Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.


Introduction
The ability of axons to transport cargo over long distances is critical for processes ranging from axon path finding and target innervation, to neuronal survival. Those processes are regulated by the retrograde transport of neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) 1 . Mutations or alterations in the expression of genes that enable axonal transport are linked to neurodegenerative diseases, including Charcot-Marie-Tooth disease type 2, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) 2, 3 .
Abnormal axonal traffic in cortical and hippocampal neurons is an early feature of both AD and HD, and a trigger for synaptic loss, neuronal death and the resultant loss of cognitive abilities [4][5][6] .
Thus, understanding the mechanisms that control axonal transport of survival and differentiation signals could provide important insights into the physiopathology of the brain.
Fast axonal transport of signaling molecules is largely mediated by endosomes. Endosomes facilitate signaling through rapid retrograde transfer of proteins such as neurotrophins, activated receptor complexes, adaptors and kinases along microtubule tracks [7][8][9] . The identity of such endosomal signaling compartments has long been debated. In hippocampal neurons, multi-vesicular bodies transport endocytic cargo retrogradely in the axon 10 . To determine the molecular identity and signaling properties of endosomes in central nervous system (CNS) neurons, it is necessary to 2 characterize their molecular machinery 11 . Different studies have provided evidence in favor of both early and late endosomal compartments to transport signaling molecules from the pre-synaptic terminal retrogradely to the cell body 12 . Key regulators of organelle tethering, docking, signaling, fusion and motility are Rab GTPases and their effectors 13,14 . Rab5 localizes to early endosomes, which sort cargo to the recycling pathway via Rab4 and Rab11, or to the degradative pathway through Rab7-positive endosomes 14 . In neurons, both Rab5 and Rab7 were shown to be important for retrograde trafficking. In dorsal root ganglion neurons, retrograde movement of NGF has been associated with Rab5-positive endosomes 9 . In contrast, Rab7-positive endosomes retrogradely transport GFP-BDNF in motor neurons 12 .
However, the identity of endosomal compartments is best defined by the combination of Rab GTPases with their specific effectors, as this can differ significantly between organelles in space and time. For example, in non-polarized cells two distinct populations of Rab5-positive early endosomes coexist and dynamically exchange cargo over time, one containing the canonical Rab5 effector EEA1 and the other harboring APPL1 15,16 . In hepatocytes, EEA1-and APPL1-positive endosomes have distinct distributions that depend on the organization of the actin cytoskeleton and the nutritional state 17 .
EEA1, which functions as a tethering factor for early endosomal membranes 18 , is localized to the somatodendritic region of hippocampal neurons 19 and therefore, cannot participate in axonal transport. Interestingly, the Rab5 effector APPL1 is an adaptor protein for Akt, a central kinase regulating cell survival 20,21 and plays a role in survival signaling from endosomes 22 . APPL1 is localized to the dendritic spines and axons of hippocampal neurons 23,24 . Its overexpression increases the amount of phospho-Akt (p-Akt) at synaptic sites, leading to formation of spines and an increase in synapse number 24 . However, the data so far are restricted to immunocytochemistry and, there is no analysis of APPL1 endosome motility. Similarly, other Rab5 effectors have been poorly studied in neurons. Rabankyrin-5 is involved in macropinocytosis and is present in the growth cone of developing hippocampal axons but it is unclear whether it localizes to other neuronal endosomal compartments 25 . Finally, the distribution of Rabenosyn-5, which plays a dual role in endocytosis and recycling 13 has not been addressed in neurons.
Here, we undertook a detailed comparison of the localization of endogenous Rab5 effectors and used live-cell imaging to quantitatively analyze the motility of axonal endosomes in pyramidal neurons, the most abundant type of neurons in the cerebral cortex and hippocampus.
3 We observed a variety of motility patterns in Rab5-positive endosomes ranging from stationary to highly processive (Fig.1A, B, C and movie 1 To analyze this difference, we fitted each speed distribution (

Characterization of Rab5 effectors in hippocampal neurons
Rab GTPases orchestrate traffic through the recruitment of effector proteins to membranes. Many Rab5 effectors have been identified and serve different functions 14 . The canonical Rab5 effector 5 EEA1, important for tethering and fusion 29 , is absent from axons 19 . Thus, we wanted to systematically identify which other Rab5 effectors were present in axons in hippocampal neurons, in order to be eligible to act in signaling endosomes. APPL1 has been localized to axons and is an adaptor protein that binds to Akt 23,30 . Another Rab5 effector, Rabankyrin-5, was found in growth cones of hippocampal neurons 25 suggesting that it may also be distributed in axons. Rabankyrin-5 is found on early endosomes and on macropinosomes, endocytic vesicles that take up fluid as well as protein aggregates, such as those generated during ALS and HD progression, and have been related to signaling endosomes via the Pincher protein 31 . Additionally, Rabenosyn-5 has not been examined in hippocampal neurons, but there is evidence for a role of Rabenosyn-5 in recycling 13 .
As we previously observed that overexpression or tagging proteins can yield localization and functional artifacts 15 , we used antibodies to detect the endogenous Rab5 effectors Rabankyrin-5, Rabenosyn-5, APPL1 and EEA1 in primary hippocampal neurons. Because all antibodies were rabbit polyclonals, the Rab5 effectors could be detected only individually without assessing their co-localization. In order to differentiate between axons and dendrites, we co-immunostained the Rab5 effectors with an axonal marker, phosphorylated neurofilament-1. For this set of experiments, neurons were grown following the Banker protocol 32 . This culture set-up consists of the co-culture of astrocytes and neurons where astrocytes provide support for neuronal growth. This astrocyte feeder layer is compatible with low density seeding of neurons, which improves the identification of axons and other neuronal structures. As expected from previous findings 19 , EEA1 labeled structures localized to the cell soma and dendrites and were absent from axons (Fig. 2B).
Rabankyrin-5, Rabenosyn-5 and APPL1 localized to the cell soma and dendrites and were also present on axons ( Fig. 2A, C and D). The EEA1 puncta in the cell body were larger than the APPL1 and Rabenosyn-5 puncta, and similar in size to the structures harboring Rabankyrin-5 (that had a high cytosolic pool and, therefore, whose puncta in axons were more difficult to resolve). In addition, a fraction of APPL1 was found in the nucleus, as previously shown for other cell types 33 .
The subcellular distribution suggests a role for Rabenosyn-5, Rabankyrin-5 and APPL1 in membrane traffic/signaling events in axons. Among the Rab5 effectors, APPL1 was previously shown to interact with signaling receptors and with the three isoforms of Akt/PKB in various cell system 23,30,[34][35][36] . Given that APPL1 is localized to axons and can be tagged without compromising its localization and function of the endosomal system 15 , we decided to focus on this protein as a candidate Rab5 effector on a signaling endosome.
APPL1 structures move bi-directionally with significant retrograde bias 6 We first validated the localization of APPL1 to Rab5 endosomes by co-expressing GFP-Rab5 and mCherry-APPL1 (validated as in 15  We also analyzed the fraction of fast retrograde motion events associated with the amounts of APPL1 and observed that it increased from 5 to 60% with the amounts of APPL1 on endosomes Altogether, these results indicate that both Rab5 and APPL1 endosomes move more frequently and processively in the retrograde than in the anterograde direction. However, the levels of APPL1 correlate more with fast retrograde movement than the levels of Rab5, arguing that they mark a population of fast retrogradely moving Rab5 endosomes.

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The preferential retrograde motility of APPL1 endosomes prompted us to ask whether they transport signaling cargo. To address this question, we co-expressed GFP-TrkB with mCherry- retrograde events). We addressed the correlation between the amount of APPL1 on TrkB endosomes and endosomal speed and observed that the velocity slightly decreases with increase APPL1 on endosomes (Fig. 4D)

Akt1 interaction with APPL1 is dependent on endocytosis
Our data suggest that Akt1 is transported retrogradely in axons in Rab5 and APPL1-positive endosomes. To determine whether APPL1 and Akt1 interact on axons, we used a FRET To measure the interaction between APPL1 and Akt1 using FRET, we analyzed live neurons expressing GFP-Akt1 alone and the pair GFP-Akt1/mCherry-APPL1. Interestingly, we observed that the lifetime of GFP-Akt1 was significantly decreased in the growth cone but not in the soma of resulting in FRET efficiencies that corresponded to zero in the cell body or values lower than 1% in the growth cone (Fig. 5B). This indicates that the decrease in the lifetime of GFP-Akt1 is specific to the APPL1-Akt1 interaction.
Signaling endosomes receive cargo and many growth factor receptors internalized via Clathrin-and Dynamin-mediated endocytosis. Since the small molecule dynasore 39 blocks Dynamin-dependent endocytosis in neurons 40 , we used it to test the requirement of endocytosis for the APPL1-Akt1 interaction. We performed the FRET/FLIM assay in neurons before and after dynasore treatment for 5 min. We observed no APPL1-Akt1 interaction in growth cones after dynasore treatment (Fig. 5C).
Lifetime measurements of cell bodies and growth cones of neurons showed that the lifetime of GFP-Akt1 was lower in the presence of mCherry-APPL1. After dynasore treatment, mCherry-APPL1 was no longer able to reduce GFP-Akt1 lifetime, indicating that under these conditions the two proteins do not interact (Fig. 5D). These data suggest that endocytosis is required for the interaction of Akt1 and APPL1, thus providing further evidence that axonal retrograde APPL1 vesicles may be signaling endosomes.

Discussion
In this study, we report a detailed quantitative characterization of Rab5 early endosome motility in axons of primary hippocampal neurons. This analysis has revealed that Rab5 endosomes follow a complex pattern of motility that can be ascribed to different populations of endosomes differing in directionality, speed and processivity. These populations correspond to endosomes with different content of Rab5, the Rab5 effector and signaling adaptor APPL1, and the signaling kinase Akt1.
Among the Rab5 endosomes, the fraction that moves retrogradely with higher frequency than in the anterograde direction, speed and processivity corresponds to a sub-population of organelles harboring APPL1. Such endosomes may participate in neurotrophin retrograde movement and signaling. Long range movement of Rab5 endosomes was also observed in motor neurons at speeds compatible to that reported for neurotrophin transport (data not shown). Consistent with this idea, APPL1 was co-transported with TrkB and Akt on endosomes, suggesting that it could bridge receptors to signaling molecules. Akt co-expression stimulated retrograde movement velocity and processivity. Finally, we found that endocytosis is required for the interaction of Akt1 and APPL1, 10 thus providing further evidence that axonal retrograde APPL1 vesicles may be signaling endosomes.
The molecular identity of signaling endosomes in neurons has been controversial. In motor neurons, Rab7 exhibited retrograde movement whereas Rab5 was reported to remain stationary 12 . On the other hand, Rab5 was found in signaling endosomes collected from sciatic nerve 7 and Rab5 endosomes transport NGF in dorsal root ganglia 9 . NGF transport via Rab5-positive endosomes has been previously visualized indirectly using quantum-dots labeled NGF 9  The different populations of motile Rab5 endosomes could also harbor Rab5 effectors other than APPL1. Whereas EEA1 is exclusively localized to the somato-dendritic compartment 19 and its 11 localization is defined by the Rab5 effector motor protein KIF16B 41 which regulates plus end motility of Rab5 endosomes 42 , Rabenosyn-5 and Rabankyrin-5 are also present in axons (Fig. 2). This suggests a differential distribution and function of Rab5 effectors on early endosomes within neurons. The underlying molecular mechanisms are unclear. Rabankyrin-5, Rabenosyn-5 and EEA1 all contain a FYVE domain which, by binding to phosphatidylinositol 3-phosphate (PI(3)P) and, together with binding to Rab5, supports recruitment to PI(3)P-positive early endosomes 14,29,44 .
These endosomes are present on axons where they recruit Ankyrin B in a PI(3)P-dependent manner 45 . Yet, the presence of FYVE domains and Rab5-binding sites are not sufficient to recruit EEA1 to these endosomes. Evidently, additional molecular interactions must play a role in the selection of Rab5 effectors present in the axon. Rabankyrin-5 participates in macropinocytosis, a process that has been related to NGF uptake and retrograde transport mediated by the pinocytic chaperone Pincher 31 . Additionally, it has been linked to retrograde transport from endosomes to the Golgi complex via interaction with the retromer protein EHD1 46 . Rabenosyn-5 links early endosomes and the recycling route via its interaction with Rab5 and Rab4 13 . Given that endocytic/recycling regulates axon elongation, it is possible that Rabenosyn-5 plays a role in this process 47   This establishes a link between APPL1/Akt interaction and endocytosis. We did not expect changes on global Akt phosphorylation levels on the basis of its interaction with APPL1, since only a small fraction of Akt is concentrated on endosomes in comparison with the magnitude of the soluble pool.

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However, the endosomal fraction must be very important, since inhibition of endocytosis with dynasore impairs Akt phosphorylation 54 . Therefore, it is conceivable that Akt phosphorylation relates to its interaction with APPL1 on endosomes. It was previously reported that TrkB colocalizes to APPL1 positive endosomes 55 . Here, we showed that these endosomes undergo bidirectional movements with a strong bias in the retrograde direction. The fluctuations of the levels of Rab5 on endosomes correlate with cargo processing 27,56 . We detected similar behavior for APPL1 on TrkB carrying endosomes with respect to retrograde speed modulation (Fig.4A-C). It is known that the APPL1/Akt signaling complex regulates synapse formation 24 and BDNF.
Neutrotrophins such as BDNF modulate synapse transmission, plasticity and growth 57  The existence of APPL1-Akt signaling endosomes raises the question of whether these endosomes and downstream signaling are affected in disease. Alzheimer's disease (AD) and other tauopathies are interesting candidates to investigate, given that they correlate with defects in the endolysosomal system 6 . It has been hypothesized that AD is caused by hyperactivation of tau by GSK-3β, a kinase downstream of Akt 58 . Akt phosphorylates and inactivates GSK-3β and our previous data suggest that APPL1 influences Akt activity towards GSK-3β, promoting its inactivation at membranes 22 . In this sense, an increase in APPL1-Akt endosomes could have a neuroprotective effect in disease.
Further exploring the role of Akt/GSK-3β activation at endosomes could contribute to the development of drugs aimed at specifically modulating GSK-3β, providing new opportunities to treatment of brain diseases.

Data Availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions
Conceptualization

Number of Events
A.