Nutrient deprivation induces α-synuclein aggregation through endoplasmic reticulum stress response and SREBP2 pathway.

Abnormal accumulation of filamentous α-synuclein (α-syn) in neurons, regarded as Lewy bodies (LBs), are a hallmark of Parkinson disease (PD). Although the exact mechanism(s) underlying LBs formation remains unknown, autophagy and ER stress response have emerged as two important pathways affecting α-syn aggregation. In present study we tested whether cells with the tetracycline-off inducible overexpression of α-syn and accumulating α-syn aggregates can benefit from autophagy activation elicited by nutrient deprivation (ND), since this approach was reported to effectively clear cellular polyglutamine aggregates. We found that nutrient deprivation of non-induced cells did not affect cell viability, but significantly activated autophagy reflected by increasing the level of autophagy marker LC3-II and autophagic flux and decrease of endogenous α-syn. Cells with induced α-syn expression alone displayed autophagy activation in an α-syn dose-dependent manner to reach a level comparable to that found in non-induced, nutrient deprived counterparts. Nutrient deprivation also activated autophagy further in α-syn induced cells, but the extent was decreased with increase of α-syn dose, indicating α-syn overexpression reduces the responsiveness of cells to nutrient deprivation. Moreover, the nutrient deprivation enhanced α-syn aggregations concomitant with significant increase of apoptosis as well as ER stress response, SREBP2 activation and cholesterolgenesis. Importantly, α-syn aggregate accumulation and other effects caused by nutrient deprivation were counteracted by knockdown of SREBP2, treatment with cholesterol lowering agent-lovastatin, or by GRP78 overexpression, which also caused decrease of SREBP2 activity. Similar results were obtained from studies of primary neurons with α-syn overexpression under nutrient deprivation. Together our findings suggested that down-regulation of SREBP2 activity might be a means to prevent α-syn aggregation in PD via reducing cholesterol levels.

Dopamine prevents lipid peroxidation-induced accumulation of toxic α-synuclein oligomers by preserving autophagy-lysosomal function.

The formation of Lewy bodies containing α-synuclein (α-syn), prominent loss of dopaminergic neurons and dopamine (DA) deficiency in substantia nigra and striatum are histopathological and biochemical hallmarks of Parkinson's disease (PD). Multiple lines of evidence have indicated that a critical pathogenic factor causing PD is enhanced production of reactive oxygen species (ROS), which reacts readily with polyunsaturated fatty acids to cause lipid peroxidation (LPO). LPO products have been shown to facilitate assembly of toxic α-syn oligomers in in vitro studies. Since DA is prone to autoxidation and cause ROS, it has been suggested that interactions among DA, LPO, and α-syn play an important role in neuronal loss in PD. However, the exact mechanism(s) remains unclear. We addressed this issue using a neuronal cell model which inducibly expresses human wild-type α-syn by the tetracycline off (Tet-Off) mechanism and stably expresses high levels of DA transporter. Under retinoic acid elicited neuronal differentiation, cells with or without overexpressing α-syn and with or without exposure to LPO inducer-arachidonic acid (AA), plus 0-500 µM of DA were assessed for the levels of LPO, α-syn accumulation, cell viability, and autophagy. AA exposure elicited similar LPO levels in cells with and without α-syn overexpression, but significantly enhanced the accumulation of α-syn oligomers and monomers only in cultures with Tet-Off induction and decreased cell survival in a LPO-dependent manner. Surprisingly, DA at low concentrations (<50 µM) protected cells from AA cytotoxicity and α-syn accumulation. Such effects were attributed to the ability of DA to preserve autophagic-lysosomal function compromised by the AA exposure. At high concentrations (>100 µM), DA exposure enhanced the toxic effects of AA. To our knowledge, this is the first report showing biphasic effects of DA on neuronal survival and α-syn accumulation.

Corticobasal degeneration with olivopontocerebellar atrophy and TDP-43 pathology: an unusual clinicopathologic variant of CBD.

Corticobasal degeneration (CBD) is a disorder affecting cognition and movement due to a progressive neurodegeneration associated with distinctive neuropathologic features, including abnormal phosphorylated tau protein in neurons and glia in cortex, basal ganglia, diencephalon, and brainstem, as well as ballooned neurons and astrocytic plaques. We identified three cases of CBD with olivopontocerebellar atrophy (CBD-OPCA) that did not have α-synuclein-positive glial cytoplasmic inclusions of multiple system atrophy (MSA). Two patients had clinical features suggestive of progressive supranuclear palsy (PSP), and the third case had cerebellar ataxia thought to be due to idiopathic OPCA. Neuropathologic features of CBD-OPCA are compared to typical CBD, as well as MSA and PSP. CBD-OPCA and MSA had marked neuronal loss in pontine nuclei, inferior olivary nucleus, and Purkinje cell layer. Neuronal loss and grumose degeneration in the cerebellar dentate nucleus were comparable in CBD-OPCA and PSP. Image analysis of tau pathology showed greater infratentorial tau burden, especially in pontine base, in CBD-OPCA compared with typical CBD. In addition, CBD-OPCA had TDP-43 immunoreactive neuronal and glial cytoplasmic inclusions and threads throughout the basal ganglia and in olivopontocerebellar system. CBD-OPCA met neuropathologic research diagnostic criteria for CBD and shared tau biochemical characteristics with typical CBD. These results suggest that CBD-OPCA is a distinct clinicopathologic variant of CBD with olivopontocerebellar TDP-43 pathology.

Adenosine monophosphate-activated protein kinase overactivation leads to accumulation of α-synuclein oligomers and decrease of neurites.

Neuronal inclusions of α-synuclein (α-syn), termed Lewy bodies, are a hallmark of Parkinson disease (PD). Increased α-syn levels can occur in brains of aging human and neurotoxin-treated mice. Because previous studies have shown increased brain lactate levels in aging brains, in PD affected subjects when compared with age-matched controls, and in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), we tested the effects of lactate exposure on α-syn in a cell-based study. We demonstrated that (1) lactate treatment led to α-syn accumulation and oligomerization in a time- and concentration-dependent manner; (2) such alterations were mediated via adenosine monophosphate-activated protein kinase (AMPK) and associated with increasing cytoplasmic phosphorylated AMPK levels; (3) AMPK activation facilitated α-syn accumulation and phosphorylation; (4) lactate treatment or overexpression of the active form of AMPK decreased α-syn turnover and neurite outgrowth; and (5) Lewy body-bearing neurons displayed abnormal cytoplasmic distribution of phosphorylated AMPK, which normally is located in nuclei. Together, our results suggest that chronic neuronal accumulation of α-syn induced by lactate-triggered AMPK activation in aging brains might be a novel mechanism underlying α-synucleinopathies in PD and related disorders.

ER stress response plays an important role in aggregation of α-synuclein.

BACKGROUND: Accumulation of filamentous α-synuclein as Lewy bodies is a hallmark of Parkinson's disease. To identify the mechanisms involved in α-synuclein assembly and determine whether the assemblies are cytotoxic, we developed a cell model (3D5) that inducibly expresses wild-type human α-synuclein and forms inclusions that reproduce many morphological and biochemical characteristics of Lewy bodies. In the present study, we evaluated the effects of several histone deacetylase inhibitors on α-synuclein aggregation in 3D5 cells and primary neuronal cultures. These drugs have been demonstrated to protect cells transiently overexpressing α-synuclein from its toxicity. RESULTS: Contrary to transient transfectants, the drug treatment did not benefit 3D5 cells and primary cultures. The treated were less viable and contained more α-synuclein oligomers, active caspases 3 and 9, as well as ER stress markers than non-treated counterparts. The drug-treated, induced-3D5 cells, or primary cultures from transgenic mice overexpressing (<2 fold) α-synuclein, displayed more α-synuclein oligomers and ER stress markers than non-induced or non-transgenic counterparts. Similar effects were demonstrated in cultures treated with tunicamycin, an ER stressor. These effects were blocked by co-treatment with salubrinal, an ER stress inhibitor. In comparison, co-treatment with a pan caspase inhibitor protected cells from demise but did not reduce α-synuclein oligomer accumulation. CONCLUSIONS: Our results indicate that an increase of wild-type α-synuclein can elicit ER stress response and sensitize cells to further insults. Most importantly, an increase of ER stress response can promote the aggregation of wild type α-synuclein.

Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells.

Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.

Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.

Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D.

Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.

Cytosine beta-D-arabinofuranoside used as a paradigm modifier to increase production of tau aggregates in a cellular model of tauopathy.

Intraneuronal deposition of filamentous tau is a hallmark of Alzheimer's disease (AD) and related tauopathies. We developed previously a cellular model recapitulating such tau anomaly and demonstrated therein consistent production of 70-kD tau. Importantly, the 70-kD species appears to derive from tau fragments with carboxy-terminal truncation and is larger than intact tau in size, suggesting the oligomeric nature in its assembly from tau. To generate the 70-kD tau in sufficient quantity for its characterization at the molecular level, we explored and demonstrated herein that cytosine beta-D-arabinofuranoside is a useful paradigm modifier to increase production of the 70-kD tau. Such oligomeric tau was enriched thereafter by immunoprecipitation to remove tau species with intact carboxy-terminus. Two-dimensional gel electrophoresis revealed that the 70-kD tau has an isoelectric point of 5.8-6.0. Future elucidation of key aggregates will provide valuable insights into the natural history of neurofibrillary degeneration and identify novel targets to develop therapeutic interventions.

Assembly of filamentous tau aggregates in human neuronal cells.

Intraneuronal deposition of microtubule-associated protein tau in filamentous aggregates constitutes a pathological hallmark of neurofibrillary degeneration that is characteristic of Alzheimer's disease (AD) and related disorders known collectively as tauopathies. Formation of such fibril inclusions, consisting of hyperphosphorylated tau in multiple isoforms, correlates with the severity of cognitive decline in AD. How neurofibrillary pathology evolves in tauopathy remains unclear at present, but availability of a cellular model with robust tau aggregation will permit experimental scrutiny of the mechanistic process leading to such neurodegeneration. Through the use of a serial transfection strategy in conjunction with a tau minigene construct, we succeeded in generating conditional transfectants of human neuronal lineage that overproduce wild-type human brain tau in isoforms 4R0N, 3R1N and 4R1N via TetOff and ecdysone inducible expression mechanisms. Such transgenic overexpression of tau in multiple isoforms facilitated the assembly of filamentous tau aggregates that exhibit immunoreactivities, physicochemical properties, and ultrastructural attributes reminiscent of those found in human tauopathies. The conditional tau transfectants thus provide us with a useful tool to elucidate the molecular and cellular events leading to neurofibrillary degeneration and a convenient means to test hypothetical mechanisms implicated in the etiopathogenesis of AD and related tauopathies.