Survival Of Dopaminergic Neurons Research Articles

Dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival

The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicer(flox/flox)). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicer(flox/flox) mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, are essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.

The role of ERK1, 2, and 5 in dopamine neuron survival during aging

Extracellular signal-regulated kinases (ERKs) 1, 2, and 5 have been shown to play distinct roles in proliferation, differentiation, and neuronal viability. In this study, we examined ERK1, 2, and 5 expression and activation in the substantia nigra (SN), striatum (STR), and ventral tegmental area (VTA) during aging. An age-related decrease in phosphorylated ERK5 was observed in the SN and STR, whereas an increase in total ERK1 was observed in all 3 regions. In primary cultures of the SN and VTA, inhibition of ERK5 but not ERK1 and 2 decreased dopamine neuronal viability significantly. These data suggest that ERK5 is essential for the basal survival of SN and VTA dopaminergic neurons. This is the first study to examine ERK1, 2, and 5 expression and activation in the SN, STR, and VTA during aging, and the relative roles of ERK1, 2, and 5 in basal survival of SN and VTA dopaminergic neurons. These data raise the possibility that a decline in ERK5 signaling may play a role in age-related impairments in dopaminergic function.

Intracellular Nogo-A facilitates initiation of neurite formation in mouse midbrain neurons in vitro

Nogo-A is a transmembrane protein originally discovered in myelin, produced by postnatal CNS oligodendrocytes. Nogo-A induces growth cone collapse and inhibition of axonal growth in the injured adult CNS. In the intact CNS, Nogo-A functions as a negative regulator of growth and plasticity. Nogo-A is also expressed by certain neurons. Neuronal Nogo-A depresses long-term potentiation in the hippocampus and modulates neurite adhesion and fasciculation during development in mice. Here we show that Nogo-A is present in neurons derived from human midbrain (Lund human mesencephalic (LUHMES) cell line), as well as in embryonic and postnatal mouse midbrain (dopaminergic) neurons. In LUHMES cells, Nogo-A was upregulated threefold upon differentiation and neurite extension. Nogo-A was localized intracellularly in differentiated LUHMES cells. Cultured midbrain (dopaminergic) neurons from Nogo-A knock-out mice exhibited decreased numbers of neurites and branches when compared with neurons from wild-type (WT) mice. However, this phenotype was not observed when the cultures from WT mice were treated with an antibody neutralizing plasma membrane Nogo-A. In vivo, neither the regeneration of nigrostriatal tyrosine hydroxylase fibers, nor the survival of nigral dopaminergic neurons after partial 6-hydroxydopamine lesions was affected by Nogo-A deletion. These results indicate that during maturation of cultured midbrain (dopaminergic) neurons, intracellular Nogo-A supports neurite growth initiation and branch formation.

Low Dose Bexarotene Treatment Rescues Dopamine Neurons and Restores Behavioral Function in Models of Parkinson’s Disease

Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinson's disease (PD).

Suppression of Inflammation with Conditional Deletion of the Prostaglandin E2EP2 Receptor in Macrophages and Brain Microglia

Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP(1-4) receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2(lox/lox) mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2(lox/lox) and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.

Therapeutic effects of repetitive transcranial magnetic stimulation in an animal model of Parkinson's disease

Repetitive transcranial magnetic stimulation (rTMS) is used to treat neurological diseases such as stroke and Parkinson's disease (PD). Although rTMS has been used clinically, its underlying therapeutic mechanism remains unclear. The objective of the present study was to clarify the neuroprotective effect and therapeutic mechanism of rTMS in an animal model of PD. Adult Sprague-Dawley rats were unilaterally injected with 6-hydroxydopamine (6-OHDA) into the right striatum. Rats with PD were then treated with rTMS (circular coil, 10Hz, 20min/day) daily for 4 weeks. Behavioral assessments such as amphetamine-induced rotational test and treadmill locomotion test were performed, and the dopaminergic (DA) neurons of substantia nigra pas compacta (SNc) and striatum were histologically examined. Expression of neurotrophic/growth factors was also investigated by multiplex ELISA, western blotting analysis and immunohistochemistry 4 weeks after rTMS application. Among the results, the number of amphetamine-induced rotations was significantly lower in the rTMS group than in the control group at 4 weeks post-treatment. Treadmill locomotion was also significantly improved in the rTMS-treated rats. Tyrosine hydroxylase-positive DA neurons and DA fibers in rTMS group rats were greater than those in untreated group in both ipsilateral SNc and striatum, respectively. The expression levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, platelet-derived growth factor, and vascular endothelial growth factor were elevated in both the 6-OHDA-injected hemisphere and the SNc of the rTMS-treated rats. In conclusion, rTMS treatment improved motor functions and survival of DA neurons, suggesting that the neuroprotective effect of rTMS treatment might be induced by upregulation of neurotrophic/growth factors in the PD animal model.

Lack of vascular endothelial growth factor receptor-2/Flk1 signaling does not affect substantia nigra development

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Lack of hypoxia-inducible factor-1α (HIF1α) impairs midbrain dopaminergic neurogenesis which could be rescued by vascular endothelial growth factor (VEGF) via VEGFR-2 signaling. Here, we conditionally inactivated the VEGFR-2, encoded by the fetal liver kinase 1 (Flk1) gene, in murine NPCs to determine its role in proliferation and survival in vitro as well as survival of dopaminergic neurons in vivo. Flk1 conditional knock-out (Flk1 CKO) mice showed no general brain phenotype. There was no midbrain-specific impairment of NPC proliferation as seen in HIF1α CKO mice. In the substantia nigra (SN) of adult Flk1 CKO mice, nonbiased stereological cell counts revealed no reduction of TH-positive neurons of Flk1 CKO mice compared with control Cre/wt mice (in which the wild-type Flk1 allele is expressed in parallel with the Cre recombinase allele). In conclusion, VEGF receptor signaling seems not to be relevant to the development and survival of substantia nigra dopaminergic neurons within the hypoxia-HIF1α signaling pathway.

Fibroblast growth factor 20 polymorphism in sporadic Parkinson’s disease in Northern Han Chinese

Fibroblast growth factor (FGF) 20 is a neurotrophic factor that exerts strong neurotrophic properties in the brain and could significantly enhance the survival of rat midbrain dopaminergic neurons. The genetic association of the FGF20 gene with Parkinson’s disease (PD) remains controversial. We used the polymerase chain reaction–restriction length polymorphism assay to assess the association of two single nucleotide polymorphisms, rs12720208 and rs1721100, within the FGF20 gene in 178 PD patients and 190 healthy controls from a Northern Han Chinese population. The results showed no significant differences in the presence of rs1721100 or rs12720208 in the FGF20 gene between PD patients and controls. This indicates the FGF20 gene might not play a major role in the genetic predisposition to PD in this population.

Autophagic Impairment Contributes to Systemic Inflammation-Induced Dopaminergic Neuron Loss in the Midbrain

BackgroundNeuroinflammation plays an important role in the pathogenesis of Parkinson’s disease (PD), inducing and accelerating dopaminergic (DA) neuron loss. Autophagy, a critical mechanism for clearing misfolded or aggregated proteins such as α-synuclein (α-SYN), may affect DA neuron survival in the midbrain. However, whether autophagy contributes to neuroinflammation-induced toxicity in DA neurons remains unknown.ResultsIntraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg) into young (3-month-old) and aged (16-month-old) male C57BL/6J mice was observed to cause persistent neuroinflammation that was associated with a delayed and progressive loss of DA neurons and accumulation of α-SYN in the midbrain. The autophagic substrate-p62 (SQSTM1) persistently increased, whereas LC3-II and HDAC6 exhibited early increases followed by a decline. In vitro studies further demonstrated that TNF-α induced cell death in PC12 cells. Moreover, a sublethal dose of TNF-α (50 ng/ml) increased the expression of LC3-II, p62, and α-SYN, implying that TNF-α triggered autophagic impairment in cells.ConclusionNeuroinflammation may cause autophagic impairment, which could in turn result in DA neuron degeneration in midbrain.

Accumulation of mitochondrial DNA deletions within dopaminergic neurons triggers neuroprotective mechanisms

Acquired alterations in mitochondrial DNA are believed to play a pathogenic role in Parkinson's disease. In particular, accumulation of mitochondrial DNA deletions has been observed in substantia nigra pars compacta dopaminergic neurons from patients with Parkinson's disease and aged individuals. Also, mutations in mitochondrial DNA polymerase gamma result in multiple mitochondrial DNA deletions that can be associated with levodopa-responsive parkinsonism and severe substantia nigra pars compacta dopaminergic neurodegeneration. However, whether mitochondrial DNA deletions play a causative role in the demise of dopaminergic neurons remains unknown. Here we assessed the potential pathogenic effects of mitochondrial DNA deletions on the dopaminergic nigrostriatal system by using mutant mice possessing a proofreading-deficient form of mitochondrial DNA polymerase gamma (POLGD257A), which results in a time-dependent accumulation of mitochondrial DNA deletions in several tissues, including the brain. In these animals, we assessed the occurrence of mitochondrial DNA deletions within individual substantia nigra pars compacta dopaminergic neurons, by laser capture microdissection and quantitative real-time polymerase chain reaction, and determined the potential deleterious effects of such mitochondrial DNA alterations on mitochondrial function and dopaminergic neuronal integrity, by cytochrome c oxidase histochemistry and quantitative morphology. Nigral dopaminergic neurons from POLGD257A mice accumulate mitochondrial DNA deletions to a similar extent (∼40-60%) as patients with Parkinson's disease and aged individuals. Despite such high levels of mitochondrial DNA deletions, the majority of substantia nigra pars compacta dopaminergic neurons from these animals did not exhibit mitochondrial dysfunction or degeneration. Only a few individual substantia nigra pars compacta neurons appeared as cytochrome c oxidase-negative, which exhibited higher levels of mitochondrial DNA deletions than cytochrome c oxidase-positive cells (60.38±3.92% versus 45.18±2.83%). Survival of dopaminergic neurons in POLGD257A mice was associated with increased mitochondrial DNA copy number, enhanced mitochondrial cristae network, improved mitochondrial respiration, decreased exacerbation of mitochondria-derived reactive oxygen species, greater striatal dopamine levels and resistance to parkinsonian mitochondrial neurotoxins. These results indicate that primary accumulation of mitochondrial DNA deletions within substantia nigra pars compacta dopaminergic neurons, at an extent similar to that observed in patients with Parkinson's disease, do not kill dopaminergic neurons but trigger neuroprotective compensatory mechanisms at a mitochondrial level that may account for the high pathogenic threshold of mitochondrial DNA deletions in these cells.

TrkB Signaling Directs the Incorporation of Newly Generated Periglomerular Cells in the Adult Olfactory Bulb

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when integration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-γ was found to be responsible for spine formation. Furthermore, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin+ PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopamine neurons mediated by the Lmx1a and neurturin in vitro: potential therapeutic application for Parkinson's disease in a rhesus monkey model.

hUC-MSCs hold great promise in vitro neuronal differentiation and therapy for neurodegenerative disorders including Parkinson’s disease. Recent studies provided that Lmx1α play an important role in the midbrain dopamine cells differentiation. Neurturin is desired candidate gene for providing a neuroprotective to DA neurons. In this study, we investigated a novel neuronal differentiation strategy in vitro with Lmx1α and NTN. We transferred these two genes to hUC-MSCs by recombinant adenovirus combined with Lmx1α regulatory factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored by using behavioral test for six months after implantation. The result showed that cells isolated from the umbilical cord were negative for CD45, CD34 and HLA-DR, but were positive for CD44, CD49d, CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western blot. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. After hUC-MSCs were induced with endogenous and exogenous factors, the mature neurons specific gene TH, Pitx3 was transcripted and the neurons specific protein TH, β-tubulinIII, NSE, Nestin, MAP-2 was expressed in those differentiated cells. In addition, the PD monkeys, transplanted with the induced cells demonstrated the animals’ symptoms amelioration by the behavioral measures. Further more, pathological and immunohistochemistry data showed that there were neuronal-like cells survived in the right brain of those PD monkeys, which may play a role as dopaminergic neurons. The findings from this study may help us to better understand the inside mechanisms of PD pathogenesis and may also help developing effective therapy for Parkinson’s disease.

Regulated Expression of Lentivirus-Mediated GDNF in Human Bone Marrow-Derived Mesenchymal Stem Cells and Its Neuroprotection on Dopaminergic Cells In Vitro

Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator - rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]- diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson’s disease (PD).

Upregulation of Glutathione Peroxidase-1 Expression and Activity by Glial Cell Line–Derived Neurotrophic Factor Promotes High-Level Protection of PC12 Cells Against 6-Hydroxydopamine and Hydrogen Peroxide Toxicities

We examined the impact of strong co-presence and function of glutathione peroxidase-1 (GPX-1) and glial cell line-derived neurotrophic factor (GDNF) on protecting the rat dopaminergic pheochromocytoma cell line PC12 against 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H₂O₂) toxicities. Primarily, GPX-1 over-expression by PC12 cells infected with pLV-GPX1 lentivirus vectors significantly increased cell survival against 6-OHDA toxicity (p<0.01). Addition of conditioned medium collected from growing wild-type astrocytes (Control astro-CM) increased survival rate of pLV-GPX1 infectants by 10% compared to their un-treated counterparts (p<0.05) and 20% compared to their treated empty vector control (p<0.01). Treatment of pLV-GPX1 cells with astro-CM of GDNF-over-secreting astrocytes (Test astro-CM) significantly induced GPX-1 expression, peroxidase enzymatic activity, and intra-cellular glutathione (GSH) levels. These changes paralleled with protection of 90% of GDNF⁺/GPX1⁺ PC12 cells against toxicity, a rate that was 37% up from their un-infected un-treated (GDNF⁻/GPX1⁻) controls (p<0.001), and 12% up from pLV-GPX1 cells that received only Control astro-CM (GPX⁺/GDNF⁻) (p<0.01). GPX-1 over-expression per se suppressed intra-cellular H₂O₂ elevation upon 6-OHDA exposure, and addition of GDNF medium significantly accelerated this suppression (p<0.01). Substitution of 6-OHDA with H₂O₂ induced similar intra-cellular changes and comparable protection levels. In all cell groups, increased cell survival against either compound was further confirmed by increased live cell counts measured by double staining. Following depletion of intra-cellular GSH, only 46% of pLV-GPX1 cells survived 6-OHDA toxicity, whereas over 70% of them were saved upon GDNF treatment (p<0.001). Moreover, capase-3 activation was reduced in pLV-GPX1 cells and maximized by addition of GDNF. Comparison analyses established correlations between GPX-1-GDNF co-presence and both enhanced cell protection and diminished levels of activated caspase-3. Our data collectively indicate that GDNF is capable of inducing anti-oxidant activities of intra-cellular GPX-1 and that growth-promoting potential of GDNF and anti-oxidant properties of GPX-1 can, in concert, maximize survival of dopaminergic neurons.

Regulation and effects of GDF-15 in the retina following optic nerve crush

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor-β superfamily and is ubiquitously expressed in the central nervous system. It is prominently upregulated in cerebral cortical and ischemic lesion paradigms. GDF-15 robustly promotes the survival of lesioned nigrostriatal dopaminergic neurons in vivo; GDF-15-deficient mice exhibit progressive postnatal motor and sensory neuron losses implying essential functions of GDF-15 in neuronal survival. We show that GDF-15 mRNA and protein are, respectively, six-fold and three-fold upregulated in the murine retina at 1 day after optic nerve crush, slightly elevated mRNA levels being maintained until day 28. However, the magnitude and time course of retinal ganglion cell (RGC) death are indistinguishable in knockout and control mice. Selected mRNAs implicated in the regulation of the death vs. survival of RGCs, including ATF3, Bad, Bcl-2 and caspase-8, were similarly regulated in both knockout and control retinae. Immunohistochemistry for tyrosine hydroxylase and choline acetyltransferase revealed no differences in staining patterns in the two genotypes. mRNA and protein levels of galanin, a putative neuroprotective factor and positive regulator of neuron survival and axonal regeneration, were prominently upregulated after crush in knockout retinae at day 3, as compared with control retinae, suggesting that GDF-15 acts as a physiological regulator of galanin. GDF-15 is therefore prominently upregulated in the retina after optic nerve crush but does not directly interfere with the magnitude and temporal progression of RGC death.

Neuroprotective effects of bee venom phospholipase 2 by suppression of neuroinflammatory responses in mouse model of Parkinson’s disease: role of CD4+CD25+Foxp3+ regulatory T cells (P3125)

Abstract In the present study, we examined whether bee venom phospholipase 2 (bvPLA2) promotes the survival of dopaminergic (DA) neurons in the MPTP-induced mouse model of Parkinson’s disease (PD). Treatment of bvPLA2 prevented degeneration of DA neurons in the substantia nigra (SN). This nueroprotection of bvPLA2 associated with the suppression of microglia and inhibition of infiltration of CD4+ T cells into the SN. Additionally, CD4+CD25+Foxp3+ Tregs were significantly increased in vivo and in vitro after treatment of bvPLA2. The increased proportion of Tregs by bvPLA2 treatement remained highly suppressive ex vivo. To determine whether this neuroprotective effects of bvPLA2 associated with Tregs, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3+ Tregs. Interestingly, bvPLA2 treatment did not prevent MPTP neurotoxicity in mice depleted of Tregs by dt treatment. Therefore, our present studies indicate that the neuro-protective effect of bvPLA2 might be associated with its anti-inflammatory properties resulting from modulation of peripheral immune tolerance by Tregs.

Mitogen-Activated Protein Kinase Phosphatase (MKP)-1 as a Neuroprotective Agent: Promotion of the Morphological Development of Midbrain Dopaminergic Neurons

A greater understanding of the mechanisms that promote the survival and growth of dopaminergic neurons is essential for the advancement of cell replacement therapies for Parkinson's disease (PD). Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Here, we show that MKP-1 is expressed in dopaminergic neurons cultured from E14 rat ventral mesencephalon (VM). When dopaminergic neurons were transfected to overexpress MKP-1, they displayed a more complex morphology than their control counterparts in vitro. Specifically, MKP-1-transfection induced significant increases in neurite length and branching with a maximum increase observed in primary branches. We demonstrate that inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in vitro is mediated by p38 and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. We further show that overexpression of MKP-1 in dopaminergic neurons contributes to neuroprotection against the effects of 6-OHDA. Collectively, we report that MKP-1 can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Thus, we propose that strategies aimed at augmenting MKP-1 expression or activity may be beneficial in protecting dopaminergic neurons and may provide potential therapeutic approaches for PD.

Profile of microRNAs in the plasma of Parkinson’s disease patients and healthy controls

The pathological hallmark of Parkinson’s disease (PD) is the loss of dopaminergic neurons and the presence of Lewy bodies and Lewy neurites in the substantia nigra pars compacta, corpus striatum and brain cortex [1]. PD is a complex disease caused by the interaction of genetic/ inherited and environmental/acquired risk factors [2]. MicroRNAs (miRNAs) are small RNAs that control gene expression by binding to the 30 UTRs of mRNAs [3]. Postmortem analysis of brain tissues and in vitro studies have identified several miRNAs implicated in PD. MiR-133b was shown to be downregulated in PD brains and to promote the survival of dopaminergic neurons [4]. MiR-433 was related with PD through targeting the FGF20, which in turn regulates the expression of a-synuclein [5]. MiR-7 targets a-synuclein and could regulate oxidative stress and cell death, while miR-184 and let-7 regulate dopaminergic neurons survival and activity [6, 7]. Recently, a miRNA profiling of PD brains identified early downregulation of miR-34b/c, modulating mitochondrial function in areas with pathological affectation [8]. Plasma (circulating) miRNAs have been proposed as biomarkers for several diseases and aging [9, 10]. Our aim was to characterize the plasma miRNA profile in PD patients and healthy controls, to determine its usefulness as a biomarker for PD. The study was approved by the Ethical Committee of Hospital Universitario Central de Asturias (HUCA) in accordance with the ethical standards of the Declaration of Helsinki and all the participants signed an informed consent. The study cohort consisted of sexand age-matched healthy controls (n = 25; mean age 67.6; 52 % males) and patients (n = 31; mean age 63.9; 55 % males) who fulfilled the PD-clinical diagnosis criteria [11]. None of the patients were receiving drugs for PD-treatment or had a diagnosis of cardiovascular or tumor disease. Full details of the experimental procedure are available as supplementary material. Briefly, blood was collected in tubes with EDTA, centrifuged and plasma was aliquoted (350 ll). A total of 2 pg of a synthetic Arabidopsis thaliana miRNA (Ath-miR-159a; 5 ll of a 0.4 pg/ll dilution) was immediately added and each aliquot stored at -80 C until use. Ath-miR-159a was used as control of the extraction process (supplementary material). Total plasma RNA was extracted (TRIzol LS Reagent, Ambion) and resuspended in 25 ll of RNAse-free water. Five ll of each sample were retrotranscribed (RT) with the Megaplex RT primers Human pool A and TaqMan microRNA Reverse transcription kit (Applied Biosystems). Three ll of the RT product were pre-amplified with the Megaplex Preamp primers Human pool A and TaqMan Universal Master Mix no AmpErase UNG (Applied Biosystems). All the preamplifications were assayed with a custom Ath-miR-159a Taqman assay in an ABI 7500 Real-Time PCR (Applied Electronic supplementary material The online version of this article (doi:10.1007/s00415-013-6900-8) contains supplementary material, which is available to authorized users.

Loss of vesicular dopamine release precedes tauopathy in degenerative dopaminergic neurons in a Drosophila model expressing human tau

While a number of genome-wide association studies have identified microtubule-associated protein tau as a strong risk factor for Parkinson’s disease (PD), little is known about the mechanism through which human tau can predispose an individual to this disease. Here, we demonstrate that expression of human wild-type tau is sufficient to disrupt the survival of dopaminergic neurons in a Drosophila model. Tau triggers a synaptic pathology visualized by vesicular monoamine transporter-pHGFP that precedes both the age-dependent formation of tau-containing neurofibrillary tangle-like pathology and the progressive loss of DA neurons, thereby recapitulating the pathological hallmarks of PD. Flies overexpressing tau also exhibit progressive impairments of both motor and learning behaviors. Surprisingly, contrary to common belief that hyperphosphorylated tau could aggravate toxicity, DA neuron degeneration is alleviated by expressing the modified, hyperphosphorylated tauE14. Together, these results show that impairment of VMAT-containing synaptic vesicle, released to synapses before overt tauopathy may be the underlying mechanism of tau-associated PD and suggest that correction or prevention of this deficit may be appropriate targets for early therapeutic intervention.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1105-x) contains supplementary material, which is available to authorized users.

The transcription factor Pitx3 is expressed selectively in midbrain dopaminergic neurons susceptible to neurodegenerative stress

The homeodomain transcription factor Pitx3 is critical for the survival of midbrain dopaminergic (mDA) neurons. Pitx3-deficient mice exhibit severe but selective developmental loss of mDA neurons, with accompanying locomotor deficits resembling those seen in Parkinson's disease (PD) models. Here, we identify specific mDA cell subpopulations that are consistently spared in adult Pitx3-hypomorphic (aphakia) mice, demonstrating that Pitx3 is not indiscriminately required by all mDA neurons for their survival. In aphakia mice, virtually all surviving mDA neurons in the substantia nigra (SN) and the majority of neurons in the adjacent ventral tegmental area (VTA) also express calbindin-D28k, a calcium-binding protein previously associated with resistance to injury in PD and in animal models. Cell-mapping studies in wild-type mice revealed that Pitx3 is primarily expressed in the ventral SN, a region particularly susceptible to MPTP and other dopaminergic neurotoxins. Furthermore, Pitx3-expressing SN cells are preferentially lost following MPTP treatment. Finally, SN mDA neurons in Pitx3 hemizygous mice show increased sensitivity when exposed to MPTP. Thus, SN mDA neurons are represented by at least two distinct subpopulations including MPTP-resistant Pitx3-autonomous, calbindin-positive neurons, and calbindin-negative Pitx-3-dependent cells that display elevated vulnerability to toxic injury, and probably correspond to the subpopulation that degenerates in PD. Impairment of Pitx3-dependent pathways therefore increases vulnerability of mDA neurons to toxic injury. Together, these data suggest a novel link between Pitx3 function and the selective pattern of mDA cell loss observed in PD.