Genetics of amyotrophic lateral sclerosis: seeking therapeutic targets in the era of gene therapy

Abstract

Amyotrophic lateral sclerosis (ALS) is an intractable disease that causes respiratory failure leading to mortality. The main locus of ALS is motor neurons. The success of antisense oligonucleotide (ASO) therapy in spinal muscular atrophy (SMA), a motor neuron disease, has triggered a paradigm shift in developing ALS therapies. The causative genes of ALS and disease-modifying genes, including those of sporadic ALS, have been identified one after another. Thus, the freedom of target choice for gene therapy has expanded by ASO strategy, leading to new avenues for therapeutic development. Tofersen for superoxide dismutase 1 (SOD1) was a pioneer in developing ASO for ALS. Improving protocols and devising early interventions for the disease are vital. In this review, we updated the knowledge of causative genes in ALS. We summarized the genetic mutations identified in familial ALS and their clinical features, focusing on SOD1, fused in sarcoma (FUS), and transacting response DNA-binding protein. The frequency of the C9ORF72 mutation is low in Japan, unlike in Europe and the United States, while SOD1 and FUS are more common, indicating that the target mutations for gene therapy vary by ethnicity. A genome-wide association study has revealed disease-modifying genes, which could be the novel target of gene therapy. The current status and prospects of gene therapy development were discussed, including ethical issues. Furthermore, we discussed the potential of axonal pathology as new therapeutic targets of ALS from the perspective of early intervention, including intra-axonal transcription factors, neuromuscular junction disconnection, dysregulated local translation, abnormal protein degradation, mitochondrial pathology, impaired axonal transport, aberrant cytoskeleton, and axon branching. We simultaneously discuss important pathological states of cell bodies: persistent stress granules, disrupted nucleocytoplasmic transport, and cryptic splicing. The development of gene therapy based on the elucidation of disease-modifying genes and early intervention in molecular pathology is expected to become an important therapeutic strategy in ALS.

Fig. 1

Racial/ethnic difference of amyotrophic lateral sclerosis (ALS) causative genes [28, 29, 93]. The pie charts of ALS causative genes in Europeans and Japan are shown, color-coded with SOD1, TARDBP, FUS, C9ORF72, and not determined (ND) in these four genes. Mutations were identified in 55.5% of Europeans with familial ALS and 43.6% of Japanese individuals with familial ALS. In sporadic ALS, only 7.4% of mutations were identified in Europe and 2.9% in Japan. The difference between European and Japanese is largely due to the difference in the frequency of C9ORF72 mutation, SOD1, and FUS being more common in Japanese and TARDBP being more common in European

Fig. 2

Overview of ALS pathology with a focus on axons. As axons are damaged from the initial stage of ALS, the dying-backward hypothesis, in which motor neurons are damaged from the distal part, has been proposed. RNA-seq of axon fraction shows the presence of intra-axonal transcription factors (e.g., AP-1), although the pathological significance remains unknown. NMJs are the key link between motor neurons and skeletal muscle, and NMJ disconnection is commonly observed in several types of ALS. The local translation is a molecular mechanism necessary for axonal homeostasis. In contrast, when the proteasome and autophagy are dysregulated, abnormal protein aggregation is triggered. Mitophagy is a form of autophagy, and mitochondrial pathology is a common feature of various neurodegenerative diseases. Furthermore, impaired axonal transport impairs the transport of RNA/Protein complex, lysosomes, and mitochondria. Many ALS-causing genes contribute to cytoskeleton function. Morphologically abnormal axon branching has been observed. The pathophysiology of the cell body, which is closely related to the axonal pathology, is also important. Cryptic exon induced nonsense-mediated decay (NMD) or aberrant proteins, persistent stress granules (SGs) formation, and nucleocytoplasmic transport defect have attracted attention as new therapeutic targets

Fig. 3

Motor neuron axons were extracted with a microfluidic device. a The experimental scheme of MN culture using the microfluidic device. HB9 reporter lentivirus infected motor precursor cells were plated onto the device. b Representative ICC images of MNs on the microfluidic device. The axons elongated in the microfluidics to the next well. c The enlarged images of nerve organoids stained with βIII-tubulin (cytoplasm) and Hoechst (nuclei). Bar: 1 mm. d Representative images of axon dividing. Axons were divided from the SDs by cutting the axon bundle at 450 μm away from the sphere to avoid contaminating the cell body and pushing out due to hydraulic pressure. Modified from ref. [181]