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).