II. Targeting G4C2 pathogenetic
expansions in C9ORF72
The GGGGCC large repeat expansions (from 250 up to >3000
repeats) present in the first intron of the C9ORF72 gene have
been identified in patients suffering from ALS or FTD (frontotemporal
dementia) (Dejesus-Hernandez et al., 2011; Renton et al., 2011). These
hexanucleotide expansions are transcribed bidirectionally and form
either RNA foci in the nucleus, that binds and sequesters RNA-binding
proteins, or are translated into dipeptide repeat proteins (DPRs) by an
abnormal translation process called repeat-associated non-ATG (RAN)
translation (Jiang et al., 2019). This results in gain-of-function that
leads to neuronal cell toxicity (Jiang et al., 2019). These GGGGCC
repeats exhibit somatic instability, thus, it is possible that repeat
expansion may not be present in DNA from blood samples although present
in CNS. Therefore, a blood test negative for GGGGCC expansion does not
necessarily indicate that the patient is not aC9ORF72ExpGGGGCC carrier, and this should be
taken into consideration when classifying ALS patients. Evidently, a
combination of genetic analysis and determination of poly-(GP) in CSF
will be required for a definite clinical diagnosis (Balendra et al.,
2017; Lehmer et al., 2017). Approaches to alleviate symptoms or
decelerate the course of the disease include targeting the RNA
transcribed from GGGGCC-repeats or targeting the DPRs. These RNAs adopt
two folded states that are in equilibrium, a hairpin structure and the
G-quadruplex structure (Su et al., 2014).
Targeting RNA G-quadruplexes/hairpins. HTS identified the
structurally similar chemicals DB1246 (XX ), DB1247
(XXI ), and DB1273 (XXII ), that exhibit high-affinity
binding on GGGGCC RNA G-quadruplexes. These chemicals significantly
reduced RNA foci in vitro in human iPSC-motor and iPSC-cortical
neuron cell lines and decreased DPRs. Decreased levels of DPRs and
improved survival of larvae reaching the pupal stage of development was
observed in vivo , in GGGGCC repeat-expressing Drosophilastreated with DB1273 (Simone et al., 2018).
The cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin
(TMPyP4)) XXIII binds to GGGGCC RNA G-quadruplexes in a
concentration-dependent manner and causes a conformational change in
their secondary structures conferring thermal instability of GGGGCC RNA
G-quadruplexes. Thus, TMPyP4 blocks the interaction of GGGGCC RNA
G-quadruplexes with RNA-binding proteins, such as ASF/SF2 and hnRNPA1
(Zamiri et al., 2014). Another group developed three compounds
(XXIV , XXV , XXVI ) targeting the
hexanucleotide repeat region of RNAs and tested them for binding to
hairpin RNA and reducing RAN in a cell-free model. XXIV andXXV reduced RNA foci and significantly decreased RAN
translation in GGGGCC repeat-expressing neurons (Su et al., 2014).
Improving the selectivity of XXIV , led to compoundXXVII that binds selectively in the internal loops of the
hairpin form of RNA. XXVII blocked polysome assembly and
reduced RNA foci and RAN translation in vitro (Wang et al.,
2019). The in vivo action of these compounds inC9ORF72 -ALS remains to be validated.
Targeting DPRs. RAN translation of C9ORF72 RNA
GGGGCC-repeats in all six-reading frames produces five
repeated polypeptides
[poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA)] that are
toxic to cells through induction of nucleolar stress and defects in mRNA
splicing (Mori et al.,
2013a; Mori et al., 2013b).
Inhibition of PIKFYVE kinase that converts
phosphatidylinositol-3-phosphate (PI3P) to
phosphatidylinositol-3,5-biphosphate [PI(3,5)P2] leads to increased
PI3P levels that regulate autophagosome formation and engulfment of
proteins for degradation. Therefore, it may be important for the
destruction of DPRs. Indeed, the PIKFYVE inhibitor apilimod
(XXVIII ) reduces DPRs in C9-BAC mice that harbor the humanC9ORF72 with 100-1000 GGGGCC repeats (Staats et al., 2019).
Another way to reduce protein aggregates, including DPRs, is through
proteosome activation. Rolipram (XXIX ) is an antidepressant
drug that acts via inhibition of phosphodiesterase 4 (PDE4) and
promotes proteasome function. Rolipram decreased poly(GA) in primary
hippocampal neurons in vitro (Khosravi et al., 2020). DPRs induce
integrated stress response (ISR) through ER implicating TMX2(Kramer et al., 2018). ISR involves hyperphosphorylation of elf2a and
increased RAN of C9ORF72 RNA GGGGCC-repeats (Cheng et al., 2018).XXX , a selective inhibitor of eIF2α dephosphorylation and
tauroursodeoxycholate (TUDCA), a chemical chaperone, showed protection
against poly(GA)-induced stress and cell death in vitro (Zhang et
al., 2014). In conclusion, there are many compounds that can target the
DPRs that need to be evaluated in animal models and then in clinical
trials.