Ginkgolic Acid Promotes Autophagy-Dependent Clearance of Intracellular Alpha-Synuclein Aggregates

Abstract

The accumulation of intracytoplasmic inclusion bodies, known as Lewy bodies, composed of aggregates of the alpha-synuclein (α-syn) protein, is the principal pathological characteristic of Parkinson’s Disease (PD) and may lead to the degeneration of dopaminergic neurons. Currently, there is no medication that can effectively promote the efficient clearance of these pathological aggregates. In this study, we assessed the effect of ginkgolic acid (GA), a natural compound extracted from Ginkgo biloba leaves that inhibits SUMOylation among other pathways, on the clearance of α-syn aggregates. The experiments were conducted in SH-SY5Y neuroblastoma cells and rat primary cortical neurons. Depolarization of SH-SY5Y neuroblastoma cells and rat primary cortical neurons with potassium chloride (KCl) was employed to induce α-syn aggregate formation. Cells pre-treated with either GA or its structurally related compound, anacardic acid, showed a significant decrease in intracytoplasmic aggregates immunopositive for α-syn and SUMO-1. Moreover, an increased frequency of autophagosomes was observed with both compounds. Treatment with GA after the formation of aggregates, 24 hours post-depolarization, also significantly reduced the number of α-syn aggregate-bearing cells, indicating the clearance of pre-formed aggregates. However, when autophagy inhibitors were used, they blocked the GA-dependent clearance of α-syn aggregates but did not prevent the increase in autophagosome frequency. Western blot analysis revealed that the reduction in α-syn aggregate frequency observed with GA pre-treatment was not associated with a significant change in the abundance of SUMO conjugates. These findings suggest that GA can promote the autophagy-dependent clearance of α-syn aggregates and may have potential as a disease-modifying therapy.

Keywords: Parkinson’s Disease, ginkgolic acid, SUMO, autophagy, alpha-synuclein.

Introduction

Parkinson’s disease is a multi-system proteinopathy characterized by the progressive loss of dopaminergic nigrostriatal neurons, resulting in a variety of motor deficits such as resting tremor, muscular rigidity, bradykinesia, and postural instability, as well as non-motor symptoms including hyposmia and autonomic dysfunctions. The neuropathological hallmark of PD is the widespread presence of intracellular inclusion bodies (Lewy bodies) and neuritic deposits (Lewy neurites), which primarily consist of phosphorylated α-synuclein. Although Lewy body formation is thought to initially serve as a protective cellular response, their maturation eventually becomes toxic and can trigger neuronal death. The abnormal accumulation of intracellular α-syn aggregates is also associated with other α-synucleinopathies, including multiple system atrophy and dementia with Lewy bodies. Various factors, such as oxidative and nitrosative stress, post-translational modifications, proteolytic stress, and calcium imbalance, contribute to the formation of these cytotoxic α-syn species and subsequent neuronal degeneration. Reversal and prevention of α-syn aggregation may offer cytoprotection in α-synucleinopathies, and recent research has focused on approaches to promote aggregate clearance.

Neuropathological and experimentally induced α-syn inclusion bodies accumulate small ubiquitin-like modifier-1 (SUMO-1). SUMOylation is involved in numerous cellular pathways by regulating protein-protein and protein-DNA interactions. Similar to ubiquitination, SUMOylation is an enzymatic cascade that includes E1 (SUMO-activating), E2 (SUMO-specific conjugating, Ubc9), and E3 (SUMO ligase, such as PIAS3) enzymes. SUMO-1 is known to associate with cytoplasmic α-syn inclusion bodies in PD, multiple system atrophy, and dementia with Lewy bodies, as well as with intranuclear inclusion bodies like polyglutamine aggregates in Huntington’s disease. Recent research indicates both direct and indirect links between SUMOylation, neurodegenerative disease pathology, and cellular responses to misfolding and protein aggregation. For example, both wild-type and disease-linked mutant α-synuclein proteins are substrates for SUMOylation, directly modulating their solubility, while connections between SUMOylation and autophagy suggest an indirect role through the cellular response to protein aggregate accumulation. Overexpression of PIAS2, a SUMO ligase for α-syn, has been shown to increase the accumulation of intracellular aggregates of PD-linked α-syn mutants. SUMO-1 has also been observed to co-localize with lysosomes clustered around or embedded in α-syn deposits in pathological specimens and cell models, where it is conjugated to Hsp90.

The degradation of aberrant proteins, including misfolded proteins and defective organelles, is achieved through two main proteolytic pathways: the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP). Dysfunction in either pathway can impede protein clearance in neuronal and glial cytoplasm, contributing to various neurodegenerative diseases. Larger protein aggregates and damaged organelles, which cannot be processed by the proteasome, are eliminated via macroautophagy. Macroautophagy, a prominent component of the ALP, is responsible for the bulk lysosomal degradation of soluble and pathological protein aggregates as well as damaged organelles. Animal models with impaired autophagy-related proteins in neuronal tissues and human neurodegenerative lysosomal storage disorders have demonstrated that defects in ALP can result in neurodegeneration.

Ginkgolic acid and the related compound anacardic acid have been shown to inhibit SUMOylation by blocking the function of Ubc9. Prior studies on the effects of inhibiting the SUMO pathway using GA have concluded that SUMOylation plays a protective role in cellular health. In this study, both GA and anacardic acid were investigated for their effects on α-syn aggregates. GA was found to promote the clearance of pre-formed α-syn aggregates at concentrations that were not toxic to cells and led to the upregulation of macroautophagy, with minimal impact on SUMO conjugates. These results suggest that GA or similar molecules could hold therapeutic potential for Parkinson’s disease.

Materials and Methods

Cell Culture

SH-SY5Y neuroblastoma cells were seeded at a density of 10,000 cells per well in 24-well plates on 10 mm glass coverslips for immunofluorescence confocal imaging and analysis. The cells were incubated for 24 hours at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.04% amphotericin B, and 1% strep/penicillin, to allow for cell adhesion. In parallel, cells were seeded and incubated in 6-well plates at 300,000 cells per well for the preparation of cell lysates for SDS-PAGE and Western blotting.

Embryonic cortical neurons were isolated from E18 Wistar rat embryos. The cortical areas were dissected and trypsinized, then plated onto poly-L-lysine-coated 25 mm glass coverslips. Cells were initially plated in plating medium (Neurobasal media with 10% horse serum, B27 supplement, 2 mM Glutamax, and 1× penicillin/streptomycin). After 24 hours, the medium was replaced with feeding medium (Neurobasal media, B27 supplement, 1.2 mM Glutamax, and 1× penicillin/streptomycin). Cortical neurons were used for treatments between seven and fourteen days in vitro.

Ginkgolic Acid and Anacardic Acid Treatments

For immunofluorescence analysis, cells were pre-treated for one hour with media containing 6 to 100 micromolar concentrations of either ginkgolic acid or anacardic acid. Potassium chloride (50 mM) was then added to these pre-treated wells and they were incubated for one additional hour to induce α-syn aggregation. After this, the wells were replenished with fresh medium containing only GA or AA and further incubated for either 24 or 48 hours. SH-SY5Y control groups included both KCl-depolarized and non-depolarized cells. For gel electrophoresis and western blotting, the experiment was repeated in 6-well plates, with GA pre-treatment at 10, 40, and 80 micromolar concentrations for one hour, followed by depolarization with potassium chloride for an hour.

For post-treatment experiments, cells were first depolarized with KCl for an hour without prior inhibitor treatment. After 24 hours, the medium was replaced with fresh medium containing 10, 40, or 80 micromolar GA for a further 24 hours. Cell lysates from 6-well plates were prepared for gel electrophoresis and Western blot analysis, and immunofluorescence results were acquired from coverslips in 24-well plates. Following incubation, post-treatment cell viability was assessed using MTT assays.

In parallel experiments, SH-SY5Y cells depolarized with KCl were incubated for 23 hours to induce α-syn aggregation and were then treated with medium containing autophagy inhibitors chloroquine and bafilomycin A1, at 40 nanomolar and 10 micromolar respectively, for one hour. The medium was then replaced with medium containing 10 micromolar GA as well as the autophagy inhibitors for a further 24 hours. All experiments were performed in triplicate.

Immunofluorescence

Prior to fixation, coverslips were briefly washed in Dulbecco’s Phosphate Buffered Saline (DPBS). Neuroblastoma cells were fixed and permeabilized with a methanol:acetone mixture (3:1 ratio) and blocked with 20% normal horse serum. Primary cortical neurons were fixed in 3.7% paraformaldehyde with 5% sucrose in PBS for one hour, quenched for ten minutes in 0.1 M glycine, and then permeabilized in 0.1% Triton X-100 with 10% normal horse serum. The primary antibodies used for immunostaining included monoclonal mouse anti-α-syn antibody, polyclonal sheep anti-SUMO-1 antibody, polyclonal rabbit Cathepsin-D antibody, and polyclonal rabbit anti-LC3 antibody. Alexa Fluor-conjugated secondary antibodies were used for immunofluorescence, and samples were mounted with DAPI-containing mounting medium. Slides were stored in the dark at 4°C.

Laser Scanning Confocal Microscopy Image Analysis and Cell Counting

Immunolabeled slides were imaged using a confocal laser scanning microscope. Negative control slides with only secondary antibody were used to determine appropriate imaging settings. A 60X oil immersion lens was employed for all imaging. Each fluorescent channel (DAPI, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 647) was imaged sequentially, and the channels were merged and exported for further editing and analysis.

Cells immunostained for SUMO-1 and cathepsin-D, a lysosomal marker, were analyzed for co-localization. Lysosomes were classified as either SUMO-1 positive or negative, and the proportion of SUMO-1-positive lysosomes was calculated for α-syn-inclusion-body-positive or -negative cells. Inclusion-body-positive cells were considered those bearing bright α-syn puncta greater than or equal to one micrometer. LC3-positive autophagosomes were manually counted as distinct, bright puncta. For autophagy inhibitor treatments, LC3 puncta were analyzed with the ImageJ software. Background labeling was excluded, and LC3 puncta were counted using the “Analyze Particles” function. Each coverslip included a standard pattern of five regions of interest, and all experiments were performed in triplicate, with approximately forty cells counted per region. For post-treated cells, immunostained samples were manually counted for large, α-syn-positive aggregates greater than or equal to two micrometers in each region. Images were de-identified to prevent bias, and results were analyzed as the proportion of the total number of DAPI-stained nuclei.

Cell Lysate Preparation

After appropriate drug treatments, whole-cell protein extracts were prepared using RIPA buffer supplemented with a protease inhibitor cocktail and N-ethylmaleimide to inhibit deSUMOylating enzymes. After harvesting, cells were washed briefly in DPBS, scraped, collected by centrifugation at two thousand revolutions per minute for five minutes, and then lysed in RIPA buffer supplemented with inhibitors to yield cytosolic proteins. The protein concentrations were measured using a BCA assay according to the manufacturer’s instructions. Equal protein concentrations were used for further analysis.

SDS-PAGE and Western Blotting

Proteins from cell lysates were separated by SDS-PAGE using standard protocols. After electrophoresis, proteins were transferred onto PVDF membranes. Membranes were blocked with 5% milk in Tris-buffered saline containing Tween-20, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies included monoclonal mouse anti-α-synuclein and polyclonal sheep anti-SUMO-1. Secondary antibodies conjugated with horseradish peroxidase were then applied. Bound antibodies were detected using chemiluminescence reagents, and the signals were visualized on an imaging system. Densitometric quantification was performed using software analysis, and data were normalized to the total protein content.

Statistical Analysis

Data are presented as mean values ± standard error of the mean. Statistical comparisons between multiple groups were performed using one-way or two-way analysis of variance followed by appropriate post hoc tests such as Tukey’s or Dunnett’s multiple comparisons. p-values less than 0.05 were considered statistically significant.

Results

Ginkgolic Acid and Anacardic Acid Reduce Alpha-Synuclein Aggregate Formation

Treatment of SH-SY5Y neuroblastoma cells and primary cortical neurons with potassium chloride induced significant accumulation of intracytoplasmic α-synuclein aggregates. Pre-treatment with ginkgolic acid or anacardic acid led to a dose-dependent reduction in the number of cells containing these aggregates. The immunofluorescent staining for α-synuclein and SUMO-1 revealed that both compounds significantly decreased the presence of double-positive aggregates compared to the depolarized controls. The reduction was observed both at 24 hours and sustained up to 48 hours after treatment.

Increased Autophagosome Frequency with Ginkgolic Acid and Anacardic Acid

The proportion of cells displaying LC3-positive autophagosomes was evaluated to assess activation of macroautophagy. Both ginkgolic acid and anacardic acid significantly increased the frequency of autophagosomes as indicated by LC3 puncta in the cytoplasm. This upregulation of autophagy was evident irrespective of whether the compounds were used before or after the induction of α-syn aggregation by potassium chloride. The increase in LC3-positive structures was confirmed by manual counting and image analysis across multiple cell fields and independent experiments.

Ginkgolic Acid Promotes Clearance of Pre-Formed Alpha-Synuclein Aggregates

To determine if ginkgolic acid could clear pre-existing aggregates, cells were first subjected to depolarization to induce α-syn aggregate formation. After 24 hours, cells were treated with ginkgolic acid and monitored for an additional 24 hours. This post-treatment approach significantly reduced the number of cells bearing large α-synuclein-positive inclusions, indicating that ginkgolic acid not only prevents aggregate formation but also facilitates the removal of established aggregates. Cell viability assays confirmed that the concentrations used were not cytotoxic.

Autophagy Inhibition Blocks Ginkgolic Acid-Dependent Clearance

Application of autophagy inhibitors, including chloroquine and bafilomycin A1, was used to assess the requirement for autophagy in the clearance process. When cells were treated with these inhibitors prior to exposure to ginkgolic acid, the clearance of α-synuclein aggregates was effectively blocked. However, the frequency of autophagosomes remained elevated, suggesting that autophagosome formation was not suppressed, but their maturation or function was impaired. This demonstrates that the degradation and clearance of aggregates mediated by ginkgolic acid are dependent on a functional autophagy-lysosomal pathway.

SUMO Conjugation Levels Are Not Significantly Altered

Western blot analysis was performed to examine the overall abundance of SUMO conjugates in cell lysates following treatment with ginkgolic acid. Despite the observed reduction in α-syn aggregate-bearing cells, the levels of SUMOylated proteins were not significantly different when compared to controls. This suggests that the effect of ginkgolic acid on α-syn aggregates is not mediated by a global decrease in SUMOylation but likely involves enhanced autophagic processes.

Discussion

The present findings provide compelling evidence that ginkgolic acid promotes the clearance of pathological intracellular α-synuclein aggregates in cellular models of Parkinson’s disease. Both ginkgolic acid and its structural analogue, anacardic acid, effectively decrease the formation of α-syn inclusions and enhance autophagic activity. They also facilitate the removal of aggregates that have already formed, highlighting their therapeutic potential for halting or reversing disease progression in α-synucleinopathies.

The autophagy-lysosomal pathway is essential for the degradation of large protein aggregates and damaged organelles that cannot be processed via the ubiquitin-proteasome pathway. Deficits in autophagy contribute to the pathological buildup of proteins such as α-synuclein and, ultimately, to neurodegeneration. Our data show that ginkgolic acid upregulates autophagosome formation, and that inhibition of autophagic flux completely abolishes its beneficial effect on α-syn aggregate clearance. This confirms that macroautophagy is the principal mechanism underlying the degradation promoted by ginkgolic acid.

In contrast, SUMOylation status is only marginally affected. Although ginkgolic acid can inhibit SUMOylation, the clearance of aggregates in this model appears to occur independently of any major change in overall SUMO conjugation. This supports the view that the main clearance mechanism involves recruitment and activation of autophagic pathways, rather than interference with global SUMOylation.

These observations have important implications for disease-modifying therapies in Parkinson’s disease and related disorders. Currently, no approved drugs directly target the clearance of α-syn aggregates. Our results suggest that ginkgolic acid and similar molecules could provide a novel approach by activating endogenous autophagic pathways to remove toxic protein inclusions.

Conclusion

Ginkgolic acid is able to promote autophagy-dependent clearance of intracellular α-synuclein aggregates without inducing cytotoxicity or broadly reducing SUMO conjugate abundance. These results demonstrate the potential of ginkgolic acid and related compounds for the development of disease-modifying therapies aimed at the pathological protein aggregation characteristic of Parkinson’s disease and other α-synucleinopathies. Further studies will be needed to explore their efficacy and safety in preclinical and clinical settings.