III. Targeting TDP-43 pathogenic variants
Transactive response DNA binding protein 43 (TDP-43) is primarily a nuclear protein that binds to UG/TG repeats in the introns of pre-mRNA (Xiao et al., 2011) and regulates transcription or RNA processing (Polymenidou et al. 2012). Under stress conditions, TDP-43 is found in stress granules inside the cell (Colombrita et al., 2009). These cytoplasmic aggregates consist of full-length TDP-43 or of its C-terminal proteolytic fragments of 35 and 25 kDa (Hergesheimer et al., 2019). Further, cytoplasmic TDP-43 is mainly ubiquitinated and phosphorylated in ALS patients (Neumann et al., 2006).
Targeting autophagy or proteasome. TDP-43 regulates the production of ATG7, an autophagy mediator, by stabilizing theATG7 mRNA. Thus, depletion of TDP-43 causes loss of ATG7 and impaired autophagy (Bose et al., 2011). Furthermore, loss of TDP-43 increases TFEB nuclear translocation and enhances autophagosomal and lysosomal biogenesis but it impairs fusion of autophagosomes with lysosomes (Xia et al., 2016). Consequently, TDP-43 aggregation observed in ALS could lead to dysregulation of autophagy. In addition, the 25 kDa TDP-43 fragment causes severe cognitive and behavioral deficits in mice, and suppression of autophagy and proteasome activation (Caccamo et al., 2015).
Inhibitors of mTOR act as activators of autophagy through induction of autophagosome formation (Zarogoulidis et al., 2014). Administration of the autophagy activators rapamycin, spermidine, carbamazepine and tamoxifen in a mouse model of ALS/FTD with TDP-43 aggregates decreased the loss of motor neurons and TDP-43 aggregates (Wang et al., 2012). Rapamycin also increased the lifespan of a Drosophila ALS/FTD model (Cheng et al., 2015). In contrast, in Tg-SOD1G93A mice, rapamycin decreased survival and induced mitochondrial dysfunction and cell death in cultured motor neurons isolated from these animals (Zhang et al., 2011). The above data further support the hypothesis that different therapies should be used for different ALS subtypes.
A HTS study found fluphenazine (FPZ), methotrimeprazine (MTM) and 10-(4′-(N-diethyl amino)butyl)-2-chlorophenoxazine (NCP) as the most potent autophagy activators. When neuronal and astrocyte ALS cell models were treated with these chemicals, the levels of TDP-43 aggregates were reduced, cell survival was improved and decreased nuclear-cytoplasmic shuttling was observed (Barmada et al., 2014).
Phosphorylation of TDP-43. Casein kinase 1 (CK-1), cell division cycle 7 (CDC7), tau and tubulin kinase 1 and 2 (TTBK1 and TTBK2, respectively), and mitogen-activated protein kinases (MAPK/ERK) phosphorylate TDP-43 that in turn promotes the aggregation in the cytoplasm (Palomo et al., 2019). Nilotinib (XXXI ) and bosutinib (XXXII ), two tyrosine kinase inhibitors (TKI), altered glutamate synaptic signaling and showed neuroprotective effects in Tg-TDP-43wt mice (Heyburn et al., 2016). Also, nilotinib reversed mitochondrial dysfunction caused by TDP-43 aggregates (Heyburn et al., 2016). In addition, bosutinib increased survival of iPSC-derived motor neurons from patients with SALS or FALS caused by mutations in TDP-43 or GGGGCC repeat expansions in C9ORF72 while it delayed disease onset in Tg-SOD1G93A for 11 days and moderately increased survival by 8 days (Imamura et al., 2017). In conclusion, TKIs may provide a more general therapeutic strategy for ALS since they showed clinical efficacy in genetically diverse ALS models.
A series of N -(benzothiazolyl)-2-phenyl-acetamide compounds were developed and optimized for CK-1δ inhibition. The compoundXXXIII exhibited the lowest IC50 of 23 nM, while the XXXIV had an IC50 of 47 nM and both could penetrate the blood brain barrier (BBB). XXXIII decreased phosphorylation of TDP-43 and increased the lifespan of the Tg-TDP-43 Drosophila (Salado et al., 2014). Both XXXIIIand XXXIV prevented TDP-43 phosphorylation and nucleus-cytoplasm shuttling in PGRN -(progranulin) deficient lymphoblasts (Alquezar et al., 2016) and in lymphoblasts from SALS patients (negative for SOD1 pathogenetic variants and one positive for expansions in C9ORF72 ) (Posa et al., 2018). The CDC7 selective inhibitor PHA767491 (XXXV ) abolished phosphorylated forms of TDP-43 in vitro and in vivo in C. elegans (Liachko et al., 2013), indicating a possible target against pathological phosphorylation of TDP-43.
Targeting stress granules. Screening of 35 kinase inhibitors against paraquat-treated SH-SY5Y cells identified inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3) to block TDP-43 accumulation in stress granules, thus alleviating intracellular cell stress (Moujalled et al., 2013). Another chemical discovered by HTS, the LDN-0130436 (XXXVI ), improved the motor behavioral deficits of Tg-TDP-43wt and Tg-TDP-43A315T C. elegans (Boyd et al., 2014).
Other compounds. Various attempts aimed to identify compounds that may display therapeutic effect in TDP-43-ALS. In a screening of 1,200 FDA-approved drugs, the PPARγ agonist pioglitazone was identified to improve the locomotor function of Tg-TDP-43wt or Tg-TDP-43G298S Drosophilas , yet it did not improve the survival of flies (Joardar et al., 2015). Unfortunately, when pioglitazone was used in an ALS clinical trial in combination with riluzole (NCT00690118), it did not increase patient survival neither it improved any of the clinical symptoms (Dupuis et al., 2012). Failure of pioglitazone may be related to the fact that it either acts on certain ALS subtypes or the above-mentioned Drosophila models do not recapitulate the corresponding human ALS subtypes. This resembles the already mentioned case of methylene blue that displays pharmacological activity in C. elegans models of ALS but not in mouse models.
Anacardic acid (XXXVII ) acts as a histone acetyltransferase inhibitor and decreases TDP-43 mRNA and protein levels in human iPSCs derived from ALS patients carrying TDP-43 pathogenetic variants (Egawa et al., 2012). A different strategy for treating TDP-43 associated ALS involves targeting nuclear exportins that control TDP-43 nucleus-to-cytoplasm shuttling. TDP-43 has a nuclear export signal (NES) recognized by XPO1. The selective inhibitor of nuclear export (SINE) that targets XPO1, KPT-350 (XXXVIII ) partially rescues the motor deficits in a rat model of ALS/FTD generated by adenoviral delivery of TDP-43 (Archbold et al., 2018).