Mutant Pink1 induces mitochondrial dysfunction in a neuronal cell model of Parkinson's disease by disturbing calcium flux

Parkinson's disease (PD) is characterized by accumulation of α-synuclein (α-syn) and degeneration of neuronal populations in cortical and subcortical regions. Mitochondrial dysfunction has been considered a potential unifying factor in the pathogenesis of the disease. Mutations in genes linked to familial forms of PD, including SNCA encoding α-syn and Pten-induced putative kinase 1 ( PINK1 ), have been shown to disrupt mitochondrial activity. We investigated the mechanisms through which mutant Pink1 might disrupt mitochondrial function in neuronal cells with α-syn accumulation. For this purpose, a neuronal cell model of PD was infected with virally-delivered Pink1, and was analyzed for cell survival, mitochondrial activity and calcium flux. Mitochondrial morphology was analyzed by confocal and electron microscopy. These studies showed that mutant (W437X) but not wildtype Pink1 exacerbated the alterations in mitochondrial function promoted by mutant (A53T) α-syn. This effect was associated with increased intracellular calcium levels. Co-expression of both mutant Pink1 and α-syn led to alterations in mitochondrial structure and neurite outgrowth that were partially ameliorated by treatment with cyclosporine A, and completely restored by treatment with the mitochondrial calcium influx blocker Ruthenium Red, but not with other cellular calcium flux blockers. Our data suggest a role for mitochondrial calcium influx in the mechanisms of mitochondrial and neuronal dysfunction in PD. Moreover, these studies support an important function for Pink1 in regulating mitochondrial activity under stress conditions.     

Seeding induced by α-synuclein oligomers provides evidence for spreading of α-synuclein pathology

Lewy bodies, α-synuclein (α-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of α-syn in the graft is that the aggregation of α-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro -generated α-syn oligomers induce transmembrane seeding of α-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that α-syn oligomers form as a dynamic mixture of oligomer types with different properties and that α-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular α-syn oligomers can induce intracellular α-syn aggregation, therefore we hypothesize that a similar mechanism might lead to α-syn pathology propagation.     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

Effects of the cholesterol-lowering compound methyl-beta-cyclodextrin in models of alpha-synucleinopathy

The aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking, probably via lipid binding, and interactions with lipids have been shown to regulate alpha-syn aggregation. In this context, the main objective of this study was to determine whether methyl-beta-cyclodextrin (MbetaCD), a cholesterol-extracting agent, interfered with alpha-syn accumulation in models of synucleinopathy. For this purpose, we studied the effects of MbetaCD on the accumulation of alpha-syn in a transfected neuronal cell line and in transgenic mice. Immunoblot analysis showed that MbetaCD reduced the level of alpha-syn in the membrane fraction and detergent-insoluble fraction of transfected cells. In agreement with the in vitro studies, treatment of mice with MbetaCD resulted in decreased levels of alpha-syn in membrane fractions and reduced accumulation of alpha-syn in the neuronal cell body and synapses. Taken together, these results suggest that changes in cholesterol and lipid composition using cholesterol-lowering agents may be used as a tool for the treatment of synucleinopathies.     

alpha-Synuclein, oxidative stress and apoptosis from the perspective of a yeast model of Parkinson's disease

The neuronal protein α-synuclein (α-syn) has been suggested to be one of the factors linked to Parkinson's disease (PD). Several organisms, including the rat, mouse, worm, and fruit fly, are being used to study α-syn pathobiology. A new model organism was recently added to this armamentarium: the budding yeast Saccharomyces cerevisiae . The yeast system recapitulates many of the findings made with higher eukaryotes. For example, yeast cells expressing α-syn accumulate lipid droplets, have vacuolar/lysosomal defects, and exhibit markers of apoptosis, including the externalization of phosphatidylserine, the release of cytochrome c , and the accumulation of reactive oxygen species. This MiniReview focuses on the mechanisms by which α-syn induces oxidative stress and the mechanisms by which yeast cells respond to this stress. Three classes of therapeutics are discussed.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.     

On the mechanism of internalization of α-synuclein into microglia: roles of ganglioside GM1 and lipid raft

α-Synuclein (α-syn) has been known to be a key player of the pathogenesis of Parkinson's disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of α-syn into cells has also been observed. In this study, we observed that dl -threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric α-syn into BV-2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of α-syn internalization in dl -threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-treated BV-2 cells. Furthermore, inhibition of clathrin-, caveolae-, and dynamin-dependent endocytosis did not prevent the internalization of α-syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of α-syn. In addition, we further confirmed these observations by co-culture system of BV-2 cells and α-syn-over-expressing SH-SY5Y cells. These findings suggest that extracellular α-syn is internalized into microglia via GM1 as well as hitherto-unknown protein receptors in clathrin-, caveolae-, and dynamin-independent, but lipid raft-dependent manner. Elucidation of the mechanism involved in internalization of α-syn should be greatly helpful in the development of new treatments of α-syn-related neurodegenerative diseases.     

Tau enhances α-synuclein aggregation and toxicity in cellular models of synucleinopathy

Background

The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinson''s disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.

Methodology/Principal Findings

We used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET–based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity.

Conclusions/Significance

Our data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.     

The Parkinson disease-associated A30P mutation stabilizes α-synuclein against proteasomal degradation triggered by heme oxygenase-1 over-expression in human neuroblastoma cells

Proteosomal degradation of proteins is one of the major mechanisms of intracellular protein turnover. Failure of the proteosome to degrade misfolded protein is implicated in the accumulation of α-synuclein in Parkinson's disease (PD). Heme oxygenase-1 (HO-1), an enzyme that converts heme to free iron, carbon monoxide (CO) and biliverdin (bilirubin precursor) is expressed in response to various stressors. HO-1 is up-regulated in PD- and Alzheimer's disease-affected neural tissues. In this study, we found that HO-1 over-expression engenders dose-dependent decreases in α-synuclein protein levels in human neuroblastoma M17 cells. When over-expression of HO-1 was silenced in HO-1 transfected cells, level of α-synuclein was restored. Likewise, treatment of HO-1 over-expressing cells with the HO-1 inhibitor, tin mesoporphyrin, the iron chelator deferoxamine or antagonist of CO-dependent cGMP activation, methylene blue, mitigated the HO-1-induced reduction in α-synuclein levels. Furthermore, when HO-1 over-expressing cells were treated with the proteosome inhibitors, lactacystin and MG132, level of α-synuclein was almost completely restored. In contrast to the effect on α-synuclein [wild-type (WT)] levels, HO-1 over-expression did not significantly impact PD-associated α-synuclein (A30P) levels in these cells. HO-1 also significantly reduced aggregation of α-synuclein (WT) but not that of A30P. Our results suggest that HO-1, which is expressed when neurons are exposed to toxic stimuli capable of inducing protein misfolding, triggers proteosomal degradation of proteins and prevents intracellular accumulation of protein aggregates and inclusions. Resistance to HO-1 induced proteosomal degradation may render the familial PD-associated A30P mutation prone to toxic intracellular aggregation.     

Dopamine and the Dopamine Oxidation Product 5,6-Dihydroxylindole Promote Distinct On-Pathway and Off-Pathway Aggregation of α-Synuclein in a pH-Dependent Manner

The deposition of α-synuclein (α-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate α-syn aggregation, it is unclear which other factors can regulate the actions of DA on α-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of α-syn by DA. We show that α-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of α-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of α-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of α-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with β-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with α-syn to generate the α-syn aggregates implicated in Parkinson's disease.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

Modulation of α-Synuclein Aggregation by Dopamine: A Review

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by (1) the selective loss of dopaminergic neurons in the substantia nigra and (2) the deposition of misfolded α-synuclein (α-syn) as amyloid fibrils in the intracellular Lewy bodies in various region of the brain. Current thinking suggests that an interaction between α-syn and dopamine (DA) leads to the selective death of neuronal cells and the accumulation of misfolded α-syn. However, the exact mechanism by which this occurs is not fully defined. DA oxidation could play a key role is the pathogenesis of PD by causing oxidative stress, mitochondria dysfunction and impairment of protein metabolism. Here, we review the literature on the role of DA and its oxidative intermediates in modulating the aggregation pathways of α-syn.     

Designed hairpin peptides interfere with amyloidogenesis pathways: fibril formation and cytotoxicity inhibition, interception of the preamyloid state

Hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites modulate the aggregation of two unrelated amyloidogenic systems, human pancreatic amylin (hAM) and α-synuclein (α-syn), associated with type II diabetes and Parkinson's disease, respectively. In the case of hAM, we have previously reported that inhibition of amyloidogenesis is observed as an increase in the lag time to amyloid formation and a diminished thioflavin (ThT) fluorescence response. In this study, a reduced level of hAM fibril formation is confirmed by transmission electron microscopy imaging. Several of the hairpins tested were significantly more effective inhibitors than rat amylin. Moreover, a marked inhibitory effect on hAM-associated cytotoxicity by the more potent hairpin peptide is demonstrated. In the case of α-syn, the dominant effect of active hairpins was, besides a weakened ThT fluorescence response, the earlier appearance of insoluble aggregates that do not display amyloid characteristics with the few fibrils observed having abnormal morphology. We attribute the alteration of the α-synuclein aggregation pathway observed to the capture of a preamyloid state and diversion to nonamyloidogenic aggregates. These β-hairpins represent a new class of amyloid inhibitors that bear no sequence similarity to the amyloid-producing polypeptides that are inhibited. A mechanistic rationale for these effects is proposed.     

Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.     

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis

To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.     

Characterization of CLP36/Elfin/PDLIM1 in the nervous system

CLP36, one of the α-Actinin Associated LIM Protein (ALP)/Enigma family proteins, has a wide tissue distribution, but little is known about its expression and role in the nervous system. We show here that CLP36 is expressed in sensory ganglia but not in the CNS of adult rats. In primary dorsal root ganglion (DRG) neurons, CLP36 is distributed in the soma and neurites with enrichment in the growth cones. CLP36 forms a complex with α-actinin and is localized to actin cytoskeleton. To examine the role of CLP36 in neuronal cells, we transfected PC12 cells with a series of CLP36 deletion mutants and found that over-expression of CLP36 PDZ domain affects neurite outgrowth. Reduction of CLP36 function in PC12 cells by RNA interference (RNAi) induced lamellipodial protrusions around cell periphery and activated growth-cone movements, resulting in an increase in the length and number of neurites. Similarly, inhibition of CLP36 in primary DRG neurons increased the rate of neurite-bearing cells. We also found that CLP36 is up-regulated in DRG neurons and facial motoneurons after nerve injury. These findings suggest that CLP36 serves as a scaffold to form a multiprotein complex that regulates actin cytoskeleton dynamics and plays a role in controlling neurite outgrowth.     

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. 相似文献