Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis

Abstract

TAR DNA binding protein 43 (TDP-43) is a versatile RNA/DNA binding protein involved in RNA-related metabolism. Hyper-phosphorylated and ubiquitinated TDP-43 deposits act as inclusion bodies in the brain and spinal cord of patients with the motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While the majority of ALS cases (90-95%) are sporadic (sALS), among familial ALS cases 5-10% involve the inheritance of mutations in the TARDBP gene and the remaining (90-95%) are due to mutations in other genes such as: C9ORF72, SOD1, FUS, and NEK1 etc. Strikingly however, the majority of sporadic ALS patients (up to 97%) also contain the TDP-43 protein deposited in the neuronal inclusions, which suggests of its pivotal role in the ALS pathology. Thus, unraveling the molecular mechanisms of the TDP-43 pathology seems central to the ALS therapeutics, hence, we comprehensively review the current understanding of the TDP-43's pathology in ALS. We discuss the roles of TDP-43's mutations, its cytoplasmic mis-localization and aberrant post-translational modifications in ALS. Also, we evaluate TDP-43's amyloid-like in vitro aggregation, its physiological vs. pathological oligomerization in vivo, liquid-liquid phase separation (LLPS), and potential prion-like propagation propensity of the TDP-43 inclusions. Finally, we describe the various evolving TDP-43-induced toxicity mechanisms, such as the impairment of endocytosis and mitotoxicity etc. and also discuss the emerging strategies toward TDP-43 disaggregation and ALS therapeutics.

Keywords: ALS therapeutics; TDP-43; amyotrophic lateral sclerosis (ALS); endocytosis; frontotemporal lobar degeneration (FTLD); liquid-liquid phase separation (LLPS); mitotoxicity; prion.

Figure 1

TDP-43 proteinopathies. TDP-43 proteinopathies refer to the diseases where TDP-43 is implicated and it includes: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy. FTLD is a group of disorders principally of the frontal temporal lobes of the brain causing dementia. Other forms of FTLD disorders are FTLD-Tau, FTLD-FUS, and FTLD-VCP. FTLD-Tau is associated with mutations in the MAPT gene which encodes microtubule associated protein, Tau. Tau's misfolding and aggregation lead to loss of microtubule-binding function and formation of neuronal and glial inclusions (Irwin et al., 2015). FTLD-FUS is associated with mutations in the RNA-binding protein FUS, which results in disruption of its nuclear localization and leads to its accumulation into inclusion bodies (Mackenzie et al., 2011). FTLD-VCP is associated with mutations in the valosin-containing protein (VCP). FTLD-VCP manifests ubiquitin and TDP-43-positive neuronal intranuclear and cytoplasmic inclusions. FUS, fused in sarcoma; TDP-43, TAR DNA binding protein 43; VCP, valosin containing protein.

Figure 2

Structural features of TDP-43. (A) TDP-43's domain organization depicting ALS and FTLD-linked mutations. TDP-43 comprises of an NTD domain, two RRM domains, a nuclear export signal (NES), a nuclear localization signal (NLS), a prion-like disordered C-terminal domain (with glutamine/asparagine-rich (Q/N) and Glycine-rich regions) and mitochondrial localization motifs (M1−35–41; M3−146–150; M5−294–300). Sporadic mutations and familial gene mutations generating amino acid substitutions in TDP-43 are classified. Several TDP-43 mutations overlap between ALS and FTLD as well as between sALS and fALS (Baumer et al., ; Xiong et al., ; Fujita et al., ; Janssens et al., ; Budini et al., ; Chiang et al., ; Cruts et al., ; Lattante et al., ; Moreno et al., 2015). PDB structures of: (B) N-terminal region (PDB id-2N4P); (C) a tandem RRM1 and RRM2 segment (PDB id-4BS2); (D) a C-terminal region (aa: 311–360) (PDB id-2N3X). Structures in the (B–D), have been adapted with permissions respectively from: John Wiley and Sons (Mompeán et al., 2016b); Springer Nature (Lukavsky et al., 2013); and Springer Nature (Jiang et al., , creative commons attribution 4.0 license). fALS, familial amyotrophic lateral sclerosis; NES, nuclear export signal; NLS, nuclear localization signal; NTD, N-terminal domain; Q/N, glutamine/asparagine; RRM, RNA recognition motif; sALS, sporadic amyotrophic lateral sclerosis; TDP-43, TAR DNA binding protein 43.

Figure 3

Functions of TDP-43. TDP-43 performs several mRNA-related processes in the nucleus, such as transcription, splicing, maintaining RNA stability as well as miRNA and lncRNA processing. It is predominantly a nuclear protein but also shuttles between the nucleus and the cytoplasm. In the cytoplasm, TDP-43 participates in the stress granule formation, ribonucleoprotein (RNP) transport granule formation, translation and other processes. lncRNA, long non-coding RNA; miRNA, microRNA; mRNA, messenger RNA; pA, poly-A mRNA tail; TDP-43, TAR DNA binding protein 43.

Figure 4

Post-translational modifications in the TDP-43 protein. TDP-43 undergoes several post-translational modifications, such as phosphorylation, ubiquitination, acetylation, PARylation, and cysteine oxidation. Phosphorylation of the full-length and C-terminal fragments of TDP-43 is a pathological hallmark of ALS and is associated with its increased cytoplasmic mislocalization. In FTLD and ALS brain inclusions, pathological TDP-43 is found in the ubiquitinated state and mutations at the ubiquitination sites decrease the TDP-43 aggregation. Acetylation promotes accumulation of the insoluble and hyper-phosphorylated TDP-43 aggregates. PARylation promotes the phase separation of TDP-43 into stress granules. Oxidative stress mediated cysteine oxidation promotes the oligomerization and aggregation. Ac, acetylation; P, phosphorylation; PARylation, poly ADP ribosylation; Ub, ubiquitination.

Figure 5

Liquid-liquid phase separation (LLPS) and liquid-solid phase separation (LSPS) of TDP-43. (A) Proteins containing low complexity/prion-like domains undergo phase-separation into membrane-less, spherical compartments, often aided by the presence of salt, pH changes or temperature changes. Persistent stress, mutations and droplet-aging, might induce irreversible aggregation into pathological structures, such as the amyloid-like aggregates. (B) Liquid droplet-like properties are manifested by the intrinsically disordered proteins, such as: the ability of the smaller droplets to freely fuse into a larger droplet; transient intermolecular interactions allowing the dynamic rearrangement of the internal structural components; and reversible reformability upon removal of the external shear forces. (C) Liquid-liquid phase separation (LLPS) of TDP-43 is influenced by both hydrophilic and hydrophobic residues. The (G/S)-(F/Y)-(G/S) motifs (highlighted in green) promote the phase separation through transient interactions in several intrinsically disordered proteins (Li et al., 2018). The tryptophan residues promote LLPS by hydrophobic interactions (Li et al., 2018). Depletion of the TDP-43's interactions with RNA molecules, upon high protein: RNA ratio, can lead to irreversible aggregation via Liquid-solid phase separation (LSPS) (Maharana et al., 2018). ALS-linked mutations are also proposed to lead to the formation of the irreversible aggregates. FRAP, fluorescence recovery after photobleaching; LCD, Low complexity domain; LLPS, liquid-liquid phase separation; LSPS, liquid-solid phase separation; NTD, N-terminal domain; PTM, post-translational modification; RRM, RNA recognition motif.

Figure 6

Schematics of TDP-43-induced pathology. Several aspects of TDP-43-linked cellular dysfunctions have been identified in ALS, such as nuclear depletion which leads to aberrant RNA metabolism and a loss of autoregulation of TDP-43 levels. Cytoplasmic accumulation of the hyper-phosphorylated and ubiquitinated TDP-43 are ALS disease hallmarks. Fragmentation of TDP-43 leads to the formation of toxic and aggregation-prone C-terminal fragments (CTFs). TDP-43 mutations can lead to abnormal stress granule assembly and release. Aberrantly increased mitochondrial localization of TDP-43 impairs its function. TDP-43 is also associated with the misregulated autophagy and proteosomal processes. TDP-43 expression perturbs the endocytosis process possibly by altering the expression of key endocytic components. Also, the TDP-43 aggregates have been identified as an inhibitor of the endolysosomal pathway. TDP-43 interacts with chromatin remodeling protein CHD2 and perturbs the chromatin dynamics which prevents the expressions of heat shock proteins. Prion-like inter-cellular propagation of detergent-resistant, β-sheet-rich aggregates of TDP-43, has also been demonstrated in the neuronal cell models. CHD2, chromodomain helicase DNA binding protein 2; CTF, C-terminal fragments; ER, endoplasmic reticulum; HSP, heat shock protein; P, phosphorylation; Ub, ubiquitination; UPS, ubiquitin-proteasome system.

Figure 7

Role of mitochondria in the TDP-43 pathology. TDP-43 mediated dysfunction of the mitochondria leads to increase in the production of ROS that causes decline in the reduced glutathione levels which in turn can increase the aggregation of TDP-43 and also inhibit TDP-43 from binding to the nucleic acid. Mutant SOD1 can cause cytoplasmic mislocalization, fragmentation, phosphorylation and aggregation of TDP-43. Inhibition of the interaction of the mitochondrial fission proteins Drp1 and Fis1 greatly reduces the mitochondrial dysfunction caused by the TDP-43 overexpression/aggregation. TDP-43 also disrupts the ER-mitochondrial contacts which can have potential implications to the calcium signaling, ATP production and lipid transport. TDP-43 is imported into mitochondria via the outer membrane complex (TOM70) and across the inner membrane via TIM22. Several factors like chaperones and mitochondrial membrane potential might play a role in the TDP-43 import. After internalization, TDP-43 is found to interact with several proteins involved in the mitochondrial translation machinery (MRPS22, 27 and MRPL12, 22, 48, 44) and the mitochondrial respiratory complex (ATPA, CIQBP, SCAMC-3). TDP-43 also perturbs the translation of ND3/6 of the respiratory complex I and thus severely impairs the mitochondrial bioenergetics and reduces the ATP production. TDP-43 overexpression alters the CHCHD10 localization from the mitochondria to the nucleus and the loss-of-function mutations in CHCHD10 are associated with MICOS complex disassembly and may negatively regulate the assembly of the respiratory complex. TDP-43 also interacts with other crucial mitochondrial proteins including the mitochondrial fusion protein Mfn2 and the mitophagy receptor PHB2. TDP-43 is depicted here by the red oval structure. TDP-43 interaction with the mitochondrial proteins are depicted via green star. Inhibition is denoted by red cross mark. ATPA, ATP synthase subunit A; CHCHD10, coiled-coil-helix-coiled-coil-helix domain containing 10; CIQBP, complement component 1Q binding protein; CTF, C-terminal fragments; Drp1, dynamin related protein 1; ER-Mito contacts, endoplasmic reticulum (ER)-mitochondria contacts; Fis1, fission 1 (mitochondrial); Mfn2, mitofusin-2; MICOS, mitochondrial contact site and cristae organizing system; MRPL, mitochondrial ribosomal protein (large subunit); MRPS, mitochondrial ribosomal protein (small subunit); ND, NADH dehydrogenase; P, phosphorylation; PHB2, prohibitin-2; ROS, reactive oxygen species; SCAMC-3, small calcium-binding mitochondrial carrier protein 3; SOD1, superoxide dismutase 1; TIM, translocase of inner membrane; TOM, translocase of outer membrane.