Mutations in the annexin A11 gene (ANXA11) contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS) by disrupting cellular calcium ion homeostasis and stress granule (SG) protein disassembly contributing to ALS neurodegeneration.

This finding suggests a multi-target approach addressing both mechanisms may be necessary to treat ANXA11 mutation-positive ALS patients, concluded a Korean study reported in the October 21, 2010, edition of Science Translational Medicine.

"This is the first study to show that ANXA11 is an important link between calcium ion homeostasis and SG dynamics," said study leader Seung Hyun Kim.

This is an important finding, as these "are two essential cellular mechanisms of emerging interest in ALS research," said Kim, a professor of neurology in the College of Medicine at Hanyang University in Seoul and president of the Korean Dementia Association.

Also known as Lou Gehrig's disease, ALS is characterized by progressive muscle weakness and spastic paralysis due to the selective loss of central nervous system motor neurons, but treatment options are limited.

Treatment options

Riluzole (Rilutek/Tiglatuk) and edaravone (Radicava/Radicat) are FDA-approved drugs for treating ALS, but "their efficacy is minimal," said Kim, adding, "several supportive treatments are also available for treating symptoms, including excessive salivation and spasticity."

Approximately 10% of ALS cases are familial, in which mutations in the FUS gene encoding for the FUS RNA binding protein (RBP) are one of the common causes.

Mutations of other genes, including SOD1, TDP43 and UBQLN2, have now been identified as being important causative factors in ALS pathogenesis and associated with cellular pathways including protein homeostasis, RNA processing and cytoskeletal organization.

"Gene-directed therapies have recently been reported and our team and [Korean biopharmaceutical company] Corestem launched the world's first stem cell therapy for ALS in Korea in 2015," Kim told BioWorld Science.

"However, we have yet to find the most effective therapy for ALS and the development of a multi-targeted and personalized strategy is urgently needed," which has been hampered by a poor understanding of ALS etiology.

ALS etiology

Dysregulation of calcium ion homeostasis and abnormal protein aggregation have both been proposed as being major pathogenic factors underlying selective motor neuron degeneration in ALS.

More recently, mutations in ANXA11, which encodes a calcium ion-dependent phospholipid-binding protein, have been identified in both familial and sporadic ALS, but its pathophysiological roles remain unknown.

The functions of ANXA11 related to intracellular calcium ion homeostasis and SG dynamics were investigated in the new study based on an exome sequencing analysis of 500 Korean cases with sporadic ALS, which identified nine ANXA11 variants in 13 patients.

Among these mutations, two amino-terminal variants in the low-complexity domain of ANXA11 were shown to enhance SG aggregation propensity, whereas two carboxyl-terminal ANX domain variants altered calcium ion responses.

"Using live imaging techniques, we have shown that ANXA11 is an SG component, and that ALS-linked variants showed significantly delayed SG disassembly compared to the wild-type genome," explained Kim.

"Calcium ion imaging in patient skin fibroblasts with ALS-linked ANXA11 variants also showed increased basal calcium ion levels and reduced release, hence calcium ion homeostasis dysregulation."

Moreover, all four ANXA11 variants were seen to undergo abnormal phase separation to form droplets with aggregates, which led to the alteration of the biophysical properties of ANXA11.

"ALS-linked variants in ANXA11 underwent abnormal phase separation to form droplets with aggregates and altered SG dynamics," Kim said.

"These functional defects caused an increase in intracellular calcium ion load and delayed or impaired SG disassembly, which are both critical components of ALS pathology."

The research further revealed that another variant reduced ANXA11 expression and impaired calcium ion homeostasis, as caused by missense variants, point mutations that lead to an amino acid change.

Moreover, calcium ion-dependent interaction and co-aggregation between ANXA11 and ALS-causing RBPs, FUS and hnRNPA1, were observed in the motor neuron cells and autopsied brain of a patient with ALS-FUS.

"The four missense ANXA11 variants did not result in reduced ANXA11 protein expression, but one frameshift variant was associated with reduced ANXA11 mRNA and protein levels, while intracellular calcium homeostasis was impaired in all patient cells carrying the missense variants and the frameshift variant," said Kim.

The expression of ALS-linked ANXA11 variants in MNs was also shown to cause cytoplasmic sequestration of endogenous FUS and trigger neuronal apoptosis.

This is a significant finding, said Kim, as "selective motor neuron degeneration is one of the major pathogenic hallmarks of ALS and our data suggest ANXA11 may physically interact with ALS-causative RBPs, including FUS," he noted.

Moreover, in ALS-linked ANXA11 mutations, such essential RBPs for maintaining ribostasis are sequestered into the pathogenic ANXA11 positive aggregates in motor neurons, inducing RBP malfunction and motor neuron apoptosis.

These findings suggest that disease-associated ANXA11 mutations can contribute to ALS pathogenesis through toxic gain-of-function mechanisms involving abnormal protein aggregation and highlight the importance of considering multivalent roles of ALS-associated genes when developing new ALS therapies.

In this regard, "we are now investigating candidate molecules with which to disaggregate ANXA11 granules in a calcium ion-dependent manner using patient induced pluripotent stem cell- (iPSC)-derived motor neurons," said Kim.

"Such candidate molecules may offer effective therapeutic targets to reduce pathological RBP aggregates and restore calcium homeostasis in ALS motor neurons." (Nahm, M. et al. Sci Transl Med 2020, 12(566): eaax3993).