Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells.

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aß) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aß secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

Sphingolipid changes in Parkinson L444P GBA mutation fibroblasts promote α-synuclein aggregation.

Intraneuronal accumulation of aggregated α-synuclein is a pathological hallmark of Parkinson's disease. Therefore, mechanisms capable of promoting α-synuclein deposition bear important pathogenetic implications. Mutations of the glucocerebrosidase 1 (GBA) gene represent a prevalent Parkinson's disease risk factor. They are associated with loss of activity of a key enzyme involved in lipid metabolism, glucocerebrosidase, supporting a mechanistic relationship between abnormal α-synuclein-lipid interactions and the development of Parkinson pathology. In this study, the lipid membrane composition of fibroblasts isolated from control subjects, patients with idiopathic Parkinson's disease and Parkinson's disease patients carrying the L444P GBA mutation (PD-GBA) was assayed using shotgun lipidomics. The lipid profile of PD-GBA fibroblasts differed significantly from that of control and idiopathic Parkinson's disease cells. It was characterized by an overall increase in sphingolipid levels. It also featured a significant increase in the proportion of ceramide, sphingomyelin and hexosylceramide molecules with shorter chain length and a decrease in the percentage of longer-chain sphingolipids. The extent of this shift was correlated to the degree of reduction of fibroblast glucocerebrosidase activity. Lipid extracts from control and PD-GBA fibroblasts were added to recombinant α-synuclein solutions. The kinetics of α-synuclein aggregation were significantly accelerated after addition of PD-GBA extracts as compared to control samples. Amyloid fibrils collected at the end of these incubations contained lipids, indicating α-synuclein-lipid co-assembly. Lipids extracted from α-synuclein fibrils were also analysed by shotgun lipidomics. Data revealed that the lipid content of these fibrils was significantly enriched by shorter-chain sphingolipids. In a final set of experiments, control and PD-GBA fibroblasts were incubated in the presence of the small molecule chaperone ambroxol. This treatment restored glucocerebrosidase activity and sphingolipid levels and composition of PD-GBA cells. It also reversed the pro-aggregation effect that lipid extracts from PD-GBA fibroblasts had on α-synuclein. Taken together, the findings of this study indicate that the L444P GBA mutation and consequent enzymatic loss are associated with a distinctly altered membrane lipid profile that provides a biological fingerprint of this mutation in Parkinson fibroblasts. This altered lipid profile could also be an indicator of increased risk for α-synuclein aggregate pathology.

The interplay between Glucocerebrosidase, α-synuclein and lipids in human models of Parkinson's disease.

Mutations in the gene GBA, encoding glucocerebrosidase (GCase), are the highest genetic risk factor for Parkinson's disease (PD). GCase is a lysosomal glycoprotein responsible for the hydrolysis of glucosylceramide into glucose and ceramide. Mutations in GBA cause a decrease in GCase activity, stability and protein levels which in turn lead to the accumulation of GCase lipid substrates as well as α-synuclein (αS) in vitro and in vivo. αS is the main constituent of Lewy bodies found in the brain of PD patients and an increase in its levels was found to be associated with a decrease in GCase activity/protein levels in vitro and in vivo. In this review, we describe the reported biophysical and biochemical changes that GBA mutations can induce in GCase activity and stability as well as the current overview of the levels of GCase protein/activity, αS and lipids measured in patient-derived samples including post-mortem brains, stem cell-derived neurons, cerebrospinal fluid, blood and fibroblasts as well as in SH-SY5Y cells. In particular, we report how the levels of αS and lipids are affected by/correlated to significant changes in GCase activity/protein levels and which cellular pathways are activated or disrupted by these changes in each model. Finally, we review the current strategies used to revert the changes in the levels of GCase activity/protein, αS and lipids in the context of PD.