Increasing hexokinase 1 expression improves mitochondrial and glycolytic functional deficits seen in sporadic Alzheimer's disease astrocytes.

Abnormalities in cellular metabolism are seen early in Alzheimer's disease (AD). Astrocyte support for neuronal function has a high metabolic demand, and astrocyte glucose metabolism plays a key role in encoding memory. This indicates that astrocyte metabolic dysfunction might be an early event in the development of AD. In this paper we interrogate glycolytic and mitochondrial functional changes and mitochondrial structural alterations in patients' astrocytes derived with a highly efficient direct conversion protocol. In astrocytes derived from patients with sporadic (sAD) and familial AD (fAD) we identified reductions in extracellular lactate, total cellular ATP and an increase in mitochondrial reactive oxygen species. sAD and fAD astrocytes displayed significant reductions in mitochondrial spare respiratory capacity, have altered mitochondrial membrane potential and a stressed mitochondrial network. A reduction in glycolytic reserve and glycolytic capacity is seen. Interestingly, glycolytic reserve, mitochondrial spare respiratory capacity and extracellular lactate levels correlated positively with neuropsychological tests of episodic memory affected early in AD. We identified a deficit in the glycolytic enzyme hexokinase 1 (HK1), and correcting this deficit improved the metabolic phenotype in sAD not fAD astrocytes. Importantly, the amount of HK1 at the mitochondria was shown to be reduced in sAD astrocytes, and not in fAD astrocytes. Overexpression of HK1 in sAD astrocytes increases mitochondrial HK1 levels. In fAD astrocytes HK1 levels were unaltered at the mitochondria after overexpression. This study highlights a clear metabolic deficit in AD patient-derived astrocytes and indicates how HK1, with its roles in both oxidative phosphorylation and glycolysis, contributes to this.

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Activation of the Keap1/Nrf2 pathway suppresses mitochondrial dysfunction, oxidative stress, and motor phenotypes in C9orf72 ALS/FTD models.

Mitochondrial dysfunction is a common feature of C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD); however, it remains unclear whether this is a cause or consequence of the pathogenic process. Analysing multiple aspects of mitochondrial biology across several Drosophila models of C9orf72-ALS/FTD, we found morphology, oxidative stress, and mitophagy are commonly affected, which correlated with progressive loss of locomotor performance. Notably, only genetic manipulations that reversed the oxidative stress levels were also able to rescue C9orf72 locomotor deficits, supporting a causative link between mitochondrial dysfunction, oxidative stress, and behavioural phenotypes. Targeting the key antioxidant Keap1/Nrf2 pathway, we found that genetic reduction of Keap1 or pharmacological inhibition by dimethyl fumarate significantly rescued the C9orf72-related oxidative stress and motor deficits. Finally, mitochondrial ROS levels were also elevated in C9orf72 patient-derived iNeurons and were effectively suppressed by dimethyl fumarate treatment. These results indicate that mitochondrial oxidative stress is an important mechanistic contributor to C9orf72 pathogenesis, affecting multiple aspects of mitochondrial function and turnover. Targeting the Keap1/Nrf2 signalling pathway to combat oxidative stress represents a therapeutic strategy for C9orf72-related ALS/FTD.

Evidence of mitochondrial dysfunction in ALS and methods for measuring in model systems.

Metabolic dysfunction is a hallmark of multiple amyotrophic lateral sclerosis (ALS) models with a majority of ALS patients exhibiting hypermetabolism. The central sites of metabolism in the cell are mitochondria, capable of utilising a multitude of cellular substrates in an array of ATP-generating reactions. With reactive oxygen species (ROS) production occurring during some of these reactions, mitochondria can contribute considerably to oxidative stress. Mitochondria are also very dynamic organelles, interacting with other organelles, undergoing fusion/fission in response to changing metabolic states and being turned over by the cell regularly. Disruptions to many of these mitochondrial functions and processes have been reported in ALS models, largely indicating compromised mitochondrial function, increased ROS production by mitochondria, disrupted interactions with the endoplasmic reticulum and reduced turnover. This chapter summarises methods routinely used to assess mitochondria in ALS models and the alterations that have been reported in these models.

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.

The Parkinson's disease related mutant VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress.

The identification of multiple genes linked to Parkinson's disease (PD) invites the question as to how they may co-operate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to PD. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinson's associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent l-leucyl-l-leucine methyl ester, we are able to dissociate phospho-RAB accumulation from membrane rupture.

Multimodal assessment of mitochondrial function in Parkinson's disease.

The heterogenous aetiology of Parkinson's disease is increasingly recognized; both mitochondrial and lysosomal dysfunction have been implicated. Powerful, clinically applicable tools are required to enable mechanistic stratification for future precision medicine approaches. The aim of this study was to characterize bioenergetic dysfunction in Parkinson's disease by applying a multimodal approach, combining standardized clinical assessment with midbrain and putaminal 31-phosphorus magnetic resonance spectroscopy (31P-MRS) and deep phenotyping of mitochondrial and lysosomal function in peripheral tissue in patients with recent-onset Parkinson's disease and control subjects. Sixty participants (35 patients with Parkinson's disease and 25 healthy controls) underwent 31P-MRS for quantification of energy-rich metabolites [ATP, inorganic phosphate (Pi) and phosphocreatine] in putamen and midbrain. In parallel, skin biopsies were obtained from all research participants to establish fibroblast cell lines for subsequent quantification of total intracellular ATP and mitochondrial membrane potential (MMP) as well as mitochondrial and lysosomal morphology, using high content live cell imaging. Lower MMP correlated with higher intracellular ATP (r = -0.55, P = 0.0016), higher mitochondrial counts (r = -0.72, P < 0.0001) and higher lysosomal counts (r = -0.62, P = 0.0002) in Parkinson's disease patient-derived fibroblasts only, consistent with impaired mitophagy and mitochondrial uncoupling. 31P-MRS-derived posterior putaminal Pi/ATP ratio variance was considerably greater in Parkinson's disease than in healthy controls (F-tests, P = 0.0036). Furthermore, elevated 31P-MRS-derived putaminal, but not midbrain Pi/ATP ratios (indicative of impaired oxidative phosphorylation) correlated with both greater mitochondrial (r = 0.37, P = 0.0319) and lysosomal counts (r = 0.48, P = 0.0044) as well as lower MMP in both short (r = -0.52, P = 0.0016) and long (r = -0.47, P = 0.0052) mitochondria in Parkinson's disease. Higher 31P-MRS midbrain phosphocreatine correlated with greater risk of rapid disease progression (r = 0.47, P = 0.0384). Our data suggest that impaired oxidative phosphorylation in the striatal dopaminergic nerve terminals exceeds mitochondrial dysfunction in the midbrain of patients with early Parkinson's disease. Our data further support the hypothesis of a prominent link between impaired mitophagy and impaired striatal energy homeostasis as a key event in early Parkinson's disease.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels.

Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin-RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α-mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early-onset mitochondrial encephalomyopathy that is supported by analysis of a disease-associated mutation. We further show that the compound MLN4924, which globally interferes with cullin-RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.

A p21-GFP zebrafish model of senescence for rapid testing of senolytics in vivo.

Senescence drives the onset and severity of multiple ageing-associated diseases and frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP+ cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP+ cells display other markers of senescence, including senescence-associated ß-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP+ cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems.

Unexpected phenotypic and molecular changes of combined glucocerebrosidase and acid sphingomyelinase deficiency.

Heterozygous variants in GBA1, encoding glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease (PD). Moreover, sporadic PD patients also have a substantial reduction of GCase activity. Genetic variants of SMPD1 are also overrepresented in PD cohorts, whereas a reduction of its encoded enzyme (acid sphingomyelinase or ASM) activity is linked to an earlier age of PD onset. Despite both converging on the ceramide pathway, how the combined deficiencies of both enzymes might interact to modulate PD has yet to be explored. Therefore, we created a double-knockout (DKO) zebrafish line for both gba1 (or gba) and smpd1 to test for an interaction in vivo, hypothesising an exacerbation of phenotypes in the DKO line compared to those for single mutants. Unexpectedly, DKO zebrafish maintained conventional swimming behaviour and had normalised neuronal gene expression signatures compared to those of single mutants. We further identified rescue of mitochondrial Complexes I and IV in DKO zebrafish. Despite having an unexpected rescue effect, our results confirm ASM as a modifier of GBA1 deficiency in vivo. Our study highlights the need for validating how genetic variants and enzymatic deficiencies may interact in vivo.

Aging, Parkinson's Disease, and Models: What Are the Challenges?

Parkinson's disease (PD) is a chronic, neurodegenerative condition characterized by motor symptoms such as bradykinesia, rigidity, and tremor, alongside multiple nonmotor symptoms. The appearance of motor symptoms is linked to progressive dopaminergic neuron loss within the substantia nigra. PD incidence increases sharply with age, suggesting a strong association between mechanisms driving biological aging and the development and progression of PD. However, the role of aging in the pathogenesis of PD remains understudied. Numerous models of PD, including cell models, toxin-induced models, and genetic models in rodents and nonhuman primates (NHPs), reproduce different aspects of PD, but preclinical studies of PD rarely incorporate age as a factor. Studies using patient neurons derived from stem cells via reprogramming methods retain some aging features, but their characterization, particularly of aging markers and reproducibility of neuron type, is suboptimal. Investigation of age-related changes in PD using animal models indicates an association, but this is likely in conjunction with other disease drivers. The biggest barrier to drawing firm conclusions is that each model lacks full characterization and appropriate time-course assessments. There is a need to systematically investigate whether aging increases the susceptibility of mouse, rat, and NHP models to develop PD and understand the role of cell models. We propose that a significant investment in time and resources, together with the coordination and sharing of resources, knowledge, and data, is required to accelerate progress in understanding the role of biological aging in PD development and improve the reliability of models to test interventions.

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

3α,7-Dihydroxy-14(13→12)abeo-5ß,12α(H),13ß(H)-cholan-24-oic Acids Display Neuroprotective Properties in Common Forms of Parkinson's Disease.

Parkinson's Disease is the most common neurodegenerative movement disorder globally, with prevalence increasing. There is an urgent need for new therapeutics which are disease-modifying rather than symptomatic. Mitochondrial dysfunction is a well-documented mechanism in both sporadic and familial Parkinson's Disease. Furthermore, ursodeoxycholic acid (UDCA) has been identified as a bile acid which leads to increased mitochondrial function in multiple in vitro and in vivo models of Parkinson's Disease. Here, we describe the synthesis of novel C-nor-D-homo bile acid derivatives and the 12-hydroxy-methylated derivative of lagocholic acid (7) and their biological evaluation in fibroblasts from patients with either sporadic or LRRK2 mutant Parkinson's Disease. These compounds boost mitochondrial function to a similar level or above that of UDCA in many assays; notable, however, is their ability to boost mitochondrial function to a higher level and at lower concentrations than UDCA specifically in the fibroblasts from LRRK2 patients. Our study indicates that novel bile acid chemistry could lead to the development of more efficacious bile acids which increase mitochondrial function and ultimately cellular health at lower concentrations proving attractive potential novel therapeutics for Parkinson's Disease.

Persistent DNA damage alters the neuronal transcriptome suggesting cell cycle dysregulation and altered mitochondrial function.

Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response (DDR) to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DDR. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration, but the effects of persistent DNA damage in neurons are not well characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sublethal concentration of hydrogen peroxide following a 'double stress' protocol and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DDR and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. Quantitative polymerase chain reaction (qPCR) and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.

The translocator protein (TSPO) is prodromal to mitophagy loss in neurotoxicity.

Dysfunctional mitochondria characterise Parkinson's Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy-lysosomal pathway during neurotoxicity.

A Parkinson's Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells.

Parkinson's disease is a devastating neurodegenerative disorder affecting 2-3% of the population over 65 years of age. There is currently no disease-modifying treatment. One of the predominant pathological features of Parkinson's disease is mitochondrial dysfunction, and much work has aimed to identify therapeutic compounds which can restore the disrupted mitochondrial physiology. However, modelling mitochondrial dysfunction in a disease-relevant model, suitable for screening large compound libraries for ameliorative effects, represents a considerable challenge. Primary patient derived cells, SHSY-5Y cells and in vivo models of Parkinson's disease have been utilized extensively to study the contribution of mitochondrial dysfunction in Parkinson's. Indeed many studies have utilized LUHMES cells to study Parkinson's disease, however LUHMES cells have not been used as a compound screening model for PD-associated mitochondrial dysfunction previously, despite possessing several advantages compared to other frequently used models, such as rapid differentiation and high uniformity (e.g., in contrast to iPSC-derived neurons), and relevant physiology as human mesencephalic tissue capable of differentiating into dopaminergic-like neurons that highly express characteristic markers. After previously generating GFP+-LUHMES cells to model metabolic dysfunction, we report this protocol using GFP+-LUHMES cells for high-throughput compound screening in a restoration model of PD-associated mitochondrial dysfunction. This protocol describes the use of a robust and reproducible toxin-induced GFP+-LUHMES cell model for high throughput compound screening by assessing a range of mitochondrial and neuronal morphological parameters. We also provide detailed instructions for data and statistical analysis, including example calculations of Z'-score to assess statistical effect size across independent experiments.

Mitochondrial Dysfunction in Alzheimer's Disease: A Biomarker of the Future?

Alzheimer's disease (AD) is the most common cause of dementia worldwide and is characterised pathologically by the accumulation of amyloid beta and tau protein aggregates. Currently, there are no approved disease modifying therapies for clearance of either of these proteins from the brain of people with AD. As well as abnormalities in protein aggregation, other pathological changes are seen in this condition. The function of mitochondria in both the nervous system and rest of the body is altered early in this disease, and both amyloid and tau have detrimental effects on mitochondrial function. In this review article, we describe how the function and structure of mitochondria change in AD. This review summarises current imaging techniques that use surrogate markers of mitochondrial function in both research and clinical practice, but also how mitochondrial functions such as ATP production, calcium homeostasis, mitophagy and reactive oxygen species production are affected in AD mitochondria. The evidence reviewed suggests that the measurement of mitochondrial function may be developed into a future biomarker for early AD. Further work with larger cohorts of patients is needed before mitochondrial functional biomarkers are ready for clinical use.