26 Apr 2024

To allow for the local translation of proteins that suit the ever-changing needs of the axon, neurons must ship transcripts from the soma to their outermost reaches. For thousands of these mRNAs, hitchhiking is their predominant mode of transport. This is according to a study led by Juan Bonifacino at the National Institutes of Health in Bethesda, Maryland, which found that traveling lysosomes give mRNAs a ride along microtubule highways. As described April 10 in Nature Neuroscience, without proteins that tether lysosomes to kinesin motors, thousands of transcripts failed to reach their destination. Among the marooned were those encoding ribosomal subunits and mitochondrial proteins. Mitochondrial biogenesis and respiration faltered, ultimately leading to misshapen, degenerating axons akin to those seen in neurodegenerative diseases. The findings uncover a fundamental biological mechanism that keeps axons flush with enough proteins and energy to support neurotransmission.

“These findings underscore the importance of understanding the basic mechanism of mRNA transport and localization, and offer insights into potential mechanisms underlying axon degeneration and neurodegeneration,” commented Eran Perlson of Tel Aviv University. He thinks it could lead to new avenues for therapeutic exploration (comment below).

Axons are dynamic hubs of synaptic activity and protein turnover that require vast amounts of energy. How does the mRNA make it that far? Previously, researchers reported that RNA granules, which are membraneless organelles comprising a mix of RNA and RNA-binding proteins, travel from the soma into axons by tethering themselves to lysosomes via the annexin-11 protein (Liao et al., 2019). Lysosomes and related vesicles travel in axons bidirectionally, using kinesin motors to move out toward axonal terminals, and dynein on the return trip.

Co-first authors Raffaella De Pace and Saikat Ghosh and colleagues wanted to investigate how this lysosomal schlepping influences the axonal RNA repertoire, and how that might support axon biology. To do this without disabling all modes of transport, the researchers knocked out BLOC-one-related complex (BORC), which, together with the small GTPase ARL8, and the adaptor protein PLEKHM2, couples lysosomes to kinesins 1 and 3. BORC is an octameric complex, and the researchers used CRISPR to generate human iPSC-derived neurons lacking either BORCS5 or BORCS8 subunits, each needed for the tie to lysosomes. When i3 neurons derived from iPSCs lacked either subunit, they appeared to mature normally relative to their BORC-replete counterparts, sprouting axons. However, a closer look revealed profound differences. While lysosomes were broadly distributed in the soma, dendrites, and axons of wild-type neurons, i3s lacking a BORC subunit housed few, if any, lysosomes in their axons (image below).

No Ride for Lysosomes. In wild-type i3 neurons (left), LAMP1+ lysosomes (white) are found in dendrites, soma, and axons. Without BORC5 (middle) or BORC7 (right), few lysosomes travel into axons. [Courtesy of de Pace et al., Nature Neuroscience, 2024.]

How would this shutdown influence the repertoire of transcripts found there? To isolate axonal transcripts, the researchers grew iPSCs in a microfluidic device in which the cells are plated on either side of a series of microgrooves that flow into a central chamber. As the iPSCs develop into neurons, only axons are long and thin enough to traverse the microgrooves into the central chamber, where the researchers harvested them for analysis (image below). In this way, researchers could isolate and sequence RNA from pure axons, and compare it to the transcripts found within the side chambers that housed all neuronal parts.

Get into the Groove. Neurons were grown in two chambers of a microfluidic device, positioned on either side of microgrooves (left). [Courtesy of de Pace et al., Nature Neuroscience, 2024.]

The upshot? BORC deficiency profoundly affected the axonal transcriptome. Focusing on wild-type neurons first, the researchers found that axons were steeped in transcripts encoding ribosomal RNAs, ribosomal subunits, and mitochondrial proteins involved in oxidative phosphorylation. In BORC-KO neurons, the axonal, but not the soma, transcriptome was dramatically altered. Ribosomal RNAs plummeted, as did more than 2,000 protein-coding transcripts. Notably, many of the depleted axonal transcripts have been implicated in neurodegenerative diseases, including Parkinson’s, Huntington’s, Alzheimer’s, and amyotrophic lateral sclerosis.

While BORC-less axons took an RNA loss, their neuronal cell bodies saw an increase in more than 3,000 transcripts relative to wild-type neurons. These hailed from numerous biological pathways and corresponded to cell body, not axonal transcripts. RNAs encoding lysosomal proteins were among those most increased in BORC KO neurons, suggesting neurons may have been attempting to compensate for the loss of lysosomes in the axons, the authors proposed.

These shifts in the axonal transcriptome exacted a steep toll: for one, a dearth of functional ribosomes, which, in turn, sapped mitochondrial protein translation. In particular, components of the mitochondrial membrane electron transport chain were reduced. Loss of these proton-pumping proteins led to a dip in the mitochondrial membrane potential, stressing the organelles. Fewer mitochondria inhabited BORC KO neurons, and those that persisted were smaller and misshapen (image below). Their cristae—the membrane folds that provide the surface area for energy-producing oxidative phosphorylation—were few and irregularly spaced.

Mitochondrial Meltdown. Compared to the large, intact mitochondria found in wild-type axons (left), those in BORCS7 KO axons were small, with deformed cristae (right). [Courtesy of de Pace et al., Nature Neuroscience, 2024.]

All this mitochondrial mayhem instigated mitophagy, a form of autophagy that serves to rid the cell of dysfunctional mitochondria. As evidence of this, the researchers spotted axonal vesicles expressing the autophagosomal marker LC3B, some of which contained the remnants of mitochondria. Both these autophagosomes and failing mitochondria accumulated within numerous axonal swellings. The lack of lysosomes entering BORC-KO axons meant that autophagosomes had no digestive vesicles to fuse with, resulting in a glut of constipated autophagosomes within the axon.

In addition to festering autophagosomes, axonal swellings were chock-full of the microtubule-binding protein tau, as well as tangled up bits of microtubules called swirls, a common morphological feature of degenerating axons (image at right). This axonal destruction ultimately spelled doom for BORC-KO neurons, which died long before their wild-type counterparts did.

The findings cast lysosomal hitchhiking as a bona fide, and crucial, mode of RNA transport into axons, and offer mechanistic insight into rare neurodevelopmental and neurodegenerative disorders that occur among carriers of BORC mutations (de Pace et al., 2023). Furthermore, because endolysosomal dysfunction factors into several neurodegenerative diseases, the study suggests that poor axonal delivery of RNAs may contribute to axonal degeneration more broadly, de Pace told Alzforum.

Wim Annaert of KU Leuven in Belgium agreed that the findings have broad implications for neurodegenerative disease. “This study may contribute to an alternative understanding of mitochondrial dysfunction in neurodegenerative diseases, including Alzheimer’s and Parkinson’s,” he wrote. “This paper provides a mechanism by which mitochondrial defects can clearly occur downstream of lysosomal dyshomeostasis, propagating defects to other organelles and leading ultimately to neuronal degeneration.”—Jessica Shugart

Comments

1. • Eran Perlson
     Tel-Aviv University
   • Posted: 26 Apr 2024

   I thoroughly enjoyed reading this manuscript, especially its elucidation of a groundbreaking mechanism crucial for maintaining axonal homeostasis. The study sheds light on the key role of lysosome-related vesicles in transporting and localizing specific mRNAs into axons far from the cell body, highlighting their significance in maintaining axonal homeostasis and the role that axonal transport and local synthesis events play in spatiotemporal signaling, metabolic events, and axonal mitochondria maintenance.

   Through the innovative approach of BORC knockout, the study uncovers a subset of axonal mRNAs, notably those encoding ribosomal and mitochondrial proteins, that depend on this transport mechanism. The depletion of these mRNAs results in mitochondrial abnormalities and eventually leads to axonal degeneration, reminiscent of pathways implicated in diverse neurodegenerative conditions. These findings underscore the importance of understanding the basic mechanism of mRNA transport and localization and offer insights into potential mechanisms underlying axon degeneration and neurodegeneration, offering possible new avenues for therapeutic exploration.

   View all comments by Eran Perlson
2. • Wim Annaert
     KULeuven & VIB
   • Posted: 26 Apr 2024

   This is an exciting study by Juan Bonifacino’s group where they explored the regulation and importance of the BORC adaptor complex in axonal transport of lysosomes using new BORC-deficient iPSC-derived human neurons. Whereas the role of lysosomes has been extended from “degradation bins” to a central signaling hub to maintain cellular (and neuronal) homeostasis, it is more recently appreciated that some RNA granules use lysosomes and late endosomes for hitchhiking. This is particularly relevant in neurons, which have decentralized their translation machinery, allowing local translation in distal axonal regions. In this way, neurons can more rapidly respond to the dynamics of synaptic changes, which requires the presence of functional mitochondria.

   Through an impressive number of high-quality assays and analyses in mature neurons, the Bonifacino group now demonstrates that an important aspect of the transported RNA transcripts relates to critical mitochondrial functions, such as oxidative phosphorylation: If the neuron does not succeed in “feeding” its distal ends with RNA granules, this strongly affects mitochondrial fitness, impacting on lysosomal/autophagy homeostasis and resulting in a neurodegenerative phenotype. While the authors connect this to rare neurodevelopmental/neurodegenerative disorders, it likely occurs in the broader range of neurodegenerative diseases. This has been demonstrated previously by the group of Michael Ward, who identified mutations in AnnexinA11, causing ALS, that impact the efficiency of lysosomes to deliver RNA granules to distal axons (Liaio et al., 2019). 

   More broadly, this study may contribute to an alternative understanding of the role of mitochondrial dysfunction in neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Whereas genetics is increasingly identifying risk loci and genes linked to endosomal and lysosomal functions (for instance Van Acker et al., 2019; Van Acker et al., 2021), genetic evidence for a primary causal role of mitochondrial defects appears to be scarcer. But this paper provides a mechanism by which mitochondrial defects can clearly occur downstream of lysosomal dyshomeostasis, propagating defects to other organelles, and ultimately leading to neuronal degeneration.

   References:

   Liao YC, Fernandopulle MS, Wang G, Choi H, Hao L, Drerup CM, Patel R, Qamar S, Nixon-Abell J, Shen Y, Meadows W, Vendruscolo M, Knowles TP, Nelson M, Czekalska MA, Musteikyte G, Gachechiladze MA, Stephens CA, Pasolli HA, Forrest LR, St George-Hyslop P, Lippincott-Schwartz J, Ward ME. RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether. Cell. 2019 Sep 19;179(1):147-164.e20. PubMed.

   Van Acker ZP, Bretou M, Annaert W. Endo-lysosomal dysregulations and late-onset Alzheimer's disease: impact of genetic risk factors. Mol Neurodegener. 2019 Jun 3;14(1):20. PubMed.

   Van Acker ZP, Perdok A, Bretou M, Annaert W. The microglial lysosomal system in Alzheimer's disease: Guardian against proteinopathy. Ageing Res Rev. 2021 Nov;71:101444. Epub 2021 Aug 12 PubMed.

   View all comments by Wim Annaert

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References

Paper Citations

1. Liao YC, Fernandopulle MS, Wang G, Choi H, Hao L, Drerup CM, Patel R, Qamar S, Nixon-Abell J, Shen Y, Meadows W, Vendruscolo M, Knowles TP, Nelson M, Czekalska MA, Musteikyte G, Gachechiladze MA, Stephens CA, Pasolli HA, Forrest LR, St George-Hyslop P, Lippincott-Schwartz J, Ward ME. RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether. Cell. 2019 Sep 19;179(1):147-164.e20. PubMed.
2. De Pace R, Maroofian R, Paimboeuf A, Zamani M, Zaki MS, Sadeghian S, Azizimalamiri R, Galehdari H, Zeighami J, Williamson CD, Fleming E, Zhou D, Gannon JL, Thiffault I, Roze E, Suri M, Zifarelli G, Bauer P, Houlden H, Severino M, Patten SA, Farrow E, Bonifacino JS. Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics. Brain. 2024 May 3;147(5):1751-1767. PubMed.

Further Reading

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