Many neurodegenerative disease-related genes/proteins such as SOD1 and OPTN in ALS (Rabizadeh et al., 1995; Kryndushkin et al., 2012) have been established as models among yeast (Braun, 2015). To develop SOD1 yeast models, various ALS-related SOD1 mutations such as A4V, G37R, H48Q, G93A, and S134N have been introduced into the yeast SOD1 gene. The SOD1 yeast model demonstrated that the mutant human SOD1 (hmSOD1) protein is unstable and reduces cell viability but does not form insoluble SOD1 protein aggregates. Moreover, the hmSOD1 toxic effects seem not to depend on mitochondrial dysfunction or oxidative stress, but rather on the inability to control central metabolic processes, which is most probably due to the severe disruption of vacuolar compartments (Bonifacino et al., 2021).

ALS-related proteins with prion-like domains have proven to be particularly powerful in yeast models because they are similar to naturally existing yeast prion-like proteins, such as FUS, TDP-43, heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), TATA-box binding protein associated factor 15 (TAF15), and Ewing sarcoma breakpoint region 1 (EWS) proteins, which have similar protein architectures. Moreover, these proteins were found in the neuronal cytoplasm of post-mortem ALS patients. In yeast models, ALS-related proteins and their mutant isoforms typically induce GOF toxicity, which is partially mediated by aggregation via prion-like domains. Yeast did not possess TDP-43 orthologous genes. Yeast models overexpressing human wild-type (WT) TDP-43 showed that TDP-43 accumulated and formed subcellular aggregates in the cytoplasm, which inhibited cell growth, disrupted cell morphology, and generated cytotoxicity. Yeast models of ALS-related TDP-43 mutations, such as K, M337V, Q343R, N345K, R361S, and N390D, accelerate protein aggregation, increase the number of cytosolic aggregates, and lead to growth arrest and cell death. Yeasts do not express FUS orthologs; thus, yeasts FUS models have been established by ectopically transforming human WT or mutant FUS genes into yeast. FUS yeast models are tightly associated with the translocation of FUS protein from the nucleus to the cytoplasm, forming aggregates co-localized with P bodies and stress granules in yeast cytoplasm and inhibiting the ubiquitin proteasome systems (Bonifacino et al., 2021).

Yeast models also have some limitations and disadvantages. Yeast protein domains are not the same as those in humans; thus, they must be carefully considered in studies using yeast models. The formation of prion-like proteins in yeast is its (True et al., 2004; Halfmann et al., 2012); however, this is not the case for human ALS proteins with prion-like domains. Almost all mammalian all mammalian prion-like proteins are harmful to humans (An and Harrison, 2016) since the properties of yeast prion-like protein domains are not completely conserved, but produce distinct alterations of constructs and functions that lead to contrary functions between yeast and humans during the human evolution process. Another limitation of yeast models is yeast cells do not have resident interacting proteins or chaperones that regulate human pathogenic proteins compared to mammalian cells. Chaperones are especially important for quality control and are integral to the propagation of endogenous prion proteins. Owing to the deficiency of resident interacting proteins and chaperones in yeast, the interaction of ectopically expressing human prion-like proteins in yeast is naturally different from that in humans; thus, the conclusions derived from studying protein interactions in yeast models may not be suitable for human diseases (Masison and Reidy, 2015).

The hSOD1 D. melanogaster models were produced by amplifying the hSOD1 gene through missense mutation technology and replacing the nitrogenous base among DNA correspondence for hSOD1 reading site with a directive mutation. The hmSOD1 D. melanogaster models exhibit movement dysfunction, local accumulation of hmSOD1 in MNs, enlargement of glial cells, neuronal degeneration, muscle contraction, decreased survival rates, and mitochondrial dysfunction (Gois et al., 2020). It reduces lifespan and fecundity and increases susceptibility to oxidative stress, movement defects, and necrotic cell death in hmSOD1 D. melanogaster models with SOD1 mutations (Phillips et al., 1989). The hmSOD1 D. melanogaster model expresses four hSOD1 mutants – G85R, H71Y, H48R and G37R. Among the endogenous D. melanogaster, SOD1 (dSOD1) produces a large amount of dSOD1 protein in neural cells (Layalle et al., 2021). Models expressing G85R, H71Y, and H48R mutations show decreased survival, developmental defects, and larval and adult dyskinesia due to muscle axon contraction (Braems et al., 2021). In addition, some antioxidant compounds have neuroprotective effects that improve exercise performance, prolong lifespan, and reduce hSOD1 cytoplasmic inclusion bodies in the hSOD1 D. melanogaster model (Liguori et al., 2021).

The transgenic D. melanogaster model expressing only hSOD1^WT in MNs has been demonstrated to extend its longevity, but does not affect the movement or survival of MNs (Parkes et al., 1998). Moreover, the hSOD1^WTtransgenic D. melanogaster model extended the longevity of the SOD1 deletion mutant and normal D. melanogaster but did not prevent age-dependent movement disorders. The D. melanogaster model's widespread downregulation of SOD1 expression accelerates age-dependent movement disorders and shortens the lifespan of D. melanogaster (Braems et al., 2021). Meanwhile, the D. melanogaster model selectively expressing WT or causative-related hSOD1 mutations (A4V and G85R) in MNs induces progressive movement dysfunction with electrophysiological defect, and the SOD1 aberrant accumulates stress responses around the glial cells (Watson et al., 2008). Moreover, the transgenic D. melanogaster model specifically expressing hmSOD1 in MNs showed a progressive motor protective effect, prevented the accumulation of hmSOD1 aggregation, and increased the glial cell stress response in the ventral nervous cord, accompanied by electrophysiological defects in neural circuits. The hmSOD1 transgenic D. melanogaster overexpressing mutated SOD1 can model both cellular and non-cellular autonomic damage (Walters et al., 2019). The transgenic D. melanogaster model expressing WT or mutant (A4V and G85R) hSOD1 among D. melanogaster MNs would lead to D. melanogaster climbing defects that progress over time and defects among neural circuits accompanied by the stress responses of glial cells and local accumulation of mutant SOD1 proteins among MNs (Braems et al., 2021). Meanwhile, the hSOD1 A4V transgenic D. melanogaster model showed synaptic transmission defects and focal mutation SOD1 accumulation in MNs, HSP upregulation in glial cells, cellular autonomic lesions in MNs, and abnormal alterations in glial cells. This D. melanogaster model reveals that ALS is not confined to damaging MNs, and toxic SOD1 transfers from neurons to glial cells (Clement et al., 2003; Boillée et al., 2006). The hSOD1 transgenic D. melanogaster models are usually established by hSOD1 gene expression among the MNs of D. melanogaster SOD1 null background applying GAL4/UAS yeast systems. In hSOD1 transgenic D. melanogaster models, very low levels of hSOD1^WT expression are sufficient to reverse the lifespan reduction, oxidative stress increase, and physiological dysfunctions related to the SOD1 null D. melanogaster model (Mockett et al., 2003). In patients with ALS, it is very difficult to identify the complex genetics involved in its pathogenesis and to functionally investigate new candidate disease genes. D. melanogaster models can allow for an in-depth study of ALS progression from the earliest signs to terminal stages. They can rapidly perform a large range of simple assays from lifespan and motor assays to anatomical screens and can provide important functional information on the potential machinery required for proper motor neuron function and how this machinery may be dysregulated during ALS, which would not be possible in human neural systems. The D. melanogaster model is useful for addressing complex genetic diseases such as ALS. The UAS/Gal4/Gal80 system can upregulate and knock down D. melanogaster genes, including SOD1, and the ectopic expression of human SOD1 genes or mutations in a tissue-specific manner, exhibiting the typical pathologies of hmSOD1 ALS, which is beneficial for further investigation to identify disease-modifying genes, mutations, and disease pathways (Walters et al., 2019).

TDP-43 is a highly conserved and ubiquitously expressed nuclear protein that participates in various cellular processes, including mRNA splicing, transcription, stability, and transportation (Langellotti et al., 2018; Lembke et al., 2019). TDP-43 is crucial for promoting the formation and growth of neuromuscular junctions (NMJ) (Strah et al., 2020). A number of D. melanogaster models generated by the TDP-43 toxicity of endogenous D. melanogaster TDP-43 (dTDP) and hTDP-43 transgenic expression revealed that the TDP-43 protein showed toxicity, and the major phenotypes were largely similar in these D. melanogaster models. The D. melanogaster model lacking dTDP appears externally normal, but presents a deficiency in locomotion behaviors, a reduction in lifespan, anatomical defects of NMJ, and a decrease in dendrite branches (Feiguin et al., 2009; Lu et al., 2009; Lin et al., 2011). Among the above-mentioned TDP-43 D. melanogaster models, these phenotypes can be recovered and repaired by expressing the normal hTDP-43 protein in neurons, such as MNs (Feiguin et al., 2009). The phenotypes of TDP-43 D. melanogaster models revealed that the dysfunction of TDP-43 might result in the pathogenesis of ALS. In addition, overexpression of either dTDP or hTDP-43 in D. melanogaster also displayed the main pathological characteristics of ALS, premature lethality, neuronal loss, and defects in NMJ architecture and movement. TDP-43 D. melanogaster models revealed that the mechanisms of toxic GOF are related to TDP-43 proteinopathy (Lu et al., 2009; Elden et al., 2010; Hanson et al., 2010; Li et al., 2010; Ritson et al., 2010; Voigt et al., 2010; Estes et al., 2011; Miguel et al., 2011).

To study and identify the possible roles and potential pathogenic mechanisms of the TDP-43 protein in the pathogenesis of ALS, several TDP-43 transgenic D. melanogaster models have been developed to further explore whether gene modification of TDP-43 can increase or decrease the generation of TDP-43 toxicity (Elden et al., 2010; Hanson et al., 2010; Ritson et al., 2010). In these TDP-43 transgenic D. melanogaster models, upregulation of the human ATXN2 gene ortholog Pab1-binding protein 1 enhanced TDP-43 toxicity, resulting in more severe TDP-43-induced phenotypes (Elden et al., 2010). Moreover, the TDP-43 interacting partner ubiquilin 1 overexpression also produces similar TDP-43-induced phenotypes (Kim et al., 2009), reduced steady expression of TDP-43, and enhanced TDP-43 phenotypes (Hanson et al., 2010). The TDP-43-induced phenotypes among TDP-43 transgenic D. melanogaster models can also be modulated by co-expressing an ATPase member, valosin-containing protein (VCP), which is related to the protein family members of cellular activities that regulate many cell processes (Ritson et al., 2010). The above-described TDP-43 transgenic D. melanogaster models may enable the development of novel therapeutic targets that regulate TDP-43 expression in TDP-43-associated ALS patients, which is expected to prevent or treat TDP-43-associated ALS patients.

Bis-(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH), a D. melanogaster TDP-43 homolog, promotes target gene transcription by combining with sequences rich in uridine guanine, thus stabilizing the binding of chromatin regulators to DNA (Zhang et al., 2018a). Overexpression of mutant or WT hTDP-43 and TBPH in transgenic D. melanogaster models affects lifespan, mobility, axonal transport, and pupal shell sealing, whereas TBPH depletion leads to movement disorders and a shortened lifespan (Walters et al., 2019; Liguori et al., 2021). Among them, hTDP-43^WT overexpression in neurons results in an increase in NMJ buttons and branches associated with hTDP-43^WT protein aggregation (Kankel et al., 2020). However, the phenotypes induced by the mutant hTDP-43 were not distinguishable from the hTDP-43^WT induced phenotypes (Feldman et al., 2022).

Increased TDP-43 expression in TDP-43 transgenic D. melanogaster models induces mitochondrial dysfunction, including ridge abnormality and loss, resulting in a decrease in mitochondrial membrane potential and an increase in reactive oxygen species (ROS) production, usually accompanied by the loss of striated muscle tissue, and affects the survival and function of neurons due to excessive ROS production. Moreover, the TDP-43 expression increase also inhibited mitochondrial complex I activation and reduced mitochondrial ATP synthesis. Compared with the D. melanogaster control model, the volume of mitochondria in the eyes of D. melanogaster expressing TDP-43^WT or TDP−43^A315T was significantly reduced. TDP-43^WT and TDP-43^A315TGOF induced mitochondrial unfolded protein responses, including Lon and LonP1. LonP1 is a major mitochondrial matrix protease that interacts with TDP-43 to reduce mitochondrial TDP-43 protein levels, which can prevent mitochondrial damage and neurodegeneration in the D. melanogaster model induced by TDP-43 (Khalil et al., 2017; Wang et al., 2019; Layalle et al., 2021). Highly fragmented mitochondria have also been found among the axons of MNs in a D. melanogaster model expressing TDP-43 (Layalle et al., 2021). Sigma-1 receptor enhances the resistance of D. melanogaster to oxidative stress and exerts positive effects on motor activity and ATP levels (Couly et al., 2020).

In TDP-43 D. melanogaster models, significant changes were observed in glucose metabolism, including an increase in pyruvic acid levels. Among the MNs of TDP-43 D. melanogaster models, the mRNA level of phosphofructose kinase (an enzyme that controls the rate of fermentation) significantly increased (Manzo et al., 2019; Layalle et al., 2021). A high sugar diet can improve the exercise and life defect induced by TDP-43 protein lesions among MNs or/and glial cells, but it does not improve the muscles lesions in the TDP-43 D. melanogaster models, indicating that the metabolic disorders participate in the neural systems damage (Manzo et al., 2019). In addition, it has been revealed that some changes in the lipid metabolism, such as the reduction of both carnitine shuttle and lipid β oxidation, occur among TDP-43 D. melanogaster models (Layalle et al., 2021).

Among TDP-43 transgenic D. melanogaster models, other factors were found to have an impact on TDP-43. For example, the inhibition of protein tyrosine kinase 2 (PTK2) significantly reduces ubiquitin aggregation and cytotoxicity of TDP-43. Non-phosphorylated sequencesome 1 (SQSTM1) inhibits the accumulation and neurotoxicity of insoluble polyubiquitin proteins induced by TDP-43 overexpression in neuronal cells. TANK-binding kinase 1 (TBK1) participates in PTK2-mediated SQSTM1 phosphorylation. Therefore, the PTK2-TBK1-SQSTM1 axis plays a key role in the pathogenesis of TDP-43 by modulating neurotoxicity caused by damage to the ubiquitin-proteasome system in the TDP-43 transgenic D. melanogaster model (Lee et al., 2020). Recently, it was found that the generation of reversible droplet-like nuclear bodies (Wang et al., 2020), the overexpression of long non-coding RNA nuclear enriched abundant transcript 1–1 (Matsukawa et al., 2021), Mucuna pruriens and Withania somnifera (Maccioni et al., 2018; Paul et al., 2021; Zahra et al., 2022), and the production of CG5445 (a previously uncharacterized D. melanogaster gene) (Uechi et al., 2018, p. 3) alleviate the cytotoxicity of TDP-43 in neurons and improve movement or eye symptoms in the TDP-43 transgenic D. melanogaster model.

Gemin3 (DDX20 or DP103) is a dead-box RNA helicase that participates in a variety of cellular processes. The combination of Gemin3 and TDP-43 or FUS destruction aggravates vitality defects, motor dysfunction, and muscle atrophy while inhibiting the overgrowth of NMJ in the TDP-43 transgenic D. melanogaster model (Cacciottolo et al., 2019). A 3-fold knockdown of the lethal gene inhibitor (elongation factor of RNA polymerase II) significantly inhibited the morphological defects of the compound eye and medial retinae induced by TDP-43 in TDP-43 transgenic D. melanogaster models. D. melanogaster has five histone deacetylases (HDAC), among which Rpd3 knockdown significantly inhibits the rough eye phenotype caused by hTDP-43 in the TDP-43 transgenic D. melanogaster model (Yamaguchi et al., 2021). TBPH deficiency leads to larval death and reduced HDAC6 levels (Braems et al., 2021). Among the many single or double hydrophilic tag conjugating peptides D1-D8, D4 has the strongest ability to degrade TDP-43 protein and can penetrate the cell wall in short periods, inducing TDP-43 protein degradation in a dose- and time-dependent manner (Gao et al., 2019).

Reverse transposable element (RTE) expression was strongly upregulated in the head of TBPH-null D. melanogaster. TBPH regulates the silencing mechanism of small interfering RNA inhibiting RTE, and TBPH modulates Dicer 2 expression levels through direct protein mRNA interaction in TDP-43 transgenic D. melanogaster models (Romano et al., 2020). TBPH also interacted with VCP to inhibit VCP-induced degeneration in a TDP-43 transgenic D. melanogaster model (Braems et al., 2021).

Although FUS mutations obviously affect MNs as well as other neurons found in FUS transgenic D. melanogaster models, its pathogenic mechanism of proteinopathy remains largely unknown. To study the alterations of FUS-associated functions in neurodegenerative diseases, including ALS, we reproduced the FUS transgenic D. melanogaster model expressing mutant human FUS (hmFUS), such as R518K, R521C, and R521H. These models cause a severe neurodegeneration in D. melanogaster eyes, whereas WT human FUS (hFUS^WT) expression only led to mild neurodegeneration (Lanson et al., 2011).

Both movement defects and premature death have been observed in mutant FUS transgenic D. melanogaster. Moreover, mutant FUS overexpression increased the accumulation of the FUS protein. Causative roles on ALS-related hFUS mutants including both R524S and P525L in the FUS transgenic D. melanogaster model have also been described (Chen et al., 2011). Among the D. melanogaster models overexpressing WT and ALS-related FUS mutations among various neuron subsets, such as photoreceptors, mushroom bodies, and MNs, progressive age-related neuronal degeneration, such as axon loss, morphological alterations, and functional impairment of MNs, has been observed. The model of Cabeza (hFUS D. melanogaster homolog)-deficient D. melanogaster is used to study FUS functions, and the results showed both reduced lifespan and locomotor defects compared to controls. It was also found that transferring hFUS^WT gene into this model could fully recover these phenotypes, but in this model co-expressing the ALS-related mutant FUS proteins, the phenotypes of reduced lifespan and locomotor defects were not recovered. This suggests that ALS-linked FUS mutations are toxic in FUS transgenic D. melanogaster models (Walker et al., 2011). In a transgenic D. melanogaster model coupled FUS with TDP-43 gene in neurons, it was demonstrated that FUS acts on ALS pathogen-associated effects together with TDP-43 via a common genetic pathway. Moreover, FUS and TDP-43 exert synergistic effects on RNA-dependent complexes. These models show that FUS and TDP-43 exert partial damage during the pathogenesis of ALS (Layalle et al., 2021).

D. melanogaster FUS homolog Cabeza is extensively expressed in the majority of tissues, including the neural system. Most Cabeza-D. melanogaster models with Cabeza functional loss are fatal, and only a few D. melanogaster are able to develop into adulthood, but exhibit severely shortened lifespans and motor disorders. Importantly, co-expression of wild-type Cabeza in the neurons of Cabeza-mutant D. melanogaster models can repair climbing and flight defects. Compared with Cabeza-mutant D. melanogaster models, larvae overexpressing Cabeza/FUS^WT exhibited opposite NMJ electrophysiological phenotypes, characterized by reduced excitatory junction potentials and miniature excitatory junction potential amplitudes. In addition, it was found that the D. Melanogaster model may have a self-inhibitory mechanism for FUS/Cabeza, as the overexpression of FUS reduces the intracellular Cabeza content (Zhang et al., 2018a). D. melanogaster overexpressing hFUS^WT exhibits abnormal eye morphology, shortened lifespan, eclosion defects, climbing defects, a reduced number of synaptic buttons, a reduced active region of the NMJ, and axonal degeneration (Azuma et al., 2018). hFUS is overexpressed in photoreceptor neurons in the eyes of D. melanogaster, resulting in mild retinal degeneration, rough eye surfaces, and a decrease in red pigment. The changes in the different FUS regions have different effects, such as in HEK293 cells, where the C-terminal truncated FUS chelates the FUS^WT protein from the nucleus to the cytoplasm, thereby exacerbating FUS-induced retinal degeneration. The FUS-P525L mutation exacerbates FUS-induced retinal degeneration by increasing the FUS cytoplasmic distribution (Matsumoto et al., 2018; Layalle et al., 2021).

The arginine residue in the low-complexity domain of the FUS C-terminus is necessary for the maturation of FUS, while the N-terminal glutamine-glycine-serine-tyrosine (QGSY)-rich region (amino acids 1–164) and the C-terminal RGG2 domain are necessary for the toxicity of FUS, including the N-terminal QGSY, prion-like domains, and C-terminal RGG2 domains. It can distinguish between FUS and Cabeza toxicity based on its QGSY domain (Bogaert et al., 2018). The expression of different FUS mutations (R521C, 521H, 518K, R524S, or P525L) resulted in more severe rough-eye phenotypes in FUS transgenic D. Melanogaster models. FUS expression triggers Hippo activation and c-Jun N-terminal kinase signaling, leading to neuronal degeneration in FUS transgenic D. Melanogaster models. In addition, the Hippo signaling pathway is a modifier of Cabeza knockdown phenotypes (Layalle et al., 2021).

In FUS transgenic D. melanogaster models, it has been shown that inhibiting nuclear output can reduce hFUS-induced toxicity, thereby confirming the involvement of nuclear-cytoplasmic transport in the pathogenesis of ALS. It is worth noting that, recently, a series of complex enhancers and inhibitors have been found for ALS modifying factors screened in two different transgenic D. melanogaster models carrying human mutant FUS genes (Liguori et al., 2021). In the Cabeza knockdown D. melanogaster models, there are 14 inhibitory mutations knockdown models that effectively inhibit rough eye phenotypes caused by Cabeza knockdown. Mutations in Chameau and N-alpha-acetyltransferase 60 inhibit locomotion and cause morphological defects in NMJ synapses caused by Cabeza knockdown (Yamaguchi et al., 2021). FUS is similar to TDP-43 gene and regulates synaptic transmission among D. melanogaster NMJ; synaptic transmission defects precede the degeneration and loss of MNs in FUS transgenic D. melanogaster models. The nuclear-cytoplasmic transport proteins exportin-1 and nucleoporin-154 are modifiers of FUS toxicity, which prevent FUS-induced toxicity and reduce apoptosis of ventral nerve cord neurons (Braems et al., 2021).

In third-instar larvae of a Cabeza mutant D. melanogaster model, it was shown that the Cabeza mutant leads to an increase in induced and spontaneous neurotransmitter release in the NMJ (Zhang et al., 2018a). In addition, overexpression of hFUS led to a decrease in presynaptic activity areas and impaired synaptic transmission in hFUS transgenic D. melanogaster model. The loss and acquisition of FUS function in FUS and Cabeza transgenic D. melanogaster models may be achieved through dominantly negative mechanisms or the downregulation of endogenous Cabeza, which are mediated by interference with vesicular and mitochondrial transportation. This indicates that the Cabeza mutation leads to developmental and motor defects, which become more severe with age (Walters et al., 2019). Mutant FUS or D. melanogaster homolog Cabeza expressing type IV dendritic neurons leads to FUS or Cabeza cytoplasm mislocalization and axon transportation toward the pre-synapse end in FUS or Cabeza transgenic D. melanogaster models. Moreover, FUS or Cabeza overexpression leads to the progressive loss of neuronal projections and the reduction of synaptic mitochondria, while a large number of calcium transients appear in synapses by controlling the expression of calcium channels. In addition, mutant FUS overexpression results in a decrease in presynaptic synaptic-binding proteins, specifically disrupting axonal transport and inducing excessive excitability. Therefore, the mutation FUS/Cabeza damages the axon transportation of synapse vesicular proteins, thereby reducing the levels of synaptic-binding proteins, which are the main components of presynaptic release mechanisms. The overexpression of FUS can disrupt mitochondrial transport in D. melanogaster MNs, and an increase in FUS transport during neural biological processes alters the focal levels of synapse transcription or induces the formation of stress particles, thereby disrupting local translation and causing synaptic hyperexcitability (Machamer et al., 2018).

Karyopherin beta-2, also known as Kap-β-2 or transportin-1, participates in proline/tyrosine nuclear localization signal (PY-NLS), which inhibits and reverses the FUS fibrosis. In addition, it is worth noting that Kap-β2 can prevent RNA binding protein (RBP) with PY-NLS from accumulating among stress granules, restore the nucleus RBP localization and function, and rescue the neurodegeneration induced by ALS-related FUS in the FUS transgenic D. melanogaster models. The increase of Kap-β2 saves neurodegeneration and lifespan related to FUS in the FUS transgenic D. melanogaster models and reduces the accumulation of FUS in stress granules (Guo et al., 2018).

Knockdown of heat shock response gense in the D. melanogaster model expressing hFUS^WT ω-LncRNA can reduce the level of hFUS^WT mRNA and induce the generation of insoluble inclusion bodies comprising non-toxic hFUS^WT proteins and lysosomal-associated membrane protein 1 in cytoplasm (Yamaguchi et al., 2021). Nuclear chromatin-binding protein (Xrp1), the main modifier of Cabeza mutative phenotypes, is strongly upregulated in the Cabeza-mutant D. melanogaster model, effectively reducing motor and life defects. Moreover, the Cabeza-mutant D. melanogaster model exhibits an imbalance in gene expression, and the Xrp1 heterozygosity can alleviate this (Mallik et al., 2018). Several genes, including TDP-43, Transportin-1, TER94/VCP, arginine methyltransferase, and the mitochondrial companion Hsp60 have been shown to interact with FUS/Cabeza genetically in the FUS/Cabeza transgenic D. melanogaster model (Zhang et al., 2018a).

C9orf72 is a common pathogenic gene involved in ALS. The present study showed that different cellular processes, including transcription, translation, nuclear-cytoplasmic transport, and protein degradation, are involved in the pathogenesis of C9orf72 ALS (Liguori et al., 2021). The D. melanogaster genome does not contain a direct homolog of human C9orf72, thus, an ALS-linked D. melanogaster model related to C9orf72 was developed by expressing the GGGGCC (G4C2) repeat (Yamaguchi et al., 2021). Although C9orf72 in ALS patients usually comprises over hundreds of G4C2 repeat sequences, the overexpression of 30–58 G4C2 repeat sequences in D. melanogaster eyes or MNs is sufficient to result in neuronal degeneration. Based on the expression level and time, the G4C2 repeat or arginine-rich dipeptide repeat (DPR) expressed among MNs leads to serious NMJ defects in the third-instar larvae of the C9orf72 G4C2 repeat transgenic D. melanogaster model (Zhang et al., 2018a). The amplification of the G4C2 hexanucleotide repeat expansion (HRE) of C9orf72 leads to the loss of C9orf72 function by inhibiting transcriptional extension and splicing of the first intron (Zhang et al., 2018a). The characteristic pathological hallmark observed among the different tissues of C9orf72 ALS, including MNs, is RNA lesions. RNA lesions are the result of HRE transcription, leading to the accumulation of repetitive RNA aggregates, usually located in the nucleus or cytosol (Layalle et al., 2021). This suggests that RNA-carrying HRE do not cause toxicity, but that the arginine-rich DPR sequence encoded by the HRE sequence mediates neurotoxicity (Moens et al., 2018). Consistent with this, the abnormal translation of HRE leads to the accumulation of DPR protein in the brains of ALS patients (Azoulay-Ginsburg et al., 2021). When comparing the effects of RNA expression among the HRE transgenic D. melanogaster model, only DPR proteins containing arginine have neurotoxicity on the G4C2 motifs encoding different dipeptide combinations (Bolus et al., 2020).

Poly (glycine-arginine) (GR) is a common genetic cause of ALS and FTD, occupying forty percent of fALS cases (Cunningham et al., 2020). Partial functional loss of an essential DNA repair protein Ku80 inhibits retinal degeneration induced by poly (GR) in poly (GR) transgenic D. melanogaster models. In the neurons of D. melanogaster model expressing poly (GR) and in patients with C9orf72, the Ku80 expression is significantly increased. Elevated Ku80 expression contributes to GR aggregation and induces neurodegeneration in a poly (GR) transgenic D. melanogaster model (Lopez-Gonzalez et al., 2019).

Arginine-rich DPR is a highly toxic product derived from the C9orf72 repeat amplification mutation and is a common cause of fALS. However, their role in synaptic regulation and excitatory toxicity remains unclear. Applying C9orf72 DPRs with different toxic intensities revealed that arginine-rich DPRs induced selective degeneration of glutamate neurons in the C9orf72-DPR transgenic D. melanogaster model. Among them, the C9orf72-DPR can stimulate synaptic overgrowth and promote glutamate release. Furthermore, an increase in glutamate release from excitatory neurons expressing DPR not only causes excitotoxicity in postsynaptic neurons, including MNs, but also produces cell-autonomous excitotoxicity in presynaptic neurons (Xu and Xu, 2018).

Up-frameshift 1 (UPF1) is an RBP with helicase activity that reduces DPR-mediated toxicity. Overexpression of UPF1 significantly reduces the severity of known neurodegenerative phenotypes and poly (GR) content without changing the amount of repeat RNA, indicating that UPF1 has a neuroprotective effect on C9orf72-ALS (Zaepfel et al., 2021). The present study shows that UPF1 expression regulates the DPR- or HRE-induced neurodegenerative phenotypes of C9orf72-ALS transgenic D. melanogaster models, including eye symptoms (Xu et al., 2019; Ortega et al., 2020; Sun et al., 2020). In addition, DPR can directly bind to nucleoporins, and knockdown of karyopherin alpha3 in the C9orf72-ALS transgenic D. melanogaster model can enhance the toxicity of DPR (Walters et al., 2019; Braems et al., 2021). Furthermore, the loss of C9orf72 function disrupts autophagy and lysosomal functions in many types of cells, including MNs (Ji et al., 2017; Shi et al., 2018; Zhu et al., 2020).

The key regulatory factor of autophagy, which is refractory to Sigma P/sequestosome 1 [Ref(2)P/p62], is an effective inhibitor of ALS induced by G4C2 HRE in C9orf72 (Cunningham et al., 2020). The microphthalmia/transcription factor E family of transcription factors is a key regulator of autophagy-lysosome function. Transcription factor EB (TFEB) is a candidate therapeutic target for ALS (Cortes and La Spada, 2019). It was found that C9orf72-HRE impaired the microphthalmia-associated transcription factor/transcription factor EB nuclear input, disrupted autophagy, and exacerbated the protein balance defect in the C9orf72 ALS transgenic D. melanogaster model. Increased TFEB activity may prevent neuronal death and promote recovery of neuronal function through different mechanisms in the C9orf72-ALS transgenic D. melanogaster model (Torra et al., 2018).

The G4C2 amplification repeat sequence and tau protein are traditionally considered to be related to partial clinical manifestations in several neurodegenerative diseases, such as ALS. Co-expression of the G4C2 repeat sequence and Tau can lead to the synergistic deterioration of rough eyes, movement functions, longevity, and abnormal NMJ morphology in the G4C2 repeat sequence and Tau co-expression transgenic D. melanogaster model. In addition, 30 G4C2 repeats increase tau phosphorylation levels (He et al., 2019). Moreover, the downregulation of D. melanogaster tau homologous protein can reduce neurodegeneration and motor lesions and prolong the lifespan of C9ORF72 G4C2 repeatedly expressing D. melanogaster model (Braems et al., 2021).

Since C. elegans ALS models were first established in 2001 (Oeda et al., 2001), a series of C. elegans models have been developed, and many potential mechanisms associated with ALS pathogenesis have been identified. Among them, the hmSOD1 C. elegans model was produced by transferring wild-type human SOD1 (WThSOD1) and familial ALS (fALS) SOD1 mutations, such as A4V, G37R, and G93A, by controlling several promoters of the heat shock protein (HSP)-16.2 and myosin heavy chain 3 (myo-3) genes. The hmSOD1 transgenic C. elegans model controlled by the inducible HSP-16.2 promoter expresses hmSOD1 in nearly all tissues, including neurons, and the hmSOD1 C. elegan models controlled by the muscle-specific promoter of myo-3 largely express hmSOD1 among all muscles besides the pharynx.

The universal expression of hmSOD1 in C. elegans blocks natural biological responses to oxidative stress and induces the aggregation of toxic proteins, while the expression of hmSOD1 in all C. elegans neural systems leads to motor defects and neuron transmission damage in C. elegans models. The establishment of a SOD1 single copy (A4V, H71Y, L84V, G93A, and G85R) mutation knockout C. elegans model can analyze the unique effect of the special mutation on the degeneration of cholinergic and glutamic MNs in C. elegans models. These findings indicate that the pathogenesis of ALS is a neuronal subtype-specific gain of toxic functions, as well as a loss of physiological functions (Liguori et al., 2021). Moreover, loss of SOD1 function is the main cause of glutamate neuron degeneration after oxidative stress in C. elegans models, and the toxic functions gaining of SOD1 protein may cause cholinergic MNs degeneration in C. elegans models (Baskoylu et al., 2018). In the ALS-like hmSOD1 C. elegans model containing the ALS G85R mutation, a relatively high level of hmSOD1 was expressed in the neurons of C. elegans, resulting in movement defects of C. elegans expressing SOD1 G85R (Baskoylu et al., 2018).

Although morphological abnormalities of neurons and discernible survival or behavioral changes were not observed in the C. elegans model of hmSOD1 expression, the hmSOD1 C. elegans model demonstrated decreased resistance to paraquat-induced oxidative stress, significantly reducing the degradation ability of hmSOD1 proteins, leading to the abnormal accumulation of hmSOD1 proteins among muscular cells. Moreover, the final pathological phenotypes of hmSOD1 proteins in the C. elegans models were similar to the pathologically altered features observed in the post-mortem tissues of ALS patients.

The transgenic C. elegans model of entire neuronal expression of hSOD1 G85R using the synaptobrevin (snb-1) gene promoter coupled with yellow fluorescent protein (YFP) exhibits serious movement defects and paralysis (Wang et al., 2019). The observed phenotypes in this C. elegans model correlated with abnormal intra-neuronal aggregation of the mutated SOD1 protein. Other hmSOD1 genetic C. elegans models expressing various SOD1 mutants, such as G85R, G93A, and 127X, can be used to directly compare the differences in the aggregation, toxicity, and cellular interactions of hmSOD1 proteins induced by different SOD1 mutants. The hmSOD1-YFP C. elegans models expressing the SOD1 G85R, G93A, and 127X mutants in muscular cells using the muscle-specific promoter of unc-54 gene did not exhibit significant cell dysfunction (Gidalevitz et al., 2009). When these SOD1 mutations were transferred into the genetic background of temperature-sensitive mutations in C. elegans, the toxins produced by the mutated SOD1 protein were significantly enhanced, resulting in a variety of toxic phenotypes. Therefore, it was suggested that the hmSOD1 produced toxic phenotypes that might not only be the aggregate toxic products of hmSOD1 proteins but are also closely associated with genetic interactions in the genetic background.

The C. elegans body locomotion is controlled by excitatory (acetylcholine, glutamate) and inhibitory (GABA) neurons. Stable overexpression of hmSOD1 G93A mutation causes the degeneration of GABAergic MNs, whereas single-copy expression of hmSOD1 G93A mutation only causes oxidative stress-induced cholinergic and glutamic neurodegeneration (Braems et al., 2021). Endogenous SOD1 mutations, such as hmSOD1 G93A, trigger glutamate neuronal death in C. elegans (Baskoylu et al., 2018).

Metformin increased daf-1 and lgg-1 gene expression in hmSOD1 C. elegans model. The effects of Metformin on the lifespan of hmSOD1 C. elegans models were modulated by daf-16 and lgg-1 genes. In lgg-1 deficient C. elegans, metformin did not restore the movement, amount, or morphology of MNs (Senchuk et al., 2018). Metformin significantly prolonged the lifespan, improved exercise performance, and enhanced the antioxidant activity of hmSOD1 C. elegans, further indicating that metformin enhanced the expression of lgg-1, daf-16, skn-1, and other genes that regulate autophagy, longevity, and oxidative stress in hmSOD1 C. elegans models. Therefore, lgg-1 or daf-16 overexpression alleviates aging and pathological abnormalities in hmSOD1 C. elegans models, whereas genetic deletion of lgg-1 or daf-16 eliminates the beneficial effect of metformin in hmSOD1 C. elegans models. The median survival period of lgg-1 overexpression of hmSOD1^G93A C. elegans increased relative to hmSOD1^G93A C. elegans, indicating that activation of the lgg-1 gene reduces the neurotoxicity induced by hmSOD1 protein aggregation by inducing hmSOD1 protein degradation, thereby promoting the lifespan of hmSOD1 C. elegans. Although both lgg-1 and daf-16 extend the hmSOD1 C. elegans, no additive effects are observed when these two genes are simultaneously overexpressed (Alberti et al., 2010). A regulatory factor, ubiquitin-specific processing protease 7 (USP7), affects the elimination of misfolded proteins such as hmSOD1 and TDP-43. The USP7 effect is modulated by both the transforming growth factor β and the small mother against decapentaplegic (SMAD) pathways, is mediated by USP7-NEDD4L-SMAD2 axis, and its activation promotes the protein quality control. Furthermore, Math-33 deletion inhibited protein toxicity induced by hmSOD1 G85R mutation in the hmSOD1 G85R C. elegans model (Zhang et al., 2020).

The TDP-43 transgenic C. elegans models were reproduced by expressing wild-type human TDP-43 (hTDP-43^WT) protein in neurons to study TDP-43 functional alterations and neurotoxicity in the pathogenesis of ALS (Ash et al., 2010). TDP-43 transgenic C. elegans models were generated using snb-1 gene promoters to drive human TDP-43 (hTDP-43) cDNA expression in whole neurons. This TDP-43 transgenic C. elegans model exhibited distinctive uncoordinated phenotypes, such as slow movement and inappropriate responses to stimuli. The C. elegans model first exhibits these phenotypes at the larval stage and constantly retains these phenotypes throughout adulthood. This C. elegans model indicated that these uncoordinated phenotypes correlated with the synaptic lesions of abnormal MNs. The mechanism by which abnormal nuclear TDP-43 activity leads to abnormal synapses is not yet known, but it indicates that excessive activation of the nuclear TDP-43 protein might change some components of RNA metabolism, such as alternative RNA splicing, resulting in the dysfunction of some special proteins necessary for normal synapse functions.

TDP-43 transgenic C. elegans models show that TDP-43 dysfunction results in insufficient chromatin processing and dsRNA accumulation, which may activate natural immunological systems and promote neuroinflammation (Milstead et al., 2023). It is currently understood that the neurodegenerative disease-related proteins, especially TDP-43 (ALS), amyloid beta and tau (Alzheimer's disease), α-synuclein (Parkinson's disease, PD), and FUS (frontotemporal dementia, FTD), partially show some characteristics of misfolding prion proteins. In addition, the TDP-43 C. elegans model also showed that the autophagy-lysosome pathway is the prion-like transmission pathway of TDP-43 protein (Sandhof et al., 2020). Meanwhile, it was discovered that the regulation of autophagy or exocytosis/endocytosis can affect the transfer of these misfolded proteins (Tyson et al., 2017). The TDP-43 protein is an intracellular protein but can promote prion-like transmission of TDP-43 by entering the lysosomal system in the TDP-43 C. elegans model (Peng et al., 2020). This suggests that the autophagy-lysosome system and endocytosis-dependent endolysosomal pathway are related to the elimination of TDP-43 protein aggregates (Liu et al., 2017).

The currently developed TDP-43 transgenic C. elegans model also showed some similar phenotypes in that normal hTDP-43^WT overexpression in all neurons caused different scales of movement defects (Liachko et al., 2010). Moreover, the overexpression of many human ALS-related TDP-43 mutations, such as G290A, A315T, and M337V, causes a series of movement dysfunction phenotypes similar to those in ALS patients. It also indicates that these phenotypes of motor dysfunction worsen over time. The above-described similar phenotypes reproduced by the mutant TDP-43 transgenic C. elegans models reproduce major features seen in ALS patients, such as progressive muscle weakness and atrophy, shortened life span, and MNs degeneration accompanied by both hyperphosphorylation, truncation, and ubiquitination of TDP-43 proteins and the accumulation of detergent-insoluble TDP-43 proteins. The TDP-43 C. elegans model provides an ideal in vivo model for exploring the cellular and molecular mechanisms underlying ALS. These TDP-43 C. elegans models may be used to search for potential alterations in TDP-43 function in the pathogenesis of ALS, possibly revealing the neurotoxic mechanisms of TDP-43 associated with ALS, and ultimately finding possible targets for developing novel therapeutic measures.

Twenty-four human homologous genes that inhibit TDP-43 motor dysfunction after a reduction in RNAi-targeted gene expression have been identified. These genes involve multiple pathways, including the energy production and metabolism genes cox-10, F23F12.3, F55G1.5, paqr-1, and cox-6A, the extracellular matrix and cytoskeleton genes col-89, glie-8, hs-5, sax-2, vab-9, and zig3, the ion transport genes cnnm-3, C13B4.1, and unc-77, the nucleic acid function genes dna-2, tbx-11, and umps-1, protein balance genes C47E12.3, gpx7, and pcp-5 and the signal transduction genes F31E3.2, F40B5.2, and Y44E3A4 (Liachko et al., 2019). Human TDP-43 and C. elegans TDP-1 are functional homologs with similar RNA-binding activity, conserved N-terminus, nuclear localization signal (NLS), and RNA recognition motifs (Mitra et al., 2019). The C. elegans models of human TDP-43 or C. elegans TDP-1 expression in entire neurons produce the uncoordinated, slow, and GABAergic MNs constriction. In addition, the C. elegans model of targeted mutant TDP-43 expression in C. elegans GABAergic MNs can lead to age-dependent progressive paralysis, GABAergic neuronal degeneration, and synaptic lesions. Mutated exogenous TDP-43 induces oxidative stress and aberrant expression of endogenous TDP-1 decreases neuronal function and lifespan in C. elegans models. Excessive phosphorylation of TDP-43 can interfere with protein homeostasis, leading to neurotoxicity in C. elegans model. Among the kinases involved, cyclin-dependent kinase 7 is directly involved in TDP-43 phosphorylation and decreases TDP-43 phosphorylation by inhibiting the produce of this enzyme in C. elegans model (Liguori et al., 2021). TDP-1 deficiency or deletion leads to dsRNA accumulation and enhances exogenous RNAi by increasing nuclear RNAi in C. elegans. C. elegans nuclear RNAi involves chromatin alterations that are regulated by heterochromatin protein like-2 (HPL-2) and the homolog of heterochromatin protein 1 (HP1). TDP-1 interacts directly with HPL-2, and the TDP-1 deletion significantly changed the chromatin association of HPL-2. These molecular changes, replicated in C. elegans models, indicate that the nuclear depletion of hTDP-43 might result in alterations in disease-related RNA metabolism during the pathogenesis of ALS (Saldi et al., 2018).

FUS is involved in various functions associated with DNA and RNA processing (Birsa et al., 2020). Prominent features of the mutant FUS C. elegans model include cytoplasmic mislocalization, generation of FUS aggregates, age-dependent motor defects, paralysis, and impaired GABAergic neurotransmission. In addition, the transmission of MNs to muscle tissues is reduced through ultrastructural and electrophysiological methods, indicating a role for FUS in synaptic vesicle tissue and nerve transmission (Braems et al., 2021). To establish the ALS-FUS C. elegans model, the FUS R524S and P525L allele mutations are inserted into the c-terminus NLS of endogenous fust-1 gene in chromosome 2 to generate an ALS-FUS animal model. Overexpression of FUS leads to severe motor defects and damages neurons and muscle autophagy without damaging the neuronal ubiquitin-proteasome pathway in FUS C. elegans models (Baskoylu et al., 2022). FUS C. elegans models have also shown that autophagy dysfunction may contribute to the development of ALS. However, the mode and degree of autophagy dysfunction in ALS pathogenesis are not fully understood (Chua et al., 2022). Excessive expression of FUS in C. elegans models affects the self-regulation of FUS, thereby affecting RNA processing, stability, and protein homeostasis. The irreversible hydrogel formed by the mutant FUS gene impairs the function of heterogeneous ribonucleoprotein particles, reducing the speed of new protein synthesis in mutant FUS C. elegans models (Markert et al., 2020). Disruption of these functions occurs by negatively affecting autophagy. The steady-state level of FUS is crucial for normal neuronal function, as increased FUS levels accelerate diseases such as ALS (Zhang et al., 2018b).

The mutative C9ORF72 gene results in the loss of its functions, gain of toxic functions, or both; however, whether these C9ORF72 dysfunctions are involved in the pathogenesis of ALS is not yet fully understood. It has been revealed that the C9ORF72 pathogenic GGGGCC (G4P2) repeat mutation significantly decreases C9ORF72 expression. The C. elegans model harboring the 3rd and 4th exon deletion in C. elegans ALS-associated gene homolog alfa-1 led to a drastic reduction in C9ORF72 expression, exhibiting a movement defect resulting in age-dependent paralysis and the degeneration of GABA-ergic MNs. Moreover, the mutant alfa-1 C. elegans model showed osmotic stress sensitivity to provoke MNs degeneration.

The causative G4P2 repeat among the C9ORF72 gene is the key cause of familial and sporadic ALS found up to date. It is not known whether the C9ORF72 functions are the same as those of other ALS-related genes such as TDP-43 and FUS. It is important to note that C9ORF72 expression is also reduced in many ALS cases with the negative pathogenic G4P2 repeat, suggesting that C9ORF72 may exert synergistic effects with other ALS-related genes. In order to further understand the C9ORF72 potential synergistic effects with other ALS-related genes, the C. elegans model carrying mutative TDP-43 A315T or FUSΔUS5 combined with the null alfa-1 mutation were established, which showed that the null alfa-1 expression decreases and the paralytic phenotypes exacerbates but does not change the toxic of mutative FUS protein, which suggests that the mutative C9ORF72 gene exerts a synergistic effect with the mutative TDP-43 gene in the same MNs toxic pathway but not the mutative FUS gene. Reduced alfa-1/C9ORF72 expression enhanced the toxicity of the mutated TDP-43 protein but did not affect the FUS protein in the C. elegans model. This suggests that the genetic network is composed of many genes involved in the pathogenesis of ALS, where ALS-related genes interact each other based on the study in this C. elegans model. The model development of genetic networks in C. elegans is helpful for understanding the synergistic toxic mechanisms of ALS-related genes in neuronal loss in ALS (Therrien and Parker, 2014a).

C. elegans models studying the mechanism associated with the gain or loss of C9ORF72 gene functions revealed that the deletion of C9ORF72 gene leads to early serious paralysis, nuclear transport impairment, lysosomal homeostasis dysregulation, neurodegeneration, and neuronal death. The C9ORF72 C. elegans model can be used to investigate endocytosis, which reveals that alfa-1 deletion causes a defect in lysosome homeostasis, such as the dysfunction of lysosome reformate and the degradation of endocytosal elements; thus, the C9ORF72 C. elegans model is the first chosen model that is used to identify whether or not C9ORF72 is involved in the endocytosis pathway (Therrien and Parker, 2014b).

Partial neurodegenerative diseases have common or overlapping pathogenic mechanisms at the cellular level, such as protein misfolding, excitotoxicity, and altered RNA homeostasis. Moreover, genetic factors contributing to common, cross, or overlapping pathogenesis imply the existence of a possible genetic network of neurodegeneration in the pathogenesis of ALS. The recently discovered overlapping pathogenic mutation C9ORF72 in both ALS and FTD is perhaps the best illustration. It was thought that the different neurodegenerative diseases were distinct entities and possessed a single genetic spectrum; however, the current study findings are changing this opinion, considering that some neurodegenerative diseases have a similar genetic spectrum. Following the progression of the related genetic discovery, there is an urgent need to establish a novel genetic model to not only investigate the causative mechanisms associated with the pathogenic mutations of neurodegeneration but also to study the possible genetic interactions among the different mutated genes, which may reveal novel pathogenic targets. Based on the evolutionary conservation of various pathogenic genes with C. elegans, the C. elegans genetic models may be the first candidates to study the genetic interactions among these pathogenic genes; therefore, C. elegans genetic models, such as the mutative C9ORF72 gene, may be used to model both ALS and FTD to study the potential genetic interactions and networks in the pathogenesis of ALS and FTD (Therrien and Parker, 2014b).