Cell Culture Models Of Parkinson's Disease Research Articles

Sex-specific neuroprotection by inhibition of the Y-chromosome gene, SRY, in experimental Parkinson’s disease

Parkinson's disease (PD) is a debilitating neurodegenerative disorder caused by the loss of midbrain dopamine (DA) neurons. While the cause of DA cell loss in PD is unknown, male sex is a strong risk factor. Aside from the protective actions of sex hormones in females, emerging evidence suggests that sex-chromosome genes contribute to the male bias in PD. We previously showed that the Y-chromosome gene, SRY, directly regulates adult brain function in males independent of gonadal hormone influence. SRY protein colocalizes with DA neurons in the male substantia nigra, where it regulates DA biosynthesis and voluntary movement. Here we demonstrate that nigral SRY expression is highly and persistently up-regulated in animal and human cell culture models of PD. Remarkably, lowering nigral SRY expression with antisense oligonucleotides in male rats diminished motor deficits and nigral DA cell loss in 6-hydroxydopamine (6-OHDA)-induced and rotenone-induced rat models of PD. The protective effect of the SRY antisense oligonucleotides was associated with male-specific attenuation of DNA damage, mitochondrial degradation, and neuroinflammation in the toxin-induced rat models of PD. Moreover, reducing nigral SRY expression diminished or removed the male bias in nigrostriatal degeneration, mitochondrial degradation, DNA damage, and neuroinflammation in the 6-OHDA rat model of PD, suggesting that SRY directly contributes to the sex differences in PD. These findings demonstrate that SRY directs a previously unrecognized male-specific mechanism of DA cell death and suggests that suppressing nigral Sry synthesis represents a sex-specific strategy to slow or prevent DA cell loss in PD.

Small-Sized mPEG-PLGA Nanoparticles of Schisantherin A with Sustained Release for Enhanced Brain Uptake and Anti-Parkinsonian Activity.

Schisantherin A (SA) is a promising anti-Parkinsonism natural product. However, its poor water solubility and rapid serum clearance impose significant barriers to delivery of SA to the brain. This work aimed to develop SA in a nanoparticle formulation that extended SA circulation in the bloodstream and consequently an increased brain uptake and thus to be potentially efficacious for the treatment of Parkinson's disease (PD). Spherical SA nanoparticles with a mean particle size of 70 nm were prepared by encapsulating SA into methoxy poly(ethylene glycol)-block-poly(d,l)-lactic-co-glycolic acid (mPEG-PLGA) nanoparticles (SA-NPs) with an encapsulation efficiency of ∼91% and drug loading of ∼28%. The in vitro release of the SA-NPs lasted for 48 h with a sustained-release pattern. Using the Madin-Darby canine kidney (MDCK) cell model, the results showed that first intact nanoparticles carrying hydrophobic dyes were internalized into cells, then the dyes were slowly released within the cells, and last both nanoparticles and free dyes were externalized to the basolateral side of the cell monolayer. Fluorescence resonance energy transfer (FRET) imaging in zebrafish suggested that nanoparticles were gradually dissociated in vivo with time, and nanoparticles maintained intact in the intestine and brain at 2 h post-treatment. When SA-NPs were orally administrated to rats, much higher Cmax and AUC0-t were observed in the plasma than those of the SA suspension. Furthermore, brain delivery of SA was much more effective with SA-NPs than with SA suspension. In addition, the SA-NPs exerted strong neuroprotective effects in zebrafish and cell culture models of PD. The protective effect was partially mediated by the activation of the protein kinase B (Akt)/glycogen synthase kinase-3β (Gsk3β) pathway. In summary, this study provides evidence that small-sized mPEG-PLGA nanoparticles may improve cross-barrier transportation, oral bioavailability, brain uptake, and bioactivity of this Biopharmaceutics Classification System (BCS) Class II compound, SA.

Efficacy of glucagon-like peptide-1 mimetics for neural regeneration.

Glucagon-like peptide 1 (GLP-1) is secreted from enteroendocrine L cells in response to nutrient ingestion and exhibits insulinotropic properties by stimulating specific G protein-linked receptors (GLP-1Rs) on pancreatic β cells. Several GLP-1 mimetics, such as exenatide (exendin-4 (Ex-4)), liraglutide, and lixisenatide, have been developed and approved as treatments for patients with type 2 diabetes. These peptides show bioactivities almost identical to those of GLP-1 and have a substantially longer plasma half-life than GLP-1 because of their resistance to dipeptidyl peptidase-4, a GLP-1 degrading enzyme. GLP-1Rs are found in not only the pancreas but also the extrapancreatic tissues, including the nervous tissues (Harkavyi and Whitton, 2010). It is important to note that GLP-1 mimetics can cross the blood brain barrier and directly act on neurons in the central nervous system. In addition to the inhibition of appetite, the neuroprotective properties of GLP-1 have been receiving increasing attention. Recent studies have suggested that GLP-1 mimetics confer beneficial effects in neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and stroke, and multiple sclerosis (Holscher, 2014). In particular, the neuroprotective properties of Ex-4 have been demonstrated in animal and cell culture models of PD. A single-blinded clinical trial with 45 PD patients revealed that the treatment with Ex-4 significantly improved the cognition and memory of patients (Aviles-Olmos et al., 2013). The beneficial effects of GLP-1 mimetics on the peripheral nervous system (PNS) have also been reported. Both GLP-1 and Ex-4 delivered via osmotic minipumps prevented axonal degeneration in a rat model of pyridoxine-induced neuropathy (Perry et al., 2007). Treatment of streptozotocin (STZ)-induced diabetic mice with Ex-4 for 4 weeks restored motor and sensory nerve conduction velocities and hypoalgesia without normalizing blood glucose levels (Himeno et al., 2011). In addition, repeated intraperitoneal injections of Ex-4 significantly promoted axonal regeneration and functional recovery following sciatic nerve crush injury in normal adult rats (Yamamoto et al., 2013). These findings are in agreement with in vitro studies that revealed that GLP-1 and Ex-4 promoted neurite outgrowth of rat pheochromocytoma-derived PC12 cells (Perry et al., 2002) and adult mouse dorsal root ganglion (DRG) neurons (Himeno et al., 2011). Together these results provide further evidence of the direct actions of Ex-4 on the PNS; however, the underlying mechanisms remain unclear. Our recent study (Tsukamoto et al., 2015) aimed to elucidate the precise localization of GLP-1R in adult rat DRG in vivo and in vitro as well as to determine the neurotrophic and neuroprotective properties of Ex-4 in adult rat DRG neurons.

Protein kinase D1 (PKD1) phosphorylation promotes dopaminergic neuronal survival during 6-OHDA-induced oxidative stress.

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.

Diapocynin prevents early Parkinson's disease symptoms in the leucine-rich repeat kinase 2 (LRRK2R1441G) transgenic mouse

The most prominent mechanism proposed for death of dopaminergic neurons in Parkinson's disease (PD) is elevated generation of reactive oxygen/nitrogen species (ROS/RNS). Recent studies suggest that ROS produced during PD pathogenesis may contribute to cytotoxicity in cell culture models of PD. We hypothesized that inhibition of ROS production would prevent PD symptoms in the LRRK2R1441G transgenic (tg) mouse model of PD. These mice overexpress a mutant form of leucine-rich repeat kinase 2 (LRRK2) and are reported to develop PD-like symptoms at approximately 10 months of age. Despite similar expression of the transgene, our colony did not recapitulate the same type of motor dysfunction originally reported. However, tests of motor coordination (pole test, Rotor-Rod) revealed a significant defect in LRRK2R1441G mice by 16 months of age. LRRK2R1441G tg mice, or wild type littermates, were given diapocynin (200mg/kg, a proposed NADPH oxidase inhibitor) three times per week by oral gavage starting at 12 weeks of age. Decreased performance on the pole test and Rotor-Rod in the LRRK2R1441G mice was prevented with diapocynin treatment. No loss in open field movement or rearing was found. As expected, tyrosine hydroxylase staining was similar in both the substantia nigra and striatum in all treatment groups. Together these data demonstrate that diapocynin is a viable agent for protection of neurobehavioral function.

Paraquat induces epigenetic changes by promoting histone acetylation in cell culture models of dopaminergic degeneration

Environmental neurotoxic exposure to agrochemicals has been implicated in the etiopathogenesis of Parkinson's disease (PD). The widely used herbicide paraquat is among the few environmental chemicals potentially linked with PD. Since epigenetic changes are beginning to emerge as key mechanisms in neurodegenerative diseases, herein we examined the effects of paraquat on histone acetylation, a major epigenetic change in chromatin that can regulate gene expression, chromatin remodeling, cell survival and cell death. Exposure of N27 dopaminergic cells to paraquat induced histone H3 acetylation in a time-dependent manner. However, paraquat did not alter acetylation of another core histone H4. Paraquat-induced histone acetylation was associated with decreased total histone deacetylase (HDAC) activity and HDAC4 and 7 protein expression levels. To determine if histone acetylation plays a role in paraquat-induced apoptosis, the novel HAT inhibitor anacardic acid was used. Anacardic acid treatment significantly attenuated paraquat-induced caspase-3 enzyme activity, suppressed proteolytic activation and kinase activity of protein kinase C delta (PKCδ) and also blocked paraquat-induced cytotoxicity. Together, these results demonstrate that the neurotoxic agent paraquat induced acetylation of core histones in cell culture models of PD and that the inhibition of HAT activity by anacardic acid protects against apoptotic cell death, indicating that histone acetylation may represent key epigenetic changes in dopaminergic neuronal cells during neurotoxic insults.

POMD11 Cannabinoids are neuroprotective in a human cell culture model of Parkinson's disease

BackgroundWe have previously shown a protective effect of Δ9-tetrahydrocannabinol (THC) in Parkinson's disease (PD) cell culture models associated with increased CB1 cannabis receptor cDNA. Here we investigate whether this protective...

Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: Relevance to etiopathogenesis of Parkinson's disease

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V 2O 5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V 2O 5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC 50 was determined to be 37 μM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (> fourfold) and caspase-3 (> ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCδ, 72–74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCδ kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCδ proteolytic activation, indicating that caspases mediate PKCδ cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V 2O 5-induced DNA fragmentation. Furthermore, PKCδ knockdown using siRNA protected N27 cells from V 2O 5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCδ cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

Calpain as a Potential Therapeutic Target in Parkinsons Disease

Pathophysiology of idiopathic Parkinson's disease (PD) is associated with degeneration of dopaminergic neurons and inflammatory responses in the mid-brain substantia nigra (SN). However, central dopaminergic replenishment therapeutic strategy with L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor for dopamine synthesis, does not fully rescue these cells in SN or improve motor function. Besides, prolonged use of L-DOPA worsens the clinical symptoms in PD patients. Thus, there is a possibility that other areas of central nervous system may also be affected in this disease. Spinal cord, the final coordinator of movement in the central nervous system, may be one such site that is critically affected during pathogenesis of this complex movement disorder. In this review, we summarize the evidence in support of involvement of calpain, a Ca(2+)-activated non-lysosomal protease, in spinal cord degeneration in two models of experimental parkinsonism induced by the neurotoxin 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine and also the environmental toxin rotenone. The key focus of this review is to discuss the role that calpain plays in disrupting the structural and functional integrity of the spinal cord in these experimental models of parkinsonism. A similar disruptive role of calpain has been reported earlier in SN of PD patients as well as in experimental PD animals. Studies in rodent and cell culture models of PD suggest that treatment with calpain inhibitors (e.g., calpeptin, MDL-28170) can prevent neuronal death and restore functions. Furthermore, the degradation of calpain substrates in both brain and spinal cord during pathogenesis of PD suggested a putative role of calpain, and calpain inhibition as a therapeutic strategy in PD.