Systemic lupus erythematosus (SLE) and sarcoidosis are both complex autoimmune disorders with varying clinical manifestations. The incidence of SLE is as low as 4.91 per 100,000 population, and that of sarcoidosis is 0.85 per 100,000 population. The prevalence of neuropsychiatric systemic lupus erythematosus (NPSLE) ranges from 17.6% to 44.5%. The concurrent occurrence of NPSLE and sarcoidosis, although rare, presents diagnostic and management challenges. The clinical picture resulting from the coexistence of NPSLE and sarcoidosis, which share a common immunological picture, is not well defined. This case report discusses a patient with coexisting NPSLE and sarcoidosis, highlighting the intricate interplay between these conditions. Systemic lupus erythematosus (SLE) is a rare, chronic multisystemic autoimmune illness that can range from mild to potentially life-threatening . The incSarcoidosis is a multisystemic disorder of unknown cause, commonly affecting young and middle-aged adults with symptoms involving various organs and diagnosed by histological evidence of noncaseating epithelioid cell granulomas and an aggravated T helper cell type 1 (Th1) immune response . The incThe clinical presentation of individuals with concurrent NPSLE and sarcoidosis is not well defined and can be challenging to differentiate due to their shared immunological and neurological features. Concurrent findings of neuropsychiatric SLE and sarcoidosis have been rarely reported in the literature .A 45-year-old African-American man, previously diagnosed with sarcoidosis 10 years ago, presented to the emergency room with complaints of double vision on the lateral gaze and experiencing numbness, tingling, and proximal muscle weakness while lifting things against gravity and climbing stairs. Additionally, he had a history of mild chronic obstructive pulmonary disease and was using an albuterol inhaler as part of his prescribed treatment plan, taking one to two inhalations (puffs) as needed every four to six hours for symptom relief. Over the past six months, he had been troubled by a persistent dry cough.During the physical examination, it was observed that the individual had a slender physique and exhibited normal vital signs. Both of his eyes displayed limitations in abduction, indicating the presence of bilateral sixth cranial nerve neuropathies. He scored a 3/5 for both proximal muscular atrophy and proximal weakness in the upper and lower extremities. The patient scored a 5/5 on distal muscle strength. The patient reported feeling a slight discomfort in his muscles, described as occasional cramping sensations that were transient and occurred intermittently. Reflexes on the left side were entirely absent, and the patient exhibited a notable decrease in sensation in the distal legs, encompassing reduced proprioception, light touch, and vibration sense. An abdominal examination was benign. The laboratory test results are listed in Table The results of a head CT scan were normal. The chest X-ray revealed mild apical pleural thickening Figure .A positron emission tomography-computed tomography (PET-CT) scan of the chest revealed several calcified nodules that were slightly enlarged Figure .A broad, primarily axonal sensory-motor length-dependent polyneuropathy and myopathy with some evidence of a mononeuritis multiplex undergoing generalized transformation were revealed by electromyography (EMG) and a nerve conduction study (NCS). After reviewing the patient's medical history, a sarcoidosis exacerbation was identified.The patient was treated with a dosage of 40 mg of prednisone for presumed sarcoidosis exacerbation, but no response was observed even after receiving five doses of the steroid. For a paraneoplastic etiology, a workup was conducted. The CT scan revealed a normal abdomen. The results of the colonoscopy and prostate-specific antigen (PSA) test were both normal, indicating a healthy colon and prostate. An inflammatory infiltrate with enhanced plasma cells was found in the lung infiltrate after a fine needle aspiration biopsy in the left upper region; however, there were no granulomas, and all cultures were negative. The laboratory findings in the rheumatology evaluation are listed in Table The patient was identified as having NPSLE with concomitant sarcoidosis based on the presence of autoantibodies, Coombs-positive hemolytic anemia, proteinuria, symmetrical progressive sensorimotor polyneuropathy with active sarcoidosis in the lung, cranial neuropathies, and a history of psychosis.The patient began receiving intravenous (IV) cyclophosphamide at a dosage of 1000 mg/m^2 about three weeks after his initial presentation, and within four weeks, both his motor strength and extraocular movement had significantly improved. He has currently had six cyclophosphamide infusions, is off prednisone, and is responding well to methotrexate. His anemia and diplopia have completely disappeared. The patient is doing well on follow-ups.The clinical picture resulting from the coexistence of NPSLE and sarcoidosis, which share a common immunological picture, is not well defined. Both SLE and sarcoidosis exhibit cellular and humoral immunological abnormalities, such as immune system hyperreactivity, hypergammaglobulinemia, impaired cell-mediated immune function, and loss of self-antigen tolerance . In our Similar clinical signs, such as polyarthritis, fever, or peripheral lymphadenopathy, can be present in both sarcoidosis and SLE. Approximately 0.5%-1% of neuropsychiatric manifestations of SLE are cranial palsies, an uncommon central nervous system expression . The eigPatients with SLE who present with interstitial lung disease and bilateral hilar lymphadenopathy should be evaluated for the presence of sarcoidosis since these are rare in SLE alone . The mosSince there are elusive signs and symptoms for diagnosis, NPSLE management is difficult. The management of NPSLE is centered on symptomatic treatment, such as antiepileptics for seizures, anxiolytics or antipsychotics for psychiatric symptoms, antihypertensive medicines for hypertension, and correction of metabolic derangements . ImmunosSarcoidosis and SLE share a genetic predisposition. Lupus is associated with the alleles HLA-B8, HLA-A1-B8-DR3, and HLA-DR2 ,15. DespIt is important to take into account the concurrence of sarcoidosis and SLE. Rheumatologists may have difficulty diagnosing NPSLE due to the lack of sensitive and precise laboratory serum or cerebrospinal (CSF) fluid biomarkers, nuclear and radiological imaging, and other formal criteria for diagnosing and directing management decisions in NPSLE. Therefore, further research should be conducted to investigate the coexistence of sarcoidosis and SLE, aiming to enhance our comprehension of this association and facilitate improved co-management of both conditions.


This game is appropriate for medical students, interns, junior and senior residentsThe COVID-19 pandemic has forced a transition from in-person to virtual learning, and educators must innovate and adapt to keep learners engaged. One way to achieve this is through gamification.By the end of this didactic, the learner will:1. Describe the basics of the presentation of each topic listed above2. Recall the basics of management of each topic listed above3. Improve learners’ preparedness for the Emergency Medicine Inservice Exam and Written Board ExaminationWilderness and environmental medicine topics were selected from the list of topics covered on the ABEM boards. Questions were then written by the authors teaching these concepts.Learners were surveyed immediately following the session using an evaluation tool containing both Likert-scale questions on quality and applicability as well as open-ended questions on strengths and areas for improvement. The response rate to this survey was 100%.Feedback was overwhelmingly positive, with 19/20 respondents rating the sessions 5/5 for effectiveness and value of teaching compared with other sessions, and 1/20 rating the session 4/5. Nineteen out of twenty respondents rated the content as “highly relevant”; 1/20 rated it as “mostly relevant.” Nineteen out of twenty respondents rated the session 5/5 for being engaging and holding their attention; 1/20 rated it as somewhat engaging.Learners rated the session as highly relevant and engaging. We hypothesize that by engaging two screens and forcing learners to work together by text message, we were able to turn what would normally be a distraction (texting co-residents during resident conference) into a tool for learning.Electrical injury, lightning strike, thermal burns, inhalational injury, chemical burns, acute radiation syndrome, snake bites, scorpion envenomation, stingray envenomation, jellyfish stings, black widow spider bites, mammalian bites, rabies, murine typhus, bear encounters, decompression sickness, arterial gas embolism, drowning, dehydration, heat stroke, exercise-associated hyponatremia, frostbite, hypothermia, CO poisoning, hydrogen sulfide poisoning, giardia. Learner Audience:Medical students, Interns, Junior Residents, Senior ResidentsTime Required for Implementation: 90 minutes if all 30 questions are used. Alternatively, educators can elect to use only level 1 and 2 questions, which shortens the duration of the game to approximately 60 minutes. We recommend setting a 2-minute time limit for teams to answer each question.Recommended number of learners per instructor: The game is designed for 3–5 teams. We divided 20 residents into five teams of four learners each, but teams of 3–5 learners would be appropriate depending on the number of participants.Topics:Electrical injury, lightning strike, thermal burns, inhalational injury, chemical burns, acute radiation syndrome, snake bites, scorpion envenomation, stingray envenomation, jellyfish stings, black widow spider bites, mammalian bites, rabies, murine typhus, bear encounters, decompression sickness, arterial gas embolism, drowning, dehydration, heat stroke, exercise-associated hyponatremia, frostbite, hypothermia, CO poisoning, hydrogen sulfide poisoning, giardia.Objectives:By the end of this didactic, the learner will:4. Describe the basics of the presentation of each topic listed above5. Recall the basics of management of each topic listed above6. Improve learners’ preparedness for the Emergency Medicine Inservice Exam and Written Board ExaminationEach board style question was designed to test a concept relating to either the presentation or management of the topic, addressing objectives one and two. The competitive nature of the game as well as the opportunity for other teams to “snipe” incorrectly answered questions encouraged engagement by all learners, not just those whose turn it was. The question was followed by a 1–2 slide summary lecture covering basic presentation and management to fill in any gaps not addressed by the question, rounding out objectives one and two.A review of the wilderness and environmental topics covered in this presentation using published guidelines , journal articles, and textbooks is advisable. One foundational text which covers many of these topics is the following:Tintinalli’s Emergency Medicine: A Comprehensive Study Guide. Eighth edition. McGraw-Hill Education; 2016.Tintinalli JE, Stapczynski JS, Ma OJ, Cline D, Meckler GD, Yealy h, eds. The sections of this text with particular relevance are: section 2 (disaster management), chapters 8 and 10, section 13 (infectious diseases), chapters 157, 159, 150, and section 16 , chapters 208 – 211, 213 – 218, and 222. Instructors are encouraged to review the references listed for any question or topic with which they are less familiar, and which can be found in the references section.Learners should prepare by reading the following before the game:Tintinalli’s Emergency Medicine: A Comprehensive Study Guide. Eighth edition. McGraw-Hill Education; 2016.Tintinalli JE, Stapczynski JS, Ma OJ, Cline D, Meckler GD, Yealy DM, eds. Section 2 (disaster management), chapters 8 and 10, section 13 (infectious diseases), chapters 157, 159, 150, and section 16 , chapters 208 – 211, 213 – 218, and 222.The exercise was presented as part of a weekly emergency medicine grand rounds series for residents and medical students. Twenty learners participated and gave feedback. Feedback was overwhelmingly positive, with 19/20 respondents rating the sessions 5/5 for effectiveness and value of teaching compared with other sessions, and 1/20 rating the session 4/5. Nineteen out of twenty respondents rated the content as “highly relevant”; 1/20 rated it as “mostly relevant.” Nineteen out of twenty respondents rated the session 5/5 for being engaging and holding their attention; 1/20 rated it as somewhat engaging. Some modifications have been made for publication, primarily the removal of copyrighted images.For successful implementation, it is vital that teams are able to communicate effectively within the team. Establishing teams ahead of time and ensuring that this asynchronous communication channel is functional is recommended.Participants must be able to log into a video meeting platform such as Zoom or WebEx  as well as an alternate channel of communication for individual teams, such as a group text message thread or a WhatsApp group. The presenter must have a PowerPoint or similar slide presentation software as well as the ability to be on presenter mode in the video chat platform. There must also be a text chat function in the video meeting platform.Learners were not formally tested or evaluated on the material following this session. However, all participants indicated in their responses that they found the session to have high relevance and teaching value.


Sarcoidosis is notorious for producing a wide variety of skin lesions, which are categorized as either specific or nonspecific. The specific lesions include primary morphologies ranging from micropapules to subcutaneous nodules. Nonspecific skin lesions include associated conditions like erythema nodosum, calcinosis cutis, and prurigo. It is not uncommon for a patient to have a combination of specific and nonspecific lesions. In contrast, it is exceedingly rare for one patient to have multiple specific sarcoidal lesions. When present, the term “polymorphous cutaneous sarcoidosis” has been used. We present the case of a patient who presented with three specific cutaneous morphologies of sarcoidosis: papular sarcoid, Darier-Roussy subcutaneous sarcoidosis, and lupus pernio. After only two months of oral minocycline, our patient demonstrated remarkable improvement with near-complete resolution of the cutaneous lesions. In addition to describing the rare polymorphous presentation, this case also highlights the challenge of relating lesion type to overall prognosis when multiple morphologies are present. There is a wide range of cutaneous manifestations of sarcoidosis. Many different morphologies have been described, including papules, plaques, subcutaneous nodules, lupus pernio, annular lesions, scar-associated, psoriasiform, and so on. While many patients with sarcoidosis will have some form of skin involvement, it is exceedingly rare to develop more than one morphology of cutaneous sarcoidosis in a single patient . To our A 65-year-old female with a past medical history of hypertension presented for evaluation of lesions on her arms and around her eyes. Asymptomatic subcutaneous nodules on her volar forearms appeared six months prior to presentation with a gradual increase in lesions over time. Moreover, during that time, she developed papules around her eyes, which were occasionally painful and caused significant eye irritation. On further review, she mentioned a rash on her neck that had been present for a longer time, unclear of exactly how long, but did not think it was of any significance. She denied arthralgias, cough, or shortness of breath. Family history was non-contributory.The examination was notable for numerous non-tender, discrete, firm subcutaneous nodules without overlying erythema or epidermal change on her bilateral volar forearms Figure . SensatiSmall, slightly erythematous papules, some coalescing to plaques, were present on the medial canthi and eyelids, and a small papule was appreciated on the nasal tip Figure .Lastly, on the posterior right neck, there were many 1-2 mm erythematous papules coalescing into plaques Figure . The patTwo biopsies were obtained: a shave biopsy from the posterior neck and a telescoping punch biopsy from the left anterior forearm. Given the clinical suspicion of sarcoidosis from the other sites, eyelid and nose biopsies were deferred. Both biopsies showed nodular aggregates of granulomatous inflammation Figures -5, no evThe patient was started on oral minocycline 100 mg twice a day and referred to rheumatology for further systemic workup. Labs were notable for an elevated angiotensin-converting enzyme of 233 U/L. A chest X-ray and CT were recommended but not completed by the patient. She was also noted to have elevated creatinine but declined further investigations. After two months on oral minocycline, she returned for follow-up and showed significant improvement with near-complete resolution of all lesions Figures -7. While the lungs are the most commonly affected organ in sarcoidosis, it is not uncommon for cutaneous sarcoidosis to be the presenting sign of sarcoidosis ,7. When There are only a few reports of polymorphous cutaneous sarcoidosis in the literature to date. Interestingly, the reported cases all present different combinations of lesion morphologies, and underlying systemic involvement seems arbitrary. The first case described a combination of papular, psoriasiform, and pigmented purpuric dermatosis-like lesions in a 31-year-old male from Bangladesh . This paHistorically, the type of skin lesion has been used as a marker for prognosis; however, polymorphous cutaneous sarcoidosis poses a challenge to that concept . When muThe ultimate outcome for our patient is difficult to determine as the patient declined the recommended systemic workup. Systemic involvement seems unlikely based on the lack of symptoms, but pulmonary, hilar lymph node, and renal involvement cannot be completely ruled out at this time.Polymorphous cutaneous sarcoidosis describes the presence of multiple specific sarcoidal lesions occurring in one patient. This phenomenon is rarely reported in the literature. Our case of polymorphous cutaneous sarcoidosis demonstrates the unique combination of sarcoid specific lesions not previously reported: papular sarcoid, Darier-Roussy subcutaneous sarcoidosis, and lupus pernio. Fortunately, our patient demonstrated remarkable improvement with a near-complete resolution of the cutaneous lesions, after only two months of oral minocycline.


In the original article, there was a spelling mistake in the name of the second author. The correct name appears above.3−” should have been “Eu3+”; “0.248 p.m.” should have been “0.248 μM”; “0.13 p1\4” should have been “0.31 μM”; “0.9 [tM” should have been “0.9 μM”; “20 n9.120 pL” should have been “20 ng/20 μL”; the reference Baiv et al. (2018) should have been In the original article, there were errors in abstract, page 1. The third sentence previously stated:In the original article, the abbreviation of DMs-PDMS was wrongly modified during production. Corrections have been made to the “The as prepared DMs embedded PDMS DMs PD MS film was integrated with a microfluidic technique to enhance the SERS signal of a liquid substrate.”The corrected sentence is as follows:“The as prepared DMs embedded PDMS (DMs-PDMS) film was integrated with a microfluidic technique to enhance the SERS signal of a liquid substrate.”abstract, page 1; Introduction, page 2, last paragraph; Material and methods sub-heading, “Fabrication of the DMs PDMS film”, page 2; as well as the Results and discussion subheading, “DERS effect investigation of the DMs PDMS film”, page 3.Additionally, all instances of the abbreviation “DMs PDMS” have been corrected to “DMs-PDMS” throughout the In the original article, there was an error in Results and discussion, “Sensing performance evaluation”, page 4, “15 randomly selected spots” should be “20 randomly selected spots”.Consequently, in The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Neurons process real-time information from axon terminals to coordinate gene expression, growth, and plasticity. Inputs from distal axons are encoded as a stream of endocytic organelles, termed signalling endosomes, targeted to the soma. Formation of these organelles depends on target-derived molecules, such as brain-derived neurotrophic factor (BDNF), which is recognised by TrkB receptors on the plasma membrane, endocytosed, and transported to the cell body along the microtubules network. Notwithstanding its physiological and neuropathological importance, the mechanism controlling the sorting of TrkB to signalling endosomes is currently unknown. In this work, we use primary mouse neurons to uncover the small GTPase Rab10 as critical for TrkB sorting and propagation of BDNF signalling from axon terminals to the soma. Our data demonstrate that Rab10 defines a novel membrane compartment that is rapidly mobilised towards the axon terminal upon BDNF stimulation, enabling the axon to fine-tune retrograde signalling depending on BDNF availability at the synapse. These results help clarifying the neuroprotective phenotype recently associated to Rab10 polymorphisms in Alzheimer’s disease and provide a new therapeutic target to halt neurodegeneration. Communication between cells depends on their ability to respond as integrated units to spatial and temporal signalling patterns. The complex morphology of neurons provides an unrivalled model to study how sorting and trafficking of signalling complexes coordinate local signalling at axon terminals and the propagation of messages to the cell body. A clear example is provided by neurotrophic factors secreted by target tissues and sensed by axon terminals, where, among other functions, they regulate local cytoskeletal dynamics to promote or cease axon elongation, induce branching, as well as synaptic maturation and plasticity . At the Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is widely expressed in the central nervous system, together with its receptor tropomyosin-related kinase B (TrkB). By activating diverse signalling cascades, comprising the phosphoinositide 3‐kinase (PI3K)‐Akt pathway, mitogen‐activated protein kinases (MAPKs), and phospholipase C‐gamma (PLCγ), BDNF and TrkB play a critical role in the formation, maintenance, and plasticity of neuronal circuits . BindingThe Rab family of monomeric GTPases plays a central role regulating post-endocytic trafficking of TrkB. Whilst early endosome formation is regulated by Rab5, maturation and processive transport of signalling endosomes in different neuronal models are controlled by Rab7 . HoweverIn this work, we specifically focused on Rab10 since it has been shown that Rab10-positive organelles are transported both anterogradely and retrogradely along axons in hippocampal neurons . PreviouRab10 . We monitored the effects of this virus on cell viability and expression levels of Rab10 and TrkB at different time points after addition of doxycycline. We observed that 48 hr of treatment consistently halved the number of cells per field compared to control group, whereas incubation for 24 hr did not affect neuronal density . Rab10 iRab10 .To study axonal TrkB dynamics, we cultured hippocampal neurons in microfluidic chambers, which allow the cellular and fluidic compartmentalisation of axon terminals. After 7 days in vitro, neurons plated in one of the compartments (designated as ‘somatic’) displayed axons reaching the axonal compartment . MaintaiRab10 and treated with doxycycline for 18–22 hr showed lower levels of endogenous Rab10  in the nucleus. pCREB is a well-established proxy for neurotrophic signalling in neurons and has been shown to be critical for global neuronal responses to neurotrophins, such as BDNF-induced dendritic branching . NeuronsGiven its critical role regulating retrograde accumulation of TrkB, we investigated whether Rab10 was present in retrograde signalling endosomes. As stated before, signalling endosomes are likely to be a heterogeneous pool of functionally related organelles with diverse molecular compositions . Most ofCT) of being transported almost exclusively in retrograde axonal organelles in neurons  post-endocytosis. As expected, the amount of TrkB in retrograde carriers increases with time (cyan). However, the proportion of TrkB present in Rab10 compartments is very low and remains constant across the duration of the experiment (white). These results provide another evidence suggesting that TrkB localises transiently on Rab10-positive membrane compartments en route to its delivery to axonal retrograde carriers. This observation is also supported by super-resolution images (CT double-positive puncta (cyan arrowheads), but very few TrkB/HCT/Rab10 triple-positive puncta (white empty triangles) at 60 and 90 min.To specifically identify retrograde signalling endosomes containing TrkB, we took advantage of the property of the non-toxic carboxy-terminal domain of the heavy chain of tetanus neurotoxin  was monitored in neurons expressing EGFP-Rab10. After depletion of trophic factors, p75NTR uptake was visualised by incubating neurons for 45 min with an Alexa Fluor647-labelled antibody against the extracellular domain of p75NTR, in the presence of BDNF (NTR (green) are shown in NTR receptor can be found in both retrograde and anterograde Rab10 carriers. On average, 29.6% of anterograde and 15.5% of retrograde p75NTR-containing organelles were found positive for Rab10 across five independent experiments  and TrkB-Flag. As illustrated in To assess the contribution of Rab10 to the recycling of TrkB in the axon, hippocampal neurons were transfected with EGFP or a dominant negative mutant of Rab10 . Rab10 associated to GLUT4-containing endosomes is kept inactive by AS160 until Akt signalling releases the brake and promotes fusion with the plasma membrane .Q68L mutant is transported predominantly in the retrograde direction in neurons depleted of BDNF  pathway in junction . This suLittle is known about the specific sorting machinery required for the biogenesis of signalling endosomes. Endophilins A1 and A3 have been shown to regulate the trafficking of TrkB across early endosomes and mediate survival signalling . Interes2 were then seeded on glass coverslips or microfluidic chambers, pre-coated with 1 mg/mL poly-L-lysine. Before coating, glass coverslips were treated overnight with NoChromix (Godax Laboratories), washed three times and sterilised in 70% ethanol. Microfluidic chambers were produced in-house as previously described  in 5% BSA, 0.05% saponin, 0.1 mM CaCl2, and 0.1 mM MgCl2 dissolved in PBS. 4′,6-diamidino-2-phenylindole (DAPI) was added with the secondary antibodies when appropriate. Finally, coverslips were washed in PBS and mounted with Mowiol.Cells were washed in phosphate buffer saline (PBS) and fixed for 15 min in 3% paraformaldehyde (PFA) and 4% sucrose dissolved in PBS. Next, they were incubated in 0.15 M glycine dissolved in PBS for 10 min and then blocked and permeabilised simultaneously by incubation in 5% bovine serum albumin (BSA) and 0.1% saponin in PBS for 1 hr. Samples were incubated at 4°C overnight with primary antibodies diluted in 5% BSA, 0.05% saponin, 0.1 mM CaClRab10 lentivirus (pTightPuro-shRNA Rab10) and its doxycycline-dependent regulator TET-ON Advance (Clontech). After 48 hr, they were treated with doxycycline 1 µg/mL and kept in the incubator for 12, 18, 24, 36, and 48 hr. Cell density, levels of Rab10 and TrkB, as well as the general health of the culture were analysed at 12, 24, and 48 hr to establish the optimal time frame for the following experiments.Rab10 was knocked down by transducing 5 days in vitro (DIV) hippocampal neurons with an inducible shRNA Addgene_13031), the plasmids for EGFP-Rab10 WT (RRID:Addgene_49472), EGFP-Rab10 T23N (RRID:Addgene_49545), and EGFP-Rab10 Q68L (RRID:Addgene_49544) were a gift from Marci Scidmore (Addgene_134626) was a gift from Marvin Bentley . Experiments were carried out after 20–24 hr. The pEGFP-C1 plasmid is from Clontech  together with 20 ng/mL BDNF for 2.5 hr. After PFA fixation, the transport of internalised antibodies was revealed by incubating the somatic compartment with fluorescently labelled secondary antibodies. The same protocol was used to study retrograde propagation of neurotrophic signalling; after 1 hr of growth factor depletion, axons were stimulated with 20 ng/mL BDNF for 2.5 hr, and after fixation, phosphorylation of CREB in the nucleus was analysed by immunofluorescence. Transduction with a myc-Rab10 containing three silent mutations was used for rescue.Hippocampal neurons were seeded in custom-made microfluidic devices and after 5 DIV they were transduced with the inducible shRNA http://www.sussex.ac.uk/gdsc/intranet/microscopy/UserSupport/AnalysisProtocol/imagej/colocalisation). All the data points were plotted and the mean and standard error are indicated for each group and compared using Student’s t-test. Statistical significance of the individual data point is colour-coded (see figure legends).To analyse the presence of two markers in the same organelle, we used confocal z-stack images (voxel size: 0.197 × 0.197 × 0.5 µm) and the confined displacement algorithm to measure Manders’ correlation index within axons and determine its statistical significance compared to random images of identical total intensity and shape . To comphttps://henriqueslab.github.io/resources/NanoJ).High-fidelity super-resolution information was extracted from time series of 1000 confocal images per channel by using super-resolution radial fluctuations (SRRF) algorithm . Super-rAB_439712). In selected samples, 20 nM HCT was also added . For these experiments, neurons were cultured on 25 mm coverslips kept in Neurobasal for 1 hr prior to cell imaging and mounted inside Attofluor chambers  with BrightCell NEUMO photostable media  supplemented with 10 mM HEPES. Speed, pausing, and direction of labelled organelles were analysed from kymographs by using Kymoanalyzer set of macros from Encalada lab  and HALT proteases and phosphatases inhibitor . After clearing by centrifugation at 21,000 × g for 10 min, the detergent concentration was adjusted to 0.5%. Pre-washed anti-HA magnetic beads  were incubated with lysates overnight at 4°C. Samples were washed in lysis buffer five times and elutes in loading buffer  at 95°C for 10 min. The levels of immunoprecipitated Rab10 and KIF13B were analysed by Western blot using mouse anti-HA  and mouse anti-GFP B-2  antibodies.Neuro-2A cells have been originally obtained from ATCC and modified to stably express TrkB-Flag . Their iData generated in independent experiments were tested for normality and homoscedasticity to apply the appropriate corrections to the statistical tests. Each specific test, its degrees of freedom, and level of significance are indicated in the respective figure legends. Further details are summarised in the statistical annex . Plots shttps://www.color-blindness.com/coblis-color-blindness-simulator/) using The Colour Blind Simulator algorithms from Matthew Wickline and the Human-Computer Interaction Resource Network. Palettes were adjusted to maximise visibility. The Orange/Green/Purple balanced look-up table was obtained from Christophe Leterrier’s GitHub repository . Updated versions of ImageJ macros and R scripts used in this article, as well as the specific implementation of Kymoanalyzer used to analyse our datasets, can be found on our GitHub repository . Figures were checked with Coblis :Here, the authors use confocal microscopy, super resolution, and live imaging to report a role for Rab10 in the sorting of internalized TrkB receptors and in propagating the BDNF signal from axon terminals to the soma. The imaging analyses, specifically the super-resolution and live imaging, are well-done. However, the manuscript appears to be preliminary at this point; the authors draw strong conclusions about the mechanism of action of Rab10 in TrkB sorting that remain to be fully supported.Concerns:1. The authors see little co-localization between endogenous Rab10 and TrkB. The conclusion that Rab10 and TrkB are retrogradely co-transported is based on Rab10 over-expression, which raises concerns about the specificity of the interactions and artifacts related to over-expressing a Rab GTPase.2. Other than P-CREB immunofluorescence experiments, the authors do not provide data to show the functional effects of Rab10 depletion on BDNF-mediated trophic effects. The authors state in the Discussion that Rab10 knockdown perturbs neuronal morphogenesis and survival, but do not show this data. These data are necessary to strengthen the conclusion that Rab10 is necessary to mediate BDNF-dependent trophic signaling. On a related note, the color intensity scales to show TrkB or P-CREB levels in cell bodies with Rab10 depletion are not adequate.3. It is interesting that BDNF enhances anterograde Rab10 transport based on live imaging. Additional analyses are needed to strengthen the conclusion that Rab10 is enriched in distal axons and to define the mechanisms (phosphorylation?) underlying Rab10 recruitment/retention4. The anterograde motor Kif13B is suggested to be involved in BDNF-induced anterograde transport of Rab10 based on confocal microscopy analyses in fixed neurons. However, this aspect is not fully developed. The authors should determine if Kif13B directly interacts with Rab10 and the necessity of Kif13B for anterograde Rab10 transport, retrograde transport of TrkB receptors or BDNF-mediated trophic effects.5. The suggestion that Rab10 regulates "sorting" of internalized TrkB receptors is very interesting. The authors show nicely  that dominant negative Rab10 (rab10DN) does not disrupt TrkB recycling in axons, but increases the co-localization of TrkB with Rab5. The images in Figure 6d also show that Rab5 domains appear to be larger in the Rab10 DN expressing neurons, which is consistent with early endosomes being perturbed. However, additional analyses are needed to define how exactly Rab10 coordinates with Rab5 and Rab7 in "handing off" TrkB from early endosomes to late endosomes in axons. Which axonal segments were imaged in the analyses in Figure 6? Was this in the distal axons? What happens to TrkB flux in mid-axons and retrograde accumulation in cell bodies in the Rab10 DN or shRNA expressing neurons? Does expression of Rab10 shRNA or Rab10 DN decrease co-localization of retrogradely transported TrkB with Rab7?6. The authors should include important controls to show the specificity of Rab antibodies, and the knockdown of Rab10 using shRNA. In Figure 1f, the Rab10 levels (assessed by Rab10 immunofluorescence) appear to not be significantly affected by the kd or rescue, which raises questions about the antibody specificity, as well as efficacy of the kd.Comments above address concerns about conclusions that are not fully supported by the data. Here are some specific suggestions for additional analyses plus other recommendations.1. The authors should use additional measures to support that Rab10 associates/co-migrates with TrkB, for example by biochemical methods, or by performing live imaging of co-transport in neurons transfected with Rab10-GFP but with endogenous Rab10 knocked down to mitigate the OE concern.2. The authors should strengthen the data to support that Rab10 accumulates in distal axons in a BDNF-dependent manner, using confocal microscopy or biochemical means. Live imaging analyses can be used to determine if Rab10 is being mobilized long-distance from cell bodies by the retrograde BDNF signal, or if this is a local effect of BDNF on Rab10 recruitment to the axon terminals.3. In Figure 1c, the authors show reduced accumulation of axon-derived TrkB receptors in neurons expressing Rab10 shRNA. Images of the axons should be included to assess whether the TrkB receptors accumulate in distal axons, as proposed in the model 4. The authors also propose a mechanism for BDNF-induced enrichment of Rab10 in axons that involves Rab10 phosphorylation. The manuscript would be strengthened by additional analyses to define mechanisms underlying Rab10 recruitment. The imaging analyses of Kif13B could be complemented with biochemical methods to show BDNF-induced association with Rab10.5. Rab10 has been proposed to play a role in generating tubular endosomes-it would be of interest to use super-resolution imaging to define the morphology of TrkB/Rab10-positive endosomes in distal axons.6. The authors should assess whether the Rab10 kd or expression of Rab10 DN has general effects on neuronal viability or affect total TrkB levels, and not specifically on retrograde transport of axonally-derived TrkB receptors.Reviewer #2 (Recommendations for the authors):Signaling from neurotrophins requires endocytosis of the ligand-bound receptors (such as NGF-TrkA or BDNF-TrkB), sorting into signaling endosomes and subsequent transport retrogradely along the axon back to the soma for signaling. Multiple Rab proteins have been implicated in this trafficking pathway, most prominently Rab5 and Rab7. This manuscript shows that Rab10 is also required for retrograde arrival of signaling endosomes in the soma and subsequent signaling. The most interesting finding is that Rab10-positive organelles show increased anterograde transport in response to BDNF and are not part of the TrkB-positive retrogradely moving signaling endosome itself. Interference with Rab10 traps TrkB in Rab5-positive early endosomes, and the authors propose that Rab10 is required for sorting of TrkB into signaling endosomes. Since Rab10 is largely in a distinct set of axonal organelles, the point of conversion of endocytosed TrkB and Rab10 is not clear. The paper stops short of decisively answering where Rab10 is active to promote sorting and what BDNF signaling does to change Rab10 motility patterns.1) Rab10 was enriched in SEs by SILAC/mass spec (from a previous paper by this group) but now is not in the retrograde carriers. Can you discuss?2) Are the anterograde Rab10 carriers endosomally derived? It would be important to characterize the cargos of these carriers. I think the observation that BDNF affects the directionality of the Rab10 carriers is very intriguing. Were these in microfluidic chambers? How is BDNF signaling conveyed to Rab10 carriers?2) There is a lot of conjecture about Rab10 vesicles delivering machinery to the distal axon early endosome, but no experiment to address this. Where is TrkB in Rab10 interference conditions? It seems to be stuck with Rab5 and not sorting back to the axonal plasma membrane or into retrograde carriers. Is it accumulating in distal axon tips? It would be good to have live imaging of TrkB in Rab10-DN conditions along axons. The recycling experiments  are very interesting, but it looks like this is happening along the axon shaft. Is this correct?3) It is not always clear what experiments are done in microfluidic chambers. Please specify in each figure.4) Is p75 not in a complex with TrkB after BDNF binding? What is the interpretation/implication of p75 being in different carriers in the axon? I find this a very interesting observation, but the relevance is not explained or further explored.5) The data with kinesins is not adding much to the understanding of the pathway. No interference with any kinesin is performed.3) All the discussion of LRRK2 and phosphorylation is besides the point since the authors do not test involvement of this regulatory mechanism. This would be very interesting, but as is, the paper stops short of decisively answering where Rab10 is active to promote sorting and what BDNF signaling does to change Rab10 motility patterns.There are several very interesting observations, but the scope of the study is somewhat limited.eLife. Your revised article has been evaluated by Suzanne Pfeffer (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. We realize that this manuscript was already reviewed and resubmitted, and the reviewers hesitated to ask for more data, but they felt strongly that some additional work would be required to support the conclusions of your study. Note that it is not always possible to secure the same reviewers with each round of submission.Essential revisions:(The other issues that follow below are worthy of text clarification)1) The fact that there is little colocalization between Rab10 and TrkB needs further characterization. That is not consistent with "a new Rab10 organelle" that mediates TrkB sorting and retrograde transport. Please test other recycling endosome Rabs, such as Rab11, Rab14, Rab10 to determine if these are really novel organelles or simply Rab11-recycling endosomes that also contain Rab10.2) Please test whether Kif13B or Kif5B knock-down affects TrkB transport.3) The IP experiment shown in Figure 6G-H is not very convincing. Can you blot for endogenous Kif13B co-precipitation with HA-Rab10? Second, it appears that there is more HA-Rab10 precipitated in BDNF-treated samples, thus, it is unclear whether slight increase in GFP-Kif13B co-precipitation is due to that rather than increased interaction with Rab10. Third, why there is some GFP-Kif13B in sample that does not express HA-Rab10? Please address.Reviewer #1 (Recommendations for the authors):In the revised manuscript, the authors faithfully addressed most of the reviewers' concerns. I have only few suggestions (see below).1. Description of "Rab10-EGFP" and "Rab10-HA" should be considered, because EGFP and HA were tagged to the N-terminus of Rab10. Indeed, the authors have used "GFP-Rab5" in their previous paper (see ref. 12).2. Several resources were missing in the Key resources table, e.g., shRNA-resistant Rab10, Rab10-Q68L, and JIP3/4 antibodies.3. (Line 564) The reference may be mis-cited. Ref. 22 is correct?Reviewer #2 (Recommendations for the authors):This is a manuscript that focuses on understanding the machinery governing generation and transport of TrkB-containing endosomes, especially the involvement of Rab10 in the process. Since we are only beginning to understand the long-range signaling by endosomes in neurons, the topic is quite interesting and potentially significant. Additional data are needed to confirm association of endogenous Rab10 with TrkB-endosomes and the potential novelty of these structures.1) Rab10 knock-down should be evaluated using western blotting or qPCR. It is much more quantitive method than microscopy. Additionally, how authors control for possible off-target effects? They used rescue in Figure 2f-g. Similar rescues should be used in other analyses in Figures 1-2.2) Rescue images in Figure 2f are not particularly convincing. Not sure how authors can tell apart the neurons that are presumably expressing rescue plasmid.3) As other reviewers pointed out there is very little colocalization between Rab10 and TrkB. That is not consistent with authors conclusions that Rab10 marks a new type of organelle that mediates TrkB sorting and retrograde transport. Authors speculate that these are transient associating but provide little evidence to support that. Overexpression of both, Rab10 and TrkB is hardly a strong evidence for that since even under these high overexpressed conditions the colocalization is still limited. Did authors try to overexpress (with TrkB) other recycling edosome Rabs, such as Rab11, Rab14, Rab10. Finally, Rab10 is related to Rab11, which is a well-established regulator of endocytic sorting and recycling. Does Rab10 colocalize with Rab11 (I suspect it will)? Are these really novel organelles or they are simply Rab11-recycling endosomes that also contains Rab10 .4) It is not clear why authors picked to analyze Kif5b and Kif13B. There are several other kinesins that were implicated in axonal transport. Why authors chose not to study them?5) The change in Kif13B and Kif5B intensity in response to BDNF is very moderate at best. It does not help that authors do not show validation of anti-Kif5B or anti-Kif13B antibodies. It would also be good to test whether Kif13B or Kif5B knock-down affects TrkB transport.6) IP experiment shown in Figure 6G-H is not very convincing. First, since authors used anti-Kif13B antibodies for immunofluorescent microscopy, why they did not blot for endogenous Kif13B co-precipitation with HA-Rab10. Second, it appears that there is more HA-Rab10 precipitated in BDNF-treated samples, thus, it is unclear whether slight increase in GFP-Kif13B co-precipitation is due to that rather than increased interaction with Rab10. Third, why there is some GFP-Kif13B in sample that does not express HA-Rab10?7) Figure 7. Dominant-negative Rab mutants causes numerous non-specific effects. Since authors has Rab10 shRNA, all recycling experiments need to be done in Rab10-KD cells. Additionally, it is very puzzling that trapping TrkB in early endosomes did not affect recycling. Most plasma membrane receptors recycle by sequential transport from early endosomes to recycling endosomes. Consequently, I would expect that trappin TrkB in early endosomes would decrease its recycling.Reviewer #3 (Recommendations for the authors):Points #3, 5 and 6 of reviewer 2 seem very important.The functional mechanism of Rab10 in BDNF signaling still remains a bit fuzzy since the mobilization of Rab10 in the axon terminal in response to BDNF is counterintuitive to explain the increased signaling to the soma, at least in the absence of further mechanistic insight. The model in Figure 8a thus remains speculative. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]eLife.We want to thank you and the Reviewers for their positive comments and very insightful suggestions with regard to our manuscript titled “Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport”. Their advice has greatly helped us to clarify the key messages of our manuscript and prompted us to perform additional experiments to make our work more impactful and interesting for the readership of We appreciate your kind words while summarising the importance of our work: “defining how neurons respond to spatial extrinsic cues, such as neurotrophins, and relay this information longdistance to influence transcriptional events is an important topic in neurobiology”. This showed us that the main message of our work has been well recognised and valued. Moreover, the Reviewers explicitly mentioned what for us is the main focus of this work: “to report a role for Rab10 (…) in propagating the BDNF signal from axon terminals to the soma” and “that Rab10 is also required for retrograde arrival of signalling endosomes in the soma and subsequent signalling”. Reviewer #2 adds that “the most interesting finding is that Rab10-positive organelles show increased anterograde transport in response to BDNF and are not part of the TrkB-positive retrogradely moving signalling endosome itself”, whilst Reviewer #1 recognises the quality of “the imaging analyses, specifically the super-resolution and live imaging”.However, there is also consensus regarding the need of a better characterisation of the specific role of Rab10 in the system and the mechanism involving KIF13B in the anterograde transport of Rab10 organelles. In this amended version of the manuscript, we have added a new figure documenting the efficacy of the Rab10 knock down, the effects of its depletion in cell survival and TrkB expression . Moreover, we have expanded our analysis of the BDNFdependent interaction between Rab10 and KIF13B , as well as provided supplementary videos and representative data to support our observations. This new evidence has been carefully calibrated to keep in focus what the Reviewers have also recognised as the key message of our paper: the novel role of BDNF-regulated axonal transport of Rab10 organelles to propagate neurotrophic signalling from the axon to the nucleus of central neurons.We address below the specific points raised by the Reviewers:1. Reviewer #1 points out that while we see ”little co-localization between endogenous Rab10 and TrkB (…), the conclusion that Rab10 and TrkB are retrogradely co-transported is based on Rab10 over-expression, which raises concerns about the specificity of the interactions and artifacts related to over-expressing a Rab GTPase”. We agree with the reviewer regarding the fact that endogenous TrkB and Rab10 are unlikely to be retrogradely co-transported in the same organelle, as they correctly conclude from Figure 3e-f. Our data suggest that there is a transient association between Rab10 and the endosome containing TrkB. Therefore, we overexpressed wild-type Rab10 to create an experimental situation in which this transient association is stabilised long enough to be observed as events of co-transport. The reviewer suggested that we should “use additional measures to support that Rab10 associates/co-migrates with TrkB, for example by biochemical methods, or by performing live imaging of co-transport in neurons transfected with Rab10-GFP but with endogenous Rab10 knocked down to mitigate the OE concern”. As mentioned in our manuscript, we obtained independent evidence of the association of Rab10 with neurotrophin signalling endosomes in our early work describing a quantitative proteomic analysis of these organelles using an affinity purification approach in mouse motor neurons (see reference 18). Consistently with the conclusions of this work, we found that Rab10 is not accumulated on the surface of these organelles during signalling endosome maturation, as found for Rab7, further supporting the view that Rab10 interacts transiently with these organelles . Furthermore, transfection of Rab10-EGFP, even when endogenous Rab10 is knocked down, results in eight fold increase on its expression levels; additionally, biochemistry may provide inconclusive results, as it will fail to reveal dynamic interactions. For these reasons, we have provided as an additional proof of principle of the association of Rab10 to TrkB-positive endosomes, an example of a stationary TrkB-organelle which transiently accumulates Rab10-EGFP for few seconds, just before the onset of processive retrograde transport . We are actively investigating the mechanism regulating the onset of motion in these organelles, and the molecular events leading to TrkB sorting into retrograde signalling endosomes.2. Reviewer #1 also indicates that “other than P-CREB immunofluorescence experiments, the authors do not provide data to show the functional effects of Rab10 depletion on BDNF-mediated trophic effects”. While the complexity of retrograde trophic effects of BDNF are well documented [see references 1-3], we have decided to use levels of phosphorylated CREB (pCREB) in the nucleus since activation of this transcription factor constitutes the first node in which the three canonical signalling pathways of TrkB, PI3K-Akt, MAPKs and PLC-γ, converge [see reference 10]. Additionally, the majority of early genes regulated by BDNF depend on the phosphorylation of CREB at serine 133 to increase their expression [see references 19 and 31], making this node a relevant proxy for BDNF signalling and gene response. We have used the signalling readout as a complement to the accumulation of the internalised receptor itself, strengthening the physiological relevance of Rab10-depletion on TrkB transport. The reviewer also makes a very interesting point when focusing on discussion of Rab10 knockdown effect in morphology and survival. They advise that “these data are necessary to strengthen the conclusion that Rab10 is necessary to mediate BDNF-dependent trophic signaling”, adding that we should “assess whether the Rab10 kd or expression of Rab10 DN has general effects on neuronal viability or affect total TrkB levels, and not specifically on retrograde transport of axonally-derived TrkB receptors”. We thank the reviewer for raising these interesting issues. We have now included a new Figure 1, in which we analyse the changes in neuronal density after 24 and 48 hours of Rab10 knockdown. We showed that after 48 hours, there is a significant decrease in survival , fully justifying our decision of performing this set of experiments between 18 and 24 hours. Importantly, we showed that the levels of endogenous TrkB remain stable even at 48 hours of Rab10 knockdown , ruling out the depletion of TrkB as a potential confounding factor for the decreased pCREB levels and retrograde TrkB. Please also see point 4 for a discussion about the effects of Rab10 depletion on neuronal morphology. In addition, we followed the suggestion of the reviewer to include “important controls to show the specificity of Rab antibodies, and the knockdown of Rab10 using shRNA. In Figure 1f, the Rab10 levels (assessed by Rab10 immunofluorescence) appear to not be significantly affected by the kd or rescue, which raises questions about the antibody specificity, as well as efficacy of the kd”. In this regard, we have validated the significant decrease of endogenous Rab10 after 24 and 48 hours of treatment with doxycycline , further optimising our immunofluorescence protocols at the same time.3. Another interesting point raised by reviewer #1 focused on what happens to the relative concentration of Rab10 and TrkB in the axon. With regard to TrkB, the reviewer points out that: “In Figure 1c (now 2c), the authors show reduced accumulation of axon-derived TrkB receptors in neurons expressing Rab10 shRNA. Images of the axons should be included to assess whether the TrkB receptors accumulate in distal axons, as proposed in the model”. However, no accumulation of TrkB in the periphery is expected upon Rab10 knockdown. Indeed, the population of receptors undergoing retrograde transport is believed to be only a fraction of the total internalised TrkB, with the majority of the receptor engaging in local signalling and recycling back to the plasma membrane [see reference 5]. Moreover, anterograde flux of TrkB in hippocampal neurons has been shown to be regulated by retrograde BDNF signalling ; therefore, Rab10 knockdown is also expected to decrease the anterograde delivery of TrkB, thus halting any potential distal accumulation of this receptor. We have incorporated these considerations into the model shown in Figure 8a and the Discussion.Regarding the distribution of Rab10, the reviewer suggests that “it is interesting that BDNF enhances anterograde Rab10 transport based on live imaging. Additional analyses are needed to strengthen the conclusion that Rab10 is enriched in distal axons and to define the mechanisms (phosphorylation?) underlying Rab10 recruitment/retention”. At this point it is important to emphasise that our model does not predict an absolute distal enrichment of Rab10 upon BDNF stimulation either, but a dynamic readjustment on demand of the availability of Rab10 at the site of endocytosis. At any given point, Rab10 carriers are moving anterogradely and retrogradely along the axon. This balance will be transiently shifted towards retrograde or anterograde upon changes in ligand concentration; therefore, the increase in the anterograde bias will proceed only until the demand has been met and it is very unlikely that it results in significant accumulation of Rab10 under physiological conditions. This is precisely why this phenomenon is best appreciated when we perform longitudinal live-cell imaging of axons before and after BDNF stimulation . Regarding the mechanism regulating Rab10 direction bias, we agree with the reviewer about phosphorylation potentially playing a crucial role. We have included this possibility in the discussion, and we have focused our experiments in providing evidence that KIF13B recruitment to Rab10 organelles is increased by BDNF. Following the suggestion of the reviewer that “additional analyses to define mechanisms underlying Rab10 recruitment” and that “the imaging analyses of Kif13B could be complemented with biochemical methods to show BDNF-induced association with Rab10”, we have performed co-immunoprecipitation experiments to show that BDNF significantly increases the interaction between Rab10 and KIF13B . In line with our interpretation of the imaging analysis in axons, these biochemical data suggest that the effect of BDNF in the recruitment of KIF13B to Rab10 organelles is robust and provides a plausible mechanism for the increase in the anterograde transport of these organelles.4. Along the same line, both reviewers point out that “the anterograde motor Kif13B is suggested to be involved in BDNF-induced anterograde transport of Rab10 based on confocal microscopy analyses in fixed neurons. However, this aspect is not fully developed. The authors should determine if Kif13B directly interacts with Rab10 and the necessity of Kif13B for anterograde Rab10 transport, retrograde transport of TrkB receptors or BDNF-mediated trophic effects” and “the data with kinesins is not adding much to the understanding of the pathway. No interference with any kinesin is performed”. While the main message of our manuscript is unravelling a new role for axonal Rab10 organelles as dynamic regulators of retrograde TrkB trafficking and signalling, in this amended version of our manuscript, we have presented evidence showing that one specific kinesin, KIF13B, is preferentially recruited to Rab10 organelles upon BDNF stimulation. In stark contrast, the interaction of KIF5B, one of the most abundant axonal kinesins, remains unaffected. We have also responded to the request of confirming the BDNF-mediated interaction between Rab10 and KIF13B with a new set of biochemical experiments .Although we considered the idea of testing the effects of overexpressing the C-terminus of KIF13B lacking the first 441 residues (KIF13B DN), which includes the motor domain, we reasoned that these experiments may be particularly difficult to adequately control, given that KIF13B has a major role in establishing neuronal polarity [see reference 59]. Our preliminary experiments indeed confirm that the expression of KIF13B DN causes a decrease in morphological complexity that is qualitatively similar to the knockdown of Rab10 see , further5. Another aspect about which both reviewers had interesting insights and suggestions was the evidence about the role of Rab10 in sorting of TrkB from early endosomes. Reviewer #1 indicates that “the suggestion that Rab10 regulates "sorting" of internalized TrkB receptors is very interesting. The authors show nicely  that dominant negative Rab10 (rab10DN) does not disrupt TrkB recycling in axons, but increases the co-localization of TrkB with Rab5. The images in Figure 6d [now Figure 7d] also show that Rab5 domains appear to be larger in the Rab10 DN expressing neurons, which is consistent with early endosomes being perturbed. However, additional analyses are needed to define how exactly Rab10 coordinates with Rab5 and Rab7 in "handing off" TrkB from early endosomes to late endosomes in axons” and then adds the following questions: “Which axonal segments were imaged in the analyses in Figure 6? Was this in the distal axons? What happens to TrkB flux in mid-axons and retrograde accumulation in cell bodies in the Rab10 DN or shRNA expressing neurons? Does expression of Rab10 shRNA or Rab10 DN decrease co-localization of retrogradely transported TrkB with Rab7?”. In the same line, reviewer #2 adds that “there is a lot of conjecture about Rab10 vesicles delivering machinery to the distal axon early endosome, but no experiment to address this. Where is TrkB in Rab10 interference conditions? It seems to be stuck with Rab5 and not sorting back to the axonal plasma membrane or into retrograde carriers. Is it accumulating in distal axon tips? It would be good to have live imaging of TrkB in Rab10-DN conditions along axons. The recycling experiments  are very interesting, but it looks like this is happening along the axon shaft. Is this correct?”. We thank to the Reviewers for these comments. Indeed, the confocal images of both experiments shown in Figure 7 show that the expression of dominant-negative Rab10 results in enlarged Rab5-positive early endosomes accumulating internalised TrkB receptor, without affecting its internalisation rate nor its recycling to the plasma membrane. In light of these results, we propose that the increase of TrkB in early endosomes accounts for a specific deficits in its sorting to retrograde signalling endosomes. These experiments were done in mass cultures and mid-axons were imaged, since they represented the more isolated segments allowing analysis at the level of individual axons, something that is rarely possible in microfluidic chambers, where axons are bundled together within microgrooves. BDNF was added to the media in these cultures, therefore, both internalisation and recycling are observed along the entire axon and not uniquely at axon terminals. Interestingly, it is under these same exact conditions that the live-cell imaging of Rab10EGFP  has been done, showing that the BDNF-induced increase in net anterograde transport is not a consequence of a gradient of ligand, but an intrinsic feature of the system which we propose is the result from an increased interaction of Rab10 organelles with the anterograde motor KIF13B. In our model , we have integrated the fact that the main source of BDNF in the nervous system are synaptic targets; therefore, this mechanism would operate mobilising Rab10 organelles towards nerve terminals or retaining them at TrkB internalisation hotspots until the system reaches equilibrium.3), which is crucial for the regulation of Akt signalling and endosomal maturation [see reference 58]. In Drosophila, the orthologue of KIF13B, Khc-73, has been also involved as a regulator of BMP retrograde signalling carriers [see reference 61]. As reviewer #2 pointed out , a more detailed and unbiased analysis of the composition of the pool of anterograde Rab10-positive organelles is warranted to identify specific components of the sorting machinery.Regarding the hypothesis that Rab10 organelles deliver molecules essential for sorting to the distal axon, it is important to notice that Rab10 has been shown to recruit TBC-2 to the membrane of early endosomes, thus regulating the sorting of cargoes towards basolateral recycling [see reference 57]. Another additional example is the cooperation between Rab10 and Rab5 for the sorting of dense core vesicles in the Golgi apparatus . Interestingly, one of the best characterised roles of KIF13B is the anterograde transport of phosphoinositide-3,4,5-triphosphate (PIP“handing off”) fits with the almost complete exclusion between Rab10 and Rab7 that we show in Figure 3a-b. However, it is important to note that not all retrograde signalling carriers are Rab7-positive, and signalling endosomes have been shown to be heterogenous in nature, including Rab5-positive compartments moving in a less processive manner in the retrograde direction and carrying signalling molecules for long distances [see references 12 and 21]. Indeed, neurons expressing dominant-negative Rab10 display less processive retrograde transport of internalised TrkB compared to neurons transduced with wildtype Rab10 , and a significant decrease in retrograde TrkB delivered to the soma . The transition between a Rab5-Rab10 hybrid domain to Rab7-positive membranes  to isolate organelles containing this cargo from mouse stem cells-derived motor neurons after 10, 30 and 60 minutes of endocytosis. Whilst organelles isolated at early time points are enriched in endocytic and early endosomal markers, fractions isolated at later time points are positive for late endosomal markers. Hence, the abundance of Rab7 increase steadily at 30 and 60 min as shown in the figure below  but now is not in the retrograde carriers. Can you discuss?”. In this work, we have used the binding fragment of tetanus toxin , Rab10-EGFP live-cell imaging have been done in mass culture, so BDNF signalling can be acting along the entire axon. However, preliminary experiments in microfluidic chambers have shown that the same change in the direction bias occur when BDNF is added only to the terminals, suggesting that retrograde propagation of BDNF signalling is sufficient to inform Rab10-positive organelles in transit. Whilst the exact mechanism is not yet clear, coupling or merge of Rab10 organelles may facilitate the anterograde mobilisation of this compartment, as exemplified by the new Supplementary video 2, where a small stationary Rab10-positive organelle merge with a larger mobile compartment and the resulting Rab10positive combined compartment continues its anterograde motion.C. elegans it has been shown that Rab10 and Arf6 reside in the same subset of organelles. Whether concentration of p75 receptor in different domains of the axon is regulated by Rab10 organelles is an interesting possibility that we will be excited to explore in the future.9. Reviewer #2 also asked the following questions: “Is p75 not in a complex with TrkB after BDNF binding? What is the interpretation/implication of p75 being in different carriers in the axon? I find this a very interesting observation, but the relevance is not explained or further explored”. We thank the reviewer for highlighting this interesting aspect of the previous Figure 4. Whereas it is not entirely surprising that TrkB and p75 can be found in different organelles given their differential mechanisms of internalisation and post-endocytic dynamics; the presence of p75 alone in anterograde Rab10 carriers results intriguing. Bruce Carter’s group has previously shown that p75 is mobilised to the plasma membrane following TrkA activation with NGF in sympathetic neurons, which results in stronger TrkA signalling capabilities [see reference 55]. They show that this mobilisation of p75 depends on the activation of Arf6. Interestingly, in 10. Reviewer 2# has pointed out that “all the discussion of LRRK2 and phosphorylation is besides the point since the authors do not test involvement of this regulatory mechanism”. We agree with the reviewer, and therefore, the discussion of this aspects has been summarised and focused on Rab10-phosphorylarion as a potential regulatory mechanism for the recruitment of motors and other effectors.[Editors’ note: what follows is the authors’ response to the second round of review.]eLife.We thank you and the reviewers for their positive comments and very insightful suggestions during the second round of revisions of our manuscript titled “Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport”. We were delighted to learn that Reviewer #1 concerns were fully satisfied, and very grateful for the advice from Reviewers #2 and #3, who not only recognised the quality and interest of our work, but also helped us to further distil the key messages of our manuscript. Their comments prompted us to add extra controls and attempt new experiments, which made our work more robust and interesting for the readership of Three main questions persisted from Reviewers #2 and #3; they are all addressed in this resubmission. The first concern was about the nature of the Rab10 axonal compartments, and whether they correspond to previously described recycling endosomes regulated by other Rab GTPases; the other two questions both focussed on the specificity of KIF13B as the kinesin mobilising anterograde axonal Rab10. In this revised version, we present novel evidence addressing these matters and other minor points raised by the Reviewers. At the same time, we were pleased to see that none of the questions concerned the key findings of our work: a novel mechanism by which BDNF regulates Rab10 anterograde delivery, and Rab10 mediates sorting of internalised TrkB to the retrograde axonal transport route.Specifically,1. Reviewers #2 and #3 agreed when saying that “the fact that there is little colocalization between Rab10 and TrkB needs further characterization”. Additionally, Reviewer #2 suggested an alternative interpretation of our data suggesting a model prescinding of a transient association of TrkB and Rab10 in axons. In figure 3, we show both by quantitative confocal microscopy (figure 3e) and by using super-resolution radial fluctuations (figure 3f) that after 30, 60 and 90 minutes upon BDNF treatment, endogenous retrograde TrkB and Rab10 partially co-localise, and this colocalisation does not increase with time. We also showed that this association can be stabilised if both proteins are overexpressed, so it can be also observed as events of retrograde co-transport (figure 4a-d). Moreover, in the last figure we documented an example where, even under overexpression conditions, the association between a TrkB-containing signalling endosome and Rab10 was still transient and linked to the processivity of the organelle (figure 8b). It is also noteworthy that Rab10 knock-down caused a significant decrease of retrograde transport of endogenous TrkB (figure 2d-e), further stressing the functional relevance of this transient association. Promted by the reviewers asking for additional proofs of this association, we attempted an unbiased proteomic approach based on the immunoisolation of Rab10-positive membranes from primary cortical neurons by using antibody-conjugated magnetic beads followed by mass spectrometry. Using this approach, we confirmed that TrkB is present in Rab10containing membranes. We are currently optimising the immunoisolation approach, and the full results will be made available in a follow-up study. Thus, we have used four different experimental setups to confirm that TrkB transiently associates with Rab10-containing organelles.J Cell Physiol, 233, 6483-94. https://doi.org/10.1002/jcp.26503].2. Along the same lines, Reviewers #2 and #3 pointed that the low level of co-localisation “is not consistent with "a new Rab10 organelle" that mediates TrkB sorting and retrograde transport”. We fully agree with this conclusion and we find very important to clarify that what we have called “Rab10 organelles” can be more precisely described as membrane domains enriched in Rab10, which eventually interact with the endo-lysosomal system. The use of a more precise description of the compartment is particularly important in light of the diverse distribution of Rab10 in multiple organelles and membrane subdomains that has been observed in different cell types . However, there are several reasons why it is unlikely that Rab10-positive compartments we observe moving in the axon are Rab11-containing recycling endosomes. First, Rab11 organelles have been shown to be infrequent in the axon of hippocampal neurons, while we and others observe a large number of Rab10-positive axonal puncta. Second, blocking Rab10 function by overexpressing a dominant negative mutant (T23N) did not alter TrkB recycling in the axon. However, we fully agree with the reviewers regarding the need of a better characterisation of the Rab GTPases that are present in the Rab10-positive axonal membranes. Our unbiased proteomic characterisation of immunoisolated Rab10 compartments revealed the presence of more than 30 different Rabs, including those associated with the endolysosomal system . Importantly, because our starting material for immunoisolation was not pure axonal membranes, we decided to assess the co-localisation of endogenous Rab10 and other relevant Rabs by immunofluorescence. Confocal microscopy analyses confirmed a significant increase of Rab10 on Rab5 domains upon stimulation with BDNF, which has been added to the revised figure 3 (panel c).We, therefore, have changed all the references to “Rab10 organelles” into “Rab10 compartments”, which better reflects the potentially heterogeneous nature of the Rab10-containing membranes in the axon. Regarding this aspect, Reviewers also pointed to Rab10 being related to Rab11 and recycling, so they suggest to “test other recycling endosome Rabs, such as Rab11, Rab14, Rab10 to determine if these are really novel organelles or simply Rab11-recycling endosomes that also contain Rab10”. Although Rab10 and Rab11 do not belong to structurally related groups of Rabs, they have been shown to converge in a sub-class of recycling endosomes ; hence, the effects of KIF5B knock down will be necessarily complex and not only affect Rab10 trafficking. Similarly, KIF13B is crucial for establishing and maintaining neuronal polarity; therefore, knocking it down would also disrupt axon integrity . Previously, we presented the results of a pilot experiment, in which we attempted to use a KIF13B mutant lacking the motor domain, which yielded a significant decrease in neurite complexity, emphasising the need to develop new tools to further explore this point. Instead, we attempted this experiment for a different reason: we wondered which interactors of Rab10 mediate its differential direction bias in response to BDNF. Since their interaction is regulated by phosphorylation, we initially focussed on the adaptors JIP3 and JIP4; however, we found little co-localisation between JIP3, JIP4 and Rab10, as documented in the supplementary material. We then looked at KIF13B because it was shown to preferentially interact with GTP-bound Rab10, and it is highly expressed in the axon of hippocampal neurons . KIF5B was chosen as control, because it is also robustly expressed in the axon of hippocampal neurons but lacks a binding site for Rab10. It is relevant to mention that, in contrast to Reviewer #2’s views, stating that “the change in Kif13B and Kif5B intensity in response to BDNF is very moderate at best”, we don’t focus on the change of intensity of the KIFs in the axon, but on their recruitment to Rab10positive domains. While KIF13B increased in Rab10 compartments compared to the rest of the cytoplasm, KIF5B displayed the opposite behaviour (figure 6b-e). As now explained in the Discussion, it is possible that this is not the only determinant of Rab10 direction bias, but the contribution of KIF13B would be enough to explain the BDNF-dependent changes of direction, providing a lead for further studies on the detailed mechanism controlling Rab10 dynamics.3. Reviewer #2 raised questions about the reasons for studying KIF5B and KIF13B. Together with Reviewer #3, they suggested to analyse the effects of kinesin down-regulation on the transport of TrkB. In this regard, it is important to mention that we have not aimed to study direct effects of these or other kinesins on the axonal transport of TrkB, but to propose a mechanism for the BDNF-dependent regulation of Rab10 direction bias, the dynamics of which is, in turn, crucial to fine-tune TrkB sorting from early endosomes. However, it is important to notice that these kinesins have many other roles and, therefore, the prediction is that knocking them down will cause disruption of axonal dynamics of multiple cargoes. For example, anterograde transport of TrkB has been shown to be regulated by kinesin 1 . If retrograde and recycling pools of TrkB were differentially regulated in early endosomes, it is expected that blocking the sorting to retrograde transport would increase colocalisation of TrkB and Rab5, with no increase on recycling. Several lines of evidence indicate the potential of Rab10 and its effectors to define membrane sub-domains important for cargo selection and regulation of membrane curvature, budding and formation of tubules. We have included and discussed this literature in the manuscript, as well as rephrased any ambiguity on the interpretation of these experiment.6. Reviewer #2 also raised the potential confounding effects of the Rab10 DN, which they suggested to mitigate by repeating all recycling experiments in neurons treated with shRNA for Rab10 . Far from showing confounding effects, our results revealed no effect of Rab10 DN in recycling of TrkB in axons. However, expression of Rab10 DN effectively induced an increase on TrkB/Rab5 colocalisation in axons, which is consistent with the reduction on retrograde accumulation observed in Rab10-KD neurons. On the same issue, Reviewer #2 argues that “it is very puzzling that trapping TrkB in early endosomes did not affect recycling. Most plasma membrane receptors recycle by sequential transport from early endosomes to recycling endosomes. Consequently, I would expect that trapping TrkB in early endosomes would decrease its recycling” We apologise for the lack of clarity explaining our model. The key assumption in this point of the reviewer is that TrkB receptors located in the early endosome are homogeneously distributed and equally available to be destined either to local recycling or retrograde trafficking. However, the sorting endosome is rich in membrane subdomains and this heterogeneity has been hypothesised to be crucial for cargo selection and trafficking control [Redpath, G.M.I, Betzler, V.M, Rosatti, P. and Rossy, J., (2020). Membrane Heterogeneity Controls Cellular Endocyctic Trafficking. Front. 7. Finally, Reviewer #1 also provided very useful comments about the nomenclature of Rab10 constructs, a couple of missing resources in the table and asked for clarification about a reference. We have amended the manuscript accordingly.