Navigating the pathways: TAR-DNA-binding-protein-43 aggregation, axonal transport, and local synthesis in amyotrophic lateral sclerosis pathology

Neurons are highly polarized cells with axons reaching over a meter long in adult humans. To survive and maintain their proper function, neurons depend on specific mechanisms that regulate spatiotemporal signaling and metabolic events, which need to be carried out at the right place, time, and intensity. Such mechanisms include axonal transport, local synthesis, and liquid–liquid phase separations. Alterations and malfunctions in these processes are correlated to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). ALS is a fast-progressing fatal motor neuron disease pathologically characterized by muscle atrophy, neuromuscular junction degeneration, and eventually, motor neuron death (Rothstein, 2009). While the precise mechanisms underlying ALS pathogenesis remain elusive, TAR DNA binding protein 43 (TDP-43) mislocalization and aggregation play a key role in the disease process (Buratti and Baralle, 2009; Lee et al., 2012; Piol et al., 2023). Concurrently, disruptions in axonal transport, and alterations in local synthesis, which are vital for neuronal function, have also been observed in ALS. Understanding the bidirectional interplay between TDP-43 aggregation and axonal transport/local synthesis events holds promise for elucidating ALS pathology and advancing therapeutic strategies. TAR DNA binding protein 43 aggregation in amyotrophic lateral sclerosis: TDP-43, a multifunctional RNA-binding protein predominantly localized in the nucleus, plays crucial roles in transcriptional regulation, RNA processing, subcellular localization, and stress response. However, in ALS, TDP-43 undergoes aberrant cytoplasmic mislocalization and accumulation and forms insoluble aggregates, a pathological hallmark observed in nearly all ALS cases, both sporadic and familial. The mechanisms driving TDP-43 aggregation remain incompletely understood, but evidence suggests a convergence of genetic mutations, cellular stressors, dysfunction in nuclear transport, and impaired proteostasis mechanisms. TDP-43 aggregates disrupt cellular homeostasis, leading to dysregulation of RNA metabolism, protein translation, and stress granule dynamics, all of which may ultimately contribute to neurodegeneration (Alami et al., 2014; Suk and Rousseaux, 2020; Ionescu et al., 2023). Recently, we identified a molecular mechanism for axon degeneration and neuromuscular junction disruption in ALS. We showed that phospho-TDP-43 (pTDP-43) co-localizes with the core stress granule component G3BP1 in aggregates within axons, where these pTDP43-G3BP1 aggregates sequester RNA and inhibit local protein synthesis, resulting in mitochondrial malfunction and neuromuscular junction disruption with subsequent axonal degeneration (Altman et al., 2021). However, whether these axonal aggregates affect axonal transport and how, is still to be discovered. Axonal transport dysfunction in amyotrophic lateral sclerosis: Axonal transport is essential for proper neuronal development and motor neuron survival, facilitating the bidirectional movement of organelles, proteins, RNAs, and signaling molecules along axons. Disruptions in axonal transport have been implicated in ALS pathogenesis, with defects observed in both anterograde and retrograde transport mechanisms. Genetic mutations affecting motor proteins, cytoskeletal components, or regulatory factors can compromise axonal transport efficiency, leading to impaired cargo delivery and neuronal dysfunction (Guedes-Dias and Holzbaur, 2019; Guo et al., 2020). Moreover, environmental stressors, protein aggregates such as TDP-43, and mitochondrial dysfunction further exacerbate axonal transport deficits in ALS (Mandal and Drerup, 2019). Nevertheless, it is also possible that alterations in axonal transport can cause TDP-43 aggregation, making the cause-and-effect interplay between these two processes unknown. Local synthesis in amyotrophic lateral sclerosis: Local protein synthesis is a fundamental mechanism by which distal axons maintain their proteome, ensuring the availability of proteins critical for axonal function, plasticity, and survival. The dynamic process of TDP-43 liquid-liquid phase separation plays an essential role in regulating this local translation by facilitating the assembly of ribonucleoprotein granules that localize to axons. However, pathological conditions can alter the biophysical properties of TDP-43, leading to aberrant phase transitions that result in the formation of stable, insoluble condensates. These TDP-43 aggregates sequester mRNA and possibly other translational machinery, effectively impairing local protein synthesis. The disruption of this finely tuned process compromises the ability of the distal axon to maintain its structural and functional integrity. The ensuing deficits in axonal transport, synaptic function, and local protein replenishment can precipitate a cascade of neurodegenerative events, culminating in axonal degeneration and neuronal death. The impairment of local translation due to aberrant TDP-43 phase separation is therefore likely to be a critical driver in the pathogenesis of ALS, contributing to the progressive loss of motor neurons that defines the disease. Intersection of TAR DNA binding protein 43 aggregation, axonal transport, and local synthesis: The relationship between TDP-43 aggregation and axonal transport dysfunction represents a critical and underexplored aspect of ALS pathology. The connection between these phenotypes is unknown and the cause-and-effect relation is also yet to be understood (Figure 1). Firstly, TDP-43 aggregates, localized within axons, may act as physical barriers directly blocking microtubule-based motor protein movement, thus, disrupting cargo transport. Consequently, vital cellular components destined for the cell body, or the synaptic terminals will not reach their intended destinations which could eventually compromise proper synaptic function and motor neuron survival. Apart from physical interruption, TDP-43 aggregates may directly interfere with the function of motor proteins. By binding or sequestering kinesins and dyneins, the motor proteins responsible for anterograde and retrograde transport respectively, or other axonal transport regulators and adaptors, these aggregates may alter the ability of the motor proteins to bind to their designated cargo and effectively transport it to their destination in the cell. TDP-43, being an RNA-binding protein, may also bind to different mRNA transcripts located along the axon and affect local protein synthesis. Some of these mRNAs might be of motor proteins and axonal transport machinery, thus preventing them from undergoing translation and minimizing the required available pool of them for maintaining proper axonal transport. Other mRNAs whose translation might be halted by their binding by TDP-43 are mitochondrial mRNAs that are needed for the maintenance of axonal mitochondria (Altman et al., 2021). Interruption of the mitochondrial proper metabolic function needed in the axons might lead to energy deficiency that might reduce the ability of motor proteins to move, being adenosine 5′-triphosphate dependent. This disruption not only impedes the transport of essential vesicles and organelles critical for neuronal homeostasis and function but also interferes with the clearance of damaged or misfolded proteins, contributing to cellular stress and further cytoplasmic protein aggregation and accumulation which creates a vicious and toxic cycle. In this sense, dysregulated axonal transport could exacerbate TDP-43 aggregation within the nervous system, promoting the fast propagation of neurodegeneration and ALS. Additionally, TDP-43 aggregates may sequester essential regulatory factors or signaling molecules, interfering with axonal transport regulation and coordination. Impaired transport of signaling molecules can compromise neuronal communication, synaptic integrity, and overall neuronal network connectivity, contributing to the widespread neurodegeneration of the corticospinal tract observed in ALS. In addition to their effects on motor proteins and signaling molecules, TDP-43 aggregates may provoke alterations in the dynamics and stability of microtubules-polarized hollow protein fibers that comprise the cytoskeleton and allow axonal transport. By disrupting microtubule organization and integrity, these aggregates exacerbate the impediment of axonal transport, leading to aberrant cargo trafficking, synaptic dysfunction, and neurodegeneration. This impediment could be caused by the binding of the aggregates to microtubule-associated proteins that regulate the stability and organization of microtubules (Clark et al., 2016). Moreover, compromised microtubule stability may cause axons to be more susceptible to mechanical stress and injury due to the fragmented cytoskeleton, exacerbating the neuronal damage and dysfunction seen in ALS. A reverse cause-and-effect connection is also possible where axonal transport alterations cause the aggregation of TDP-43 seen in ALS patients leading to axonal toxicity and neurodegeneration, for example, by reduction of retrograde clearance, axonal aggregates accumulate and can lead to cell toxicity and death. Another possibility is pathological induction of anterograde transport of the components of the TDP-43 aggregates, or the aggregates themselves, leading to their accumulation in the distal axon while exacerbating aggregation and resulting in cell toxicity. Understanding the complex bidirectional interplay between TDP-43 aggregation and axonal transport dysfunction may offer novel insights into ALS pathogenesis and therapy.Figure 1: Scheme presenting possible bidirectional cause-and-effect connections between axonal transport and axonal TDP-43 aggregation.TDP-43 axonal aggregation may impair axonal transport by binding to motor proteins, directly interfering with their physical movement or sequestering of the motor proteins’ mRNAs. Alternatively, TDP-43 axonal aggregation may be the result of poor retrograde clearance or induced anterograde transport. Created with Adobe Illustrator. TDP-43: TAR DNA binding protein 43.Mechanistic insights and therapeutic implications: Investigating the molecular mechanisms underlying the interplay between TDP-43 aggregation and axonal transport dysfunction holds significant promise for developing targeted therapeutic interventions for ALS. Approaches aimed at modulating TDP-43 aggregation, enhancing protein clearance pathways, or restoring axonal transport efficiency represent potential therapeutic avenues. Small molecule inhibitors targeting TDP-43 aggregates and promoting their disaggregation could alleviate axonal transport deficits and mitigate neurodegeneration. Furthermore, strategies aimed at restoring mitochondrial function, promoting axonal regeneration, or enhancing neuronal resilience to stressors may offer complementary therapeutic benefits in ALS management. Future directions and challenges: Despite recent advances, several key questions and challenges remain in deciphering the intricate relationship between TDP-43 aggregation, axonal transport dysfunction, and ALS pathogenesis. Elucidating the precise mechanisms driving TDP-43 aggregation and its impact on axonal transport dynamics requires comprehensive interdisciplinary approaches, integrating advanced imaging techniques, omics technologies, and disease modeling systems. Moreover, the development of translational preclinical models that recapitulate ALS pathology and therapeutic responses accurately is essential for evaluating the efficacy and safety of potential therapeutic interventions. Addressing these challenges will be instrumental in advancing our understanding of ALS and translating scientific discoveries into effective clinical therapies. Conclusion: The intersection between TDP-43 aggregation and axonal transport dysfunction represents a compelling focal point in ALS research, offering novel insights into the disease mechanisms and its therapeutic strategies. By elucidating the bidirectional interplay between these processes, it will be possible to uncover key molecular targets and pathways driving ALS pathology, paving the way for the development of innovative therapeutic interventions. Continued collaborative efforts across disciplines and sustained investment in basic research are essential for harnessing the full potential of this promising avenue in ALS therapeutics. C-Editors: Zhao M, Sun Y, Qiu Y; T-Editor: Jia Y