Compartmentalized regulation of organelle integrity in neurodegenerative diseases: lessons from the Drosophila motor neuron

Neurons are highly polarized, morphologically asymmetric, and functionally compartmentalized cells that contain long axons extending from the cell body. For this reason, their maintenance relies on spatiotemporal regulation of organelle distribution between the somatodendritic and axonal domains. Although some organelles, such as mitochondria and smooth endoplasmic reticulum, are widely distributed throughout the neuron, others are segregated to either the somatodendritic or axonal compartment. For example, Golgi outposts and acidified lysosomes are predominantly present in the somatodendritic domain and rarely distributed along the axon, whereas newly formed autophagosomes and synaptic vesicles are mainly distributed in the distal axon (Britt et al., 2016). To establish these polarized features, neurons regulate the axonal transport of organelles to maintain their distribution, integrity, and dynamics. Thus, it is not surprising that neurons in neurodegenerative diseases display early and diverse defects in organelle structure, distribution, and dynamics.

Over the past couple of decades, Drosophila has been used as a practical in vivo model system to study organelle dynamics and axonal transport related to human neurological diseases. Here, I briefly review recent studies in Drosophila motor neurons that demonstrate the importance of organelle integrity in the pathogenesis of neurodegenerative diseases. This article highlights distinct features of organelle integrity in the compartmentalized neuron and its importance to neuronal physiology.

Mitochondrial quality control in Parkinson’s disease (PD): Impaired mitochondrial integrity has long been suggested to be a major cause of PD pathology, and this concept has been strongly supported by the studies of two PD-associated genes, PINK1 and Parkin, involved in mitochondrial quality control. In 2006, Drosophila studies demonstrated that PINK1 acts upstream of Parkin to maintain mitochondrial homeostasis. The physiological importance of PINK1/Parkin in mitochondrial quality control became the center of our attention, as PINK1 accumulates on the outer membrane of depolarized mitochondria, and Parkin promotes mitochondrial elimination via mitophagy. Though PINK1 is dispensable for the basal mitophagy in the mammalian system and neither PINK1 nor Parkin knockout mice recapitulate PD pathologies, the Parkin-mediated mitophagy through PINK1 was detected in fly dopaminergic neurons. Indeed, this epistatic role of the PINK1/Parkin pathway was additionally validated in mitochondria axonal transport by showing that knockdown of Parkin restores PINK1-induced mitochondrial arrest in fly neuropeptidergic neurons (Wang et al., 2011). Of note, two mitochondrial GTPases are phosphorylated and ubiquitinated by PINK1/Parkin: Miro, a mitochondrial motor adaptor protein, and Mitofusin, a regulator of mitochondrial fusion. In fact, PINK1/Parkin targets Miro to inhibit mitochondrial trafficking (Wang et al., 2011), while it regulates Mitofusin to prevent mitochondrial elongation (Ziviani et al., 2010). These findings suggest that neurons control mitochondrial quality by integrating mitochondrial movement with their morphology to properly distribute them within neuronal compartments. Parkin’s highly compartmentalized in vivo role in mitochondrial quality control was further monitored spatiotemporally in Drosophila motor and sensory neurons. This study suggests that Parkin plays a restricted role in the cell body where it modulates mitochondrial fission-fusion balance and indirectly affects axonal mitochondria composition (Sung et al., 2016). Thus, neurons maintain mitochondrial quality control in a compartmentalized manner, emphasizing the intricate interdependence of mitochondrial integrity and axonal distribution.

It is plausible that appropriate mitochondrial morphology is required for the proper distribution within the axon independent of their bioenergetic efficiency (Trevisan et al., 2018). In fly neurons, mitochondria possess distinct features in their structure and dynamics between the soma and the axon. For example, axonal mitochondria show a uniform oval morphology and travel bidirectionally over long distances, whereas somatodendritic mitochondria display tubular and rod-like structures and move remarkably restricted distances. The underlying cellular mechanisms of how neurons keep these polarized features and whether these distinctions are important for neuronal physiology remain unclear. However, it has become clear that mitochondrial morphology and dynamics are directly coupled to ensure the proper distribution of mitochondria into the different neuronal compartments.

Endoplasmic reticulum (ER) integrity in hereditary spastic paraplegia: The ER is an interconnected network of tubules and sheets that form a distinct membrane architecture, and it is the most abundant and widespread organelle in neurons. The importance of neuronal ER integrity is highlighted by studies of ER-shaping proteins associated with hereditary spastic paraplegia. For the last decade, Drosophila motor neurons have been widely utilized to study the impact of hereditary spastic paraplegia-associated proteins, such as Atlastin, Reticulon, and REEPs, on ER organization and continuity. In Drosophila, loss of atlastin disrupts the tubular ER network in motor axons and presynaptic boutons and decreases evoked neurotransmitter release (Summerville et al., 2016). In addition, loss of Drosophila Reticulon or REEP orthologs, Rtnl1 or ReepB, alters ER tubule continuity in the distal motor axon (Yalcin et al., 2017). In Rtnl1 mutants, there is an alteration of the presynaptic ER network, impaired Ca²⁺ dynamics, and disrupted neurotransmission, highlighting the importance of ER integrity to neuronal physiology (Perez-Moreno et al., 2023). Importantly, the observation of synaptic vesicle accumulation in Rtnl1 null fly motor axons (Yalcin et al., 2017) suggests a potential role for the ER in regulating organelle distribution, which in turn is critical to presynaptic physiology. Indeed, the ER maintains organelle integrity not only through its structure but also in its motion. Although the relationship between ER morphology and dynamics is not yet fully understood, studies in cultured Drosophila neurons show that altered ER morphology is tightly coupled with disrupted ER dynamics (Del Castillo et al., 2019). Thus, the complex morphology and dynamics of the ER are tightly linked with compartmentalized neuronal functions.

In fly motor neurons, live imaging clearly indicates that ER morphology and dynamics are distinctly regulated between the cell body and axonal compartment (Sung and Lloyd, 2023). In the perinuclear soma, ER forms a net-like reticular structure and shows fluctuating movement. However, axonal ER forms a ladder-like tubular structure with constant outgrowth and retraction, and these dynamic features are even observed within distal synaptic boutons of the neuromuscular junction. Interestingly, these differences in ER dynamics are similar to those observed in the perinuclear versus periphery of non-neuronal cells. As axons have uniformly arranged microtubules with plus-ends oriented toward the synaptic termini, the asymmetric features of the ER suggest an involvement of microtubule polarity in ER structure, dynamics, or both. Remarkably, ER remodeling can affect organelle distribution within axons without disturbing microtubules-based fast axonal transport. Though ER tubule discontinuity is caused by the depletion of Rtnl1 and Reeps in the fly motor axon, retrogradely moving vesicles were detected across the ER gap, suggesting continuous ER is not essential for vesicle transport (Yalcin et al., 2017). The underlying mechanism of how ER controls organelle distribution is not yet clear. However, ER-organelle membrane contact sites have recently been suggested to act as a hub for distributing organelles. Thus, maintenance of ER integrity appears to be crucial for organelle positioning in neurons to support compartmentalized functions.

Autophagy and endo-lysosomal pathway in amyotrophic lateral sclerosis (ALS): Like other cells, neurons need proper degradation machinery to remove unwanted materials, and macroautophagy (hereafter called autophagy) is a major cellular process responsible for the clearance of damaged organelles and protein aggregates. Unlike in other cell types in which autophagy is induced upon starvation, neuronal autophagy is constitutively regulated and spatiotemporally controlled. The formation of autophagosomes is initiated at the distal axon from phagophore assembly sites, and newly formed autophagosomes travel retrogradely to the soma where acidified lysosomes are enriched. Besides autophagy, the endo-lysosomal pathway also contributes to the neuronal degradation process, though its pathway typically receives cargo from the early endosomes originating in the plasma membrane. Since both pathways, autophagy and the endo-lysosomal system, eventually result in the degradation of contents in the lysosome, both autophagy and the endo-lysosomal pathway are intricately linked and implicated in neurodegenerative diseases. In motor neuron degenerative disease ALS, impairments in both autophagy and the endo-lysosomal pathway have been suggested to play key roles in disease pathogenesis, likely contributing to the pathological hallmark of ubiquitinated cytoplasmic protein aggregates. The most common genetic cause for ALS is an expansion in the C9orf72 gene, and Drosophila has been widely used to model GGGGCC repeat toxicity. In Drosophila motor neurons, expression of an expanded GGGGCC repeat (30 or more repeats) leads to the accumulation of the autophagy adaptor protein p62 and ubiquitinated protein aggregates, in addition to lysosome impairments (Cunningham et al., 2020). By the in vivo axonal transport analyses, fly motor neurons expressing 30 repeats of GGGGCC show specific alterations in endo-lysosomal mobility and distribution (Sung and Lloyd, 2022). Importantly, these same neurons show a severe reduction in autophagosomes, and our recent study shows that this phenotype is caused by impaired ER tubule dynamics (Sung and Lloyd, 2023). These findings suggest that in a fly C9orf72-associated ALS model, both autophagy and endo-lysosomal trafficking are specifically disrupted by an implication of ER tubule defects, leading to an accumulation of toxic protein aggregates and organelles.

In fly motor neurons, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons of the neuromuscular junction, and the majority of axonal autophagosomes travel retrogradely toward the cell body (Sung and Lloyd, 2023). Indeed, Atg9-positive autophagosome precursor vesicles are exclusively detected in larval synaptic terminal boutons, while autophagosomes are detected throughout the neuron with a distinct motility behavior in the cell body and axonal compartment. As the polarized features of autophagosomal biogenesis and trafficking are consistently observed in other neuronal cell types, a similar degradation system is likely utilized in all neurons to maintain organelle integrity.

Conclusion and future perspectives: Our current understanding is that neurons orchestrate intracellular organelles spatiotemporally to keep their compartmentalized features (Figure 1). In addition, it is plausible that disease affects the multiple organelles in keeping their integrities. Thus, it has become apparent that the dynamics of disparate organelles are inextricably intertwined, limiting the knowledge gained from studies of individual organelles. However, our understanding of how organelle integrity contributes to compartmentalized neuronal functions is still limited. What are the underlying mechanisms by which neurons maintain compartmentalized organelle functions? To what extent do neurons regulate the compartmentalized organelle integrity in response to intrinsic and/or extracellular cues? Hopefully, our efforts to answer these questions will lead to insights into the pathogenesis of neuronal disorders. At the same time, it will aid in our understanding of neuronal physiology, such as neuronal plasticity and regeneration.

Figure 1.

Compartmentalized features of organelle dynamics and morphology in Drosophila motor neurons.

In Drosophila motor neurons, multiple organelles display distinct features in each neuronal compartment. Mitochondria have rod-like structures in the cell body but show an oval-shaped structure in the axonal compartment. The endoplasmic reticulum (ER) is detected as a reticular form in the cell body but observed as a tubular form in the axon and synaptic terminals. While acidified lysosomes are predominantly localized in the somatodendritic compartment, the phagophore assembly site is exclusively detected at the distal axon. Within the axon, organelles, such as mitochondria, move bi-directionally, whereas autophagosomes and lysosomes predominantly travel retrogradely. It seems likely that the polarized features of organelle dynamics are linked to compartmentalized organelle integrity, such as ER morphology, ER tubule dynamics, and autophagosomal biogenesis. Created with BioRender.com.

I thank Thomas E. Lloyd (Johns Hopkins University, USA) for his feedback on the manuscript.

This work was supported by the Merkin PNNR Center (23-DF/C2/261) (to HS).