Graphical abstract graphic file with name fx1.jpg [45]Open in a new tab Highlights * • Rab26 deficiency causes depression/anxiety-like behavior and cognitive impairment * • Rab26 regulates the trafficking of serotonin (5-HT) transporter (SERT) * • Rab26 interacts with SERT and regulates the endocytosis of SERT * • Loss of Rab26 results in 5-HT uptake to impair synaptic transmission __________________________________________________________________ Natural sciences; Biological sciences; Neuroscience; Behavioral neuroscience; Molecular neuroscience; Cellular neuroscience Introduction Membrane trafficking along with endocytic and exocytic pathways is essential for protein sorting and membrane homeostasis in all eukaryotic cells.[46]^1 Therefore, alterations in membrane trafficking are associated with multiple human diseases, including cancer, infections, metabolic diseases, and neurodegeneration.[47]^2 Major neurodegenerative diseases are closely associated with disordered membrane trafficking, which typically results in the accumulation of undegraded proteins due to aberrant endosomal sorting, lysosomal degradation, or autophagy.[48]^3^,[49]^4 For example, intracellular Aβ accumulations are toxic and induce cellular stress to neurons.[50]^5 Thus, neurons may degrade accumulated substances through degradative pathways to protect neural cells. Moreover, the degradation of surface receptors may reduce the response to neurotransmitters.[51]^6 The Rab family of small GTPases is the master regulator of vesicle-mediated trafficking and regulates almost every trafficking step, along with endocytosis and exocytosis pathways.[52]^7^,[53]^8 Rab GTPases switch between GTP-bound active and GDP-bound inactive states.[54]^9 They were initially discovered in brain tissue.[55]^10 Therefore, many Rab proteins are linked to neurological diseases, such as Alzheimer’s, Parkinson’s, and mental illness.[56]^11^,[57]^12 Rab3 and Rab27 regulate neuronal function by mediating synaptic vesicle transport and neurotransmitter release.[58]^13^,[59]^14^,[60]^15 Although Rab26 evolutionarily forms a subgroup with Rab3 and Rab27, its role in the neuronal system remains to be elucidated. Rab26 orchestrates multiple membrane trafficking events.[61]^16^,[62]^17 Rab26 was first identified on the surface of secretory granules in the acinar cells of rat parotid glands.[63]^18 MIST1 transcription factor, having Rab3D and Rab26 as its target genes, regulates secretory granule maturation.[64]^19 Alterations in Rab26 and effectors are associated with multiple human diseases, including cancers, diabetes, and acute lung injury.[65]^20^,[66]^21 Rab26 mediates the autophagic degradation of p-Src, resulting in the suppression of the migration and invasion of breast cancer cells.[67]^22 It also serves as a negative regulator of insulin secretion by sequestering Syt1 to inhibit insulin granule fusion with the plasma membrane.[68]^23 Recent studies provided the link between synaptic vesicle-associated Rab26 and the autophagic pathway.[69]^24 Plekhg5 regulates the autophagy of synaptic vesicles by activating Rab26 to direct synaptic vesicles to preautophagosomal structures.[70]^25^,[71]^26 Accordingly, Rab26 overexpression leads to synaptic vesicle accumulation in autophagosomes and the dysregulation of Rab26-dependent autophagy promotes the accumulation of impaired synaptic vesicles, and causes synaptotoxicity and cognitive and memory deficits in mice.[72]^27 In addition, Rab26 interacts with GPCRs and coordinates transport of GPCRs from the Golgi to the cell surface.[73]^28^,[74]^29 Although these studies suggest a role of Rab26 in the nervous system, its precise function remains unclear. In this study, we found that Rab26 deficiency resulted in depression and anxiety-like behaviors and cognitive impairment, which was likely due to the aberrant trafficking of serotonin transporter (SERT). SERT belongs to the solute carrier (SLC) 6 gene family that also includes transporters for the neurotransmitters dopamine, glycine, norepinephrine, and γ-aminobutyric acid (GABA), and they share a predicted structure with 12 transmembrane domains and intracellular N and C termini.[75]^30 Furthermore, SERT has been the subject of intensive research efforts as the target for antidepressants, but little is known about its trafficking. Our results showed that Rab26 interacts with SERT and regulates its endocytosis and autophagic degradation. These investigations define a physiological role of Rab26 in the brain and shed new light on the trafficking of SERT. Results Rab26 is highly expressed in the brain and potentially involved in neurological function Rab26 gene was first cloned and identified in rat pancreas, brain and parotid gland tissues.[76]^16 Rab26 plays a role in exocytosis-related intracellular vesicular transport along with Rab3, Rab27, and Rab37.[77]^16 However, its role in the brain remains to be elucidated. To define the physiological role of Rab26, we examined the mRNA level of Rab26 in different tissues by RT-PCR. The results demonstrated that the mRNA level of Rab26 was relatively higher in brain tissues ([78]Figure 1A, 1B). The protein level of Rab26 in different brain regions was analyzed by Western blot analysis, indicating that Rab26 was highly expressed in the hippocampus and hypothalamus ([79]Figures 1C, 1D). Rab26 was also highly expressed in human neuroblastoma SH-SY5Y and PC12 cells, which are commonly used neuronal cell lines to study neuronal function ([80]Figures S1A and S1B). Figure 1. [81]Figure 1 [82]Open in a new tab Rab26 is highly expressed in the brain and potentially involved in neurological function (A) mRNA expression level of Rab26 in different tissues was detected by RT-PCR. (B) Quantitative analysis of the results of (A) from 3 independent experiments. (C) Protein level of Rab26 in different brain tissues was investigated by Western blotting. (D) Quantitative data of (C) from 3 independent experiments. (E) Primary hippocampal neurons co-expressing GFP-Rab26WT with mCherry-PSD95 (dendritic spine marker) or mCherry-Syt1 (synaptic vesicle marker). Scale bars, 20 μm. (F) Cultured primary hippocampal neurons were immuno-stained with anti-Rab26 and anti-Syt-1 antibodies. Scale bars, 20 μm. (G) Western-blot using lysates of brains isolated from WT, Rab26^−/+ and Rab26^−/− mice to confirm the depletion of Rab26 (n = 3). (H) RNA-sequencing data of 247 differentially expressed genes in WT and Rab26^−/− hippocampal tissues. (I) KEGG pathway enrichment analysis of differentially expressed genes. Although Rab26 was found to associate with the synaptic vesicle,[83]^24 the intracellular distribution of Rab26 in the neuron is not clear. When Rab26 was co-expressed with PSD95 or Synaptotagmin-1 (Syt-1) in the cultured primary hippocampal neurons, it was observed that Rab26 co-localized with dendritic spine marker PSD95 and synaptic vesicle marker Syt-1 ([84]Figure 1E), which was verified by the co-localization of the endogenous Rab26 with Syt-1 ([85]Figure 1F). Interestingly, fluorescent microscopy showed that Rab26WT or Rab26Q123L (active), but not Rab26T77N mutant (inactive), were enriched within or at the base of dendritic spines ([86]Figure S1C). These results indicate that Rab26 is associated with synaptic vesicles at the dendritic spines (synapse area). To investigate the physiological role of Rab26, we generated Rab26 gene knockout mice by CRISPR/Cas9 technology. The depletion of Rab26 was confirmed by detecting the Rab26 protein in brain tissue using Western blot analysis. As shown in [87]Figure 1G, heterozygous (Rab26^−/+) mice had reduced Rab26 protein as compared to wild-type (WT) mice, and homozygous (Rab26^−/−) mice did not express Rab26. Immunofluorescence staining of Rab26 in mouse brain tissues revealed that there was dramatically reduced staining of Rab26 in cortical, hippocampal regions in Rab26^−/− mice ([88]Figure S1D). These results established that the Rab26 gene was efficiently depleted in Rab26^−/− mice. Next, we analyzed the transcriptome in hippocampal tissues of WT and Rab26^−/− mice by RNA sequencing (RNA-seq). 128 differentially expressed genes (DEGs) were identified in the knockout group as compared with the WT group, and there were 185 genes upregulated, while 62 genes were down-regulated in Rab26 knockout mice ([89]Figures 1H, [90]Table S2). KEGG analysis showed that these DEGs in the hippocampus were mainly enriched in neurological diseases, such as Huntington's disease, Parkinson's disease, and Alzheimer's disease ([91]Figure 1I). These results suggest Rab26 may play a patho-physiological role in the nervous system. Rab26 deficiency causes depression and anxiety-like behaviors and cognitive impairment in mice Although Rab26 is highly expressed in the brain and potentially associates with neuronal diseases, Rab26-deficient mice are viable and exhibit no major difference in appearance compared to WT mice. In addition, histological analysis through H&E staining and Nissl staining of the coronal sections of the brain revealed that Rab26-deficient mice did not show major morphological changes in the cortical and hippocampal regions ([92]Figures S2A and S2[93]B). Moreover, there were no significant differences in the number of neurons in cortical, hippocampal, and amygdala regions between WT and Rab26^−/− mice ([94]Figure S2C). Loss of Rab26 did not show major changes in dendritic lengths compared with control ([95]Figure S2D). These data suggest that Rab26 deficiency may not influence the development of the mice or the neuronal system. Therefore, we carried out behavioral experiments to examine if there are any behavioral abnormalities in Rab26-deficient mice. We performed behavioral tests to assess the impact of Rab26 deficiency on depression and anxiety-like behaviors. In the open field test (OFT), Rab26-deficient mice displayed a decreased number of entries into the center and spent less time in the center than the WT mice ([96]Figure 2A). Consistently, these Rab26-deficient mice were obviously afraid to enter into the open arms and spent less time in the open arms in the high elevated plus maze test (EPM) ([97]Figure 2B), but no significant differences in their body weight and total travel distance ([98]Figures 2A and 2B). Furthermore, Rab26-deficient mice stopped struggling earlier in the tail suspension test (TST) and forced swim test (FST) and showed that immobility times were significantly increased compared to WT mice, exhibiting depression-like behaviors. After treatment with the antidepressant escitalopram (selective serotonin reuptake inhibitor, SSRI), the immobility times for WT mice decreased significantly in both TST and FST. However, the immobility times for Rab26-deficient mice did not decrease significantly ([99]Figures 2C and 2D), indicating that the depletion of Rab26 in mice resulted in less sensitive to antidepressant drug. Collectively, these data suggest that Rab26 deficiency induces depression/anxiety like behaviors in mice. Figure 2. [100]Figure 2 [101]Open in a new tab Rab26 deficiency causes depression, anxiety-like behaviors, and cognitive dysfunction in mice (A) Representative tracks of WT or Rab26^−/− mice in open-field test (OFT), as well as body weight, travel distance, the number of center entries, and time spent in center (n = 12 mice, data were analyzed by Mann-Whitney test). (B) Representative tracks of WT or Rab26^−/− mice in elevated plus maze tests (EPM), as well as travel distance, the time spent in open arms, and the number of entries into open arms (n = 10 mice, data were analyzed by Mann-Whitney test). (C and D) Tail suspension test (TST) and forced swim test (FST) in WT mice or Rab26^−/− mice after treatment with escitalopram (10 mg/kg or 15 mg/kg). (n = 10 mice, data were analyzed by one-way ANOVA). (E and F) Y/Z-maze tests were used to evaluate the spontaneous alternation behaviors and thus the short-term working memory of mice. In T maze and Y maze tests, spontaneous alternation triplet percentage was calculated for comparison (n = 9–12 mice, data were analyzed by the Mann-Whitney test). (G) A schematic diagram shows a training timeline of CS-US pairing implementations. In fear conditioning tests, mice were trained and analyzed for freezing behavior under both contextual and cued tests. Freezing behavior was expressed as a percentage of the total duration (n = 10–12 mice, data were analyzed by the Mann-Whitney test). (H) Escape latency of WT and Rab26^−/− mice was recorded in Morris Water Maze (MWM) tests within a 6-day training period. WT and Rab26^−/− mice were assayed for their time spent in the target and the other three quadrants, latency to platform, the number of crossing target and total distance in target were recorded at the 7th day (n = 12 mice for each group, statistically significant differences were assessed by the Mann-Whitney test). Data represent mean ± SD, ns: not significant, ∗p < 0.05, ∗∗p < 0.01. To address whether Rab26 deficiency affects cognitive function, we performed behavioral tests with 3-month-old mice. Rab26-deficient mice exhibited significantly fewer spontaneous alternations than WT mice in Y maze test ([102]Figure 2E), and tended to reduce the alternations (though not significant) in the T maze test compared with control mice ([103]Figure 2F). These results indicate that knockout of Rab26 significantly impairs short-term working memory in mice. In addition, the freezing percentage of contextual test and cued test with Rab26 deficient mice was significantly decreased compared to that of respective WT mice in fear conditioning tests (FCT) ([104]Figure 2G). During Morris water maze testing, Rab26-deficient mice spent a longer time to locate the platform than the WT mice on the last training day. Then, the probe trial was assessed 24 h after the training phase, when the platform was removed, the mice were tasked to find the removed platform. The results showed that Rab26-deficient mice displayed significantly fewer platform crossings and spent less time in the target quadrant compared to WT mice ([105]Figure 2H), but there were no significant differences in the total distance in the target and latency to the platform between the WT and Rab26-deficient mice. Together, these results demonstrate that a deficiency of Rab26 impairs cognitive function in mice. Deficiency of Rab26 reduces synaptic transmission Ultra-structural analysis of the cortex and hippocampus by transmission electron microscopy showed there are no major differences in synapse number between WT and Rab26^−/− mice ([106]Figures 3A–3C and 3E). However, we observed increased accumulation of synaptic vesicles in presynaptic terminals of both cortical ([107]Figure 3A) and hippocampal neurons ([108]Figure 3B) from Rab26-deficient mice compared to WT mice ([109]Figures 3D and 3F). The protein levels of several synaptic markers showed no significant differences in cortical and hippocampal tissues from Rab26-deficient mice compared to WT mice ([110]Figure S3A). These data suggest that Rab26 deficiency may inhibit the synaptic vesicle trafficking and neurotransmitter release, resulting in the accumulation of synaptic vesicles. Figure 3. [111]Figure 3 [112]Open in a new tab Neuronal knockout of Rab26 reduces synaptic transmission in mice (A) Transmission electron microscopy showing the ultra-structures of cortical regions from WT and Rab26^−/− mice. Scale bars, 500 nm, red asterisk represents the synapse. (B) Transmission electron microscopy showing the ultra-structures of hippocampal regions from WT and Rab26^−/− mice. Scale bars, 500 nm, red asterisk represents the synapse. (C and D) Quantification of the number of synapses and number of synaptic vesicles in the cortex (n = 3 mice, data were analyzed by unpaired Student’s t test). (E and F) Quantification of the number of synapses and number of synaptic vesicles in the hippocampus (n = 3 mice, data were analyzed by unpaired Student’s t test). (G) Representative traces of mEPSCs in WT and Rab26^−/− mice, as well as quantitative analysis of the frequency and amplitude of mEPSCs (n = 9 neurons from n = 3 mice, data were analyzed by the Mann-Whitney test). (H) Representative traces of mIPSCs in WT and Rab26^−/− mice, as well as quantitative analysis of the frequency and amplitude of mIPSCs (n = 9 neurons from n = 3 mice, data were analyzed by the Mann-Whitney test). (I) LTP was induced using high-frequency stimulation (HFS) in WT or Rab26^−/− slices. Percentage of change of the fEPSP slopes was normalized from baseline, and the representative fEPSP recording traces were shown. (J) The comparisons of mean potentiation from the fEPSP slopes were calculated between 50 and 60 min after HFS (n = 12 neurons from n = 4 mice, data were analyzed by the Mann-Whitney test). Data represent mean ± SD. ns: not significant, ∗∗p < 0.01, ∗∗∗p < 0.001. Dysregulated synaptic vesicle trafficking and neurotransmitter release will disrupt synaptic function.[113]^31 Since deficiency of Rab26 caused depression/anxiety like behaviors and impaired cognition, we carried out electrophysiological experiments to detect the neurotransmission of the synapses using whole-cell patch-clamp recordings. The results demonstrated that the frequency, but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly decreased in Rab26-deficient mice compared to WT mice ([114]Figure 3G). However, both the frequency and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) showed no difference between Rab26-deficient mice and WT mice ([115]Figure 3H). To more directly evaluate the role of Rab26 in synaptic plasticity, we induced LTP and measured field excitatory postsynaptic potentials (fEPSPs) in the Schaffer collateral-CA1 pathway using acute hippocampal slices from WT and Rab26-deficient mice. The results indicated that LTP was dramatically reduced in Rab26 knockout mice compared to WT mice ([116]Figure 3I). Analysis of the degree of potentiation at 50–60 min after HFS confirmed the less of LTP response in Rab26 knockout slices, thereby providing evidence for the important role of Rab26 in synaptic potentiation ([117]Figure 3J). Together, these results suggest that Rab26 plays an important role in sustaining synaptic transmission. Rab26 deficiency leads to the defective endocytosis of serotonin (5-HT) transporter Serotonin (5-hydroxytryptamine, 5-HT) plays a major role in regulating the mood, cognition, and motor behavior, and the dysregulation of serotonergic transmission is implicated in several neurological disorders.[118]^32 Given the observed depression/anxiety-like behaviors and cognitive impairment in Rab26-deficient mice, we examined the levels of 5-HT in brain tissues and serum in mice using ELISA assay. However, no significant alterations in 5-HT levels were detected in brain tissues ([119]Figure 4A). Figure 4. [120]Figure 4 [121]Open in a new tab Rab26 deficiency inhibits the endocytosis of SERT (A) 5-HT concentrations in serum, cerebellum, cortex, and hippocampus were quantified by ELISA (n = 3 mice, data were analyzed by the unpaired Student’s t test). (B) Protein levels of SERT and Rab26 in cortical and hippocampal tissues were measured by Western blotting. GAPDH was used as a loading control. (C) Quantitative analysis of B from 4 independent experiments, data were analyzed by unpaired Student’s t test. (D) 293T cells stably expressing shRNA-Ctrl or shRNA-Rab26 were transfected with myc-SERT, cell surface proteins were biotin-labeled, and bound to streptavidin agarose beads; the bound SERT was analyzed by Western blot analysis. (E) 293T cells stably expressing PCDH-GFP-C1 and PCDH-GFP-Rab26WT were transfected with myc-SERT, then processed for analyzing the level of SERT as mentioned in D. Data represent the mean ± SD of three independent experiments. Unpaired Student’s t test was used to determine the significant difference. (F) SH-SY5Y cells stably expressing shRNA-Ctrl or shRNA-Rab26 were transfected with myc-SERT. Confocal microscopy images showing the surface and intracellular SERT signals (arrowheads indicate the differential localization of SERT in cells expressing shRNA-Ctrl or shRNA-Rab26). Scale bars, 20 μm. (G) Intracellular SERT signal was quantified within regions of interest (ROIs) using ImageJ software, and the results were compared using an unpaired Student’s t test (shRNA-Ctrl group n = 20, shRNA-Rab26 group n = 26). (H) SH-SY5Y cells were treated with 10 μM Serotonin (5-HT), then the intracellular 5-HT was detected through ELISA assay (n = 3, data were analyzed by unpaired Student’s t test). Data represent mean ± SD. ns: not significant, ∗p < 0.05, ∗∗p < 0.01 ∗∗∗∗p < 0.0001. Considering the potential role of Rab26 in presynaptic neurons, we examined the levels of several synaptic proteins in the presynaptic neurons. Western blot analysis revealed no changes in synaptophysin, Syt-1, Rph3A, or SNAP25 expression upon Rab26 knockout ([122]Figure S3A). Interestingly, the level of SERT, a transporter for 5-HT, was significantly increased in the hippocampus and cortex of Rab26-deficient mice compared to WT mice ([123]Figures 4B and 4C). Considering that Rab26-deficient mice exhibited less sensitive to the SSRI antidepressant ([124]Figures 2C and 2D), the increasing level of SERT may be physiologically related to the function of Rab26. Next, we examined the surface levels of SERT in 293T cells using a cell surface biotinylation assay. The results revealed that SERT level was increased on the cell surface after shRNA-mediated knockdown of Rab26 ([125]Figure 4D). Conversely, upon overexpressing Rab26 in 293T cells, the amount of SERT at the surface or total SERT was significantly reduced ([126]Figure 4E). In addition, confocal microscopy revealed that the intracellular pool of SERT was significantly reduced in Rab26-depleted cells when compared with control cells ([127]Figures 4F and 4G). Furthermore, when stimulated with serotonin (5-HT), the amount of serotonin in cell lysates of Rab26-knockdown SH-SY5Y cells was significantly higher than that of control cells ([128]Figure 4H), indicating that Rab26 knockdown promoted serotonin uptake due to increased SERT at the plasma membrane. The above data suggest that Rab26 may regulate SERT trafficking. Therefore, we examined the role of Rab26 in mediating the endocytosis of SERT. As serotonin can induce SERT endocytosis,[129]^33^,[130]^34^,[131]^35 SH-SY5Y cells were cultured under starvation in serum free media overnight, then incubated with serotonin to stimulate SERT internalization in a time course dependent experiment. Immunofluorescence microscopy analysis showed that the majority of SERT was associated with the plasma membrane, whereas a subset of SERT translocated to the cytoplasm in the cells over-expressing Rab26 in a time dependent manner, indicating that SERT was indeed internalized ([132]Figures 5A and 5C; [133]S4A and S4C). However, in SH-SY5Y cells expressing lentivirus-transduced shRNA-Rab26, no apparent internalization of SERT was observed upon Rab26 knockdown ([134]Figures 5B and 5D; [135]S4B and S4D). Figure 5. [136]Figure 5 [137]Open in a new tab Rab26 regulates the endocytosis of SERT (A) SH-SY5Y cells stably expressing PCDH-GFP-Vector or PCDH-GFP-Rab26WT were transfected with myc-SERT, cells were starved overnight in serum-free medium, then incubated with 1 μM 5-HT at 37°C for different times. Internalized SERT was visualized by immunofluorescence. Scale bars, 20 μm. (B) SH-SY5Y cells stably expressing shRNA-Ctrl or shRNA-Rab26 were transfected with myc-SERT, then processed for endocytosis experiments as mentioned in (A). Scale bars, 20 μm. (C) Intracellular SERT signal in (A) was measured using ImageJ software, the dot plot in the panel shows values from individual cells deriving from three independent experiments. (D) Quantitative analysis of (B) from three independent experiments. Data represent mean ± SD. The statistically significant differences were assessed by the Mann-Whitney test, ns: not significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. The above results suggest that Rab26 regulates the endocytosis of SERT, and Rab26 deficiency resulted in defective endocytosis of SERT. The surface accumulation of SERT may promote serotonin reuptake into neurons, thus reducing serotonin level in the synaptic cleft to inhibit synaptic transmission. This mechanistic link between defective SERT endocytosis and Rab26 deficiency aligns with the depression/anxiety-like behaviors and cognitive deficits observed in Rab26-deficient mice. Rab26 interacts with serotonin (5-HT) transporter to facilitate the endocytosis of serotonin (5-HT) transporter Previous studies have shown that Rab26 interacts with α2B-adrenergic receptor (α2B-AR) to mediate its trafficking,[138]^29 and Rab5 and Rab18 are potential partners for SERT,[139]^36 suggesting that Rab26 may also regulate SERT endocytosis through direct interaction. To test this hypothesis, we examined the interaction of Rab26 with SERT. Co-immunoprecipitation assays using cell lysates of 293T cells co-transfected with mCherry-Rab26 and myc-SERT revealed a specific Rab26-SERT interaction ([140]Figure 6A). Co-IP experiments also demonstrated that SERT preferentially interacted with Rab26WT and Rab26Q123L (GTP-bound), indicating Rab26 interacts with SERT dependent on its guanine nucleotide-binding activity, as the interaction between Rab26T77N mutant and SERT was dramatically weakened ([141]Figures 6B and 6C). Figure 6. [142]Figure 6 [143]Open in a new tab Rab26 interacts with SERT to facilitate the endocytosis of SERT (A) 293T cells were co-transfected with mCherry-Rab26WT and myc-SERT, cell lysates were subjected to co-immunoprecipitation experiment, precipitated proteins were analyzed by Western-blot. (B and C) 293T cells were co-transfected with myc-SERT and mCherry-C1, mCherry-Rab26WT, mCherry-Rab26Q123L or mCherry-Rab26T77N, respectively. The cell lysates were subjected to co-immunoprecipitation experiment, precipitated proteins were analyzed by Western blot. (n = 3, data were analyzed using one-way ANOVA with Tukey’s multiple comparison test). (D and F) SH-SY5Y cells were transfected with GFP-C1 or GFP-Rab26WT and immuno-stained with anti-SERT antibody to show the co-localization of Rab26 with endogenous SERT. Scale bars, 20 μm. (E and G) SH-SY5Y cells were transfected with myc-SERT and GFP-C1 or GFP-Rab26WT, respectively, then processed for immunofluorescence microscopy. Scale bars, 20 μm. The graph represents the mean of three independent experiments with 9 images each, data were analyzed by the Mann-Whitney test. Data represent mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Immunofluorescence microscopy revealed that GFP-Rab26WT co-localizes with endogenous SERT in SH-SY5Y cells ([144]Figures 6D and 6F). In addition, when co-expressing myc-SERT and GFP-Rab26WT, significant co-localization of Rab26 and SERT was observed ([145]Figures 6E and 6G), which is consistent with the interaction of Rab26 with SERT. It is a prerequisite that SERT and Rab26 are endogenously co-expressed in the same cell if the putative interaction of SERT is of physiological relevance. Therefore, we analyzed serotonergic neurons of the mouse dorsal raphe nucleus for the presence of Rab26. We identified serotonergic neurons by staining cryosections of the mouse brain covering the dorsal raphe by double immunofluorescence with antibodies against NeuN and Rab26. We confirmed that the existence of Rab26 in dorsal raphe neurons ([146]Figure S3F). We next examined the fate of internalized SERT in the SH-SY5Y cells. GFP-Rab26WT and myc-SERT were co-transfected with mCherry-Rab5 (an early endosomal marker) or mCherry-Rab7 (a late endosomal marker). Confocal microscopy analysis showed that a pool of SERT clearly co-localized with Rab5 and Rab7 ([147]Figures 7A and 7B). In addition, the intracellular SERT also co-localized with the endogenous Lamp1 (late endosomal/lysosomal marker) ([148]Figures 7C and 7D). Notably, the co-localization of SERT with Rab5 or Rab7 was markedly decreased in cells transfected with shRNA-Rab26 compared to the shRNA-Ctrl ([149]Figures 7E–7H). These results suggest that Rab26 interacts with SERT and facilitates its endocytosis into the endocytic compartments. Figure 7. [150]Figure 7 [151]Open in a new tab Rab26 facilitates SERT endocytosis in endocytic compartments (A and B) SH-SY5Y cells were co-transfected with GFP-Rab26WT, myc-SERT, and mCherry-tagged Rab5 (early endosome marker) or Rab7 (late endosome marker), then processed for immunofluorescence microscopy. Pearson correlation coefficients were calculated from 9 images across three replicates, data were analyzed by the Mann-Whitney test. Scale bars, 20 μm. (C and D) SH-SY5Ycells were co-transfected with GFP-Rab26WT and myc-SERT and immuno-stained with anti-Lamp1 (lysosomal marker), then processed for immunofluorescence microscopy. Pearson’s correlation coefficients were calculated from 9 images across three replicates, data were analyzed by the Mann-Whitney test. Scale bars, 20 μm. (E and F) SH-SY5Y cells stably expressing shRNA-Ctrl and shRNA-Rab26 were co-transfected with myc-SERT and mCherry-tagged Rab5, then processed for immunofluorescence microscopy. The results were quantitatively analyzed as above. (G and H) SH-SY5Y cells stably expressing shRNA-Ctrl and shRNA-Rab26 were co-transfected with myc-SERT and mCherry-tagged Rab7, then processed for immunofluorescence microscopy. The results were quantitatively analyzed as above. Scale bars, 20μm. Data represent mean ± SD. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Rab26-dependent autophagy is involved in the degradation of serotonin (5-HT) transporter Recent studies indicate that SERT undergoes constitutive internalization and is preferentially sorted to the late endosomes and lysosomes for degradation.[152]^30 Given Rab26’s established role in neuronal autophagy,[153]^24 we hypothesized that Rab26 may mediate the autophagic degradation of the internalized SERT under stress conditions. Western blot demonstrated that the overexpression of Rab26 promoted autophagy by inducing the expression of LC3-II in SH-SY5Y cells upon starvation treatment ([154]Figures 8A and 8B). Conversely, knockdown of Rab26 by lentivirus-transduced shRNA-Rab26 markedly decreased the level of LC3-II, suggesting that the downregulation of Rab26 impairs autophagy in SH-SY5Y cells ([155]Figures 8C and 8D). Figure 8. [156]Figure 8 [157]Open in a new tab Rab26-dependent autophagy is involved in the degradation of SERT (A) SH-SY5Y cells were infected with PCDH-GFP-vector or PCDH-GFP-Rab26 lentivirus, after 72 h, cells were cultured in nutrient starvation medium for different time courses. The protein levels of LC3 and Rab26 were analyzed by Western blot. (B) Quantitative analysis of the result (A) from 3 independent experiments, data were analyzed by unpaired Student’s t test. (C) SH-SY5Y cells were infected with shRNA-Ctrl or shRNA-Rab26 lentivirus, then processed for starvation and analysis of LC3 and Rab26 as mentioned in (A). (D) Quantitative analysis of the result (C) from 3 independent experiments, data were analyzed by unpaired Student’s t test. (E) SH-SY5Y cells were co-transfected with GFP-C1 or GFP-Rab26WT and myc-SERT and mCherry-LC3, cells were processed for immunofluorescence microscopy. Scale bars, 20 μm. (F) Pearson correlation coefficients were calculated for multiple individual images of panel E using ImageJ software. The graph represents the mean of three independent experiments, the statistically significant differences were assessed by the Mann-Whitney test. (G) SH-SY5Y cells were infected with PCDH-Vector or PCDH-Rab26WT lentivirus, after 48 h, and the cells were starved for 12 h. The protein levels of SERT were analyzed by Western blot. (H) SH-SY5Y cells were infected shRNA-Ctrl or shRNA-Rab26 lentivirus, after 48 h, and cells were starved for 12 h. The protein levels of SERT were analyzed by Western blot. (I and J) Quantitative analysis of (G) and (H) from 3 independent experiments. Data represent mean ± SD. One-way ANOVA was employed to analyze the possible differences between groups, ns: not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Next, we examined whether Rab26-dependent autophagy is responsible for the degradation of SERT. We performed confocal microscopy in SH-SY5Y cells co-expressing GFP-Rab26WT, mCherry-LC3, and myc-SERT. Rab26 overexpression promoted SERT colocalization with LC3-positive autophagosomes ([158]Figures 8E and 8F). Consistent with this observation, Western blot demonstrated that Rab26 significantly decreased the amount of the SERT protein in SH-SY5Y cells upon starvation treatment ([159]Figures 8G and 8I), indicating Rab26 promotes the autophagic degradation of SERT during starvation. Moreover, Rab26 knockdown inhibited the degradation of SERT ([160]Figures 8H and 8J). As deficiency of Rab26 increased the protein level of SERT in mouse brain tissues ([161]Figures 4B and 4C), it is proposed that the deficiency of Rab26 impairs both the endocytosis and autophagy-mediated degradation of SERT, resulting in increased levels of SERT. Discussion Over 70 Rab proteins have been characterized in mammalian cells, and most of them are expressed in brain tissues.[162]^10 It is known that alterations of Rab GTPases or Rab-mediated membrane trafficking are associated with virtually all neuronal activities.[163]^37 However, only a few Rab proteins are well characterized in neuronal function and disorders. Rab3 and Rab27 regulate synaptic vesicle exocytosis to mediate neurotransmitter release.[164]^38 Recent studies indicated that Rab39B deficiency leads to neuronal dysfunction and behavioral changes in mice.[165]^39 Although Rab26 was found to regulate synaptic vesicle trafficking, and is probably related to cognitive impairment under manganese stress,[166]^27 its role in neuronal patho-physiology remains to be elucidated. In this study, we examined the behaviors of Rab26 gene knockout mice and found that Rab26 deficiency resulted in depression/anxiety-like behavior and cognitive impairment. Mechanistically, we reveal that Rab26 modulates synaptic transmission by regulating SERT endocytosis and degradation. This finding provides direct evidence linking between Rab proteins dysfunction and mood disorders/neurological diseases. Previous studies have demonstrated that Rab26-dependent autophagy promoted accumulation of impaired synaptic vesicles,[167]^24^,[168]^27 suggesting that the dysregulation of Rab26 may be associated with neuronal disorders. We found that Rab26 deficiency increased depression and anxiety in mice, accompanied by defects in short-term working memory, spatial memory, and impaired synaptic plasticity, suggesting the loss of Rab26 results in aberrant synaptic transmission. However, the levels of neurotransmitters such as 5-HT were not significantly altered in our experiments, suggesting that the loss of Rab26 does not affect the production of neurotransmitters, but probably affects the transport events of neurotransmitters. Mechanistically, we found that Rab26 interacts with SERT and regulates its endocytosis and degradation, which consequently modulates the transport of 5-HT. SERT/Slc6a4 is a serotonin transporter playing a vital role in regulating serotonergic signaling, and alterations in its function have been linked to several neuropsychiatric disorders such as depression, anxiety, obsessive-compulsive disorder, and autism.[169]^40^,[170]^41 These diseases are characterized not only by severe emotional and affective impairments but also by disturbed cognitive functions.[171]^42 Consistently, our results revealed that Rab26 deficiency resulted in aberrant SERT trafficking and accumulation at the plasma membrane, which may lead to reduced synaptic transmission. Serotonin signaling is mediated by serotonin receptors and regulated by SERT, which induces cellular uptake of serotonin, removing it from circulation, thereby limiting its availability for signaling.[172]^33^,[173]^43^,[174]^44 A recent review suggests that SERT endocytosis plays an important role in the regulation of serotonergic signaling.[175]^33 SERT endocytosis can be induced by serotonin and selective serotonin reuptake inhibitors (SSRIs) to reduce surface levels of SERT.[176]^34^,[177]^35^,[178]^45 Notably, the depletion of MAD2 in cultured serotonergic rat neurons reduced endocytosis of SERT.[179]^46 SERT transports serotonin from the extracellular space into neurons, thereby inhibiting the availability of serotonin for receptor binding and signaling.[180]^33 SSRIs exert their antidepressant effects by promoting SERT endocytosis, thereby sustaining synaptic serotonin levels.[181]^45^,[182]^47^,[183]^48 Consistent with this mechanism, our study demonstrates that loss of Rab26 impairs SERT endocytosis and degradation, resulting in its surface accumulation. We further demonstrated that Rab26 is a direct binding partner that facilitates SERT internalization in SH-SY5Y cells. Collectively, these findings establish that Rab26 deficiency induces depression/anxiety behaviors and cognitive impairment through inhibiting SERT endocytosis and degradation, resulting in the surface accumulation of SERT to enhance serotonin (5-HT) re-uptake into the neurons, consequently reducing the synaptic transmission, and showing less sensitive to SSRI. Following endocytosis of a surface protein, endosomal sorting controls the receptor/transporter fate and modulates the receptor signaling outcome.[184]^33 Recent studies indicate that SERT is primarily sorted to the late endosomes and lysosomes for degradation rather than to recycling.[185]^30 While Rab26 was previously shown to regulate lysosomal trafficking and autophagic function during secretory granule maturation.[186]^16 Moreover, Rab26 directly binds to Atg16L1, resulting in the clustering of synaptic vesicles for degradation in mammalian neurons.[187]^24 Here, we found that the over-expression of Rab26 induced autophagy and decreased SERT level in SH-SY5Y cells under starvation conditions, and Rab26 knockdown inhibited the degradation, indicating the degradation of SERT depends on the Rab26-induced autophagy. Thus, we propose that the inhibition of autophagy and the blockage of endocytosis together lead to elevated SERT level in Rab26 deficient brain or cells. Depression and anxiety disorders are leading causes of disability worldwide affecting the health of billions of people.[188]^49^,[189]^50 SERT is a target for antidepressants and SSRIs are the main class of pharmacologic agents used to treat depression and anxiety but the mode of their therapeutic action is hotly debated.[190]^51 Moreover, these drugs are accompanied by many side effects, including sexual dysfunction, systemic metabolic disorder, and persistent hypertension.[191]^52 Our results demonstrate that Rab26 deficiency causes depression/anxiety-like behaviors and cognitive dysfunction, due to synaptic dysfunction. These findings indicate that the behavioral defects in mice are associated with the impaired trafficking of SERT caused by loss of Rab26. Our study uncovers a novel regulation pathway for SERT by Rab26 (for example small molecules capable of increasing the expression levels and/or functionality of Rab26), providing novel therapeutic targets and strategies for these neuronal disorders. Limitations of the study A knock-in experiment to rescue the phenotypes would be beneficial to comprehensively understand the physiological function of Rab26 in regulating emotional behaviors. In addition, we did not screen small molecules which regulate the expression of Rab26. The expression of Rab26 in the depression animal model and the effects of small molecules on regulating the expression of Rab26 and emotional behaviors deserve further examination. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tuanlao Wang, E-mail: xmuibrwtl@xmu.edu.cn. Materials availability Plasmids generated specifically for this study can be obtained from the lead contact. Data and code availability * • Data: The authors declare that data supporting the findings of this study are available within the article and its [192]supplemental information files. All data reported in this article will be shared by the [193]lead contact upon request. Any additional information required to reanalyze the data reported in this article is available from the [194]lead contact upon request. * • Code: This article does not report the original code. Acknowledgments