C9orf72 plays a central role in Rab GTPase-dependent regulation of autophagy

Abstract

A GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 gene is the most common genetic defect associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Haploinsufficiency and a resulting loss of C9orf72 protein function has been suggested as a possible pathogenic mechanism in C9ALS/FTD. C9ALS/FTD patients exhibit specific ubiquitin and p62/sequestosome-1 positive but TDP-43 negative inclusions in the cerebellum and hippocampus, indicating possible autophagy deficits in these patients. In a recent study, we investigated this possibility by reducing expression of C9orf72 in cell lines and primary neurons and found that C9orf72 regulates the initiation of autophagy. C9orf72 interacts with Rab1a, preferentially in its GTP-bound state, as well as the ULK1 autophagy initiation complex. As an effector of Rab1a, C9orf72 controls the Rab1a-dependent trafficking of the ULK1 initiation complex prior to autophagosome formation. In line with this function, C9orf72 depletion in cell lines and primary neurons caused the accumulation of p62/sequestosome-1-positive inclusions. In support of a role in disease pathogenesis, C9ALS/FTD patient-derived iNeurons showed markedly reduced levels of autophagy. In this Commentary we summarise recent findings supporting the key role of C9orf72 in Rab GTPase-dependent regulation of autophagy and discuss autophagy dysregulation as a pathogenic mechanism in ALS/FTD.

Keywords: ALS; C9orf72; FTD; Rab GTPase; ULK1; autophagy.

Figure 1.

Autophagy and the Rab GTPases. The four stages of autophagy are indicated with the different Rabs involved at each stage. 1) Translocation of the ULK1 initiation complex to the phagophore is the first step in autophagy initiation. Rab1a mediates trafficking of the ULK1 complex to the phagophore and is involved in delivery of ATG9 positive membranes to the site of phagophore formation. Rab5 is involved in translocation of the Class III PI3 kinase complex and delivery of LC3-II. 2) Elongation of the phagophore membrane requires the Class III PI3 kinase complex. Rab8a, Rab24, Rab32, Rab33b and Rab39b are all involved in autophagosome formation and may aid in elongation by the delivery of additional membrane via ATG9/ATG2/WIPI1/2. Autophagy substrates are recruited to the growing phagophore by autophagy receptors such as p62/sequestosome-1 and optineurin. Autophagy receptors bind ubiquitin on the substrates and LC3-II on the nascent phagophore resulting in substrate delivery. 3) After completion and closure, autophagosomes are transported to allow fusion with the lysosome. Rab7 is involved in autophagosome transport while Rab11 delivers multi-vesicular bodies (MVBs) to the autophagosome, which appears to be required for maturation. 4) Autophagosome fusion with the lysosome allows degradation of the autophagic substrates. Rab7, Rab8b and Rab9 are involved in the fusion of autophagosomes and lysosomes, a process that may also require Rab24. Finally, autophagic substrates are degraded by the acid hydolases of the lysosome and recycled back to the cytoplasm.

Figure 2.

C9orf72 regulates the Rab1a dependent trafficking of the ULK1 complex. Upon inhibition of mTOR, the inhibitory phosphorylation of ULK1 is lost, leading to the activation of ULK1. Active ULK1 phosphorylates the other members of the initiation complex, including FIP200 and ATG13. Functioning as a Rab1a effector, C9orf72 mediates the interaction between the active ULK1 initiation complex and GTP-Rab1a within its target membrane. Thus C9orf72 controls the Rab1a dependent trafficking of the ULK1 initiation complex to the phagophore. How Rab1 is activated in response to autophagy induction is not yet known.

Figure 3.

C9orf72 is a central hub in a Rab cascade pathway during autophagy induction. 1) C9orf72 controls the Rab1a-dependent translocation of the ULK1 initiation complex to the site of phagophore formation by interacting with the ULK1 complex and active GTP-Rab1a. 2) At the site of phagophore formation, the C9orf72/SMCR8/WDR41 complex functions as a GEF for Rab8a and Rab39b. 3) Active Rab8a and Rab39b may be involved in delivery of additional membrane to the site of phagophore formation by retrieval of ATG9 positive membranes. Additional membrane allows elongation of the nascent phagophore and formation of an autophagosome. 4) The autophagy receptors p62/sequestosome-1 and optineurin interact with Rab8a and Rab39b, promoting nucleation and site specific recruitment of autophagic substrates to the growing phagophore. Furthermore, Rab8a also recruits TBK1, leading to phosphorylation of optineurin, SMCR8 and ULK1. The phosphorylation of optineurin enhances its interaction with LC3-II, facilitating substrate delivery and the progression of autophagy. 5) The Class III PI3 Kinase complex is also required for autophagosome formation. Rab5, activated by Alsin, delivers the PI3 Kinase complex to the phagophore and also facilitates the recruitment of the ATG7-ATG5 conjugation system required for LC3-II formation.

Figure 4.

Autophagy and ALS/FTD. A number of genes, indicated in red, implicated in ALS/FTD have been linked to the autophagy/lysosomal pathway.