. Metabolomic profiles of LRRK2 PD patients were well separated from their family members, but there was a slight overlap between family members with and without LRRK2 mutations. Both LRRK2 and idiopathic PD patients showed significantly reduced uric acid levels. We also found a significant decrease in levels of hypoxanthine and in the ratios of major metabolites of the purine pathway in plasma of PD patients.

Conclusions/Significance

These findings show that LRRK2 patients with the G2019S mutation have unique metabolomic profiles that distinguish them from patients with idiopathic PD. Furthermore, asymptomatic LRRK2 carriers can be separated from gene negative family members, which raises the possibility that metabolomic profiles could be useful in predicting which LRRK2 carriers will eventually develop PD. The results also suggest that there are aberrations in the purine pathway in PD which may occur upstream from uric acid.     

Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease

Background

Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson''s disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.

Methodology/Principal Findings

Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.

Conclusions/Significance

LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.     

Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions

Background

Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.

Methodology and Principal Findings

The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis.

Conclusions/Significance

These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages.     

A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells

Background

Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions.

Methods and Findings

This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal differentiation in stem cells could be found in the media supernatants of the co-cultures.

Conclusions

The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications.     

Dual Beneficial Effects of (-)-Epigallocatechin-3-Gallate on Levodopa Methylation and Hippocampal Neurodegeneration: In Vitro and In Vivo Studies

Background

A combination of levodopa (L-DOPA) and carbidopa is the most commonly-used treatment for symptom management in Parkinson''s disease. Studies have shown that concomitant use of a COMT inhibitor is highly beneficial in controlling the wearing-off phenomenon by improving L-DOPA bioavailability as well as brain entry. The present study sought to determine whether (-)-epigallocatechin-3-gallate (EGCG), a common tea polyphenol, can serve as a naturally-occurring COMT inhibitor that also possesses neuroprotective actions.

Methodology/Principal Findings

Using both in vitro and in vivo models, we investigated the modulating effects of EGCG on L-DOPA methylation as well as on chemically induced oxidative neuronal damage and degeneration. EGCG strongly inhibited human liver COMT-mediated O-methylation of L-DOPA in a concentration-dependent manner in vitro, with an average IC ₅₀ of 0.36 µM. Oral administration of EGCG moderately lowered the accumulation of 3-O-methyldopa in the plasma and striatum of rats treated with L-DOPA + carbidopa. In addition, EGCG also reduced glutamate-induced oxidative cytotoxicity in cultured HT22 mouse hippocampal neuronal cells through inactivation of the nuclear factor κB-signaling pathway. Under in vivo conditions, administration of EGCG exerted a strong protective effect against kainic acid-induced oxidative neuronal death in the hippocampus of rats.

Conclusions/Significance

These observations suggest that oral administration of EGCG may have significant beneficial effects in Parkinson''s patients treated with L-DOPA and carbidopa by exerting a modest inhibition of L-DOPA methylation plus a strong neuroprotection against oxidative damage and degeneration.     

Nf2/Merlin Controls Spinal Cord Neural Progenitor Function in a Rac1/ErbB2-Dependent Manner

Objective

Individuals with the neurofibromatosis type 2 (NF2) cancer predisposition syndrome develop spinal cord glial tumors (ependymomas) that likely originate from neural progenitor cells. Whereas many spinal ependymomas exhibit indolent behavior, the only treatment option for clinically symptomatic tumors is surgery. In this regard, medical therapies are unfortunately lacking due to an incomplete understanding of the critical growth control pathways that govern the function of spinal cord (SC) neural progenitor cells (NPCs).

Methods

To identify potential therapeutic targets for these tumors, we leveraged primary mouse Nf2-deficient spinal cord neural progenitor cells.

Results

We demonstrate that the Nf2 protein, merlin, negatively regulates spinal neural progenitor cell survival and glial differentiation in an ErbB2-dependent manner, and that NF2-associated spinal ependymomas exhibit increased ErbB2 activation. Moreover, we show that Nf2-deficient SC NPC ErbB2 activation results from Rac1-mediated ErbB2 retention at the plasma membrane.

Significance

Collectively, these findings establish ErbB2 as a potential rational therapeutic target for NF2-associated spinal ependymoma.     

ABC transporters B1, C1 and G2 differentially regulate neuroregeneration in mice

Background

ATP-binding cassette (ABC) transporters are essential regulators of organismic homeostasis, and are particularly important in protecting the body from potentially harmful exogenous substances. Recently, an increasing number of in vitro observations have indicated a functional role of ABC transporters in the differentiation and maintenance of stem cells. Therefore, we sought to determine brain-related phenotypic changes in animals lacking the expression of distinct ABC transporters (ABCB1, ABCG2 or ABCC1).

Methodology and Principal Findings

Analyzing adult neurogenesis in ABC transporter-deficient animals in vivo and neuronal stem/progenitor cells in vitro resulted in complex findings. In vivo, the differentiation of neuronal progenitors was hindered in ABC transporter-deficient mice (ABCB1^0/0) as evidenced by lowered numbers of doublecortin⁺ (−36%) and calretinin⁺ (−37%) cells. In vitro, we confirmed that this finding is not connected to the functional loss of single neural stem/progenitor cells (NSPCs). Furthermore, assessment of activity, exploratory behavior, and anxiety levels revealed behavioral alterations in ABCB1^0/0 and ABCC1^0/0 mice, whereas ABCG2^0/0 mice were mostly unaffected.

Conclusion and Significance

Our data show that single ABC transporter-deficiency does not necessarily impair neuronal progenitor homeostasis on the single NSPC level, as suggested by previous studies. However, loss of distinct ABC transporters impacts global brain homeostasis with far ranging consequences, leading to impaired neurogenic functions in vivo and even to distinct behavioral phenotypes. In addition to the known role of ABC transporters in proteopathies such as Parkinson''s disease and Alzheimer''s disease, our data highlight the importance of understanding the general function of ABC transporters for the brain''s homeostasis and the regeneration potential.     

Non-Motor and Motor Features in LRRK2 Transgenic Mice

Background

Non-motor symptoms are increasingly recognized as important features of Parkinson’s disease (PD). LRRK2 mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively evaluated in LRRK2 transgenic mouse models.

Objective

Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor phenotypes precede motor symptoms.

Methodology

We investigated the onset of motor and non-motor phenotypes on the LRRK2^R1441G BAC transgenic mice and their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction), and learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate controls.

Conclusions

LRRK2^R1441G BAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory mechanisms modulating the progression of PD in these models should be further evaluated.     

Nonmotor Symptoms in LRRK2 G2019S Associated Parkinson’s Disease

Background

Idiopathic Parkinson’s disease (IPD) and LRRK2-associated PD (LRRK2-PD) might be expected to differ clinically since the neuropathological substrate of LRRK2-PD is heterogeneous. The range and severity of extra-nigral nonmotor features associated with LRRK2 mutations is also not well-defined.

Objective

To evaluate the prevalence and time of onset of nonmotor symptoms (NMS) in LRRK2-PD patients.

Methods

The presence of hyposmia and of neuropsychiatric, dysautonomic and sleep disturbances was assessed in 33 LRRK2-G2019S-PD patients by standardized questionnaires and validated scales. Thirty-three IPD patients, matched for age, gender, duration of parkinsonism and disease severity and 33 healthy subjects were also evaluated.

Results

University of Pennsylvania Smell Identification Test (UPSIT) scores in LRRK2-G2019S-PD were higher than those in IPD (23.5±6.8 vs 18.4±6.0; p = 0.002), and hyposmia was less frequent in G2019S carriers than in IPD (39.4% vs 75.8%; p = 0.01). UPSIT scores were significantly higher in females than in males in LRRK2-PD patients (26.9±4.7 vs 19.4±6.8; p<0.01). The frequency of sleep and neuropsychiatric disturbances and of dysautonomic symptoms in LRRK2-G2019S-PD was not significantly different from that in IPD. Hyposmia, depression, constipation and excessive daytime sleepiness, were reported to occur before the onset of classical motor symptoms in more than 40% of LRRK2-PD patients in whom these symptoms were present at the time of examination.

Conclusion

Neuropsychiatric, dysautonomic and sleep disturbances occur as frequently in patients with LRRK2-G2019S-PD as in IPD but smell loss was less frequent in LRRK2-PD. Like in IPD, disturbances such as hyposmia, depression, constipation and excessive daytime sleepiness may antedate the onset of classical motor symptoms in LRRK2-G2019S-PD.     

Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system

Background

Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.

Methodology/Principal Findings

RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle''s loop and distal tubules.

Conclusions/Significance

Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.     

miR-29a Modulates Neuronal Differentiation through Targeting REST in Mesenchymal Stem Cells

Objective

To investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.

Methods

Rat bone marrow derived–MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.

Results

MSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.

Conclusion

miR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.     

LMNA Knock-Down Affects Differentiation and Progression of Human Neuroblastoma Cells

Background

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.

Methodology/Principal Findings

Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.

Conclusions/Significance

We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.     

Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells

Background

Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it''s estimated to be 2.5 to 3%.

Methodology/Principal Findings

We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.

Conclusions/Significance

These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson''s disease, multiple sclerosis, and Alzheimer''s disease.     

Short Term Morphine Exposure In Vitro Alters Proliferation and Differentiation of Neural Progenitor Cells and Promotes Apoptosis via Mu Receptors

Background

Chronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved.

Methods

Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry.

Results

Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.

Conclusions

Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.     

Maintenance and Neuronal Cell Differentiation of Neural Stem Cells C17.2 Correlated to Medium Availability Sets Design Criteria in Microfluidic Systems

Background

Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes.

Methodology/Principal Findings

A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10⁴ µm³/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10⁴ µm³/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length.

Significance

MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in maintaining the stemness of NSCs.