Midbrain Cultures Research Articles

A comparative study of Lrrk2 function in primary neuronal cultures

Objective To assess the contribution of wild-type, mutant and loss of leucine-rich repeat kinase-2 ( LRRK2; Lrrk2) on dendritic neuronal arborization. Background LRRK2 mutations are recognized as the major genetic determinant of susceptibility to Parkinson’s disease for which a cellular assay of Lrrk2 mutant function would facilitate the development of targeted molecular therapeutics. Methods Dendritic neuronal arborization (neurite length, branching and the number of processes per cell) was quantified in primary hippocampal and midbrain cultures derived from five lines of recombinant LRRK2 mice, including human BAC wild-type and mutant overexpressors (Y1699C and G2019S), murine knock-out and G2019S knock-in animals. Results Neuronal arborization in cultures from BAC Lrrk2 wild-type animals is comparable to non-transgenic littermate controls, despite high levels of human transgene expression. In contrast, primary neurons from both BAC mutant overexpressors presented with significantly reduced neuritic outgrowth and branching, although the total number of processes per cell remained comparable. The mutant-specific toxic gain-of-function observed in cultures from BAC mutant mice may be partially rescued by staurosporine treatment, a non-specific kinase inhibitor. In contrast, neuronal arborization is far more extensive in neuronal cultures derived from murine knock-out mice that lack endogenous Lrrk2 expression. In Lrrk2 G2019S knock-in mice, arguably the most physiologically relevant system, neuritic arborization is not impaired. Conclusions Impairment of neuritic arborization is an exaggerated, albeit mutant specific, consequence of Lrrk2 over-expression in primary cultures. The phenotype and assay described provides a means to develop therapeutic agents that modulate the toxic gain-of-function conferred by mutant Lrrk2.

KATP channel blockade protects midbrain dopamine neurons by repressing a glia‐to‐neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis

While K(ATP) channels serve primarily as metabolic gatekeepers in excitable cells, they might also participate in other important cellular functions. Here, we demonstrate that K(ATP) channel blockade with the sulfonylurea derivative glibenclamide provided robust protection to dopamine neurons undergoing spontaneous and selective degeneration in midbrain cultures. Unexpectedly, glibenclamide operated not by a direct effect on dopamine neurons but instead by halting the proliferation of a population of immature glial cells lacking astrocytic and microglial markers. The antimitotic effect of glibenclamide appeared essential to unmask a prosurvival phosphoinositide 3-kinase (PI3K)/Akt-dependent signaling pathway that controlled shuttling of calcium from endoplasmic reticulum to mitochondria in dopamine neurons. Preventing integrin-ligand interactions with a decoy ligand, the Arg-Gly-Asp-Ser sequence peptide, reproduced survival promotion by glibenclamide via a mechanism that also required PI3K/Akt-dependent regulation of mitochondrial calcium. Noticeably, Arg-Gly-Asp-Ser did not cause a reduction in glial cell numbers indicating that it prevented the death process downstream of the level at which glibenclamide intervenes. Based on these results, we propose that K(ATP) channel blockade protected dopamine neurons by inhibiting a glia-to-neuron signaling pathway that propagates through integrin/ligand interactions and ultimately disrupts PI3K/Akt-dependent signaling and mitochondrial calcium homeostasis.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson’s disease models

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Neuromelanin Activates Microglia and Induces Degeneration of Dopaminergic Neurons: Implications for Progression of Parkinson’s Disease

In Parkinson's disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H₂O₂), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H₂O₂ production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

Glutamate Controls Growth Rate and Branching of Dopaminergic Axons

Dopamine-releasing neurons of the substantia nigra pars compacta produce an extraordinarily dense and expansive plexus of innervation in the striatum converging with glutamatergic corticostriatal and thalamostriatal axon terminals at dendritic spines of medium spiny neurons. Here, we investigated whether glutamatergic signaling promotes arborization and growth of dopaminergic axons. In postnatal ventral midbrain cultures, dopaminergic axons rapidly responded to glutamate stimulation with accelerated growth and growth cone splitting when NMDA and AMPA/kainate receptors were activated. In contrast, when AMPA/kainate receptors were selectively activated, axon growth rate was decreased. To address whether this switch in axonal growth response was mediated by distinct calcium signals, we used calcium imaging. Combined NMDA and AMPA/kainate receptor activation elicited calcium signals in axonal growth cones that were mediated by calcium influx through L-type voltage-gated calcium channels and ryanodine receptor-induced calcium release from intracellular stores. AMPA/kainate receptor activation alone elicited calcium signals that were solely attributable to calcium influx through L-type calcium channels. We found that inhibitors of calcium/calmodulin-dependent protein kinases prevented the NMDA receptor-dependent axonal growth acceleration, whereas AMPA/kainate-induced axonal growth decrease was blocked by inhibitors of calcineurin and by increased cAMP levels. Our data suggest that the balance between NMDA and AMPA/kainate receptor activation regulates the axonal arborization pattern of dopamine axons through the activation of competing calcium-dependent signaling pathways. Understanding the mechanisms of dopaminergic axonal arborization is essential to the development of treatments that aim to restore dopaminergic innervation in Parkinson's disease.

Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinson's disease. Bipyridyl herbicides, such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H2O2 production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H2O2 production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (< or = 300 microM) concentrations, PQ-induced H2O2 production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H2O2 production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H2O2 production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H2O2 production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H2O2 differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H2O2 production with all three compounds. These data identify a novel role for complex III dependence of mitochondrial ROS production by redox cycling herbicides, while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK-KO) midbrain neuronal cultures are resistant to epoxomicin-induced cell death. This resistance is due to increased GSH and DJ-1 protein levels in PK-KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro-apoptotic Bax/Bcl-2 ratio, the phosphorylation of tau, and the levels of chaperones heat-shock protein 70 and C-terminal Hsc-interacting protein, but none of these effects took place in epoxomicin-treated PK-KO cultures. Poly-ubiquitinated proteins increased more in WT than in PK-KO-treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK-KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK-KO neurons triggers the enhancement of autophagy. The treatment with l-buthionine-S,R-sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK-KO cultures. We also found that PK-KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat-shock protein 70 response, and to activation of p-serine/threonine kinase protein signaling pathway as well as to increased poly-ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK-KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition-induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK-KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old-WT.

Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF‐mediated signaling

Functions of retinoic acid receptors (RARs) in adult CNS have been poorly characterized. Here we investigated potential neuroprotective action of tamibarotene (Am80), an RARalpha/beta agonist available for the treatment of acute promyelocytic leukemia, on midbrain dopaminergic neurons. Am80 protected dopaminergic neurons in rat midbrain slice culture from injury mediated by lipopolysaccharide-activated microglia, without affecting production of nitric oxide, a key mediator of cell injury. The effect of Am80 was mimicked by another RAR agonist, TAC-101, but not by a retinoid X receptor agonist, HX630, and HX630 did not synergize with Am80. We observed neuronal expression of RARalpha and RARbeta in midbrain slice culture and also found that Am80 increased tissue level of brain-derived neurotrophic factor (BDNF) mRNA. Exogenous BDNF prevented dopaminergic neurodegeneration, and the neuroprotective effect of Am80 was suppressed by a TrkB inhibitor, K252a, or by anti-BDNF neutralizing antibody. These results reveal a novel action of RARs mediated by enhancement of BDNF expression. Finally, oral administration of Am80 prevented dopaminergic cell loss in the substantia nigra induced by local injection of lipopolysaccharide in mice, indicating that RARs are a promising target of therapeutics for neurodegenerative disorders.

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

Comparison of Gene Expression Profile in Embryonic Mesencephalon and Neuronal Primary Cultures

In the mammalian central nervous system (CNS) an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA) primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis

The floor plate, an essential ventral midline organizing center that produces the morphogen Shh, has distinct properties along the neuraxis. The neurogenic potential of the floor plate and its underlying mechanisms remain unknown. Using Shh as a driver for lineage analysis, we found that the mouse midbrain, but not the hindbrain, floor plate is neurogenic, giving rise to dopamine (DA) neurons. Distinct spatiotemporal Shh and Wnt expression may distinguish the neurogenetic potential of these structures. We discovered an inhibitory role for Shh: removal of Shh resulted in neurogenesis from the hindbrain midline and, conversely, high doses of Shh inhibited proliferation and DA neuron production in midbrain cultures. We found that Wnt/beta-catenin signaling is necessary and sufficient for antagonizing Shh, DA progenitor marker induction and promotion of dopaminergic neurogenesis. These findings demonstrate how the dynamic interplay of canonical Wnt/beta-catenin signaling and Shh may orchestrate floor plate neurogenesis or a lack thereof.

Vascular endothelial growth factor B (VEGF-B) is up-regulated and exogenous VEGF-B is neuroprotective in a culture model of Parkinson's disease

Parkinson's disease (PD) results from the degeneration of dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine released in the striatum. Current oral dopamine replacement or surgical therapies do not address the underlying issue of neurodegeneration, they neither slow nor halt disease. Neurotrophic factors have shown preclinical promise, but the choice of an appropriate growth factor as well as the delivery has proven difficult. In this study, we used a rotenone rat midbrain culture model to identify genes that are changed after addition of the neurotoxin. (1) We challenged rat midbrain cultures with rotenone (20 nM), a pesticide that has been shown to be toxic for dopaminergic neurons and that has been a well-characterized model of PD. A gene chip array analysis demonstrated that several genes were up-regulated after the rotenone treatment. Interestingly transcriptional activation of vascular endothelial growth factor B (VEGF-B) was evident, while vascular endothelial growth factor A (VEGF-A) levels remained unaltered. The results from the gene chip array experiment were verified with real time PCR and semi-quantitative western analysis using β-actin as the internal standard. (2) We have also found evidence that exogenously applied VEGF-B performed as a neuroprotective agent facilitating neuron survival in an even more severe rotenone culture model of PD (40 nM rotenone). VEGF-B has very recently been added to the list of trophic factors that reduce effects of neurodegeneration, as was shown in an in vivo model of motor neuron degeneration, while lacking potential adverse angiogenic activity. The data of an in vivo protective effect on motor neurons taken together with the presented results demonstrate that VEGF-B is a new candidate trophic factor distinct from the GDNF family of trophic factors. VEGF-B is activated by neurodegenerative challenges to the midbrain, and exogenous application of VEGF-B has a neuroprotective effect in a culture model of PD. Strengthening this natural protective response by either adding exogenous VEGF-B or up-regulating the endogenous VEGF-B levels may have the potential to be a disease modifying therapy for PD. We conclude that the growth factor VEGF-B can improve neuronal survival in a culture model of PD.

NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice

Parkin mutations produce Parkinson’s disease (PD) in humans and nigrostriatal dopamine lesions related to increased free radicals in mice. We examined the effects of NP7, a synthetic, marine derived, free radical scavenger which enters the brain, on H2O2 toxicity in cultured neurons and glia from wild-type (WT) and parkin null mice (PK-KO).NP7, 5–10μM, prevented the H2O2 induced apoptosis and necrosis of midbrain neuronal and glial cultures from WT and PK-KO mice. NP7 suppressed microglial activation and the H2O2 induced drop-out of dopamine neurons. Furthermore, NP7 prevented the increased phosphorylation of ERK and AKT induced by H2O2. NP7 may be a promising neuroprotector against oxidative stress in PD.

Atypical parkinsonism in the Caribbean island of Guadeloupe: Etiological role of the mitochondrial complex I inhibitor annonacin

On the French West Indian island of Guadeloupe, atypical parkinsonian patients represent two-thirds of all cases of parkinsonism, which is exceptionally frequent compared to epidemiological data from European countries where atypical parkinsonism accounts for only approximately 5% of all cases. The clinical entity was a unique combination of levodopa-resistant parkinsonism, tremor, myoclonus, hallucinations, REM sleep behavior disorder and fronto-subcortical dementia. Based on the presence or the absence of supranuclear gaze palsy, two subgroups of patients were distinguished. In patients with oculomotor signs that came to autopsy, neuronal loss was found to predominate in the substantia nigra and the striatum but other brain areas were also affected, including the frontal cortex. In addition, tau-containing lesions were detected throughout the brain. Epidemiological data suggested a close association of the disease with the regular consumption of soursop, a tropical annonaceous plant. Experimental studies performed in midbrain cell cultures identified annonacin, a selective mitochondrial complex I inhibitor contained in the fruit and leaves of soursop, as a probable etiological factor. Consistent with this view, chronic administration of annonacin to rats through Alzet osmotic minipumps showed that annonacin was able to reproduce the brain lesions characteristic of the human disease.

Improvement of embryo midbrain cell cultures for testing developmental neurotoxicity in vitro

Improvement of embryo midbrain cell cultures for testing developmental neurotoxicity in vitro

Gender differences and estrogen effects in parkin null mice

Estrogens are considered neurotrophic for dopamine neurons. Parkinson's disease is more frequent in males than in females, and more prevalent in females with short reproductive life. Estrogens are neuroprotective against neurotoxic agents for dopamine neurons in vivo and in vitro. Here, we have investigated the role of estrogens in wild-type (WT) and parkin null mice (PK-/-). WT mice present sexual dimorphisms in neuroprotective mechanisms (Bcl-2/Bax, chaperones, and GSH), but some of these inter-sex differences disappear in PK-/-. Tyrosine hydroxylase (TH) protein and TH+ cells decreased earlier and more severely in female than in male PK-/- mice. Neuronal cultures from midbrain of WT and PK-/- mice were treated with estradiol from 10 min to 48 h. Short-term treatments activated the mitogen-activated protein kinase pathway of WT and PK-/- neurons and the phosphatidylinositol 3'-kinase/AKT/glycogen synthase kinase-3 pathway of WT but not of PK-/- cultures. Long-term treatments with estradiol increased the number of TH+ neurons, the TH expression, and the extension of neurites, and decreased the level of apoptosis, the expression of glial fibrillary acidic protein, and the number of microglial cells in WT but not in PK-/- cultures. The levels of estrogen receptor-alpha were elevated in midbrain cultures and in the striatum of adult PK-/- male mice, suggesting that suppression of parkin changes the estrogen receptor-alpha turnover. From our data, it appears that parkin participates in the cellular estrogen response which could be of interest in the management of parkin-related Parkinson's disease patients.

Reduced vesicular storage of dopamine exacerbates methamphetamine‐induced neurodegeneration and astrogliosis

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5-10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.

脂多糖预处理改善脂多糖诱导的中脑脑片神经元损伤

To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA). In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05). Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Pitx3‐transfected astrocytes secrete brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures

The transcription factor Pitx3 is crucial for the development and differentiation of dopamine (DA) neurons. Our previous work has shown the Pitx3 can up-regulate the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in neuroblastoma cell line SH-SY5Y. Primary astrocytes are the major nonneuronal cells and can be easily modified genetically to deliver therapeutic molecules into the brain, so we investigated whether Pitx3 can increase the expression and secretion of BDNF and GDNF in primary astrocytes. We first transfected Pitx3 plasmid in purified rat astrocytes and collected the conditioned medium (CM) from the Pitx3-transfected cultures, and then we measured the BDNF and GDNF levels from the CM and tested the protective effect of the CM against rotenone-induced DA neuron injury in ventral mesencephalon (VM) cultures. We found that the BDNF and GDNF levels were 1.4-fold and 1.5-fold higher in the CM from Pitx3-transfected astrocytes than empty vectors-transfected controls. Incubation with the CM from Pitx3-transfected astrocytes significantly attenuated the rotenone-induced DA neuron injury, and such protection can be significantly blocked by preincubation with antibodies against either BDNF or GDNF, whereas preincubation with purified BDNF or GDNF replicated the neuroprotection against rotenone-induced injury in VM cultures. These results demonstrate that Pitx3-transfection in astrocytes can up-regulate BDNF and GDNF expression and produce protective benefit to DA neurons, which might be a potential therapeutic alternative for Parkinson's disease.

Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons

Parkinson's disease (PD) is characterized by the selective and progressive loss of dopaminergic (DA) neurons in the midbrain substantia nigra. Currently, available treatment is unable to alter PD progression. Previously, we demonstrated that valproic acid (VPA), a mood stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, increases the expression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in astrocytes to protect DA neurons in midbrain neuron-glia cultures. The present study investigated whether these effects are due to HDAC inhibition and histone acetylation. Here, we show that two additional HDAC inhibitors, sodium butyrate (SB) and trichostatin A (TSA), mimic the survival-promoting and protective effects of VPA on DA neurons in neuron-glia cultures. Similar to VPA, both SB and TSA increased GDNF and BDNF transcripts in astrocytes in a time-dependent manner. Furthermore, marked increases in GDNF promoter activity and promoter-associated histone H3 acetylation were noted in astrocytes treated with all three compounds, where the time-course for acetylation was similar to that for gene transcription. Taken together, our results indicate that HDAC inhibitors up-regulate GDNF and BDNF expression in astrocytes and protect DA neurons, at least in part, through HDAC inhibition. This study indicates that astrocytes may be a critical neuroprotective mechanism of HDAC inhibitors, revealing a novel target for the treatment of psychiatric and neurodegenerative diseases.