RTP801 Mediates Transneuronal Toxicity via Extracellular Vesicles by Affecting Their Protein Cargo and Abrogating Their Trophic Effect

doi:10.21203/rs.3.rs-2736462/v1

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RTP801 Mediates Transneuronal Toxicity via Extracellular Vesicles by Affecting Their Protein Cargo and Abrogating Their Trophic Effect

https://doi.org/10.21203/rs.3.rs-2736462/v1

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Background

Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in the paracrine trophic support or in the propagation of toxic molecules, including proteins. RTP801 is a stress-regulated protein, whose levels are elevated during neurodegeneration and induce neuron death. However, whether RTP801 toxicity is transferred trans-neuronally via EVs remains unknown.

Methods

We overexpressed or silenced RTP801 protein in cultured cortical neurons and isolated the neural-derived EVs (RTP801-EVs, or shRTP801-EVs respectively). We characterized their protein content by mass spectrometry (MS) and western blotting (WB). RTP801-EVs toxicity was assessed by treating cultured neurons with these EVs and quantifying apoptotic neuron death and branching. We also tested shRTP801-EVs functionality in the pathologic in vitro model of 6-Hydroxydopamine (6-OHDA) by biochemical analyses.

Results

Expression of RTP801 increased the number of EVs released by neurons. Moreover, RTP801 led to a distinct proteomic signature of neural-derived EVs, containing more pro-apoptotic markers. Hence, we observed that RTP801 toxicity was transferred to neurons via EVs, activating apoptosis and impairing neuron morphology complexity. In contrast, EVs derived from neurons where RTP801 was silenced were able to increase the arborization of recipient neurons. We also showed that 6-OHDA neurotoxin elevated protein levels of RTP801 in both cell lysates and EVs. Furthermore, neural-derived EVs induced phosphorylation of Ser473-Akt and Ser235/236-RPS6 in recipient neurons, and this effect was lost when EVs were derived from neurons treated with 6-OHDA. Interestingly, EVs derived from neurons where RTP801 was silenced prior to exposing them to 6-OHDA maintained Akt and RPS6 transactivation in recipient neurons.

Conclusion

Taken together, these results suggest that RTP801 toxicity can be spread via EVs and therefore, it could contribute to the progression of neurodegenerative diseases in which RTP801 is involved.

Extracellular vesicles

RTP801/REDD1

neuron

toxicity

6-OHDA

mTOR signaling

Cell cultures

HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (all from Thermo Fisher Scientific) in a 5% CO₂ atmosphere at 37ºC. EVs-depleted FBS was obtained from 18 h of ultracentrifugation at 100,000 x g in a 70.1 Ti rotor and was used to prepare complete media (10% FBS).

Rat primary cortical cultures were prepared as previously described^1,2. Briefly, cortices from embryonic (E18) Sprague-Dawley rats were dissected out, dissociated in 0.05% trypsin, and plated at a density of 750 cells/mm² on poly-L-lysine-coated plates or at a density of 300 cells/mm² on poly-L-lysine-coated coverslips. Neurons were maintained in Neurobasal medium supplemented with B27 (1:50), 2mM GlutaMAX, and penicillin/streptomycin (all from Thermo Fisher Scientific). One-third of the medium was replaced every 7 days. Cell cultures were maintained in a 5% CO₂ atmosphere at 37ºC.

Lentiviral preparation

Lentiviruses to knockdown the expression of RTP801 were produced in HEK293 cells transfected with a pLL3.7 vector expressing RTP801 (shRTP801, 5’-AAGACTCCTCATACCTGGATG-3’) or scramble (shCtr, 5’- GTGCGTTGCTAGTACCAAC-3’) shRNA. Lentiviruses to overexpress RTP801 were produced in HEK293 cells transfected with a FUGW vector expressing only eGFP or eGFP-RTP801 (RTP801 tagged with eGFP at the N-terminal, sequence in³). Transfections were carried out using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Seventy-two hours post-transfection, cell media was collected and centrifuged to remove debris. Virus-containing media was incubated with PEG 6000 8.5% (Panreac) and NaCl 0.35M for at least 90 minutes at 4ºC. Lentiviruses were finally concentrated by centrifuging the media at 7,500 x g for 15 minutes and resuspended in sterile 1 X PBS/Ca²⁺.

Transfection, lentiviral infection, and treatments

HEK293 cells were transfected with pEGFP-C3 or pEGFP-RTP801-C3 and primary rat cortical neurons with pEGFP-C1bos or pEGFP-C1bos-CD63 (a kind gift from Dr. Giovanna Bossi’s laboratory), and with a pCMS-eGFP plasmid (Addgene). Transfections were done using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s protocol.

Rat primary cortical neurons were transduced at DIV11 with lentiviral particles containing eGFP or eGFP-RTP801 constructs. 48 h hours later (DIV13), cultures were harvested, and EVs were isolated from the culture media. For loss of function experiments, neurons were transduced at DIV6-7 with lentiviral particles containing an shRNA construct against RTP801 (shRTP801) or a scrambled control sequence (shCT) as previously described¹. Six days later (DIV13-14) cultures were harvested and EVs were isolated.

Half of the medium was kept aside right before the transduction and was used to replace all the media 24 h prior EVs isolation, to avoid lentivirus contamination during EVs isolation.

6-Hydroxydopamine (6-OHDA, Tocris Bioscience) treatments were performed in cortical primary neurons after 13-14 days of differentiation at a concentration of 50 µM in H₂O for 16 h, as previously described⁴. One-third of the media was replaced right before exposure to the toxin.

Extracellular vesicles isolation, characterization, labeling, and treatment

EVs were purified from depleted-DMEM media or Neurobasal medium as previously described with minor modifications⁵. To compare the number, size, and content of EVs produced between different conditions, the exact same volume of culture media was subjected to the EVs isolation method. Briefly, cell culture was collected and centrifuged at 300 x g for 5 min at room temperature to remove cell debris and centrifuged again for 20 min at 2,500 x g in a centrifuge Beckman GPR and 4ºC. After, cell media was centrifuged for 30 minutes at 10,000 x g in a SS-34 rotor in a centrifuge Sorvall (4ºC) to remove other microvesicles of bigger diameter. Finally, the supernatant was ultracentrifuged at 4 ºC for 2 hours at 100,000 x g in a 70.1 Ti rotor, in an Ultracentrifuge Optima L-100XP with Polypropylene Centrifuge Tubes (Beckman 355630), final supernatant was discarded, and the resulting pellet was washed with 1X PBS-Ca²⁺ and ultracentrifuged again for 1 h at 100,000 x g (4ºC). The resulting pellet contained the EV fraction. For western blot (WB) analysis, the pellet was resuspended in loading buffer (Thermo Fisher Scientific) and loaded into the WB gel. For EVs quantification, the pellet was resuspended in 1X PBS, and vesicle size and quantity were determined using the Nanosight nanoparticle tracking LM10 system (NTA) (Malvern). For transmission electron microscopy (TEM) observation, the pellet was resuspended in 1X PBS-2% paraformaldehyde (PFA) and then subjected to negative staining. Briefly, the suspension was adsorbed on copper grids for 25 min and contrasted with 2% uranyl acetate for 2 min and finally washed in water. Samples were observed with JEOL J1010 80kV microscope.

For the generation of red fluorescent-labeled EVs, the EVs in 1X PBS were incubated with 200 nM MemGlow-560 (Cytoskeleton Inc.) for 1 h at RT. The excess of dye was removed with two centrifuges at 10,000 x g for 15 min at 4ºC. The resulting supernatant contained the labeled EVs. For EVs internalization assay, neuronal cultures were treated with labeled EVs for 4 h. As a negative control, the same protocol was followed using the MemGlow-560 suspension without EVs (only 1X PBS).

Western blotting

Whole-cell extracts were collected and processed as previously described⁶. The following primary antibodies were used (1:1,000 it not stated otherwise): rabbit monoclonal anti-GFP (Cell Signaling, #2956), rabbit polyclonal anti-RTP801 (1:500, Proteintech, #10638-1-AP), mouse monoclonal anti-TSG101 (Abcam, #ab83), mouse monoclonal anti-Flotillin-1 (BD Bioscience, #610821), mouse monoclonal anti-Alix (Cell Signaling Technology, #2171), rabbit polyclonal anti-VGLUT1 (Synaptic Systems, #135303), rabbit polyclonal anti-Calnexin (Abcam, #ab22595), rabbit polyclonal ant i-Nedd4 (Santa Cruz Biotechnologies, #sc-25508), rabbit polyclonal anti-Phospho-Akt-Ser473 (Cell Signaling Technology, #4060S), rabbit polyclonal anti-Phospho-RPS6-Ser235/236 (Cell Signaling Technology, #4858S), rabbit polyclonal anti-Akt (Cell Signaling Technology, #4691S), mouse monoclonal anti-RPS6 (Cell Signaling Technology, #2317), rabbit polyclonal anti-Phospho-p70S6K (Cell Signaling Technology, #9208), rabbit polyclonal anti-p70S6K (Cell Signaling, #9202), rabbit monoclonal anti-Phospho-4EBP1 (Cell Signaling Technology, #2855), rabbit monoclonal anti-4EBP1 (Cell Signaling Technology, #9644). The loading control was obtained by incubation with an anti-α-actin-Peroxidase antibody (1:100,000; Merk, #A3854). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:10,000) were obtained from Thermo Fisher Scientific (1:10,000, #31430 and #31460, respectively). Chemiluminescent images were acquired using a Chemidoc imager (BioRad) or a LAS-4000 Imager (Fujifilm, Valhalla, NY, USA) and quantified by computer-assisted densitometric analysis (ImageJ).

Immunofluorescence and image analyses

Rat primary cortical neurons were fixed with 4% PFA in 1X PBS and stained as previously described⁷. The following primary antibodies were used: rabbit polyclonal anti-RTP801 antibody (1:200; Proteintech, #10638-1-AP), mouse monoclonal anti-MAP2 (1:500, Abcam, #ab11268), chicken polyclonal anti-GFP (1:500, Synaptic Systems, #132006) and rabbit polyclonal anti-cleaved caspase-3 (1:200; Cell Signaling Technologies, #9661). Goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 555 or Alexa 633 were purchased from Thermo Fisher Scientific (1:500). For nuclear staining, Hoechst 33342 (1:5,000; Thermo Fisher Scientific, #H3570) was used.

For EVs uptake assay, images were acquired with ZEISS LSM880 confocal microscope as z-stacks (5 slices in a range of 5 μm) with a 63x magnification. Orthogonal views (YZ and XZ) were obtained with ImageJ to show the colocalizing EVs inside the neuron.

RTP801 staining intensity and eGFP-CD63-RTP801 puncta colocalization analyses were performed with ImageJ software. Images for Figure 2 were taken with a Leica TCS SP5 confocal microscope.

For the toxicity and branching analysis, images were acquired with a ZEISS Axio Observer Z1 fluorescence microscopy with a 20x magnification. Using Cell Profiler cell image analysis software (www.cellprofiler.org, Broad Institute)^8,9, nuclei and neuronal somas were identified as independent objects. A mask with neuronal soma was used to obtain only neuronal nuclei. Cleaved caspase-3 positive cells were identified as independent objects and were related with neuronal nuclei, with the RelateObjects module, to distinguish nuclei positive or negative for cleaved caspase-3. Measures of mean intensity of cleaved caspase-3 per neuron were obtained. Nuclei classification into viable or apoptotic was based on intensity, intensity distribution, size and shape, texture, and granularity parameters, using machine learning in Cell Profiler Analyst software^10,11.

For the branching analysis, neurites were enhanced and turned into a binary image. From this image, and using the neuronal soma as input, neurons were identified as independent objects. Neurons touching the border of the image were discarded. Neuron objects were used to mask the binary image of enhanced neurites. From this image, the morphological skeleton was generated with Morph module. Measurements of endpoints (termini branches), trunks (primary dendrites), non-trunk (intermediate branches), and total tree length were obtained per neuron, using the MeasureObjectSkeleton module. Neurons from 5 fields per coverslip were analyzed and the mean of all neurons per field was acquired. At least 3 independent cultures were analyzed.

Proteomics

Samples were processed and analyzed at the Proteomics Platform of Navarrabiomed-IdiSNA Center for Biomedical Research. For sample preparation, protein extracts were diluted in Laemmli sample buffer (4%) and were then loaded into a 0.75-mm-thick polyacrylamide gel containing a 4% stacking gel cast over 12.5% resolving gel. To concentrate the entire proteome at the stacking/resolving gel interface, the run was stopped as soon as the front entered 3 mm of the resolving gel. Gel was then stained using Coomassie Brilliant Blue and bands were excised and digested using 1:20 trypsin solution at 37ºC for 16h as previously described¹². Peptide fragments were purified and concentered using C18 Zip Tip Solid Reverse Phase columns (Millipore). Samples were then separated by reverse phase LC-MS/MS using an UltiMate 3000 UHPLC System (ThermoFisher) fitted with a column in an acetonitrile gradient coupled to the Orbitrap Exploris 480 MS (ThermoFisher). Mass range was set to 375-1500 ppm. All the other acquisition parameters were set as previously described¹³. The MaxQuant computing platform v.1.6.17.0¹⁴. and the environment-integrated Andromeda search engine¹⁵ were used to process the raw files. For peptide identification, a target-decoy search strategy¹⁶ was performed against a target/decoy version of the rat UniProt database without isoforms with a maximum peptide mass of 7500 Da. The false discovery rate limit was set to 1% on both the peptide and protein identification levels. The Perseus software v.1.6.14.0¹⁷ was used for statistical and differential expression analyses. Only proteins with at least two identified peptides were considered for further analyses. The option “two samples t-test” was used to compare experimental conditions. Here, comparisons were considered to be statistically different if the following conditions were met: (i) Benjamini-Hochberg adjusted p-values under 0.05 and a (ii) log2 fold-change over 0.3 and under -0.3. R (v.4.2.1) packages ComplexHeatmap¹⁸, EnhancedVolcano and mixOmics¹⁹, were used for multivariate data analysis and visualization.

Statistics

All experiments were performed at least in three independent neuronal cultures, and data of each replicate is reported as mean+SEM. Normal distribution was considered when all the data passed one of the following normality tests: D’Agostino-Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov. When normality was assumed, parametric statistical analyses were performed. When two conditions were compared, analyses were performed using the unpaired two-sided t-test (95% confidence). Before pairs of comparisons, we performed the F test to compare variances. T-test with Welch’s correction was performed when variances were unequal. When multiple groups were compared, one-way ANOVA or two-way ANOVA with Bonferroni’s post hoc test was performed. To detect significant outlier values, Grubb’s and ROUT tests were used. Values of P<0.05 were considered statistically significant. The statistics used in each experiment are specified in figure legends.

Cell-to-cell transmission is a common mechanism for the progression of several neurodegenerative disorders including Parkinson’s disease (PD)or Alzheimer’s disease (AD)²⁰.Mounting evidence indicates the existence of prion-like transmission of protein aggregates in interconnected brain areas, such as alpha(α)-synuclein, or amyloid-beta(β) (reviewed in ^21,22). For example, the pathological staging process in PD proposed by Braak and co-workers²³ is based on the caudo-rostrally distribution of α-synuclein aggregates in post-mortem brains and supports the hypothesis that α-synuclein may spread to neighbor cells. Therefore, transneural propagation of toxic proteins could contribute to the early stages of neurodegenerative disorders. However, the precise mechanisms of this neuron-to-neuron communication and the specific effects that exert on the recipient cells are not yet completely elucidated.

EVs act as vehicles for transcellular communication in general and have a crucial role in the central nervous system (CNS)²⁴. There are different types of EVs, distinguished by size, biogenesis, and composition. Among them, exosomes are 50-100/150 nm vesicles (depending on the method used for the isolation and characterization, reviewed in ²⁵), that are delivered into the extracellular space upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies (MVBs). In contrast, microvesicles are 0.1-1 μm released by outward budding from the plasma membrane^26,27. EVs transport active proteins, lipids, RNAs, and signaling complexes that modulate gene and protein expression in the receiving cells (reviewed in ^28,29). EVs are biologically active, and, in neurons, they can be both protective and toxic ^30,31. Moreover, EVs have a crucial role in neuronal development and connectivity^32,33. In the mammalian nervous system, cortical neurons secrete EVs in a process that depends on synaptic glutamatergic activity and post-synaptic calcium rise³⁴. Recently, given that EVs can directly fuse to the plasma membrane of the recipient cell and release their content into the cytosol, they have been proposed as vehicles to transfer toxic proteins trans-synaptically, such as α-synuclein or amyloid-β, which are associated with neurodegenerative disorders^35,36.

RTP801, also known as REDD1, is a protein that results toxic for neurons. RTP801 is sufficient to induce cell death in NGF-differentiated PC12 cells^3,37and cultured sympathetic³ and cortical neurons^1,4. RTP801 is coded by the stress-responsive gene DDIT4, whose expression is induced in response to hypoxia, nutrient deprivation³⁸, dexamethasone, thapsigargin, tunicamycin, heat shock³⁹ or cigarette smoke in lung cells⁴⁰. In the CNS, RTP801 expression is increased in response to ischemia³⁷, β-amyloid peptide^41,42, mutant huntingtin (mhtt)², and both PD-mimetic toxins 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-OHDA³. Importantly, RTP801 is not only accumulated in cellular and animal pathologic models, but also in samples from patients suffering from neurodegenerative diseases. In fact, RTP801 protein is elevated in dopaminergic neurons from the substantia nigra pars compacta in sporadic and mutant parkin PD patients^3,4, in postmortem striata and neuron-derived iPS cells from Huntington´s disease (HD) patients^2,43 and lymphocytes and postmortem hippocampi from AD patients^44,45. Remarkably, DDIT4 is one of the top three common upregulated transcripts in PD and HD⁴⁶. Some studies show that RTP801 modulates cytotoxicity in vitro in the context of neurodegeneration^47–49. In line with this, downregulation of RTP801 in AD and HD mouse models prevents cognitive deficits and neuroinflammation^45,50.

The action mechanism of RTP801 involves a sequential regulation of the mechanistic target of rapamycin (mTOR) and Akt survival kinases. mTOR exists in two complexes, each one with different specificity over substrates. The mTORC1 complex regulates protein translation by phosphorylating substrates such as ribosomal protein S6 kinase/S6 (RPS6) and 4EBP1^51–53 and the mTORC2 complex regulates survival signals by phosphorylating Akt at Serine residue 473, among other substrates^54,55. In PD, RTP801 acts upstream of tuberous sclerosis complex 1 and 2 (TSC1/TSC2), which in turn inhibits mTOR⁵⁶. As consequence, RPS6/4EBP1 cannot be phosphorylated by mTORC1, and protein translation is tuned down. But most importantly, the neuronal survival kinase Akt cannot be phosphorylated by mTORC2 and so it is unable to enhance pro-survival signals, thereby triggering neuronal death⁴⁹.

Here, we investigated whether RTP801 toxicity can be transferred to neurons via EVs. Moreover, we assessed whether EVs can modulate the mTOR/Akt pathway transneuronally in cultured neuronal models exposed to the PD mimetic 6-OHDA. Our results reveal that RTP801 is present in neural-derived EVs and promotes their loading with pro-apoptotic proteins, which could contribute to propagate toxicity to neurons. Transneuronal RTP801 impairs neuron arborization and activates apoptosis in recipient neurons. Moreover, neural-derived EVs exerted a trophic effect on neurons, activating the mTOR/Akt signaling pathway, an effect that was lost via RTP801 when EVs were derived from 6-OHDA-stressed neurons.

RTP801 is present and released in EVs

To investigate whether endogenous RTP801 is present in neural EVs, neuronal culture media (DIV14) was subjected to sequential ultracentrifugation. The presence of EVs within the exosomal range was confirmed using NTA, with a size average of 135 nm (Figure 1A). We also confirmed the expected size and shape of small EVs using TEM (Figure 1B). We observed by WB that EVs fraction was enriched in EVs markers such as Alix, Flotillin-1 and TSG101, whereas they showed little signal for the negative control calnexin, in comparison with neuronal lysates. Furthermore, we detected that endogenous RTP801 was present in low levels in EVs derived from primary neurons (Figure 1C). To test whether these EVs could be internalized by neurons, we labeled them with a fluorogenic membrane probe (Memglow-560, in red) and treated cortical cultured neurons with these labeled EVs. We confirmed the presence of internalized EVs under confocal microscope. Negative control using vehicle, confirmed that Memglow-560 specifically binds to EVs’-membrane, as no fluorescent signal was detected in the absence of the vesicles (1X PBS) (Figure 1D).

Next, to confirm that ectopic RTP801 could be incorporated in EVs, we transduced cortical cultured neurons with lentiviral particles containing the ectopic form of RTP801 tagged with eGFP at the N-terminal (eGFP-RTP801). We also transduced sister cultures with lentiviral particles only containing eGFP as control. EVs released in the culture media were collected 48 h after transduction and the EVs-producing neurons were lysated. EVs per each condition were isolated starting from the exact volume of culture media collected from eGFP or eGFP-RTP801 overexpressing neurons and then resolved in a WB. We observed that ectopic RTP801 (with a molecular weight of around 55 KDa due to the eGFP tag) was found and enriched in EVs (Figure 1E).

We also observed ectopic control protein eGFP in the EVs fraction. Furthermore, ectopic RTP801 elevated TSG101 and Flotillin-1 in the EVs fraction, suggesting an increase in the EVs release compared to the control eGFP condition (Figure 1 E & F). NTA measurements confirmed that ectopic RTP801 enhanced EVs release (4.38x10¹⁰±1.329x10⁹particles/ml in eGFP-overexpressing cultures and 1.56x10¹¹±4.24x10⁹particles/ml in RTP801-overexpressing cultures; P-value=0.0076 Student’s T-test). Although the increased amount of EVs in eGFP-RTP801 condition elevated the levels of actin in the EVs fraction, it must be mentioned that actin is a proper loading control for the cell lysates but not for the EVs fraction since actin, and other cytoskeletal proteins, can be loaded also in EVs⁵⁷. In fact, the increased amount of EVs is also reflected as an increase in actin levels.

We confirmed this finding in HEK293T cells, a proliferating non-neuronal cell model, where overexpression of RTP801 does not induce cell death⁵⁸. Like our results in neurons, RTP801 was present in the EVs fraction and induced an elevation of TSG101 and Flotillin-1 (Figure S1 A & B). NTA measurements corroborated that ectopic RTP801 increased the release of EVs in the cultured media (1.11x10¹¹±2.46x10¹⁰particles/ml in eGFP-overexpressing cultures and 2.36x10¹¹±1.69x10¹⁰particles/ml in eGFP-RTP801-overexpressing cultures; P-value=0.0273 Student’s T-test). Moreover, we observed that ectopic RTP801 was promoting the presence of the endogenous RTP801 in the EVs fractions in both neurons (Figure 1E) and HEK293 cells (Figure S1). Altogether, these results support that ectopic RTP801 increases the release of EVs compared to the control conditions.

RTP801 modulates the protein cargo of neural-derived EVs

Next, to investigate whether RTP801 could influence EVs protein cargo, we overexpressed or silenced RTP801 in cultured cortical neurons. Neurons were transduced with lentiviral particles containing eGFP or eGFP-RTP801 constructs or with scrambled shRNA (shCT) or shRNA against RTP801 (shRTP801). Three days later, we isolated EVs from the respective culture media (eGFP-, RTP801-, shCT- or shRTP801-EVs) and assessed EVs protein content by MS/proteomics (Figure 2).

PLS-DA plots revealed that both RTP801 overexpression and silencing changed the protein composition of EVs, as judged by sample group clustering in both comparisons (Figure S2). Pairwise comparisons of the proteomic data were used to identify enriched proteins and showed differentially expressed proteins in RTP801-derived EVs (Figure 2A & B) and in shRTP801- derived EVs (Figure 2C & D).

Volcano plots showed that the most overexpressed proteins in RTP801-EVs were SrSf2 (Serine/arginine-rich splicing factor 2), Glod4 (Glyoxalase domain-containing protein 4), Uqcrc1 (Cytochrome b-c1 complex subunit 1), Hspe1 (10kDa heat shock protein) and Ak1 (Adenylate kinase isoenzyme 1). SrSf2, Uqcrc1 and Hspe1 have been linked to apoptotic processes, as shown in Table 1. Ak1 has been described to have a neuropathogenic role in Aβ-mediated tau phosphorylation⁵⁹. The most downregulated proteins in RTP801-EVs were H2afy (Core histone macro-H2A.1), RPS6 (40S ribosomal protein S6), Qki (protein quaking), Rps13 (40S ribosomal protein S13), and Ndgr3 (protein NDGR3). Strikingly, H2afy and Ndgr3 have been linked to anti-apoptotic processes (Table 1), and RPS6 is a substrate of mTOR activation which regulates protein translation, that in turn, is associated with cell survival^51–53(Figure 2B).

In contrast, in the case of shRTP801-EVs, the most overexpressed protein was Slc38a3 (sodium-coupled neutral amino acid transporter 3) and the most downregulated were Gnb4 (guanine-nucleotide-binding protein subunit beta-4) and Txn (thioredoxin) (Figure 2D). All three proteins have been associated with apoptotic processes, and remarkably, Slc38a3, the most enriched one, has been linked to anti-apoptotic roles, as shown in Table 2.

Taking into account all the differentially expressed proteins (Figure 1A & C), and based on a bibliographic research of its apoptotic role (Table 1 & 2), we confirmed our first observation that RTP801-EVs cargo was enriched in proteins involved in apoptosis and seemed to have higher levels of pro-apoptotic proteins (in red, such as cytochrome b-c1, cytochrome c and alpha-internexin). The same cargo showed lower levels of anti-apoptotic proteins (in green, such as core histone macro-H2A, NDRG3 protein, and endophilin), in comparison with its control eGFP-EVs. In contrast, EVs derived from RTP801-silenced neurons (shRTP801-EVs) showed high levels of anti-apoptotic proteins (in green, such as sodium-coupled neutral aminoacid transporter 3, ADP/ATP traslocase 2 or pleiotropic regulator 1) and lower levels of other apoptotic-related proteins (Figure 3A & C, Table 1 & 2).

Therefore, although we did not get statistically significant alterations in pathway analyses, our bibliographic research of the differentially expressed proteins points towards an apoptotic role of RTP801-EVs. The fact that elevated levels of RTP801 have been reported to be toxic to neurons^1,4, and that EVs derived from neurons overexpressing RTP801 contain high levels of this protein along with apoptotic-related proteins, may suggest that RTP801 toxicity could be propagated via EVs.

Table 1. Significantly altered proteins involved in apoptosis found in EVs derived from RTP801-overexpressing neurons compared to the control. Pro-apoptotic proteins are shown in red, anti-apoptotic proteins in green, and proteins with a dual role as pro- and anti-apoptotic in orange. Up-arrows indicate that the protein is upregulated and down-arrows that the protein is downregulated. (Cas, abbreviated form os caspase).

Protein ID		Protein name	Gene name	FC	Described function in apoptosis	References
▲	Q6PDU1	Serine/arginine-rich splicing factor 2	SrSf2	3.50	SrSf2 down-regulation inhibits apoptotic pathways in renal cancer	Kedzierska et al., (2016)⁶⁰
					SrSf2 is necessesary for the survival of hematopoietic cells and its loss results in apoptosis	Komeno et al., (2015)⁶¹
					SrSf2 promotes the pre-mRNA splicing of apoptosis-related genes, such as Cas8, Cas9, c-Flip and Bcl-x, in response to DNA-damaging agents	Li and Wang (2021)⁶²
▲	Q68FY0	Cytochrome b-c1 complex subunit 1, mitochondrial	Uqcrc1	2.32	Uqcrc1 regulates cytocrome c (cyt-c)-induced apoptosis	Hung et al., (2021)⁶³
▲	P26772	10 kDa heat shock protein, mitochondrial	Hspe1	2.19	The function of Hspe1 and Hspd1 in apoptosis is complex, as they can regulate both positively and negatively the intrinsic pathway	Samali, et al., (1999)⁶⁴
						Lau, et al., (1997)⁶⁵
						Lin, et al., (2001)⁶⁶
▲	P62898	Cytochrome c, somatic	Cycs	1.92	Cycs triggers the activation of apoptosis-protease activating factor 1, which is necessary for the maturation of Cas9 and Cas3	Garrido et al., (2006)⁶⁷
					In neurons, the block of cyt-c activity prevents apoptosis	Neame, et al., (1998)⁶⁸
▲	P02773	Alpha-fetoprotein	Afp	1.91	Afp induces apoptosis in tumor cells by the activation of Cas3	Dudich, et al., (1999)⁶⁹
					Knock-down of Afp inhibits hepatocellular carcinoma cell proliferation and tumor growth by inducing apoptosis	Chen et al., (2020)⁷⁰
▲	A7VJC2	Heterogeneous nuclear ribonucleoproteins A2/B1	Hnrnpa2b1	1.82	Loss of hnrnpa triggers apoptosis in ovarian cancer	Yang et al., (2020)⁷¹
					Knock-down of hnrnpa2B1 promotes apoptosis of breast cancer cells	Yin et al., (2020)⁷²
▲	P23565	Alpha-internexin	Ina	1.77	Over-expression of Ina in PC12 cells results in apoptosis-like cell death	Chien, et al., (2005)⁷³
					High levels of Ina in Purkinje cells lead to degeneration and death of these neurons	Ching, et al., (1999)⁷⁴
▲	P13383	Nucleolin	Ncl	1.70	Ncl inhibits apoptosis in B-cell lymphomas Ncl is a negative regulator of H2O2 induced apoptosis Ncl antisense oligonucleotides induce apoptosis Chemical inhibition of human Ncl induces early apoptosis in càncer cells	Wise et al., (2013)⁷⁵ Zhang et al., (2010)⁷⁶ Wu et al., (2012)⁷⁷ Bellone et al., (2022)⁷⁸
▲	Q9QX69	LanC-like protein 1	Lancl1	1.56	Over-expression of LanCL1 preserves cell viability after oxygen and glucose deprivation in neuronal HT22 cells	Xie et al., (2018)⁷⁹
					Over-expression of LanCL1 protects motor neurons from apoptosis in mice models of amyotrophic lateral sclerosis	Tan et al., (2019)⁸⁰

Table 1. cont.

▲	P04256	Heterogeneous nuclear ribonucleoprotein A1	Hnrnpa1	1.48	HnRNP A1 is a nucleo-cytoplasmic shuttling protein which is known to either up- or down-regulate the IRES-dependent translation of varios mRNAs that encode proteins related to apoptosis and proliferation, such as XIAP, c-Myc, FGF2, Apaf-a, cyclin-D1 and Bcl-xL.		Lewis et al., (2007)⁸¹
							Bonnal et al., (2005)⁸¹
							Jo et al., (2008)⁸²
							Cammas et al., (2007)⁸³
							Bevilacqua et al., (2010)⁸⁴
					Knock-down of hnRNP A1 in mature neurons induce neuronal dysfunction and cell death		Anees, et al., (2021)⁸⁵

▲	Q4KLJ8	Phosducin-like protein 3	Pdcl3	1.25	Pdcl3 regulates the activation of caspases during apoptosis		Wilkinson, et al., (2004)⁸⁶
▲	P30904	Macrophage migration inhibitory factor	Mif	1.08	Over-expression of Mif leads to a deficient deletion of apoptotic cells in photocarcinogenesis		Honda et al., (2009)⁸⁷
					Knock-down of Mif increases apoptotic cells in perivascular-resident macrophage-like melanocytes		Zhang et al., (2017)⁸⁸
					In cortical neurons, after a traumatic brain injury, Mif promotes neuronal cell death in vivo		Ruan et al., (2021)⁸⁹
					Mif controls cytokine release in neuroinflammation and inhibition of MIF significantly reduced cytokine production and cell death.		Nasiri, et al., (2020)⁹⁰
▲	P97603	Neogenin	Neo1	0.98	Inhibition of neogenin promotes neuronal survival after spinal cord injury		Chen and Shifman (2019)⁹¹
					Neogenin overexpression induces apoptosis in cerebellar neurons		Fujita et al., (2008)⁹²
▲	P63004	Platelet-activating factor acetylhydrolase IB subunit alpha	Pafah1b1	0.80	Deletion of Pafah1b1 in mice provokes germ cell apoptosis		Feng et al., (2022)⁹³
▲	F1LTR1	WD repeat-containing protein 26	Wdr26	0.62	Over-expression of WDR26 reduces cell death and cas-3 mediated apoptosis in H2O2 treated cells		Liu et al., (2022)⁹⁴
					WDR6 inhibits apoptosis of cardiomyocytes after oxidative stress		Feng et al., (2012)⁹⁵
▲	Q63524	Transmembrane emp24 domain-containing protein 2	Tmed2	0.62	Tmed2 is related to apoptosis in MM.1S and RPMI 8226 cell lines		Ge et al., (2020)⁹⁶
▼	P85971	6-phosphogluconolactonase	Pgls	-0.62		Pgls knockdown increases ROS and apoptosis rate in hepatocellular carcinoma cell lines		Li et al., (2021)⁹⁷
▼	B2DD29	Serine/threonine-protein kinase BRSK1	Brsk1	-1.58		Loss of Brsk1 results in neuronal apoptosis and reduces the number of neural progenitors		Dhumale et al., (2018)⁹⁸
▼	Q641Z6	EH domain-containing protein 1	Ehd1	-1.69		Ehd1 knockout mice show increased number of apoptotic cells in the lens epithelium		Arya et al., (2015)⁹⁹
▼	Q7TP98	Interleukin enhancer-binding factor 2	Ilf2	-1.80		Ilf2 overexpression increases cell viability and decreases cell apoptosis, whereas knockdown shows opposite trends in human vascular smooth muscle cells.		Wei et al., (2021)¹⁰⁰
						Ilf2 downregulation increases the number of apoptotic cells in liver cancer cells		Cheng et al., (2016)¹⁰¹

Table 1. cont.

▼	O35964	Endophilin-A2	Sh3gl1	-1.85	Endophilin A2 protects muscle cells from H2O2 induced apoptosis	Liu et al., (2016)¹⁰²
▼	Q6AYR2	Protein NDRG3	Ndrg3	-2.06	NDRG3 promotes cell proliferation and anti-apoptosis during hypoxia	Lee et al., (2015)¹⁰³
▼	Q6AYR2	Protein NDRG3	Ndrg3	-2.06	Knockdown NDRG3 promotes cell apoptosis via ERK1/2 signaling pathway in gastric cancer cells	Liu et al., (2022)¹⁰⁴
▼	Q02874	Core histone macro-H2A.1	H2afy	-3.11	H2AFY knockdown suppresses cell proliferation and promotes apoptosis of HCC cells in vitro.	Huang et al., (2021)¹⁰⁵

Table 2. Significantly altered proteins involved in apoptosis in EVs derived from neurons where RTP801 was silenced with an shRNA compared to the control. Pro-apoptotic proteins are shown in red, anti-apoptotic proteins in green, and proteins with a dual role as pro- and anti-apoptotic in orange. Up-arrows indicate that the protein is upregulated and down-arrows that the protein is downregulated. (Cas, abbreviated form of caspase).

Protein ID		Protein name	Gene name	FC	Described function in apoptosis	References
▲	Q9JHZ9	Sodium-coupled neutral amino acid transporter 3	Slc38a3	2.22	Slc38a3 promotes metastasis by activation of PDK1	Wang et al., (2017)¹⁰⁶
					Slc38Aa3 levels increase during stress	Krokowski et al., (2013)¹⁰⁷
					Slc38a3 knock-down disrupts mTOR/S6K pathway	Chan et al., (2015)¹⁰⁸
▲	Q09073	ADP/ATP translocase 2	Slc25a5	1.68	Inhibition of Slc25a5 in neuroblastoma reduces cell viability	Seneviratne et al., (2022)¹⁰⁹
					Slc25a5 inhibits apoptosis	Chevrollier, et al., (2011)¹¹⁰
▲	Q9WUC8	Pleiotropic regulator 1	Plrg1	1.53	Plrg1 deficiency increases the rate of apoptosis	Kleinridders, et al., (2009)¹¹¹
▲	Q5RJQ4	NAD-dependent protein deacetylase sirtuin-2	Sirt2	1.51	Sirt2 knock-down inhibits cell proliferation and promotes cell apoptosis in multiple myeloma	Ding and Hao (2021)¹¹²
					Sirt2 inhibition sensitizes lung cancer cells to etoposide-mediated apoptosis	Hoffmann et al., (2014)¹¹³
					Sirt-2 induced apoptosis in MPP+-treated neuronal cells	Liu et al., (2014)¹¹⁴
▲	P13383	Nucleolin	Ncl	1.12	Ncl inhibits apoptosis in B-cell lymphomas	Wise et al., (2013)⁷⁵
					Ncl is a negative regulator of H2O2 induced apoptosis	Zhang et al., (2010)⁷⁶
					Ncl antisense oligodeoxynucleotides induce apoptosis	Wu et al., (2012)⁷⁷
					Chemical inhibition of human Ncl induced early apoptosis in cancer cells	Bellone et al., (2022)⁷⁸
▲	Q8R478	WW domain-binding protein 2	Wbp2	1.07	Downregulation of Wbp2 results in apoptosis of glioma cells	Gao et al., (2020)¹¹⁵
						Chen et al., (2018)¹¹⁶

Table 2. cont.

▲	P28073	Proteasome subunit beta type-6	Psmb6	0.98	Psmb6 along with Psmb5 inhibition induce cytotoxicity	Britton et al., (2009)¹¹⁷
▲	Q9Z2L0	Voltage-dependent anion-selective channel protein 1	Vdac1	0.79	Vdac1 prevents cell death during hypoxic conditions.	Brahinm-Horn et al., (2012)¹¹⁸
					Overexpression of Vdac1 induce apoptosis in many cell types	Weisthal et al., (2014)¹¹⁹
						Abu-Hamad et al., (2008)¹²⁰
						Ghosh et al., (2007)¹²¹
						Godbole et al., (2003)¹²²
						Zaid et al., (2005)¹²³
▼	P49911	Acidic leucine-rich nuclear phosphoprotein 32 family member A	Anp32a	-1.25	Anp32a knockdown induces apoptosis in myeloid leukemia cell lines	Yang et al., (2018)¹²⁴
					Anp32a activates the Apaf apoptosome and therefore induces cas-9 dependent apoptosis	Hill et al., (2004)¹²⁵
						Jiang et al., (2003)¹²⁶

▼	O35353	Guanine nucleotide-binding protein subunit beta-4	Gnb4	-2.07	Ectopic Gnb4 induces or inhibits apoptosis dependent on the cell line	Wang et al., (2018)¹²⁷
▼	P11232	Thioredoxin	Txn	-2.78	Txn-1 attenuates neuronal apoptosis in ischemia conditions	Ma et al., (2012)¹²⁸
						Zhou et al., (2009)¹²⁹
					Txn depletion induces oxidative stress	Yang et al., (2011)¹³⁰

Overexpression of RTP801 promotes apoptosis and impairs neuron arborization complexity via EVs in cultured cortical neurons

To evaluate the transneuronal pro-apoptotic role of RTP801, we used EVs from RTP801 overexpressing neurons (RTP801-EVs) or RTP801 silenced neurons (shRTP801-EVs), along with its controls (eGFP-EVs and shCT-EVs respectively). We treated sister cortical neuronal cultures with three different doses per conditions: 400, 800 or 1600 EVs per cell (EVs:cell ratio). After 24 h, untreated (UT) or EVs-treated neuronal cultures were fixed and immunostained with antibodies against MAP2 and cleaved caspase-3 (ClvCas3), and nuclei were stained with Hoechst 33342 (Figure 3A & B). Neurons (MAP2+ cells, in green) were segregated as ClvCas3 positive (ClvCas3+, in red) or negative (ClvCas3-), and neuronal nuclei were into viable, condensed, or fragmented, considering the last two as apoptotic. To apply this classification, we used a machine learning pipeline (Figure 3C), which showed an accuracy of 95,65% (Figure 3D). We observed that ClvCas3 intensity levels were increased in neurons treated at the highest dose of RTP801-EVs (Figure 3E & F), which highly suggests their putative pro-apoptotic role. No changes were observed with the treatment with the other EVs (shCT-EVs, shRTP801-EVs, or eGFP-EVs), or at lower doses of treatment (Figure 3E).

Interestingly, we also observed an increase in the number of cells with fragmented chromatin and negative for ClvCas3 (Figure 3G, Figure S3), combinatory readouts of last stage of the apoptotic process^131,132, confirming the toxicity of RTP801 at the highest dose of RTP801-EVs treatment (Figure 3G, Figure S3). Together, our findings strongly suggest that RTP801 is propagated via EVs and, along with a pro-apoptotic cargo induces apoptosis in recipient neurons.

To further gain insight into the toxic effect of RTP801-EVs, we next evaluated neuron arborization complexity after 24 h of treatment. To do so, neuron skeleton was extracted to quantify primary dendrites, intermediate branches, termini branches (endpoints), and total tree length (the length of all the branches in a neuron) (Figure 4A). RTP801-EVs treatment (at a ratio of 1600 EVs:cell) reduced primary dendrites, intermediate branches, and endpoints in mature neurons. Strikingly, shRTP801-EVs at the same dose produced the opposite effect in cultured sister neurons. Indeed, shRTP801-EVs increased the total tree length with no effect in the primary dendrites (Figure 4B & C, Figure S4). These results further confirm the toxicity of RTP801-EVs and hint the potential neurotrophic function of shRTP801-EVs.

6-OHDA elevates RTP801 protein levels in the EVs.

Next, to test the transneuronal toxicity of endogenous RTP801 via EVs, we turned to the 6-OHDA in vitro model, a PD neurotoxin that mediates neuronal cell death via RTP801³.

To test our hypothesis, cortical neurons at DIV 14 were exposed to 50µM 6-OHDA for 16 h. Then, released EVs and cell lysates were collected for analyses. NTA analysis revealed that 6-OHDA increased the number of extracellular particles in the treated cultures versus the non-treated ones (∅= 3.60x10¹⁰±9,00x10⁹particles/ml versus 6-OHDA=1.52x10¹¹±3.88x10⁰⁹particles/ml; P-value=0.0036 Student’s T-test). No variation in the vesicles size was found (145.05±10.11 nm in untreated cultures vs. 134.54±7.31 nm in 6-OHDA treated cultures). By TEM, we confirmed that 6-OHDA induced cortical neurons to release more EVs, and that these particles had the expected size and shape of exosomes and small microvesicles (Figure 5A). The EVs protein content was examined by WB analysis, and showed that 6-OHDA exposure increased the levels of the EVs markers NEDD4¹³³, TSG101, and Flotilin-1, in the EVs fraction. In this line, TSG101 and Flotillin-1 were decreased in the neuronal lysates (Figure 5B & C). Importantly, 6-OHDA elevated RTP801 protein levels in both cell lysates and EVs fractions (Figure 5B & C). These results indicate that 6-OHDA increases the release of EVs and elevates the endogenous levels of RTP801 in EVs, suggesting a toxic propagation of RTP801.

6-OHDA-induced EVs do not mediate pro-survival signals in recipient cortical neurons.

As the previous results demonstrated that RTP801 mediates neuron death via EVs, here we investigated whether the increased levels of endogenous RTP801 in 6-OHDA-derived EVs could mediate transcellular toxicity and affect intracellular signaling in recipient naïve neurons.

First, EVs were isolated from cortical neuronal cultures exposed or not to 6-OHDA, and sister neuronal cultures were treated with these isolated EVs for 4 h, 16 h, or 24 h, at a ratio of 400 EVs:cell (Figure 6 A). We observed that after 24 h, control (CT) EVs stimulated the mTOR/Akt survival pathway. We observed that CT-EVs increased the phosphorylation of the Ser235/236 residues in RPS6, as a readout of mTORC1 activity, and the phosphorylation of Ser473 residue of Akt, as a readout of mTORC2 activity (Figure 6 B & C). No changes were observed at earlier time-points. In contrast, 6-OHDA-derived EVs were not able to stimulate either phosphorylation (Figure 6 B & C).

To prove that 6-OHDA-induced RTP801 mediated the loss of mTOR/Akt survival pathway activation via EVs, we first exposed or not to 6-OHDA cultures transduced with shCT or shRTP801. Consistent with our previous work, we observed an increase in RTP801 levels in control neurons exposed to 6-OHDA but not in neurons with a partial knockdown of RTP801 (Figure S5).

We next isolated EVs from these conditions, and from non-transduced neuronal cultures (CT), and these vesicles from the six different conditions were used to treat naïve sister neuronal cultures. 24 h later, recipient neuron lysates were analyzed by WB (Figure 7A). Like our previous results (Figure 6), EVs derived from control, shCT or shRTP801-expressing neurons elevated levels of phospho-Akt and phospho-RPS6. However, EVs derived from neurons exposed to 6-OHDA (CT and shCT) did not exert this activation. Strikingly, when RTP801 was silenced (shRTP801-neurons), EVs could activate of phospho-Ser473-Akt and phospho-Ser235/236-RPS6 (Figure 7B & C). We also tested the effect over other mTOR/Akt readouts, such as phospho-Ser371-p70S6K and phospho-T37/46-4EBP-1, but no difference was observed between conditions (Figure S6). Our findings are consistent with the trophic effect previously observed at the level of neuron arborization of EVs derived from shRTP801-expressing neurons.

These results confirm that RTP801 toxicity can be propagated via neural-derived EVs and in pathological conditions, RTP801 elevation can specifically block EVs-induced activation of Akt and RPS6 in recipient neurons.

This study describes for the first time that endogenous RTP801 toxicity can be spread via EVs to recipient neurons. We found that endogenous RTP801 is present in EVs, and ectopic expression led to an enrichment of RTP801 in EVs in primary neurons and cell lines. Both RTP801 overexpression and silencing altered the proteomic profile of EVs. Expression of RTP801 knockdown promoted the loading of apoptotic-related proteins whereas RTP801 knockdown increased in the levels of anti-apoptotic proteins. RTP801-EVs were able to transfer toxicity to recipient neurons, impairing neuron arborization and inducing apoptotic death. Moreover, in cultured neurons, endogenous RTP801 was elevated in response to 6-OHDA, and the EVs isolated from these stressed neurons lost the ability to activate the mTOR/Akt pathway via Akt and RPS6. In line with this, the knockdown of RTP801 in EV-producing cells exposed to 6-OHDA rescued this activation of mTOR/Akt pathway in recipient neurons.

Intercellular transfer of bioactive molecules by EVs has been widely investigated in the nervous system^28,29,134. One of the mechanisms for the progression of neurodegenerative diseases is the spreading of disease-causing proteins from one brain area across other interconnected regions¹³⁵. EVs have been suggested to play a role in the propagation of aggregation-prone proteins, as several pathogenic proteins, such as β-amyloid³⁵, Tau¹³⁶, SOD1¹³⁷, and α-synuclein³⁶ can be released through this pathway. Our data revealed that both endogenous and ectopic RTP801 are present in EVs isolated from cultured cortical neurons.

We also observed that expression of RTP801 increased EVs release in both HEK293T cells and cultured neurons. However, the mechanism by which RTP801 could potentiate EVs release is not known yet. There is evidence that RTP801 induces an elevation of cytoplasmic calcium levels from mitochondrial reservoirs, as RTP801 is associated with ROS mitochondrial production³⁷ and endoplasmic reticulum stress¹³⁸. Another potential mechanism is that RTP801 could affect autophagy and therefore EVs release since these two cellular events are tightly intertwined¹³⁹. In this line, RTP801 elevation in mice overexpressing α-synuclein A53T and exposed to chronic restraint stress is associated with autophagy inhibition¹³⁸.

Our proteomic results indicated that overexpression or knock-down of RTP801 changed the protein content of neural-derived EVs. Increasing evidence shows that EVs’ molecular composition is highly influenced by environmental challenges. Indeed, intracellular stress can modulate the specific sorting of proteins and other components, and this, modify the biological response induced in recipient cells^140–142. In line with these observations, and the fact that RTP801 induces neuron-death^1,4, we found that EVs derived from neurons overexpressing RTP801 (RTP801-EVs) contained higher levels of pro-apoptotic proteins (such as cytochrome b-c1, cytochrome c and alpha-internexin) and lower levels of anti-apoptotic proteins (such as core histone macro-H2A, NDRG3 protein, and endophilin). When the apoptotic cascade is initiated, cytochrome c is released to the cytosol^67,143. Other apoptotic-related proteins, such as alpha-internexin^73,74, phosducin-like protein 3⁸⁶, neogenin^91,92, and transmembrane protein 2¹⁴⁴, might be activated also in response to RTP801 to accelerate the cell death.

Not only apoptotic-related proteins were found enriched, but also proteins involved in neurodegeneration such as adenylate kinase 1, which is involved in the neuropathology of AD⁵⁹. Moreover, RPS6 was found in low levels in RTP801-EVs, in line with the inhibitory role of RTP801 of the mTOR pathway.^51–53.

Strikingly, in shRTP801-EVs we found an enrichment of proteins associated with anti-apoptotic processes (such as sodium-coupled neutral aminoacid transporter 3, ADP/ATP traslocase 2 or pleiotropic regulator 1), highly indicating a potential trophic role of these EVs derived from RTP801 silenced neurons.

In line with our proteomic results, RTP801-EVs treatment increased the levels of active caspase-3 in neurons, highly suggesting that RTP801-EVs were activating apoptosis in recipient cells. We also observed a visible effect on late apoptosis, since a higher number of neurons presented fragmented nuclei with no cleaved caspase-3. In this line, Takemoto et al (2003) showed that caspase-3 inactivation preceded nuclear fragmentation¹³¹. Hence our observations support that RTP801 triggers apoptotic neuron death via EVs in a dose-dependent manner, but we cannot discard that other molecules, such as lipids or RNAs sensitive to RTP801 levels, could contribute to this effect as well.

In addition, RTP801-EVs significantly impaired dendritic arborization in recipient neurons, further confirming RTP801 toxicity role via EVs and supporting the apoptotic role of RTP801-EVs. Dendritic branching defects in mature neurons have devastating consequences on neuronal function and survival and are common features in patients suffering from neurodegenerative diseases¹⁴⁵. The fact that increased levels of RTP801 are found in PD, AD and HD human postmortem brains, and contribute to neuron impairment in disease mouse models^45,50, suggests that RTP801 could be an active player in spreading neurodegeneration via EVs and therefore, a potential future target in the treatment of neurodegeneration.

Importantly, EVs derived from neurons with silenced RTP801 (shRTP801-EVs) led to an increased dendritic arborization complexity in recipient neurons. This suggests that RTP801 is negatively modulating neuron branching in mature neurons. RTP801 is a negative regulator of mTORC1¹⁴⁶, and Nakai et al (2020) described that the activation of mTORC1 induced neurite outgrowth and branching in cultured cortical and hippocampal neurons, while the inhibition abolished these effects¹⁴⁷. In line with our results, Ota K.T. et al (2014) reported an atrophy of primary apical dendrites in neurons overexpressing RTP801 present at cortical layer V, measured by a decrease in spine density¹⁴⁸.

Our previous work showed that RTP801 protein is involved in PD pathogenesis^1,4. Here, we also found that RTP801 was enriched in EVs derived from cortical neurons treated with the neurotoxin 6-OHDA. Hence, our results suggest that oxidative stress induced by 6-OHDA can contribute to EVs release potentiation, exacerbating neurodegeneration in PD.

As abovementioned, EVs not only act as vehicles to transfer toxic proteins but also can exert a neuroprotective function^149,150. Our findings indicate that neural EVs play a trophic role by promoting specifically the phosphorylation of Ser235/236 residues of RPS6 and the Ser473 residue of Akt, readouts of the mTORC 1 and 2 kinase activities, respectively. The enhancement of the mTOR pathway by EVs in different recipient cell types has been previously pointed out^151–153. For instance, in a neuronal system, oligodendroglia derived EVs induced neuroprotection by the phosphorylation of Akt and ERK under oxidative stress or starvation¹⁵⁴. Furthermore, Tassew N.G. et al (2017) reported that fibroblasts derived EVs were able to activate mTOR in cortical neurons by Wnt recruiting¹⁵⁵. Interestingly, we observed that 6-OHDA-induced EVs were not able to promote mTOR/Akt pro-survival signals in recipient neurons. On the contrary, when RTP801 expression was suppressed, the derived EVs (shRTP801-EVs) could partially restore the phosphorylation of both Akt and RPS6. Hence, these data suggest that 6-OHDA-induced RTP801 impairs this signaling cascade transneuronally. Therefore, our results indicate that RTP801 interferes with the ability of EVs to promote trophic support to recipient neurons.

Our results indicate that the modulation of RTP801 expression alters the protein composition of neural-derived EVs. Furthermore, high levels of RTP801 in EVs exert a toxic effect on recipient neurons triggering apoptosis and branching alterations. In contrast, silencing RTP801 in EVs may have a trophic effect on neurite complexity. Moreover, we showed that neural-derived EVs enhance the mTOR pathway via Akt and RPS6 activation. However, under pathological conditions, RTP801-enriched EVs lost this signaling activation. Altogether, our results place RTP801 as an important contributor in neurodegeneration via EVs.

6-OHDA: 6-Hydroxydopamine

AD: Alzheimer’s disease

ClvCas3: Cleaved caspase-3

CNS: Central nervous system

CT: Control

DIV: Day in vitro

EVs: Extracellular vesicles

FBS: Fetal bovine serum

HD: Huntington’s disease

ILV: Intraluminal vesicles

Mhtt: Mutant huntingtin

MPTP: 1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine

MS: Mass spectometry

mTOR: Mechanistic target of rapamycin

MVB: Multivesicular bodies

NTA: Nanoparticle tracking analysis

PBS: Phosphate buffered saline

PD: Parkinson’s disease

PFA: Paraformaldehyde

PLS-DA: Partial least squares-discriminant analysis

RPS6: Ribosomal protein S6

TEM: Transmission electron microscopy

TSC1/TSC2: Tuberous sclerosis complex 1 and 2

UT: Untreated

WB: Western blot

Ethics approval

All experiments involving rat embryos for culturing neurons were approved by the local animal care committee of Universitat de Barcelona following European (2010/63/UE) and Spanish (RED53/2013) regulations for the care and use of laboratory animals.

Consent for publication

Not applicable

Availability of data and materials

Mass-spectrometry data and search results files were deposited in the Proteome Xchange Consortium via the JPOST partner repository (https://repository.jpostdb.org) (Okuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, Takami T, Kobayashi D, Araki N, Yoshizawa AC et al: jPOSTrepo: an international standard data repository for proteomes. Nucleic Acids Res 2017, 45(D1):D1107-D1111) with the identifier PXD040082 for ProteomeXchange and JPST002037 for jPOST (for reviewers: https://repository.jpostdb.org/preview/170310131463ea007f81c18; Access key: 3507).

Compelling interests

All authors declare to have no competing interests.

Funding

Supported in part by grants from Ministerio de Ciencia e Innovación /AEI/10.13039/501100011033/ and “FEDER”: SAF2017-88812-R and PID2020-119236RB-I00, from CM; PID2020-119386RB-I00 from JA and PID2019-106447RB-I00 from EP-N. JS-B was supported by a FPU grant from the Spanish Ministry of Science and Innovation (grant #FPU18/00194). NM-F was supported by a FPI grant from the Spanish Ministry of Science and Innovation (grant IBES-2015-072727). PG-S was supported by a FPU grant from the Spanish Ministry of Science and Innovation (grant #FPU21/02928). GC-C was funded by a FI-2021 grant from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (grant #FI-B-00378). AC-G was supported by a Michael J. Fox Foundation grant (MJFF-000858). Supported also by the crowdfunding campaign “SOS recerca en Parkinson” via Goteo.org and Portal d’Avall, S.L.

Author’s contributions

NM-F, JS-B, and CM designed this study. JS-B and NM-F performed the experiments. NM-F, JS-B, PG-S, LP-S, JF-I and ES analyzed the data. NM-F, JS-B, LP-S, PG-S and GC-C prepared the figures. NM-F and JS-B wrote the first draft of the manuscript. CM, JS-B, LP-S, PG-S, GC-C, AC-G, JF-I, ES, EP-N, and JA revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We are grateful to M. Calvo and J.M. Rebled (Centres Científics i Tecnològics, Universitat de Barcelona) for help in confocal and electronic microscopy, respectively. We thank Dr. Hernando del Portillo, Joan Seguí (Institut d’Investigació Germans Trias i Pujol, IGTP) and Amable Bernabé (Instituto de Ciencia de Materiales de Barcelona, ICMAB) for technical assistance from the NanoSight measurements. We thank Dr. J. Romaní for technical assistance. We thank Dr. Miguel Segura (Vall d’Hebron Research Institute, VHIR) for technical advice and helpful discussion.

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RTP801 Mediates Transneuronal Toxicity via Extracellular Vesicles by Affecting Their Protein Cargo and Abrogating Their Trophic Effect

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