Spectrums of Amyotrophic Lateral Sclerosis. Группа авторов

molecular biology techniques have made it possible to insert gene mutations and express mutant proteins in a number of animal models (see details in Chapter 8). Small animals such as Drosophila melanogaster and Danio rerio (zebrafish), have been widely used due to the simplicity and rapidity of manipulations, especially for drug screening. The zebrafish has many advantages in this sense, mostly because it is a vertebrate and has high genetic homology with humans. The zebrafish can be used at the embryonic stage, taking advantage of egg transparency and its rapid development, which can be followed in real time; and also at the adult stage, using transgenic lines. Motor phenotypes can be easily detected and analyzed, and in vivo imaging can be promptly performed. Genetic interactions can be tested as well as mechanisms of action of pathogenesis. High‐throughput drug screening can be done to test libraries containing thousands of chemical compounds at the same time. Since the zebrafish is not a mammal and does not have UMNs (the corticospinal and rubrospinal tracts are absent), it can be considered a very useful tool to study cellular dynamics in vivo and may be used prior to other models, such as rodents [81, 82].

A wide range of murine models has been created [83] but the most commonly used remains the first one developed: a transgenic strain carrying the SOD1^G93A pathogenic variant [84]. This model has been used to test most drugs in preclinical phases. It should be noted that these treatments are administrated at the pre‐symptomatic stage, whereas ALS patients are treated after a disease onset that seems to be preceded by a long pre‐symptomatic period. To better investigate the pre‐symptomatic stage, a SOD1 pig model has recently been obtained [85]. Since pigs have a long lifespan, transgenic pigs, stably expressing the human pathological allele SOD1^G93A, have a pre‐symptomatic phase of about 27 months. After this period, gait abnormality and concomitant dysphagia appear and progress rapidly with severe respiratory impairment. SOD1 animal models have been used in preclinical investigations of almost all drugs used in clinical trials. However, the principal limit of this model is that TDP‐43 pathology, which is present in about 97% of all ALS subtypes, is not detected in SOD1 mutated patients, suggesting different disease mechanisms. In addition, preclinical studies performed in mice have failed to be transferred to humans [86].

      Considerable efforts have been undertaken to study the biological role of C9orf72, because its pathogenic expansion is the most frequent cause of ALS and FTD in populations of European descent. Drosophila, zebrafish, and rodents have been used to test various hypotheses of the C9orf72 mechanism, including loss of function, leading to haploinsufficiency of the gene, and gain of function, with the accumulation of RNA foci and dipeptide repeats (DPR) resulting from non‐conventional repeat translation. TDP‐43 inclusions are detectable in mice expressing the C9orf72 expanded allele, suggesting that TDP‐43 is downstream of C9orf72. Knockout mice show an inflammatory phenotype, thus implicating C9orf72 in immune regulation and the autophagic pathway [87]. Mice expressing the repeat expansion present with RNA foci and DPR, but they do not have a behavioral phenotype, suggesting that the gain of function is not sufficient to cause the disease [88, 89]. A combination of different mechanisms is probably required for disease development [90].

      Different animal models, reproducing mutations in different genes, are needed to investigate ALS in its complexity along with the clinical overlap with other diseases of the spectrum. For example, a transgenic mouse has recently been described, carrying the MATR3^S85C variant. This model shows myopathic histological changes: TDP‐43 aggregates in muscles, and respiratory problems occur due to myopathic changes in diaphragm muscles. Interestingly, the observed myotoxicity recapitulates the clinicopathological features of distal myopathy and ALS [91]. Also, a TBK1 mouse, recently developed, reproduces the main symptoms of ALS/FTD. Mice carrying the conditional neuronal deletion of TBK1 show memory deficits and reduced locomotor activity. Interestingly, TBK1 overexpression extended the lifespan of symptomatic mice not only for TBK1 knockout strains but also for SOD1^G93A mice, thus suggesting that TBK1 and SOD1 are probably part of the same pathway and can be targeted by the same drugs [92].

      By comparing phenotypes across ALS models carrying mutations in different genes, it is possible to study the disease as broadly as possible.

      In Vitro Models

      The combination of in vivo and in vitro models can be a good strategy to investigate disease mechanisms in depth. In recent years, a number of studies have been performed on commercial cells engineered to carry mutations in ALS‐associated genes. In recent years, the innovative possibility of reprogramming somatic cells obtained from patients opened new avenues for ALS research. Hopefully it will lead to significant improvements in the future of regenerative medicine. Generating cells from patients has two significant advantages that are unique in this model:

      1 It is possible to obtain human motor neurons, glial cells, and microglia, the cell types that are primarily affected by the disease and that have been studied in the past only as post‐mortem samples.

      2 Cells obtained from patients carry exactly the same genetic background as the patient. This means there is no need to insert the genetic mutation artificially: it is possible to study cells as they are in nature. In this context, it is possible to investigate the disease mechanism in all ALS subtypes, including those with known and unknown genetic defects. Moreover, a genetic mutation that arises spontaneously can be corrected using gene‐editing techniques to revert the phenotype.

      Two different strategies have been set up to reprogram cells from patients. The most commonly used is the generation of induced pluripotent stem cells (iPSCs) from skin fibroblasts [93] and their subsequent differentiation into motor neurons. The second strategy is the direct conversion of skin fibroblasts into motor neurons or glial cells [94].

      Fibroblasts can be easily obtained through a skin biopsy, which is not invasive and is very well tolerated by patients. Specific transcription factors (Oct3/4, Sox2, Klf4, and c‐Myc) can be introduced by retroviral transduction into somatic cells to convert them into iPSCs [93]. To avoid side effects caused by the use of retroviruses that integrate in the genome, various tools have been developed, such as non‐integrating virus and mRNA transcription factors. The iPSCs have the ability to self‐renew in culture and can differentiate into cell types of all three germ layers while maintaining the patient's genetic background. Direct conversion of neuronal cells from fibroblasts allows us to bypass the pluripotent stage and can be obtained by overexpressing a combination of transcription factors [94]. Thanks to this strategy, the maturity of the cell, as well as its epigenetic signatures, are preserved; and stem cells can be a better method to study late‐onset diseases.

      Once obtained, iPSCs can be differentiated into every kind of cell. The most recent innovative approach consists of generating three‐dimensional cell cultures called organoids, with the aim of better reproducing intercellular interactions and physiological properties. Organoids are particularly useful for drug testing since they better mimic patient's response and tolerability.

CONCLUSION

      Recent genetic discoveries and progress in neuropathology have completely changed the perspective on ALS. The current idea is that ALS cannot be considered a single entity (as it was until a few years ago) but rather is part of a clinical spectrum of disease. Various clinical manifestations can be described depending on familial history, age of onset, site of onset, disease duration, and overlap with other conditions as cognitive impairment or myopathies. Animal and cellular models have been established to better characterize the disease pathogenesis and to link the disease to different biological pathways. All these models have the same goal: looking for treatments that can stop or at least significantly slow the disease progression.

CONFLICT OF INTEREST

      The authors declare no potential conflict of interest with respect to research, authorship, and/or publication of this manuscript.

COPYRIGHT AND PERMISSION STATEMENT