https://orcid.org/0000-0002-7828-8118
ABSTRACT
The unique cellular organization and metabolic demands of neurons pose a challenge in the maintenance of neuronal homeostasis. A critical element in maintaining neuronal health and homeostasis is mitochondrial quality control via replacement and rejuvenation at the axon. Dysregulation of mitochondrial quality control mechanisms such as mitophagy has been implicated in neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis. To sustain mitophagy at the axon, a continuous supply of PINK1 is required; however, how do neurons maintain a steady supply of this protein at the distal axons? In the study highlighted here, Harbauer et al. show that axonal mitophagy is supported by local translation of Pink1 mRNA that is co-transported with mitochondria to the distal ends of the neuron. This neuronal-specific pathway provides a continuous supply of PINK1 to sustain mitophagy.
Neurons are special types of cells. As neurons develop, they exhibit dramatic polarization by undergoing complex morphological rearrangements to establish distinct compartments such as the cell body, and the axonal and dendritic arbors. Engagement of differential biochemical, transcriptional, and translational programs in these compartments sculpt functional neuronal roles, ultimately leading to synaptic activity. Accurate localization, sorting, and trafficking of cellular components is critical for the establishment of neuronal polarity. Following biogenesis in the cell body, nuclear-encoded mRNA, proteins, and organelles are targeted to the synaptic terminal with persistent directionality [Citation1]. Further, to maintain optimal neuronal function, aged materials from the distal tips are either degraded locally or transported back to the cell body for degradation [Citation2]. Therefore, the cell body serves as a site for much of the synthesis and degradation of cellular components while axons and dendrites serve primarily as platforms for transmitting and receiving electrochemical signals, respectively [Citation3,Citation4].
Synaptic activity in neurons requires significant energy investments, and oxidative phosphorylation in the mitochondria is the primary supplier of this energy in the form of ATP [Citation5]. To meet the local energy demands, neurons maintain a healthy mitochondrial pool by continuous replacement and rejuvenation at the distal tips. This is achieved by a combination of mitochondrial transport, removal of damaged mitochondria by mitophagy, and local translation of nuclear-encoded mitochondrial proteins [Citation6]. Several questions remain unanswered in understanding the role of quality control of mitochondria for neuronal survival and neurodegeneration. Harbauer et al. [Citation7] specifically ask, how are nuclear-encoded mitochondrial mRNA transported to the distal tips of neurons?
To answer this question, the authors chose Pink1 mRNA as a model in their investigation for the following reasons: 1) PINK1 is a nuclear-encoded mitochondrial protein. 2) Axonal mitochondria undergo PINK1-PRKN/parkin-mediated mitophagy upon damage. This pathway requires the continuous supply, import and degradation of PINK1. 3) PINK1 has a short half-life, on the order of a few minutes, making co-transport of the protein with mitochondria problematic. Therefore, they hypothesized that Pink1 mRNA is transported along the axon and locally translated to sustain mitophagy [Citation7].
The authors systematically determined the requirement of local translation of Pink1 in the axons using a combination of live microscopy and microfluidics. First, they showed that Pink1 mRNA is enriched in axonal compartments both in vitro and in vivo. To observe translation of Pink1 in axons, the authors used the photoconvertible protein Kaede coding region, downstream of amino acids 1–225 of PINK1. Pre-existing PINK1-Kaede is irreversibly photoconverted from green to red using 405-nm laser light. Any green signal observed post-photoconversion indicates local translation of the PINK1-Kaede fusion protein. The authors observe the appearance of a green signal in axons 45 min after photoconversion, suggesting that local translation of the transcript supplies PINK1 protein.
RNAscope in-situ hybridization revealed that endogenous Pink1 colocalizes with the mitochondrial marker protein ATP5A1 in both axons and dendrites. Due to technical challenges in live imaging of axons, the authors analyzed colocalization and cotransport of Pink1 mRNA with mitochondria in the dendrites. Live imaging of Pink1 mRNA using the MS2-PP7-splitVenus system shows significant overlap with mitochondria in dendrites. Furthermore, whereas Pink1 mRNA associates with mitochondria and moves in synchrony with this organelle, the lack of preference in directionality suggests that tethering of Pink1 mRNA with the mitochondria is an ongoing association and not just limited to delivery of the transcript to the distal regions of the neuron.
Harbauer et al. next sought to determine the mechanism involved in tethering Pink1 mRNA to the mitochondria. Accordingly, they identified the specific region of Pink1 mRNA associated with mitochondria by expressing shortened versions of the transcript fused to blue fluorescent protein (BFP). Again, using the MS2-PP7-splitVenus system, they demonstrated that the 5' UTR and sequence encoding the N-terminal part of PINK1 (amino acids 1–225) localize the mRNA to the mitochondria. This part of PINK1 encompasses both the start codon and mitochondrial targeting sequence (MTS). The authors first determined that translation is required for the sequence to be localized to mitochondria by observing a shift in Pink1 from mitochondria to the cytosol upon treatment with puromycin, a translation inhibitor. The authors then asked if the MTS causes PINK1 to localize with the organelle. To test this hypothesis, they expressed constructs consisting of the Pink1 5' UTR fused to the sequence for BFP lacking or including the MTS. They observed that neither of these transcripts is sufficient to drive mitochondrial localization of PINK1, prompting them to modify their hypothesis and investigate the role of RNA binding proteins in Pink1 mRNA localization to the mitochondria.
The authors observed that Pink1 mRNA association with mitochondria is reduced upon SYNJ2BP depletion. Under these conditions, Pink1 mRNA colocalizes with RFP-DDX6, a P-body marker, whereas the control RNA for Actb (actin, beta) colocalization with RFP-DDX6 remains unchanged. Furthermore, SYNJ2BP depletion reduces antimycin A-induced mitophagy in axons. These results indicate that SYNJ2BP selectively enriches Pink1 mRNA on the mitochondria and enables its subsequent transport to the distal axons along with the maintenance of mitophagy. Interestingly, the authors found that this axis is specific to neurons. They observed that Pink1 mRNA does not associate with mitochondria in non-neuronal cells such as COS-7, HeLa cells and mouse embryonic fibroblasts. This finding raises the possibility of an additional component that confers the neuronal specificity of this pathway. To dissect this potential specificity, the authors considered the binding partners of SYNJ2BP, SYNJ2A (a SYNJ2 splice isoform) and RRBP1. They found that Synj2a is neuronally enriched compared to Rrbp1. Overexpression of SYNJ2A in COS-7 cells localizes Pink1 mRNA, specifically, to the mitochondria. In addition, SYNJ2 depletion in neurons reduces Pink1 mRNA localization to mitochondria, suggesting its role as a mitochondrial anchor for Pink1 mRNA.
SYNJ2 consists of an RNA recognition motif and the authors, based on homology and mutation analysis, identified V909, Q951 and L953 to be involved in RNA binding. The authors created an artificial tether, SYNJ2mito, that can localize to the mitochondrial outer membrane, even in the absence of SYNJ2BP, and asked if wild-type SYNJ2 compared to the SYNJ2V909,Q951,L953A mutant showed a difference in Pink1 mRNA localization. Overexpressed wild-type SYNJ2mito successfully colocalizes with Pink1 mRNA and mitochondria whereas with the mutant, the Pink1 mRNA remains cytosolic. Furthermore, the interaction of SYNJ2 and SYNJ2BP is dependent on the translation of PINK1.
In this study, Harbauer et al., demonstrate the neuronal-specific mechanism of Pink1 mRNA localization to, and cotransport with, the mitochondria via the SYNJ2-SYNJ2BP axis. Neurons provide an interesting arena to study spatial control of translational regulation. Harbauer et al. recognized the need for local translation at the distal tips of the neurons, especially for proteins with a short half-life. While the existing model argues that transcripts are transported to the axons and dendrites with the help of RNA granules, Harbauer et al. provide compelling evidence to suggest a different model of transport. They demonstrated that mitochondria function as carriers of mitochondrial transcripts, befitting their role as translational hubs in the axon.
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References
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