Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies

Abstract

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer's disease, Parkinson's disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

Keywords: Alternative splicing; Alzheimer’s disease; Antisense oligonucleotides; Disease-modifying treatment; Neurodegenerative diseases; Parkinson’s disease; Splice-switching; Splicing defects.

Fig. 1

Schematic of the process of pre-mRNA splicing and major spliceosome assembly. Initial assembly into Complex E involves binding of the U1 snRNP (U1) to the 5’splice site (ss), while non-snRNP splicing factor 1 (SF1) and U2AF bind to the branchpoint sequence and polypyrimidine tract, respectively [16]. Subsequently, U2 snRNP is recruited by SF1 and U2AF, replaces SF1 to bind to the branchpoint, and initiates the formation of Complex A. The recruitment of U2 then facilitates enlistment of the U4/U6-U5 tri-snRNP that is pre-assembled from the U4/U6 and U5 snRNPs, thus forming the pre-catalytic Complex B. Next, destabilisation of U4 and U1 leads to the dissociation of U4, while U6 replaces U1 at the 5’ss and gives rise to the activated spliceosome. This catalytically activated Complex B initiates the first step in splicing, giving rise to Complex C that then cleaves the 5’ss, releasing the first exon to fold and the 5’ss can now join to the branchpoint, forming a lariat within the intron. Following the lariat formation is the second step in splicing; cleavage of the intron at the 3’ss, release of the lariat and the ligation of the two bordering exons. Upon completion of the final step, the spliceosome dissociates so that the snRNPs may be recycled and splicing of a subsequent intron occurs. This is repeated until all the introns from the mRNA are removed, thus giving rise to the formation of the mature mRNA transcript [17, 18]. Following intron excision and ligation of the exons, the U snRNPs are recycled. 5’ss, 3’ss, bp and polypyrimidine tracts are shown in the line representing the intron. Exons are shown as magenta boxes. Adapted from Pitout (2019) [19].

Fig. 2

Schematic of the most common forms of alternative splicing. a Exon skipping. b Intron retention. c Alternative 5’ splice site (ss) selection. d Alternative 3’ ss selection. e Alternative polyadenylation (polyA) sites. f Mutually exclusive exons. Light blue boxes denote segments included in the final message, while green boxes denote segments excluded in the mature mRNA transcript. Dotted lines show the splicing pattern. Note: mechanisms are not mutually exclusive, and combinations can often occur.

Fig. 3.

Alternative transcripts of SNCA and MAPT, and the stem loop near MAPT exon 10 donor splice site. a Five SNCA alternative transcripts resulting from skipping of exon(s) 3, 4, and/or 5. b Tau isoforms with three (3R) or four (4R) C-terminal microtubule binding repeats due to alternative splicing of MAPT exon 10. Self-complementary stem loop at the 3’-end of exon 10 and the 5’-end of intron 10 and a strong intron splicing silencer (ISS) interfere with the pairing of U1 small nuclear RNA to MAPT exon 10, weakening exon 10 inclusion. The intronic mutation IVS10+16 C>T (as indicated by arrows) disrupts the ISS encoded by sequence 5’-ucacacgu-3’ and increases MAPT exon 10 inclusion. Exonic sequences are shown in capital letters; intronic sequences are in lower cases. Ex: exon; R: repeat.

Fig. 4

Milestones of the development of antisense oligonucleotide therapeutics (excluding siRNA) from bench to bedside. Approved drugs in red are splice-switching antisense oligomers. AO: antisense oligonucleotides; FDA: US Food and Drug Administration; CMV: cytomegalovirus retinitis (in immunocompromised patients); HoFH: Homozygous familial hypercholesterolemia; DMD: Duchenne muscular dystrophy; SMA: spinal muscular atrophy; HTA: Hereditary transthyretin-mediated amyloidosis; BD: Batten disease.

Fig. 5

Mechanisms of action of splice-switching antisense oligonucleotides. a Stimulating splicing factors (SF) shown in pink circles such as SR proteins binding to exon splicing enhancers (ESE) promote the inclusion of an exon, while inhibitory SF in green circles such as hnRNPs binding to intron splicing silencers (ISS) inhibit exon inclusion. When promoting outweighs inhibiting actions, exons are included to generate a full-length transcript and wild-type protein. b Antisense oligomers (AOs) annealing to ESE blocks the interaction between SF and ESE and induces targeted (i) in-frame exon skipping, thus inducing in-frame transcripts and correspondingly new protein isoforms; and (ii) out-of-frame exon skipping and disrupts the reading frame and creates premature stop codon (PTC) in a downstream exon, that may lead to nonsense-mediated mRNA decay of the targeted transcript and downregulation of the protein. (iii) AOs anneal to ISS to increase targeted exon inclusion and generate a full-length transcript and wild-type protein.