Lysophosphatidylcholine acyltransferase 1 promotes pathology and toxicity in two distinct cell-based alpha-synuclein models.

Alpha-synuclein (αSyn) pathology is a hallmark of Parkinson's disease. Here we show that lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a regulator of αSyn pathology and cytotoxicity. LPCAT1 is upregulated by αSyn E35K E46K E61K (3K) in human M17 neuroblastoma cells and primary rat cortical neurons, and in postmortem brain tissue from PD patients with confirmed αSyn aggregate pathology. Suppression of LPCAT1 reduces αSyn accumulations and toxicity in our neuroblastoma αSyn 3K overexpression model. Further overexpression of LPCAT1 promotes pS129 αSyn positive aggregation in primary neurons in the αSyn pre-formed fibril (PFF) model. A phospholipid product of LPCAT1 enzymatic activity, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, similarly promotes neuronal PFF seeded aggregation. Using a pH sensitive PFF model we provide evidence that αSyn fibrils have altered endo-lysosomal processing under LPCAT1 enhancement, suggesting less aggregate degradation. Our data demonstrates that LPCAT1 and associated phospholipids can regulate αSyn pathology.

SCD Inhibition Protects from α-Synuclein-Induced Neurotoxicity But Is Toxic to Early Neuron Cultures.

Here, we report the independent discovery and validation of stearoyl-CoA desaturase (SCD) as a modulator of α-synuclein (αSyn)-induced pathology and toxicity in cell-based Parkinson's disease (PD) models. We identified SCD as top altered gene from transcriptional profiling in primary neurons exogenously expressing αSyn with the amplified familial PD mutation 3K. Thus, we sought to further explore SCD as a therapeutic target in neurodegeneration. We report that SCD inhibitors are toxic to early human and rat neuron cultures while displaying minimal toxicity to late cultures. The fatty acid product of SCD, oleic acid (OLA), fully rescues this toxicity in early cultures, suggesting on-target toxicity. Furthermore, SCD inhibition rescues αSyn 3K-induced toxicity in late primary neurons. We also confirm that SCD inhibitors reduce formation of αSyn accumulations, while OLA increases these accumulations in an αSyn 3K neuroblastoma model. However, we identify a caveat with this model where αSyn 3K levels can be suppressed by high SCD inhibitor concentrations, obscuring true effect size. Further, we show that both SCD1 or SCD5 knock-down reduce αSyn 3K accumulations and toxicity, making both a putative drug target. Overall, we confirm key findings of published data on SCD inhibition and its benefits in αSyn accumulation and stress models. The differential neurotoxicity induced by SCD inhibition based on neuron culture age must be accounted for when researching SCD in neuron models and has potential clinical implications. Lastly, our gene profiling studies also revealed novel putative genes connected to αSyn neurotoxicity that are worth further study.

The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

SIRT1 accelerates the progression of activity-based anorexia.

Food consumption is fundamental for life, and eating disorders often result in devastating or life-threatening conditions. Anorexia nervosa (AN) is characterized by a persistent restriction of energy intake, leading to lowered body weight, constant fear of gaining weight, and psychological disturbances of body perception. Herein, we demonstrate that SIRT1 inhibition, both genetically and pharmacologically, delays the onset and progression of AN behaviors in activity-based anorexia (ABA) models, while SIRT1 activation accelerates ABA phenotypes. Mechanistically, we suggest that SIRT1 promotes progression of ABA, in part through its interaction with NRF1, leading to suppression of a NMDA receptor subunit Grin2A. Our results suggest that AN may arise from pathological positive feedback loops: voluntary food restriction activates SIRT1, promoting anxiety, hyperactivity, and addiction to starvation, exacerbating the dieting and exercising, thus further activating SIRT1. We propose SIRT1 inhibition can break this cycle and provide a potential therapy for individuals suffering from AN.

Cellular energetics and mitochondrial uncoupling in canine aging.

The first domesticated companion animal, the dog, is currently represented by over 190 unique breeds. Across these numerous breeds, dogs have exceptional variation in lifespan (inversely correlated with body size), presenting an opportunity to discover longevity-determining traits. We performed a genome-wide association study on 4169 canines representing 110 breeds and identified novel candidate regulators of longevity. Interestingly, known functions within the identified genes included control of coat phenotypes such as hair length, as well as mitochondrial properties, suggesting that thermoregulation and mitochondrial bioenergetics play a role in lifespan variation. Using primary dermal fibroblasts, we investigated mitochondrial properties of short-lived (large) and long-lived (small) dog breeds. We found that cells from long-lived breeds have more uncoupled mitochondria, less electron escape, greater respiration, and capacity for respiration. Moreover, our data suggest that long-lived breeds have higher rates of catabolism and ß-oxidation, likely to meet elevated respiration and electron demand of their uncoupled mitochondria. Conversely, cells of short-lived (large) breeds may accumulate amino acids and fatty acid derivatives, which are likely used for biosynthesis and growth. We hypothesize that the uncoupled metabolic profile of long-lived breeds likely stems from their smaller size, reduced volume-to-surface area ratio, and therefore a greater need for thermogenesis. The uncoupled energetics of long-lived breeds lowers reactive oxygen species levels, promotes cellular stress tolerance, and may even prevent stiffening of the actin cytoskeleton. We propose that these cellular characteristics delay tissue dysfunction, disease, and death in long-lived dog breeds, contributing to canine aging diversity.

Nicotine promotes neuron survival and partially protects from Parkinson's disease by suppressing SIRT6.

Parkinson's disease is characterized by progressive death of dopaminergic neurons, leading to motor and cognitive dysfunction. Epidemiological studies consistently show that the use of tobacco reduces the risk of Parkinson's. We report that nicotine reduces the abundance of SIRT6 in neuronal culture and brain tissue. We find that reduction of SIRT6 is partly responsible for neuroprotection afforded by nicotine. Additionally, SIRT6 abundance is greater in Parkinson's patient brains, and decreased in the brains of tobacco users. We also identify SNPs that promote SIRT6 expression and simultaneously associate with an increased risk of Parkinson's. Furthermore, brain-specific SIRT6 knockout mice are protected from MPTP-induced Parkinson's, while SIRT6 overexpressing mice develop more severe pathology. Our data suggest that SIRT6 plays a pathogenic and pro-inflammatory role in Parkinson's and that nicotine can provide neuroprotection by accelerating its degradation. Inhibition of SIRT6 may be a promising strategy to ameliorate Parkinson's and neurodegeneration.

SIRT6 knockout cells resist apoptosis initiation but not progression: a computational method to evaluate the progression of apoptosis.

Apoptosis is essential for numerous processes, such as development, resistance to infections, and suppression of tumorigenesis. Here, we investigate the influence of the nutrient sensing and longevity-assuring enzyme SIRT6 on the dynamics of apoptosis triggered by serum starvation. Specifically, we characterize the progression of apoptosis in wild type and SIRT6 deficient mouse embryonic fibroblasts using time-lapse flow cytometry and computational modelling based on rate-equations and cell distribution analysis. We find that SIRT6 deficient cells resist apoptosis by delaying its initiation. Interestingly, once apoptosis is initiated, the rate of its progression is higher in SIRT6 null cells compared to identically cultured wild type cells. However, SIRT6 null cells succumb to apoptosis more slowly, not only in response to nutrient deprivation but also in response to other stresses. Our data suggest that SIRT6 plays a role in several distinct steps of apoptosis. Overall, we demonstrate the utility of our computational model to describe stages of apoptosis progression and the integrity of the cellular membrane. Such measurements will be useful in a broad range of biological applications.

Coordinated regulation of protein synthesis and degradation by mTORC1.

Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

Chronic activation of mTOR complex 1 is sufficient to cause hepatocellular carcinoma in mice.

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient-sensitive protein kinase that is aberrantly activated in many human cancers. Whether dysregulation of mTORC1 signaling in normal tissues increases the risk for cancer, however, is unknown. We focused on hepatocellular carcinoma, which has been linked to environmental factors that affect mTORC1 activity, including diet. Ablation of the gene encoding TSC1 (tuberous sclerosis complex 1), which as part of the TSC1-TSC2 complex is an upstream inhibitor of mTORC1, results in constitutively increased mTORC1 signaling, an effect on this pathway similar to that of obesity. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes that accompany the primary etiologies of this cancer in humans, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling led to unresolved endoplasmic reticulum stress and defects in autophagy, factors that contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we conclude that increased activation of mTORC1 can promote carcinogenesis and may thus represent a key molecular link between cancer risk and environmental factors, such as diet.