Autophagy and Its Impact on Neurodegenerative Diseases: New Roles for TDP-43 and C9orf72

Abstract

Autophagy is a catabolic mechanism where intracellular material is degraded by vesicular structures called autophagolysosomes. Autophagy is necessary to maintain the normal function of the central nervous system (CNS), avoiding the accumulation of misfolded and aggregated proteins. Consistently, impaired autophagy has been associated with the pathogenesis of various neurodegenerative diseases. The proteins TAR DNA-binding protein-43 (TDP-43), which regulates RNA processing at different levels, and chromosome 9 open reading frame 72 (C9orf72), probably involved in membrane trafficking, are crucial in the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Additionally, recent studies have identified a role for these proteins in the control of autophagy. In this manuscript, we review what is known regarding the autophagic mechanism and discuss the involvement of TDP-43 and C9orf72 in autophagy and their impact on neurodegenerative diseases.

Keywords: ALS; C9orf72; FTLD; TDP-43; autophagy; neurodegenerative diseases.

Figure 1

Autophagic pathway. The omegasome expands its membrane to form a double membrane organelle called autophagosome, which sequesters intracellular material such as proteins and organelles. Then, the autophagosome fuses with the lysosome to form the autolysosome. The lysosome supplies hydrolytic enzymes, which are activated following the autophagosome-lysosome fusion, promoting the degradation of the autolysosome cargo. The digestion of the cargo generates new metabolites that turn back to the cytosol and that will be re-used by the cell.

Figure 2

The autophagic machinery. The energetic status of the cell is sensed by the proteins mTOR and AMPK. When nutrients are high mTOR, which, together with Raptor, Deptor, mLST8, and PRAS 40 forms the mTORC1 complex, inhibits the ULK1 kinase activity phosphorylating its Ser638 and Ser757. ULK1, ATG13, and FIP200 form the ULK complex. Upon nutrient deprivation, AMPK is activated and induces autophagy phosphorylating different target proteins. Unlike mTOR, AMPK activates ULK1 by phosphorylation on Ser317 and Ser777, leading to the activation of the PI3KC3 complex, which is formed by AMBRA, PI3KC3, Beclin 1 and ATG14L. AMPK can also induce autophagy by direct suppression of the mTOR pathway phosphorylating Raptor (Ser722 and Ser792) or TSC2 (Thr1227). Active PI3KC3 increases the production of phosphatidylinositol 3-phosphate (PtdIns3P) in specific membrane micro domains of the plasma membrane, the ER, mitochondria and/or MAMs. Accumulation of PtdIns3P recruits FYVE domains proteins such as WIPIs and DFCP1. The elongation and expansion of the phagophore membrane are mediated by ATG proteins through two ubiquitination-like systems: the complex ATG12-ATG5-ATG16L and the conjugate ATG8 (LC3)-phosphatidylethanolamine (PE) (also called LC3-II). Finally, while the ATG12-ATG5-ATG16L conjugate is disassembled from the autophagosomal membrane, LC3-II remains enclosed to the inner and the external membrane of the vacuole. Other proteins such as SNARES and RABs are also required for the trafficking of membranes and vacuole elongation.

Figure 3

Physiology and pathology of TDP-43. (A) In physiological conditions, thanks to its capacity to shuttle between the nucleus and the cytoplasm, TDP-43 regulates several processes that impact on the cellular fate of different RNAs. These processes are: (i) RNA transcription: it includes the regulation of RNA transcription through the direct binding of TDP-43 to promoter regions. (ii) mRNA alternative splicing: the most studied and known function of TDP-43 is the regulation of alternative mRNA splicing. TDP-43 binds to exon or intron regions and recruits other RNA binding proteins (RBPs) such as hnRNPs or SRs proteins (heterogeneous nuclear ribonucleoproteins and serine-arginine proteins, respectively). (iii) RNA stabilization: RNA stabilization has been proposed as a function of TDP-43. TDP-43, binding to different mRNAs or miRNAs inhibits their premature degradation and possibly favors their translocation from the nucleus to the cytoplasm. Different studies report a decrease in the half-life of several mRNAs and miRNAs after TDP-43 down-regulation. (iv) miRNAs synthesis and processing: TDP-43 has been implicated in the synthesis and processing of different miRNAs by interacting with Drosha and Dicer complexes. It has been reported that TDP-43 down-regulation affects the cellular levels of several miRNAs. (v) Stress granules localization/regulation: In different conditions of cellular stress, it has been shown that TDP-43 is located in the stress granules (SGs) where it interacts with classical SGs proteins such as TIAR and G3BP. Although the real function of TDP-43 in SGs is still unknown, it has been reported that TDP-43 down-regulation affects SGs formation by decreasing G3BP mRNA levels. (vi) RNA transport: Data indicate TDP-43 is required for the transport of different RNAs along the neuronal axon, by a mechanism that might involve microtubules and kinesin complexes. (B) In a pathological situation, TDP-43 forms cellular aggregates, mainly in the cell cytoplasm, through a mechanism that is currently unknown. It has been proposed that TDP-43 aggregates can be toxic for the cell. However, a generally accepted hypothesis suggests that TDP-43 aggregates could act as “sink” that continuously sequesters functional TDP-43 protein, thus, increasing the aggregation process by a positive feed-back loop. This would lead to a generalized TDP-43 loss-of-function, which promotes cellular stress and eventually leads to cell death. Gray color: represents attenuated processes due to TDP-43 loss-of-function.

Figure 4

Regulation of autophagy by TDP-43. It has been reported that TDP-43 regulates basal autophagy by stabilizing three different mRNAs: atg7, raptor, and dynactin 1. The cellular levels of these autophagic mRNAs decrease after TDP-43 down-regulation. Thus, data indicate TDP-43 can participate in autophagy regulation at two different steps: autophagic initiation and autophagic flux progression. (I) Autophagy initiation: TDP-43 stabilizes the mRNA that codes for the protein Raptor, thus ensuring sufficient levels of this protein and keeping mTORC1 active (i). In turn, mTORC1 allows the phosphorylation of TFEB decreasing its nuclear translocation. Thus, by stabilizing mRNA raptor levels, TDP-43 ensures the maintenance of basal autophagy. (ii) TDP-43 stabilizes atg7 mRNA and thus its protein levels. In this way, TDP-43 regulates the initiation step and the formation of new autophagosomes (ii). (II) Autophagic flux progression: by stabilizing dynactin 1 mRNA levels and Dynactin 1 protein levels, TDP-43 allows the progression of the autophagic flux (iii). The latter, is supported by data showing that Dynactin 1 is required in the process of autophagosome-lysosome fusion.

Figure 5

TDP-43 aggregation dysregulates autophagy. In a pathological condition, TDP-43 aggregates might sequester functional TDP-43 causing a complete loss-of-function of this protein. Loss of TDP-43 activity decreases the levels of raptor, atg7 and dynactin 1 mRNAs (i). This situation can affect the autophagic pathway in two different ways. (I) Autophagy induction: loss of Raptor protein destabilizes the mTOR complex decreasing the phosphorylation that inhibits TFEB nuclear translocation. Dephosphorylated TFEB in now able to translocate into the nucleus, where it increases the transcription of genes that stimulate autophagy (ii). Loss of TDP-43 activity negatively impacts on the Atg7 protein level, thus decreasing autophagosomes formation (iii). (II) Autophagy progression: this step can be reduced following TDP-43 aggregation as Dynactin 1 protein levels are lower due to TDP-43 loss-of-function (iv). Overall, although the loss of TDP-43 activity increases the expression of genes related with autophagy progression, autophagy is blocked due to the failure in both, autophagosomes formation and autophagosome-lysosome fusion. This situation can promote cellular stress and cell death. Gray color: represents down-regulated proteins and attenuated processes due to TDP-43 loss-of-function.

Figure 6

Regulation of autophagy by C9orf72. Studies indicate C9orf72 protein is implicated in autophagy regulation. Specifically, data show C9orf72 is involved in the progression of the autophagic process by three different mechanisms. (i) C9orf72 interacts with SMCR8 forming a heterodimer that recruits WDR4. The complex C9orf72/SMCR8/WDR41 activates several RAB proteins, which participate in autophagy. (ii) The C9orf72/SMCR8/WDR4 complex recruits and activates the ULK complex by its phosphorylation in S575. (iii) The heterodimer C9orf72/SMCR8 interacts with the proteins SINTBAD, NAP, and TANK. These proteins, in turn, recruit TBK1 to the complex allowing the phosphorylation of SMCR8, which is required for autophagy progression.

Figure 7

Autophagy dysregulation in C9orf72-dependent neurodegenerative diseases. In a pathological situation, it has been observed that the presence of GGGGCC repeats (n ≥ 30) in c9orf72 region can be toxic for the cell. Data indicate that GGGGCC repeats can internally interact to form RNA foci (i) that are toxic by recruiting different RNA binding proteins (RBPs) such as hnRNPH, hnRNPA2, ADARB2, and Purα. (ii) In addition to RNA foci, it has been shown that GGGGCC repeats interfere with the normal transcription (iii), splicing (iv), and translation (v) processes of C9orf72, thus reducing C9orf72 protein levels in the cell. Moreover, several studies showed that GGGGCC repeats can form polypeptides composed by dipeptides repetitions (DRPs). These DRPs are synthetized through a non-classical mechanism of protein translation (vi). To our knowledge, there is no evidence indicating that DRPs might interfere with the autophagic process. Overall, in pathological conditions, GGGGCC repeats decrease C9orf72 protein levels, thus affecting the normal autophagic process. Autophagy dysregulation culminates with the accumulation of autophagosomes, caused by impaired autophagosomal-lysosomal fusion, which might contribute to the formation and accumulation of protein aggregates like Ataxin and eventually TDP-43. This causes cellular stress and might lead to cell death. Gray color: represents down-regulated proteins and attenuated processes due to TDP-43 loss-of-function.