The debated toxic role of aggregated TDP-43 in amyotrophic lateral sclerosis: a resolution in sight?

Transactive response DNA-binding protein-43 (TDP-43) is an RNA/DNA binding protein that forms phosphorylated and ubiquitinated aggregates in the cytoplasm of motor neurons in amyotrophic lateral sclerosis, which is a hallmark of this disease. Amyotrophic lateral sclerosis is a neurodegenerative condition affecting the upper and lower motor neurons. Even though the aggregative property of TDP-43 is considered a cornerstone of amyotrophic lateral sclerosis, there has been major controversy regarding the functional link between TDP-43 aggregates and cell death. In this review, we attempt to reconcile the current literature surrounding this debate by discussing the results and limitations of the published data relating TDP-43 aggregates to cytotoxicity, as well as therapeutic perspectives of TDP-43 aggregate clearance. We point out key data suggesting that the formation of TDP-43 aggregates and the capacity to self-template and propagate among cells as a 'prion-like' protein, another pathological property of TDP-43 aggregates, are a significant cause of motor neuronal death. We discuss the disparities among the various studies, particularly with respect to the type of models and the different forms of TDP-43 used to evaluate cellular toxicity. We also examine how these disparities can interfere with the interpretation of the results pertaining to a direct toxic effect of TDP-43 aggregates. Furthermore, we present perspectives for improving models in order to better uncover the toxic role of aggregated TDP-43. Finally, we review the recent studies on the enhancement of the cellular clearance mechanisms of autophagy, the ubiquitin proteasome system, and endocytosis in an attempt to counteract TDP-43 aggregation-induced toxicity. Altogether, the data available so far encourage us to suggest that the cytoplasmic aggregation of TDP-43 is key for the neurodegeneration observed in motor neurons in patients with amyotrophic lateral sclerosis. The corresponding findings provide novel avenues toward early therapeutic interventions and clinical outcomes for amyotrophic lateral sclerosis management.

Aß1-42 and Tau as Potential Biomarkers for Diagnosis and Prognosis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease, but its definitive diagnosis delays around 12 months. Although the research is highly active in the biomarker field, the absence of specific biomarkers for diagnosis contributes to this long delay. Another strategy of biomarker identification based on less specific but sensitive molecules may be of interest in clinical practice. For example, markers related to other neurodegenerative diseases such as Alzheimer's disease (AD) could be fully explored. Here, we compared baseline levels of amyloidß1-42 (Aß1-42), total Tau, and phosphorylated-Tau (phospho-Tau) protein in the cerebrospinal fluid (CSF) of ALS patients to controls and correlated it with clinical parameters of ALS progression collected over 12 months. We observed increased levels of Aß1-42 (controls: 992.9 ± 358.3 ng/L; ALS: 1277.0 ± 296.6 ng/L; p < 0.0001) and increased Aß1-42/phospho-Tau ratio and Innotest Amyloid Tau Index (IATI) (both p < 0.0001). IATI and the phospho-Tau/total Tau ratio correlated positively with ALSFRS-R and weight at baseline. Multivariate analysis revealed that baseline ALSFRS-R was associated with Aß1-42 and phospho-Tau/total Tau ratio (p = 0.0109 and p = 0.0013, respectively). Total Tau and phospho-Tau levels correlated negatively with ALSFRS-R variation at months 6 and 9, respectively (p = 0.02 and p = 0.04, respectively). Phospho-Tau/total Tau ratio correlated positively with ALSFRS-R variation at month 9 (p = 0.04). CSF levels of Aß1-42 could be used as a complementary tool to ALS diagnosis, and total Tau and phospho-Tau levels may help establishing the prognosis of ALS. Further studies merit exploring the pathophysiological mechanisms associated with these markers. Despite their lack of specificity, phospho-Tau/total Tau and Aß1-42 should be combined to other biological and clinical markers in order to improve ALS management.

TAR DNA-binding protein of 43 kDa (TDP-43) and amyotrophic lateral sclerosis (ALS): a promising therapeutic target.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that lacks an effective treatment. Aggregates of the TAR DNA-binding protein-43 (TDP-43) are observed in 97% of all ALS cases, thus making this protein a major therapeutic target in ALS. . AREAS COVERED: The authors describe the major cellular functions of TDP-43 and the features and consequences of TDP-43 proteinopathy. Drawing from fundamental and preclinical studies on cellular and animal TDP-43 models of ALS and selected clinical trials, the major pathways that have been targeted for the mitigation of TDP-43 pathology in ALS are discussed. The authors provide insights on the approaches targeting the tendency of TDP-43 for aggregation, defective nucleocytoplasmic transport, dysfunctional proteostasis, abnormal stress granule dynamics, and pathological post-translational modifications of TDP-43. EXPERT OPINION: The complexity of ALS and TDP-43 proteinopathy generates challenges for the development of novel therapeutic approaches. However, the critical involvement of TDP-43 in the initiation and progression of ALS, makes it a promising therapeutic target. Further research should be centered on the development of precision strategies, consideration of patient subgroups, the prevention of the mislocalization of TDP-43 and restoration of the lost functions of TPD-43. .

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2-5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a "prion-like" protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism.

Understanding and managing metabolic dysfunction in Amyotrophic Lateral Sclerosis.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron disease that leads to death after a median survival of 36 months. The development of an effective treatment has proven to be extremely difficult due to the inadequate understanding of the pathogenesis of ALS. Energy metabolism is thoroughly involved in the disease based on the discoveries of hypermetabolism, lipid/glucose metabolism, the tricarboxylic acid (TCA) cycle, and mitochondrial impairment. AREA COVERED: Many perturbed metabolites within these processes have been identified as promising therapeutic targets. However, the therapeutic strategies targeting these pathways have failed to produce clinically significant results. The authors present in this review the metabolic disturbances observed in ALS and the derived-therapeutics. EXPERT OPINION: The authors suggest that this is due to the insufficient knowledge of the relationship between the metabolic targets and the type of ALS of the patient, depending on genetic and environmental factors. We must improve our understanding of the pathological mechanisms and pay attention to the subtle hidden effects of changing diet, for example, and to use this strategy in addition to other drugs or to use metabolism status to determine subgroups of patients.

TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis.

Cytoplasmic TDP-43 aggregates are a hallmark of amyotrophic lateral sclerosis (ALS). Today, only two drugs are available for ALS treatment, and their modest effect prompts researchers to search for new therapeutic options. TDP-43 represents one of the most promising targets for therapeutic intervention, but reliable and reproducible in vitro protocols for TDP-43-mediated toxicity are lacking. Here, we used HEK293T cells transfected with increasing concentrations of TDP-43-expressing plasmid to evaluate different parameters of toxicity and alterations in cellular metabolism. Overexpression of TDP-43 induced aggregates occurrence followed by the detection of 25- and 35-kDa forms of TDP-43. TDP-43 overexpression decreased cell viability and increased cells arrested at G2/M phase and nuclear fragmentation. Analysis of the energetic metabolism showed a tendency to decrease oxidative phosphorylation and increase glycolysis, but no statistical differences were observed. Metabolomics revealed alterations in different metabolites (mainly sphingolipids and glycerophospholipids) in cells overexpressing TDP-43. Our data reveal the main role of TDP-43 aggregation in cellular death and highlight novel insight into the mechanism of cellular toxicity induced by TDP-43. Here, we provide a simple, sensitive, and reliable protocol in a human-derived cell line to be used in high-throughput screenings of potential therapeutic molecules for ALS treatment.

Metabo-lipidomics of Fibroblasts and Mitochondrial-Endoplasmic Reticulum Extracts from ALS Patients Shows Alterations in Purine, Pyrimidine, Energetic, and Phospholipid Metabolisms.

Amyotrophic lateral sclerosis (ALS) is characterized by a wide metabolic remodeling, as shown by recent metabolomics and lipidomics studies performed in samples from patient cohorts and experimental animal models. Here, we explored the metabolome and lipidome of fibroblasts from sporadic ALS patients (n = 13) comparatively to age- and sex-matched controls (n = 11), and the subcellular fraction containing the mitochondria and endoplasmic reticulum (mito-ER), given that mitochondrial dysfunctions and ER stress are important features of ALS patho-mechanisms. We also assessed the mitochondrial oxidative respiration and the mitochondrial genomic (mtDNA) sequence, although without yielding significant differences. Compared to controls, ALS fibroblasts did not exhibit a mitochondrial respiration defect nor an increased proportion of mitochondrial DNA mutations. In addition, non-targeted metabolomics and lipidomics analyses identified 124 and 127 metabolites, and 328 and 220 lipids in whole cells and the mito-ER fractions, respectively, along with partial least-squares-discriminant analysis (PLS-DA) models being systematically highly predictive of the disease. The most discriminant metabolomic features were the alteration of purine, pyrimidine, and energetic metabolisms, suggestive of oxidative stress and of pro-inflammatory status. The most important lipidomic feature in the mito-ER fraction was the disturbance of phosphatidylcholine PC (36:4p) levels, which we had previously reported in the cerebrospinal fluid of ALS patients and in the brain from an ALS mouse model. Thus, our results reveal that fibroblasts from sporadic ALS patients share common metabolic remodeling, consistent with other metabolic studies performed in ALS, opening perspectives for further exploration in this cellular model in ALS.

How Can a Ketogenic Diet Improve Motor Function?

A ketogenic diet (KD) is a normocaloric diet composed by high fat (80-90%), low carbohydrate, and low protein consumption that induces fasting-like effects. KD increases ketone body (KBs) production and its concentration in the blood, providing the brain an alternative energy supply that enhances oxidative mitochondrial metabolism. In addition to its profound impact on neuro-metabolism and bioenergetics, the neuroprotective effect of specific polyunsaturated fatty acids and KBs involves pleiotropic mechanisms, such as the modulation of neuronal membrane excitability, inflammation, or reactive oxygen species production. KD is a therapy that has been used for almost a century to treat medically intractable epilepsy and has been increasingly explored in a number of neurological diseases. Motor function has also been shown to be improved by KD and/or medium-chain triglyceride diets in rodent models of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and spinal cord injury. These studies have proposed that KD may induce a modification in synaptic morphology and function, involving ionic channels, glutamatergic transmission, or synaptic vesicular cycling machinery. However, little is understood about the molecular mechanisms underlying the impact of KD on motor function and the perspectives of its use to acquire the neuromuscular effects. The aim of this review is to explore the conditions through which KD might improve motor function. First, we will describe the main consequences of KD exposure in tissues involved in motor function. Second, we will report and discuss the relevance of KD in pre-clinical and clinical trials in the major diseases presenting motor dysfunction.

The Relevancy of Data Regarding the Metabolism of Iron to Our Understanding of Deregulated Mechanisms in ALS; Hypotheses and Pitfalls.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the loss of motor neurons. Its etiology remains unknown, but several pathophysiological mechanisms are beginning to explain motor neuronal death, as well as oxidative stress. Iron accumulation has been observed in both sporadic and familial forms of ALS, including mouse models. Therefore, the dysregulation of iron metabolism could play a role in the pathological oxidative stress in ALS. Several studies have been undertaken to describe iron-related metabolic markers, in most cases focusing on metabolites in the bloodstream due to few available data in the central nervous system. Reports of accumulation of iron, high serum ferritin, and low serum transferrin levels in ALS patients have encouraged researchers to consider dysregulated iron metabolism as an integral part of ALS pathophysiology. However, it appears complicated to suggest a general mechanism due to the diversity of models and iron markers studied, including the lack of consensus among all of the studies. Regarding clinical study reports, most of them do not take into account confusion biases such as inflammation, renal dysfunction, and nutritional status. Furthermore, the iron regulatory pathways, particularly involving hepcidin, have not been thoroughly explored yet within the pathogenesis of iron overload in ALS. In this sense, it is also essential to explore the relation between iron overload and other ALS-related events, such as neuro-inflammation, protein aggregation, and iron-driven cell death, termed ferroptosis. In this review, we point out limits of the designs of certain studies that may prevent the understanding of the role of iron in ALS and discuss the relevance of the published data regarding the pathogenic impact of iron metabolism deregulation in this disease and the therapeutics targeting this pathway.