Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.     

The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson''s disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.     

DJ-1 Modulates α-Synuclein Aggregation State in a Cellular Model of Oxidative Stress: Relevance for Parkinson's Disease and Involvement of HSP70

Background

Parkinson''s disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (α-syn) (PARK1) has been investigated as α-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too.

Methodology/Principal Findings

The present study addressed the question whether α-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and α-syn aggregation and toxicity. We developed an in vitro model of α-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and α-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-α-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-α-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H₂O₂ or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-α-syn neurotoxic at nanomolar scale, with the appearance of TAT-α-syn aggregates.

Conclusion/Significance

DJ-1 inactivation may thus promote α-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of α-syn fibril formation.     

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease

Background

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation.

Methods

To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.

Results

We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.

Conclusion

Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.     

α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel,Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease

Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.     

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson''s disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.The discovery of α-synuclein (α-syn) as the main component of Lewy bodies (LBs) and the identification of gene duplication and missense mutations in the α-syn gene in some familial forms of Parkinson''s disease (PD) have reinforced the central role of α-syn in the etiology of both sporadic and familial cases of PD.¹ Nevertheless, the relationship between α-syn aggregation and neurodegeneration in PD remains elusive.²A possible role for extracellular α-syn in the pathogenicity of PD emerged from the observation that newly grafted neurons in PD patients exhibit α-syn pathology similar to that of neighboring diseased cells.^{3, 4} Despite the consensus that α-syn is mainly an intracellular protein, α-syn has been detected in the cerebrospinal fluid under both pathological and healthy conditions.⁵ In addition, in vivo rodent models and cellular studies have shown that monomers⁶ and aggregated forms^{6, 7} of α-syn are secreted into the extracellular space via several mechanisms,^{7, 8} including the nonclassical endoplasmic reticulum/Golgi-independent exocytosis⁸ or the exosomal route,^{9, 10} and are then internalized by neighboring cells.⁷ This suggests that extracellular α-syn may play a critical role in the spreading of α-syn pathology throughout the brain and contributes to PD progression.Additional evidence for a causal role of extracellular α-syn in PD come from in vivo studies and cell culture models: (1) intracranial injections of pathological forms of α-syn, isolated from LBs or old mice, as well as recombinant α-syn fibrils, were shown to nucleate further α-syn aggregation, pathology spreading and trigger neurodegeneration in vivo in wild-type (WT) or transgenic mice^{11, 12, 13, 14} and rhesus monkeys;¹⁵ (2) recombinant extracellular α-syn aggregates are internalized in cultured cells and seed the aggregation of endogenous α-syn;^{12, 16, 17, 18} and (3) extracellular α-syn activates microglia that initiates or enhances nigral neurodegeneration.^{19, 20, 21} Although the toxic effects of exogenous recombinant α-syn have been thoroughly investigated in different cellular models,^{7, 17, 18, 22, 23, 24, 25} the relative contribution of monomeric, oligomeric and fibrillar forms of α-syn to the overall toxicity remains controversial. Therefore, the identification of toxic α-syn species and the molecular mechanisms by which they contribute to neurodegeneration is required to better understand how extracellular α-syn contributes to PD pathogenesis and to develop novel strategies for the diagnosis and treatment of PD and other synucleinopathies. In our study we explored the relationship between neurotoxicity and the aggregation state or amyloid formation propensity of α-syn in various cellular models.     

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines,in Yeast,and in Vivo

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.     

Differential mitochondrial roles for α-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation

Mitochondria are highly dynamic organelles with strict quality control processes that maintain cellular homeostasis. Within axons, coordinated cycles of fission-fusion mediated by dynamin related GTPase protein (DRP1) and mitofusins (MFN), together with regulated motility of healthy mitochondria anterogradely and damaged/oxidized mitochondria retrogradely, control mitochondrial shape, distribution and size. Disruption of this tight regulation has been linked to aberrant oxidative stress and mitochondrial dysfunction causing mitochondrial disease and neurodegeneration. Although pharmacological induction of Parkinson’s disease (PD) in humans/animals with toxins or in mice overexpressing α-synuclein (α-syn) exhibited mitochondrial dysfunction and oxidative stress, mice lacking α-syn showed resistance to mitochondrial toxins; yet, how α-syn influences mitochondrial dynamics and turnover is unclear. Here, we isolate the mechanistic role of α-syn in mitochondrial homeostasis in vivo in a humanized Drosophila model of Parkinson’s disease (PD). We show that excess α-syn causes fragmented mitochondria, which persists with either truncation of the C-terminus (α-syn^1–120) or deletion of the NAC region (α-syn^ΔNAC). Using in vivo oxidation reporters Mito-roGFP2-ORP1/GRX1 and MitoTimer, we found that α-syn-mediated fragments were oxidized/damaged, but α-syn^1–120-induced fragments were healthy, suggesting that the C-terminus is required for oxidation. α-syn-mediated oxidized fragments showed biased retrograde motility, but α-syn^1–120-mediated healthy fragments did not, demonstrating that the C-terminus likely mediates the retrograde motility of oxidized mitochondria. Depletion/inhibition or excess DRP1-rescued α-syn-mediated fragmentation, oxidation, and the biased retrograde motility, indicating that DRP1-mediated fragmentation is likely upstream of oxidation and motility changes. Further, excess PINK/Parkin, two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, rescued α-syn-mediated membrane depolarization, oxidation and cell death in a C-terminus-dependent manner, suggesting a functional interaction between α-syn and PINK/Parkin. Taken together, our findings identify distinct roles for α-syn in mitochondrial homeostasis, highlighting a previously unknown pathogenic pathway for the initiation of PD.Subject terms: Mechanisms of disease, Parkinson''s disease     

Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 10¹³) or varying titers (2.2 x 10¹², 1.0 x 10¹³, 5.9 x 10¹³, or 1.0 x 10¹⁴) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 10¹⁴ titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 10¹³ titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 10¹³ and 1.0 x 10¹⁴ rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 10¹³ rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.     

Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration

Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.     

A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.     

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.     

FABP3 Protein Promotes α-Synuclein Oligomerization Associated with 1-Methyl-1,2,3,6-tetrahydropiridine-induced Neurotoxicity

α-Synuclein (αSyn) accumulation in dopaminergic (DA) neurons is partly regulated by long-chain polyunsaturated fatty acids. We found that fatty acid-binding protein 3 (FABP3, H-FABP), a factor critical for arachidonic acid (AA) transport and metabolism in brain, is highly expressed in DA neurons. Fabp3 knock-out (Fabp3^−/−) mice were resistant to 1-methyl-1,2,3,6-tetrahydropiridine-induced DA neurodegeneration in the substantia nigra pars compacta and showed improved motor function. Interestingly, FABP3 interacted with αSyn in the substantia nigra pars compacta, and αSyn accumulation following 1-methyl-1,2,3,6-tetrahydropiridine treatment was attenuated in Fabp3^−/− compared with wild-type mice. We confirmed that FABP3 overexpression aggravates AA-induced αSyn oligomerization and promotes cell death in PC12 cells, whereas overexpression of a mutant form of FABP3 lacking fatty-acid binding capacity did not. Taken together, αSyn oligomerization in DA neurons is likely aggravated by AA through FABP3 in Parkinson disease pathology.     

Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease

14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson''s disease (PD) and the ability of 14-3-3s to interact with α-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, -ɛ, and -γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, -ɛ, or -γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium, whereas other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homologue (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression – particularly of the 14-3-3θ, -ɛ, and -γ isoforms – in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD.     

A Highlights from MBoC Selection: Drosophila Histone Deacetylase 6 Protects Dopaminergic Neurons against α-Synuclein Toxicity by Promoting Inclusion Formation

Parkinson''s disease (PD) is associated with progressive degeneration of dopaminergic (DA) neurons. We report for the first time that the Drosophila histone deacetylase 6 (dHDAC6) plays a critical role in the protection of DA neurons and the formation of α-synuclein inclusions by using a Drosophila PD model constructed by ectopic expression of human α-synuclein. Depletion of dHDAC6 significantly enhances the effects caused by ectopic expression of α-synuclein, namely, loss of DA neurons, retinal degeneration, and locomotor dysfunction. Expression of α-synuclein in the DA neurons leads to fewer inclusions in the brains of dHDAC6 mutant flies than in wild-type flies. Conversely, overexpression of dHDAC6 is able to suppress the α-synuclein–induced DA neuron loss and retinal degeneration and promote inclusion formation. Furthermore, mutation of dHDAC6 reinforces the accumulation of oligomers that are suggested to be a toxic form of α-synuclein. We propose that α-synuclein inclusion formation in the presence of dHDAC6 protects DA neurons from being damaged by oligomers, which may uncover a common mechanism for synucleinopathies.     

Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer's and Parkinson's diseases

Background

Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer''s disease (AD) and Parkinson''s disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.

Methodology/Principal Findings

In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.

Conclusion/Significance

These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.     

The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson’s disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers.     

Traversing a wormhole to combat Parkinson’s disease

Human movement disorders represent a significant and unresolved societal burden. Among these, the most prevalent is Parkinson’s disease (PD), a disorder afflicting millions worldwide. Despite major advances, stemming primarily from human genetics, there remains a significant gap in our understanding of what factors underlie disease susceptibility, onset, and progression. Innovative strategies to discern specific intracellular targets for subsequent drug development are needed to more rapidly translate basic findings to the clinic. Here we briefly review the recent contributions of research using the nematode roundworm Caenorhabditis elegans as a model system for identifying and characterizing gene products associated with PD. As a microscopic but multicellular and genetically tractable animal with a well-defined nervous system and an experimentally tenable lifespan, C. elegans affords significant advantages to researchers attempting to determine causative and therapeutic factors that influence neuronal dysfunction and age-associated neurodegeneration. The rapidity with which traditional genetic, large-scale genomic, and pharmacological screening can be applied to C. elegans epitomizes the utility of this animal for disease research. Moreover, with mature bioinformatic and functional genomic data readily available, the nematode is well positioned to play an increasingly important role in PD-associated discoveries.Physicists and astronomers have long posited the concept of a ‘wormhole’ as a means of rapid interstellar travel by analogy with how a worm could eat a hole from one end of an apple, through the center to the other end, and create a shortcut through the intervening space. Unfortunately, bending the fabric of space and time is not typically considered a viable option to more rapidly explore cures for neurodegenerative diseases (and would likely result in a poorly received grant application). The biological equivalent of the space exploration program, the human genome project, has unleashed an age of genomic, proteomic, metabolomic, and bioinformatic analyses that has generated a wealth of datasets primed for subsequent discovery. This exponential growth demands the development of functional means for exploiting this treasure trove of biological information. In this regard, biomedical researchers are literally turning to a worm to accelerate the path toward therapeutic advances and get to the core of mechanisms underlying Parkinson’s disease (PD).While drugs to treat the symptoms of PD have been prescribed for decades (e.g. L-DOPA), an unmet need for innovative strategies to discern disease etiology and treatments that either halt or reverse progression remains. Application of a microscopic nematode roundworm toward gaining insights into a human neurodegenerative disorder may seem impractical; yet, C. elegans affords many advantages for such research, as it has a defined cell lineage, completed genome sequence, and lifespan of only 2 weeks. As opposed to the human brain, which is estimated to have over 100 billion nerve cells, this nematode contains precisely 302 neurons for which a defined neuronal connectivity map has been determined. In this regard, C. elegans is ideal for investigation of diseases associated with neuronal dysfunction and ageing, and represents a model system that is poised to bridge the gap between basic and translational research.Interpretation of disease-associated data obtained in invertebrate systems requires downstream validation in mammals prior to establishing the therapeutic significance of any findings. The likelihood of a positive outcome is significant, however, due to the evolutionary conservation of metazoan genomes. For example, human homologs have been identified for at least 50% of C. elegans genes. Remarkably, these include all orthologs of reported genes linked to familial forms of PD (Fig. 1), with the one exception being the gene encoding α-synuclein. Despite this difference, ectopic overexpression of both wildtype and mutant (A53T) human α-synuclein led to motor deficits or DA neuron loss in C. elegans (Lasko et al., 2003). Subsequent reports also demonstrated phenotypic changes associated with driving α-synuclein expression from a variety of pan-neuronal and specific neuronal promoters (Cao et al., 2005; Ved et al., 2005; Kuwahara et al., 2006). The translational utility of C. elegans is perhaps best demonstrated by the characterization of conserved neuroprotective genes that block α-synuclein-associated degeneration in nematodes (Cooper et al., 2006; Gitler et al., 2008; Hamamichi et al., 2008).Open in a separate windowFig. 1PD-associated gene products and their prospective sites of cellular functionPD is hypothesized to be a consequence of the dysfunction of intersecting and compensatory protein degradation components, including those associated with the lysosome and autophagy, as well as those associated with the ubiquitin proteasome system (UPS). Inefficient clearance and enhanced misfolding, or expression, of α-synuclein has been shown to block intracellular trafficking and increase cytotoxicity. Accordingly, mutations in Parkin (an E3 ubiquitin ligase), ubiquitin C-terminal hydrolase-1 (UCHL-1), as well as in a lysosomal ATPase termed ATP13A2 and the Gaucher’s disease-related protein glucocerebrosidase (GBA), have further implicated protein degradation pathways in PD. PD-associated mutations in genes linked with mitochondrial function, such as those encoding DJ-1 and the PTEN-induced kinase (PINK1), presumably affect the production of reactive oxygen species (ROS), which accelerate protein damage and neurodegeneration. While mutations in the leucine-rich repeat kinase 2 (LRRK2) protein now represent the most common heritable cause of PD, the function of this large, 2527 amino acid multidomain-containing protein remains undefined.The rich, 40-year history of C. elegans genetics provides a legacy of behavioral and neuroanatomical information that researchers draw upon to elucidate relationships between gene function and PD-associated mechanisms (Bargmann, 1998). Using traditional genetic suppressor and enhancer screening, investigators have begun to dissect genetic contributors to DA neuron development and function (Chase et al., 2004). A limbless simple invertebrate will certainly never recapitulate the phenotypic behaviors associated with the tremor and dyskinesia of PD, yet this nematode does afford opportunities for accurate quantification of factors that influence DA neuron survival. Loss of DA neurons in C. elegans is not lethal and leads only to a subtle behavioral deficit where affected animals display an inability to discriminate local mechanosensory cues (Sawin et al., 2000). Unfortunately, this behavior, while quantifiable, is not robust enough to be easily used in extensive screening paradigms.Mammalian and primate modeling approaches to PD have traditionally involved use of neurotoxins to induce DA neuron loss and evaluate the consequences of neurodegeneration. Among these toxins are 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), as well as the pesticides rotenone and paraquat. Worms are susceptible to these toxins (Nass et al., 2002; Braungart et al., 2004; Cao et al., 2005); moreover, the defined and transparent neuroanatomy of C. elegans includes precisely eight neurons that produce DA, thereby enabling an unparalleled level of quantitative analysis of neurodegeneration and protection by use of fluorescent protein labeling (Fig. 2). Furthermore, these animals can be evaluated in specific genetic or transgenic backgrounds to screen for factors that confer either neuroprotection or enhancement of degeneration. One neuroprotective gene product termed TOR-2, a human torsinA-related protein with molecular chaperone-like activity (Caldwell et al., 2008), was shown to deter multiple forms of toxic insults to DA neurons, including 6-OHDA, excess intracellular DA production, and α-synuclein overexpression (Cao et al., 2005). The neuroprotective capacity of torsin-like proteins renders them intriguing candidates for subsequent drug targeting and validation in mammalian systems, where human torsinA is endogenously expressed in DA neurons (Augood et al., 1998).Open in a separate windowFig. 2Anterior dopamine neurons of C. elegansCell bodies and processes of the six anterior DA neurons including two pairs of the CEP (cephalic) neurons and one pair of ADE (anterior deirid) neurons, as illuminated using GFP driven from the dopamine transporter promoter (P_dat-1::GFP). Two additional DA neurons (PDEs) are found in the posterior (not shown).Directed and comprehensive screens have been undertaken to define chemical and genetic effectors of worm DA neuron sensitivity to 6-OHDA (Nass et al., 2005; Maranova and Nichols, 2007). These studies identified a collection of intragenic mutations in the gene sequence encoding the key protein required for DA reuptake into presynaptic neurons, the DA transporter (DAT-1). Structural and functional relationships revealed through this work have implications not only for PD, but also for other disorders associated with DAT function (e.g. depression, ADHD). It has also been shown that worm dat-1 knockout mutants exhibit diminished α-synuclein-dependent degeneration, which indicates a possible role for DAT in maintaining an important balance of intraneuronal levels of DA, the dysregulation of which may contribute to cytotoxicity (Cao et al., 2005).Elegant studies in Drosophila have demonstrated the capacity for model systems to reveal unsuspected relationships between PD gene products, such as PINK1 and Parkin, and their impact on mitochondrial integrity and morphology (Clark et al., 2006; Park et al., 2006; Poole et al., 2008). Likewise, the ability of worm research to link genotype to phenotype in this manner is accelerating our understanding of the underlying nature of PD. Ved et al. investigated the functional consequences of rotenone-induced stress and reported that pan-neuronal overexpression of human αsynuclein (wild-type or A53T mutant), RNAi knockdown of a C. elegans DJ-1 ortholog (B0432.2), or a deletion mutant of the C. elegans parkin ortholog (pdr-1), all produced similar patterns of mitochondrial vulnerability in response to pharmacological challenges associated with complex I inhibition (Ved et al., 2005). Furthermore, the effect of rotenone, which led to mortality in these animals, was more severe in α-synuclein-expressing strains and the pdr-1 deletion mutant than in control animals of wild-type background. Initial work on worm PDR-1 revealed that this Parkin ortholog, an E3 ubiquitin ligase, cooperates with conserved degradation machinery to mediate ubiquitin conjugation (Springer et al., 2005). Co-expression of C. elegans pdr-1 and α-synuclein variants in human cell cultures showed that a truncated protein derived from an in-frame pdr-1 (lg103) deletion allele causes aggregation of α-synuclein, similar to Parkin variants isolated from PD patients. This altered gene product also resulted in proteotoxicity and hypersensitivity to ER stress (Springer et al., 2005). These examples demonstrate how mutant analysis in C. elegans offers a powerful strategy for uncovering functional relationships between gene products with key roles in PD pathogenesis in mammalian cells.C. elegans has been recently used to show a protective role for human LRRK2 (leucine-rich repeat kinase 2) against mitochondrial toxicity induced by rotenone, an effect potentiated by knockdown of an endogenous worm ortholog, lrk-1 (Wolozin et al., 2008). These data are surprising when considering a PD-associated G2019S mutant version of LRRK2 that exhibits increased kinase activity was also found to be protective; kinase activity has been suggested to contribute to the pathogenesis associated with LRRK2 neurotoxicity (West et al., 2007). An initial report on lrk-1 function in C. elegans indicated a role for the product of this gene in establishing neuronal polarity (Sakaguchi-Nakashima et al., 2007). As mutations in LRRK2 now represent the most common genetic cause of PD (Haugarvoll et al., 2008), further insights gleaned from C. elegans on this important, yet relatively uncharacterized, protein family will undoubtedly be informative.The capacity for genomic-scale screening has brought C. elegans to the forefront of modern functional analysis (Kamath and Ahringer, 2003). In contrast to mammals, where individual knockouts of mice are expensive and time consuming, worms are an efficient and economical alternative. Hamamichi et al. took a hypothesis-driven approach toward identification of putative genetic modifiers of PD via a multi-tiered screen for C. elegans genes that impact the misfolding of transgenic human α-synuclein, as well as neuroprotection, in vivo (Hamamichi et al., 2008). This study encompassed RNAi knockdown of nearly 900 bioinformatically prioritized gene targets, comprising components of cellular pathways implicated in protein folding or degradation, as well as gene products that are either co-expressed or interact with worm orthologs of familial PD genes. From this varied, but biased, ‘guilt by association’ list of targets, 20 candidate genes emerged as having the greatest propensity to influence α-synuclein misfolding. Internal validation was evident within this short list, as included were the worm homologs of two established recessive PD genes (DJ-1, PINK1), as well as a gene (ULK2) shown to be one of only six identified in a genome-wide association study of polymorphisms in PD patients (Fung et al., 2006). Importantly, overexpression of select cDNAs in C. elegans DA neurons revealed that five of seven gene products tested, chosen from the primary effectors of α-synuclein misfolding identified in the RNAi screen, exhibited significant protection from age- and dose-dependent neurodegeneration induced by α-synuclein (Hamamichi et al., 2008). Thus, application of C. elegans in this context uncovered functionally evaluated targets identified by the two primary clinical criteria associated with PD: α-synuclein accumulation and DA neurodegeneration.These genes, which included uncharacterized proteins, as well as regulators of autophagy, lysosomal function, cellular trafficking, and G-protein signaling, now represent outstanding candidates for strategically targeted drug development and validation in mammalian systems. A more recently conducted unbiased genome-wide RNAi screen for genetic factors that influence αsynuclein inclusion formation in C. elegans also yielded an over-representation of genes encoding components of vesicular trafficking, as well as specific age-associated genes (e.g. sir-2.1) (van Ham et al., 2008). Similar validation of the effect that these additional targets may have on DA neuron survival will likely reveal functionally significant relationships as well. Associations between PD and lysosomal degradation are of growing interest following the identification of a lysosomal ATPase, ATP13A2 (PARK9), as a gene product linked to hereditary early-onset PD with dementia (Ramirez et al., 2006), in addition to the discovery of a higher incidence of PD in patients with mutations in the Gaucher’s disease-associated gene, GBA, encoding the lysosomal storage-enzyme glucocerebrosidase. (Aharon-Peretz et al., 2004; Clark et al., 2007). It is easy to envision an expansion of such gene-specific data and the application of C. elegans toward investigating the functional consequences of single-nucleotide polymorphisms (SNPs) found in human populations, as these may potentially lead to genetic biomarkers of disease susceptibility.The burgeoning promise that a microscopic worm may contribute toward the goal of drug discovery for PD has become a tangible reality. Containing a rudimentary nervous system, unlike single-celled organisms, but more amenable to transgenic analysis and drug screening than mammals, C. elegans serves as an excellent intermediary bottleneck in translational research pipelines to characterize therapeutic gene and drug targets across animal models. Concerted efforts toward therapeutic development have been initiated that exploit the high-throughput screening capabilities of yeast cells to define numerous gene targets of interest, followed by subsequent evaluation of their neuroprotective capacity in worms, fruit flies and rat neuronal cultures (Cooper et al., 2006; Gitler et al., 2008). This approach has already revealed that overproduction of αsynuclein leads to a cytotoxic blockage in intracellular vesicular trafficking that can be alleviated by specific members of the Rab protein family (Cooper et al., 2006; Gitler et al., 2008). Thus, through the employment of a combination of powerful model systems, not only has a prospective cellular cause of PD been illuminated, but targeted screens for small molecules that protect against underlying functional aspects of neurodegeneration are now possible.When envisioning future directions whereby C. elegans will benefit PD research, several inescapable advantages of this model system should be considered. The most obvious of these is the well-established use of the worm for investigating mechanisms of aging (Kenyon, 2005). Indeed, the only definitive risk factor for PD is aging, where PD symptomatically affects over 2% of people above age 65. Extensive studies on worm longevity mutants (e.g. daf-2 or age-1) have demonstrated that evolutionarily conserved mechanisms are shared between invertebrates and mammals. Microarray and genomic-scale RNAi analyses conducted in age-extending backgrounds have yielded significant insights into gene sets that implicate dietary restriction and insulin-like signaling pathways as crucial mediators of lifespan (Murphy et al., 2003; Panowski et al., 2007). The vital, but poorly understood, interface between aging and age-associated neurodegenerative disease represents an exciting frontier that is readily explored using C. elegans.Furthermore, in the advent of the microRNA (miRNA) revolution, our nascent understanding of putative regulatory roles for miRNAs in neuron function will likely soon interface with our understanding of neurological disease and PD (Kosik, 2006; Kim et al., 2007). Considering the pioneering role worm research has played in defining miRNA function, a universe of possibilities exist, as worm researchers are well-poised to explore relationships between miRNAs, ageing and neuroprotective gene activity (Ibanez-Ventoso, 2007).

Advantages of C. elegans as a model for Parkinson’s disease

• All orthologs of genes linked to familial PD have been identified in C. elegans, except α-synuclein
• Ectopic overexpression of α-synuclein has neurotoxic effects in C. elegans, which are blocked by neuroprotective genes
• C. elegans has a simple nervous system (302 neurons compared with over 100 billion in humans) that is amenable to quantitative analysis of neurodegeneration
• C. elegans is susceptible to toxins commonly used to model neurodegeneration
• C. elegans studies predict relationships between cellular signaling, trafficking and protein degradation pathways, which are being tested for their susceptibility to targeted drug development for PD

Finally, although the primary advances in PD etiology have come through human genetics, the largely idiopathic nature of PD remains linked to inexplicable environmental causes. C. elegans research into innate immunity and cellular stress response has already provided mechanistic insights into organismal defenses and environmental influences on homeostasis (Kim et al., 2002; Mohri-Shiomi and Garsin, 2008). An expansion of neurotoxicity studies conducted in C. elegans is warranted and may yield a greater understanding of the interplay between genetic composition and environmental factors such as heavy metals, pesticides and other untested exposures.The worm is unquestionably a powerful system, yet it has its limitations: the anatomical and functional connectivity of the neuronal circuitry within this simple nematode cannot recapitulate the complex features of mammalian dopamine neurons or mimic the precise behavioral deficits associated with their loss. Likewise, as a cautionary caveat to inferring conservation of function from genetic interaction data, Tischler et al. demonstrated that synthetic lethal gene interactions between yeast and worm genes are not significantly conserved (Tischler et al., 2008). In this context, as the march toward systems biology proceeds and integrated analyses of gene or protein activity continue to lead to an increasing number of complex datasets, our ability to eventually define causes and cures will depend on functional strategies for wading through the emerging ‘information overload’. Science will benefit from the efficient manner by which C. elegans research can contribute to the quest for translational and personalized medical breakthroughs, by boldly going where no worm has gone before.