Wei-Yi Ong

Objective:While Spinal Muscular Atrophy (SMA) is classically defined by motor neuron loss, increasing evidence points to broader systemic involvement, including the liver. Current innovative therapies do not fully address this as their main aim is to improve motor function and overall survival by targeting the motor neurons in SMA patients. We investigated shared and tissue-specific molecular changes in liver and motor neurons to understand systemic effects of SMA in SMA patients and carriers.Methods:Using patient-derived stem cells from SMA patients, we generated induced liver (iHeps) and motor neuron (iMNs) cells. Using genome editing, we further created isogenic carrier and wild-type lines from the same SMA patient. We studied the proteomic differences in SMA across liver and motor neurons and determined shared and tissue-specific effects.Results:A total of 432 proteins (9.3% iHep-specific, 64.8% iMN-specific and 24.9% shared) were found dysregulated in either tissue. Commonly dysregulated proteins in both tissues included those upregulated in cell structure and extracellular matrix organization; and downregulated in mitochondrial function and metabolism-related processes. We further narrowed down to 36 proteins showing SMN-dependent effects involved in protein hydroxylation, tissue organization, wound healing and cell adhesion. In isogenic SMA carriers, cross-tissue dysregulation was limited to five proteins, with broader pathway perturbations largely tissue-specific.Interpretation:Our findings reinforce SMA’s multi-system nature, even in apparently asymptomatic carriers. Patient-derived stem cell models offer a powerful platform to understand the multi-systemic nature of SMA and gain insights into the full spectrum of its biology. These insights could inform systemic treatment approaches for SMA and SMA carriers.

Ergothioneine (ET) is a naturally occurring thiol/thione that possesses several cytoprotective properties. Multiple studies suggest a potential neuroprotective role for ET. Here, we show in various Parkinson's disease (PD) models that ET is indeed neuroprotective. Firstly, using Drosophila genetic PD models, we demonstrated that ET treatment ameliorates the pathological phenotypes of parkin and LRRK2 PD mutant flies. This includes an improvement in their climbing score and the preservation of their dopaminergic neuronal number and mitochondrial integrity. Similarly, we observed the rescue of PD phenotypes by ET in mice treated with the Parkinsonian neurotoxin 6‐OHDA. This protective effect of ET is abolished in mice lacking OCTN1. Finally, we found that ET protects human LRRK2‐G2019S patient‐derived dopaminergic neurons from rotenone‐induced neurotoxicity; the action of ET is again OCTN1‐dependent. Collectively, our results strongly support a neuroprotective role for ET in PD and suggest that ET may be useful in the prevention and/or treatment of PD. image

Cell death involving oxidative stress and mitochondrial dysfunction is a major cause of dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson’s disease patients. Ergothioneine (ET), a natural dietary compound, has been shown to have cytoprotective functions, but neuroprotective actions against PD have not been well established. 6-Hydroxydopamine (6-OHDA) is a widely used neurotoxin to simulate the degeneration of dopaminergic (DA) neurons in Parkinson’s disease. In this study, we investigated the protective effect of ET on 6-OHDA treated iPSC-derived dopaminergic neurons (iDAs) and further confirmed the protective effects in 6-OHDA-treated human neuroblastoma SH-SY5Y cells. In 6-OHDA-treated cells, decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial reactive oxygen species (mROS), reduced cellular ATP levels, and increased total protein carbonylation levels were observed. 6-OHDA treatment also significantly decreased tyrosine hydroxylase levels. These effects were significantly decreased when ET was present. Verapamil hydrochloride (VHCL), a non-specific inhibitor of the ET transporter OCTN1 abrogated ET’s cytoprotective effects, indicative of an intracellular action. These results suggest that ET could be a potential therapeutic for Parkinson’s disease.

Spinal muscular atrophy (SMA) is typically characterized as a motor neuron disease, but extraneuronal phenotypes are present in almost every organ in severely affected patients and animal models. Extraneuronal phenotypes were previously underappreciated, as patients with severe SMA phenotypes usually died in infancy; however, with current treatments for motor neurons increasing patient lifespan, impaired function of peripheral organs may develop into significant future comorbidities and lead to new treatment-modified phenotypes. Fatty liver is seen in SMA animal models, but generalizability to patients and whether this is due to hepatocyte-intrinsic survival motor neuron (SMN) protein deficiency and/or subsequent to skeletal muscle denervation is unknown. If liver pathology in SMA is SMN dependent and hepatocyte intrinsic, this suggests SMN-repleting therapies must target extraneuronal tissues and motor neurons for optimal patient outcome. Here, we showed that fatty liver is present in SMA patients and that SMA patient–specific induced pluripotent stem cell–derived hepatocyte-like cells were susceptible to steatosis. Using proteomics, functional studies, and CRISPR/Cas9 gene editing, we confirmed that fatty liver in SMA is a primary SMN-dependent hepatocyte-intrinsic liver defect associated with mitochondrial and other hepatic metabolism implications. These pathologies require monitoring and indicate the need for systematic clinical surveillance and additional and/or combinatorial therapies to ensure continued SMA patient health.

Oxidative stress-mediated cell death is a major cause of dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson’s disease patients. Ergothioneine (ET), a natural dietary compound, has been shown to have cytoprotective functions, but neuroprotective actions against PD have not been well established. 6-Hydroxydopamine (6-OHDA) is a widely used neurotoxin to simulate the degeneration of dopaminergic (DA) neurons in Parkinson's disease. In this study, we investigated the protective effect of ET on 6-OHDA treated iPSC-derived dopaminergic neurons (iDAs) and further confirmed the protective effects in 6-OHDA treated human neuroblastoma SH-SY5Y cells. In 6-OHDA treated cells, metabolic stress in the form of decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial reactive oxygen species (mROS), reduced cellular ATP levels, and increased total protein carbonylation levels was observed. 6-OHDA treatment also significantly decreased tyrosine hydroxylase levels. These effects were significantly abrogated when ET was present. Verapamil hydrochloride (VHCL), a non-specific inhibitor of the ET transporter, OCTN1, abolished ET’s cytoprotective effects, indicative of an intracellular action. These results suggest that ET could be a potential therapeutic for Parkinson’s disease.

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1 -KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1 -KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1 -KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A₂ that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A₂ IIA (sPLA₂-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA₂-IIA levels together with eicosanoids in the sera of COVID patients. sPLA₂ could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A₂, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA₂-IIA in COVID-19-associated coagulopathy (CAC).

Recent findings have suggested that the natural compound ergothioneine (ET), which is synthesised by certain fungi and bacteria, has considerable cytoprotective potential. We previously demonstrated the anti-inflammatory effects of ET on 7-ketocholesterol (7KC)-induced endothelial injury in human blood-brain barrier endothelial cells (hCMEC/D3). 7KC is an oxidised form of cholesterol present in atheromatous plaques and the sera of patients with hypercholesterolaemia and diabetes mellitus. The aim of this study was to elucidate the protective effect of ET on 7KC-induced mitochondrial damage. Exposure of human brain endothelial cells to 7KC led to a loss of cell viability, together with an increase in intracellular free calcium levels, increased cellular and mitochondrial reactive oxygen species, a decrease in mitochondrial membrane potential, reductions in ATP levels, and increases in mRNA expression of TFAM, Nrf2, IL-1β, IL-6 and IL-8. These effects were significantly decreased by ET. Protective effects of ET were diminished when endothelial cells were coincubated with verapamil hydrochloride (VHCL), a nonspecific inhibitor of the ET transporter OCTN1 (SLC22A4). This outcome demonstrates that ET-mediated protection against 7KC-induced mitochondrial damage occurred intracellularly and not through direct interaction with 7KC. OCTN1 mRNA expression itself was significantly increased in endothelial cells after 7KC treatment, consistent with the notion that stress and injury may increase ET uptake. Our results indicate that ET can protect against 7KC-induced mitochondrial injury in brain endothelial cells.

Ergothioneine (ET) is a naturally occurring antioxidant and cytoprotective agent that is synthesized by fungi and certain bacteria. Recent studies have shown a beneficial effect of ET on neurological functions, including cognition and animal models of depression. The aim of this study is to elucidate a possible effect of ET in rodent models of stroke. Post-ischemic intracerebroventricular (i.c.v.) infusion of ET significantly reduced brain infarct volume by as early as 1 day after infusion in rats, as shown by triphenyltetrazolium chloride (TTC) assay. There was a dose-dependent increase in protection, from 50 to 200 ng of ET infusion. These results suggest that ET could have a protective effect on CNS neurons. We next elucidated the effect of systemic ET on brain infarct volume in mice after stroke. Daily i.p. injection of 35 mg/kg ET (the first dose being administered 3 h after stroke) had no significant effect on infarct volume. However, daily i.p. injections of 70 mg/kg, 100 mg/kg, 125 mg/kg and 150 mg/kg ET, with the first dose administered 3 h after stroke, significantly decreased infarct volume at 7 days after vessel occlusion in mice. In order to elucidate at what time interval during the 7 days there could be effective protection, a second set of experiments was carried out in mice, using one of the effective loading protocols, i.e. 125 mg/kg i.p. ET but the brains were analyzed at 1, 4 and 7 days post-stroke by MRI. We found that ET was already protective against neuronal injury and decreased the size of the brain infarct from as early as 1 day post-stroke. Behavioral experiments carried out on a third set of mice (using 125 mg/kg i.p. ET) showed that this was accompanied by significant improvements in certain behaviors (pole test) at 1 day after stroke. Together, results of this study indicate that i.c.v. and systemic ET are effective in reducing brain infarct volume after stroke in rodent models.

Research advances have shed new insight into cellular pathways contributing to PD pathogenesis and offer increasingly compelling therapeutic targets. In this review, we made a broad survey of the published literature that report possible disease-modifying effects on PD. While there are many studies that demonstrate benefits for various therapies for PD in animal and human studies, we confined our search to human “randomised controlled trials” and with the key words “neuroprotection” or “disease-modifying”. It is hoped that through studying the results of these trials, we might clarify possible mechanisms that underlie idiopathic PD. This contrasts with studying the effect of pathophysiology of familial PD, which could be carried out by gene knockouts and animal models. Randomised controlled trials indicate promising effects of MAO-B inhibitors, dopamine agonists, NMDA receptor antagonists, metabotropic glutamate receptor antagonists, therapies related to improving glucose utilization and energy production, therapies related to reduction of excitotoxicity and oxidative stress, statin use, therapies related to iron chelation, therapies related to the use of phytochemicals, and therapies related to physical exercise and brain reward pathway on slowing PD progression. Cumulatively, these approaches fall into two categories: direct enhancement of dopaminergic signalling, and reduction of neurodegeneration. Overlaps between the two categories result in challenges in distinguishing between symptomatic versus disease-modifying effects with current clinical trial designs. Nevertheless, a broad-based approach allows us to consider all possible therapeutic avenues which may be neuroprotective. While the traditional standard of care focuses on symptomatic management with dopaminergic drugs, more recent approaches suggest ways to preserve dopaminergic neurons by attenuating excitotoxicity and oxidative stress.