Neuroimaging has revealed robust interactions between the prefrontal cortex and the hippocampus when people stop memory retrieval. Efforts to stop retrieval can arise when people encounter reminders to unpleasant thoughts they prefer not to think about. Retrieval stopping suppresses hippocampal and amygdala activity, especially when cues elicit aversive memory intrusions, via a broad inhibitory control capacity enabling prepotent response suppression. Repeated retrieval stopping reduces intrusions of unpleasant memories and diminishes their affective tone, outcomes resembling those achieved by the extinction of conditioned emotional responses. Despite this resemblance, the role of inhibitory fronto-hippocampal interactions and retrieval stopping broadly in extinction has received little attention. Here we integrate human and animal research on extinction and retrieval stopping. We argue that reconceptualising extinction to integrate mnemonic inhibitory control with learning would yield a greater understanding of extinction’s relevance to mental health. We hypothesize that fear extinction spontaneously engages retrieval stopping across species, and that controlled suppression of hippocampal and amygdala activity by the prefrontal cortex reduces fearful thoughts. Moreover, we argue that retrieval stopping recruits extinction circuitry to achieve affect regulation, linking extinction to how humans cope with intrusive thoughts. We discuss novel hypotheses derived from this theoretical synthesis. Thoughts and emotions, like actions, often need to be controlled. For example, when reminded of an unpleasant event, people often try to reduce distress by limiting awareness of the unwanted memory. Attempts to limit awareness are often achieved by retrieval stopping, an intentional effort to terminate the retrieval of content elicited by the reminder. Over the last two decades, evidence has mounted that the prefrontal cortex plays a key role in retrieval stopping. Retrieval stopping recruits a prefrontally-mediated inhibitory control mechanism that, over repeated exposures to reminders, suppresses unwanted traces, rendering them less intrusive, less unpleasant, and less likely to be retrieved –3. RetriIn the last two decades, knowledge about the neural mechanisms of fear extinction also has rapidly expanded –13. ExtiOur central thesis asserts that extinction recruits prefrontally-mediated inhibitory control mechanisms to suppress retrieval, contributing to the affective adaptations normally observed. Consider an example of retrieval suppression, reported by an acquaintance of the first author. One afternoon, the acquaintance was driving down a pleasant residential street when a boy ran out from behind a bush to retrieve a ball before it rolled into the road. Panicking, the friend nearly wrecked his car, even though there was no danger of hitting the boy. He overreacted because the boy’s abrupt appearance reminded him of roadside bomb attacks from his time in the Iraq war. In the ensuing seconds, he suppressed the unpleasant reminding, enabling him to calm down and resume driving. After being home from Iraq for several months, situations like this rarely reminded him of roadside attacks. This illustrates how cues can elicit unwelcome memories automatically, and how important it is to have the ability to stop such intrusions in the immediate and longer-term.This incident illustrates the kind of example researchers have in mind when arguing for extinction’s relevance to post-traumatic stress disorder (PTSD). Yet, a key aspect of the person’s coping behavior that goes unacknowledged in the extinction analogy is their effort to control retrieval of the intrusive thought through retrieval stopping. The person not only needed to adjust their emotional state, but also how their memories responded to environmental cues, a situation referred to as a memory adaptation problem . If peopTo illustrate the analogy, consider the structural similarities between action and retrieval stopping on the one hand, and extinction on the other Fig. . During The extinction of conditioned fear resembles action and retrieval stopping. On each trial, the person attends to a stimulus that has become associated to a threat (the conditioned stimulus), leading them to experience fear, and make defensive responses . On initial trials, these responses are appropriate, given the prior pairing of the conditioned and unconditioned (aversive) stimuli. But repeated exposures without the unconditioned stimulus make it clear that the conditioned stimulus no longer predicts threat. Correspondingly, the fear reaction ceases to be useful and so it declines. The formal similarity to stopping is unmistakable: in extinction learning, as with action and retrieval stopping, repeated exposures to a cue reduce its tendency to elicit the associated response, under conditions designed to communicate that the response is not needed. Extinction differs from stopping, however, in the presumed intentionality of the process reducing the response. Whereas retrieval and action stopping are accompanied by an explicit goal to terminate retrieval or action production, in extinction, no stopping instruction occurs; rather, any decision about the wisdom of stopping the emotional response, if it occurs, is left to the individual, based on their detection, over exposures to the stimulus, of a decrease in the likelihood of threat.Some differences among these three situations are more apparent than real. For example, because extinction training provides no overt stopping cue, declines in emotional responding with repetition are easily construed as reflecting an automatic learning process that incrementally adjusts the cue-response association. This interpretation may seem desirable because extinction arises in animals, for which many might be reticent to attribute intentionality; indeed, extinction in invertebrates indicates that some cases of extinction may reflect automatic processes. But all manifestations of extinction need not be painted with the same brush, simply because stopping is not signaled by the task. Absence of stopping cues is a poor reason to assume intentional stopping does not occur. Overt stopping cues rarely exist when action or retrieval stopping occurs “in the real world”; rather, the impetus to stop comes from changes detected by the organism. Stopping oneself from catching a hot pot is initiated by perceptual feedback indicating a threatening outcome; and stopping retrieval of an unwelcome thought begins when we detect a memory or thought that threatens our emotional state. Gradual declines in either conditioned responding or intrusive thoughts may reflect increasing certainty that these responses are no longer relevant and greater willingness to stop them. Under natural conditions, then, extinction and stopping do not intrinsically differ in overt cuing or their apparent intentionality.Extinction also appears to differ in the content that is controlled, with a strong focus on fear and threat responses. Here again, these differences are more apparent than real. For example, as the example of the Iraq war veteran suggests, episodic memories for conditioning experiences may accompany affective learning and be elicited by conditioned stimuli, at least for mammals with intact hippocampi. Indeed, in human participants, fear conditioning can lead people to develop intrusive images as conditioned responses –19, consGiven the foregoing parallels, it becomes important to consider whether retrieval suppression contributes to extinguishing conditioned fear. In our retrieval stopping model, we introduce several assumptions that bridge these phenomena. First, we assume that organisms find fear aversive for several reasons. Fear responses  can interfere with rewarding or survival-relevant pursuits such as seeking food, shelter, or mates. Fear states also undermine or bias high-level processes, such as planning, decision making, and behavioral control that are important for pursuing goals , 22. FinBehavioral and imaging studies usually measure retrieval stopping with the Think/No-Think paradigm . This pResearch using the TNT procedure shows that people can stop retrieval. Several notable effects support this conclusion. First, retrieval stopping abolishes the benefits of reminders on memory, as reflected by often large differences between Think and No-Think items on delayed tests Fig. . Indeed,Beyond its effects on explicit memory, repeatedly presenting a cue and stopping retrieval gradually reduces the memory’s tendency to intrude and people’s affective responses to the suppressed content. Because intrusions are, by nature, involuntary retrievals yielding no observable behavior, they must be probed with self-reports. For example, many studies identify intrusions by asking, after each No-Think trial, whether the cue elicited the memory, despite participants’ efforts to stop retrieval. Studies often collect these reports during an fMRI or EEG protocol to determine whether they identify distinct neural mechanisms of the intrusion response . Remarkably, whereas participants commonly experience involuntary intrusions on early trials , their incidence declines robustly after repeated stopping, showing proportional reductions of nearly 50% , 33–38. fMRI studies have revealed contributions of the prefrontal cortex to retrieval stopping, and how this region modulates brain activity relating to memory and emotion. Here we summarize which prefrontal regions suppression engages, and under what conditions, and evidence for how it modulates the hippocampus and the amygdala.To isolate prefrontal regions involved in retrieval stopping, neuroimaging studies have scanned participants as they performed the Think/No-Think task. All trials in this task present a cue from a trained pair. Some trials ask participants to retrieve the paired item , whereas others cue them to stop retrieval , typically signaled by green or red colored task cues, respectively. Regions more activated during No-Think than Think trials reflect recruitment of stopping-related processes above and beyond processes involved in cue processing and retrieval, as reflected in increased blood-oxygenation-level-dependent signal . Retrieval stopping studies have examined word pairs, face-scene pairs, word-scene pairs, word-face, and word-object pairings of both neutral and negative valence , 3, 29. Retrieval stopping engages right-lateralized prefrontal regions, including dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex (VLPFC), posterior middle frontal gyrus (pMFG), and bilateral insula. Amongst these, the most extensive activations arise in right DLPFC, which often extend the anterior–posterior length of the MFG, in a region spanning Brodmann’s areas (BA) 9 and 46. Anteriorly, the right DLPFC activation usually extends into the posterior aspect of BA10, bordering 9/46. Indeed, DLPFC activations sometimes are restricted to this anterior BA9/46/10 area, suggesting it is a key DLPFC region of retrieval stopping ).Another benefit of the retrieval stopping model lies in its potential to forge stronger connections between translational neuroscience on extinction and the human psychiatric conditions it is intended to address. An uncomfortable and not always acknowledged disconnect exists between research on extinction and disorders such as PTSD and anxiety: Whereas extinction is thought to model how experience changes fear responses to conditioned stimuli, the real symptoms faced by patients also include intrusive memories, thoughts, and images, the content of which distress the patient , 147. InOne final implication of the retrieval stopping model concerns its consequences for clinical treatment. There is enthusiasm for the value of extinction learning as an experimental model of processes that might be deficient in clinical populations suffering from anxiety, PTSD and OCD , 148–150Although support for the retrieval stopping model is promising, further evidence is needed to evaluate its predictions, and to elaborate the mechanisms enabling the prefrontal cortex to regulate hippocampal activity. To encourage work on this hypothesis, we discuss areas of development in animal and human studies that could evaluate this model’s potential in translating foundational neuroscience to mental health.One area for development in animal research is to determine whether IL PFC exerts an inhibitory influence over hippocampal activity during extinction learning, and if so, whether it suppresses neurons representing the original conditioning event. Work by Lacagnina et al.  shows thFuture research also should compare retrieval stopping and extinction in humans more precisely, using imaging and behavioral experimentation. Is the rDLPFC region engaged during extinction also involved during retrieval stopping? Moreover, although the hippocampus shows reduced activity during extinction learning, as does retrieval stopping, we do not know whether these reductions are causally related to rDLPFC activity, an issue that could be examined with effective connectivity analyses. If the hippocampus is downregulated, does this disrupt memory for the conditioning events, as retrieval stopping predicts? Translational implications can also be examined. For example, if extinction spontaneously engages retrieval stopping, can the impact of extinction be improved by instructing people to stop retrieval during extinction trials, ensuring that it always contributes (cf. )? To whaWhen organisms stop actions, the result is visible to the naked eye; but when they stop thoughts or feelings, the outcome is not observable. This simple difference contributes to vastly different theoretical traditions in neuroscience relevant to stopping actions and thoughts. On one hand, the objective reality of response stopping has triggered a vast and detailed neurobiological account of how organisms stop actions, centered around constructs such as inhibitory control, which posit a dynamic, goal-related interruption of neural processes underlying action production. On the other hand, neuroscience paradigms such as extinction, are not conceptualized as stopping, even though incentives for organisms to stop memories and affect are ubiquitous. Rather, accounts of extinction focus on simple learning mechanisms applied incrementally over extinction trials. That conditioned emotional responses decrease gradually over unreinforced repetitions of a CS without special instructions or demands, in organisms simple and complex invites a basic learning account, especially given the understandable reticence to assign intentionality and control to animals. But if species such as mice and rats can engage the prefrontal cortex to stop their actions, why would they not be able to do so for affective and cognitive responses, especially when the persistence of those responses may be detrimental?Here we built a case for the value in reconceptualizing extinction as inhibitory control over memory. We argued that extinction naturally recruits retrieval stopping to downregulate activity in the hippocampus and amygdala during extinction trials and gradually adapts mnemonic responses to conditioned stimuli. In essence, extinction reflects the organism solving a memory adaptation problem . Accordi


Background. Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease (ILD). This prospective observational study aimed at the evaluation of any correlation between genetic variants associated with IPF susceptibility and high-resolution computed tomography (HRCT) patterns. It also aimed at evidencing any differences in the HRTC pattern between the familial and sporadic form at diagnosis and after two years. Methods. A total of 65 IPF patients (mean age at diagnosis 65 ± 10) were enrolled after having given written informed consent. HRCT and genetic evaluations were performed. Results. A total of 19 familial (mean age 62 ± 15) and 46 sporadic (mean age 70 ± 9) IPF patients were enrolled. A statistically significant difference was evidenced in the HRTC pattern at diagnosis between the two groups. Sporadic IPF patients had a predominantly usual interstitial pneumonia (UIP) pattern compared with those patients with familial IPF . Moreover, familial IPF patients had more alternative diagnoses than those with sporadic IPF . Furthermore, there was a slight increase in the typical UIP pattern in the familial IPF group at two years from diagnosis. Conclusions. Genetic factors play a pivotal role in the risk of developing IPF. However, further studies are required to clarify how these genetic factors may guide clinical treatment decisions. Idiopathic pulmonary fibrosis (IPF) is a progressive and almost invariably fatal interstitial lung disease (ILD), of unknown etiology, which is characterized by progressive fibrosis and loss of lung function ,2. EvideAs the likelihood that specific radiologic features reflect IPF is not absolute but depends on the clinical context, the high-resolution computed tomography (HRCT) pattern may be inconclusive, and clinical information may further improve HRCT scan interpretation ,10,11,12Several mutations have been associated with an increased risk of IPF, including those in genes for surfactant proteins , telomerase reverse transcriptase (TERT), the RNA component of telomerase (TERC), and TOLLIP and MUC5B, all of which play important roles in lung host defense ,32,33,34This study aimed at evidencing any correlations between genetic variants associated with IPF susceptibility and HRCT patterns and at determining any differences between familial and sporadic IPF. A total of 65 patients affected by familial and sporadic forms of IPF were enrolled in the study diagnosed according to the 2018 criteria  during rThe exclusion criteria were: subjects with insufficient clinical information  and lack of two-year follow-up. Patients with inadequate HRCT examinations, i.e., HRCT, who could not be evaluated due to motion artifacts were also excluded. HRCT scanning , 280–400 depending on patient size; and field of view, 40 cm. The HRCT scans were performed with a 256-row multidetector CT system , and the technical parameters were: rotation time, 270 ms; beam collimation, 128 × 2 × 0.625 mm; normalized pitch, 0.975; The images were analyzed with consensus by two senior experienced thoracic imaging radiologists (16 and 10 years of experience); any discrepancies in imaging interpretation were resolved by obtaining the opinion of a third radiologist with 20 years of experience. The CT images were analyzed at standard lung window settings (a window level of −600 HU and a window width of 1600 HU) on a picture archiving and communications system (PACS)-integrated workstation  ,12. Usual interstitial pneumonia (UIP): the presence of honeycombing in association with a reticular pattern with or without traction bronchiectasis/bronchiolectasis, with a subpleural and basal predominance;Probable UIP: the presence of a reticular pattern in association with traction bronchiectasis/bronchiolectasis without honeycombing and with a subpleural and basal predominance;Indeterminate UIP: the presence of subtle reticulation or ground-glass opacities, with a subpleural and basal predominance;Alternative diagnosis: the presence of findings suggestive of an alternative diagnosis included mosaic attenuation, predominant ground-glass opacities, consolidations or signs of fibrosis with an atypical distribution in the upper-mid lung ,10,11.The first task of the radiologists was to classify the HRCT pattern into four categories according to the Fleischner Society classification of fibrotic lung diseases: Then, radiologists evaluated a follow-up HRCT, performed two years after the first HRCT scan, to assess the current radiological pattern and whether it had remained unchanged, was stable or had worsened.DNA extraction and quantification of blood DNA was prepared by QIAamp Blood Mini kit . DNA quality was further evaluated by PCR analysis by the BIOMED 2 PCR multiplex protocol with PCR products analyzed by DNA 1000 Assay  on the Agilent 2100 Bioanalyzer on-chip electrophoresis , as previously described by Simbolo et al. . https://www.github.com/ekg/vcflib accessed on 31 Decembre 2018), SnpSift [Next-Generation Sequencing of Multiplex PCR Amplicons Ampliseq multigene custom panel was designed to explore all exons of ABCA3, DKC1, PARN, RTEL1, SFTPA2, SFTPC, TERC and the TERT genes. Two additional regions were added to investigate the putative promoter of MUC5B and TERT (chr11: 1241210-1241230 and chr5: 1295105-1297000). Twenty nanograms of DNA was used for multiplex PCR amplification, followed by ligation of a specific barcode sequence to each sample for identification. Emulsion PCR was performed to construct the libraries of clonal sequences using the Ion 27 OneTouchTM OT2 System (Life Technologies). The quality of the libraries obtained was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies). Sequencing was run on the Ion Torrent Personal Genome Machine  loaded with 318v2 chips. Data analysis, including alignment to the hg19 human reference genome and variant calling, was carried out by the Torrent Suite Software v.5.0 (Life Technologies). Filtered variants were annotated by a custom pipeline, based on vcflib . Descriptive statistics of patient demographics and clinical characteristics were reported as frequencies (proportions) for categorical variables and mean (standard deviation) (SD) for continuous variables. The two groups  were compared for age by the Student’s t-test, whilst the Chi-Squared test or Fischer Exact test was performed to identify differences in categorical variables . A The study population included 65 patients affected by IPF (mean age at diagnosis 65 ± 10), 19/65 the familial form (mean age at diagnosis 62 ± 15) and 46/65 the sporadic form (mean age at diagnosis 70 ± 9) see .(a)the age at onset of the disease was lower in patients with the familial form than in those with the sporadic form;(b)there was no significant difference in gender for the familial form, whilst there was a prevalence of males in the sporadic form;(c)moreover, no current smokers were observed in the group of patients affected by the familial form, whilst there were numerous current smokers in the sporadic form group.The analysis of the clinical characteristics of the two populations showed that:At diagnosis, there was a statistically significant difference in the HRTC pattern between the two groups: sporadic IPF patients had a predominantly typical UIP pattern compared to familial IPF patients . There was a predominance of an alternative diagnosis HRTC pattern in familial IPF compared to sporadic IPF  see . The HRCFurthermore, when we evaluated the baseline HRTC patterns versus HRCT patterns at 2 years from diagnosis (paired comparisons of patients in two different time points), both groups had similar percentages of patients with a worsened HRTC evaluation  see .p-value = 0.033). Furthermore, patients with SFTPC mutation had a frequent, but not statistically significant, indeterminate or alterative pattern . Rare mutations were observed in our study groups, as listed in tern see   patterns and any differences between familial and sporadic IPF on the basis of the HRCT with a long follow-up (2 years). Although the number of patients enrolled is small, our data do reflect those in international literature, i.e., patients with the familial form were younger at diagnosis, the sporadic form is more frequent in males and this form has a higher proportion of smokers ,41,42,43After 2 years, the lung HRCT pattern of most patients with genetic mutations moved towards a more typical UIP pattern. These observations lead us to postulate that the etiology and pathogenesis of IPF could change in the early phases of the disease according to the presence of specific driver mutations, but it becomes more similar when pulmonary fibrosis progresses to a more advanced stage. Thus, an integrated approach to patients with IPF combining genetic variants and HRCT scan patterns could help to better identify different natural history and clinical evolution, suggesting a need for more targeted treatment to ameliorate disease prognosis and outcomes. At the time of diagnosis and during the follow-up, an HRCT scan, performed and interpreted by an expert radiologist, still plays a key role. However, the likelihood that specific radiologic features reflect IPF is dependent on the clinical context, and according to our data, probably also by the genetic variants. Although both radiologists and pulmonologists can identify findings suggestive of a fibrosing ILD, both need the input of one from the other to improve the differential diagnosis process. When there is doubt regarding the diagnosis, an integrated approach is required and a working diagnosis may be appropriate. However, as new information pertinent to the patient’s diagnosis becomes available over time, it should be reviewed at regular intervals. Furthermore, genetic tests for common or rare mutations should be offered to patients with a history of familial pulmonary fibrosis to stratify disease course and risk. The genetic evaluation could be also helpful for estimating the risk of IPF for patients’ family members ,11,12,44Updated diagnostic guidelines for IPF will hopefully reflect the growing knowledge of genetic components of the disease in a way that ameliorates the clinical course and the therapeutic approach of these patients. In this study, we described the association between HRCT patterns and genetic variants in IPF. We confirmed the central role of HRCT scans in the diagnosis of IPF, and we demonstrated that HRTC evaluation may be enriched by genetic information to ameliorate the diagnosis, the follow-up and the therapeutic approach in this complex disease.


A repeat expansion in C9orf72 is the major cause of both frontotemporal dementia and amyotrophic lateral sclerosis, accounting for approximately 1 in 12 cases of either disease. The expansion is translated to produce five dipeptide repeat proteins (DPRs) which aggregate in patient brain and are toxic in numerous models, though the mechanisms underlying this toxicity are poorly understood. Recent studies highlight nucleocytoplasmic transport impairments as a potential mechanism underlying neurodegeneration in C9orf72-linked disease, although the contribution of DPRs to this remains unclear. We expressed DPRs in HeLa cells, in the absence of repeat RNA. Crucially, we expressed DPRs at repeat-lengths found in patients (> 1000 units), ensuring our findings were relevant to disease. Immunofluorescence imaging was used to investigate the impact of each DPR on the nucleus, nucleocytoplasmic transport machinery and TDP-43 localisation. DPRs impaired the structural integrity of the nucleus, causing nuclear membrane disruption and misshapen nuclei. Ran and RanGAP, two proteins required for nucleocytoplasmic transport, were also mislocalised in DPR-expressing cells. Furthermore, DPRs triggered mislocalisation of TDP-43 to the cytoplasm, and this occurred in the same cells as Ran and RanGAP mislocalisation, suggesting a potential link between DPRs, nucleocytoplasmic transport impairments and TDP-43 pathology. The expanded region of C9orf72 is transcribed to produce long, repetitive G4C2 RNA transcripts which form intraneuronal nuclear foci in patient brain and sequester RNA-binding proteins1. The repeat region is also translated via unconventional repeat-associated non-ATG translation to generate five distinct dipeptide repeat proteins (DPRs): glycine–alanine (GA), glycine–arginine (GR), proline–arginine (PR), alanine–proline (AP) and glycine–proline (GP). These peptides form inclusions in patient brain4 and are highly toxic in various cell culture models8, as well as in Drosophila10, zebrafish11 and mice16. However, the mechanisms underlying DPR toxicity are unknown.Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two devastating neurodegenerative diseases with considerable clinical, genetic and pathological overlap. A large G1. TDP-43 is mislocalised from the nucleus to cytoplasm where it becomes abnormally hyperphosphorylated and forms insoluble intraneuronal inclusions. TDP-43 pathology correlates well with neuronal loss in C9FTD/ALS, leading to the proposal of an “amyloid-like cascade” hypothesis of neurodegeneration, whereby the C9orf72 expansion produces RNA foci and DPRs which somehow trigger TDP-43 pathology and this causes neuronal death17. However, the link between RNA foci/DPRs and TDP-43 mislocalisation is not well established, and there are likely multiple mechanisms through which products of the C9orf72 expansion impair cellular function which converge to cause neurodegeneration in FTD/ALS.In addition to DPR pathology, the FTD/ALS-related protein TDP-43 (transactive response DNA-binding protein 43) also aggregates in C9orf72-positive patient brain22. These studies led to the novel hypothesis that the C9orf72 expansion causes neurodegeneration by disrupting nucleocytoplasmic transport, with a wide range of downstream consequences for cellular processes necessary for normal function and survival. However, the exact mechanisms underlying nucleocytoplasmic transport impairments in C9FTD/ALS remain unclear. Much of the literature published to date utilises models which express pure G4C2 repeat sequences, and therefore all five DPRs as well as G4C2 repeat RNA will be produced in those models, rendering it impossible to determine the specific cause of any observed phenotypes. Furthermore, those models which use alternative-codon sequences to express DPRs in the absence of G4C2 repeat RNA do so at extremely short repeat-lengths which are not found in patients. We have previously shown that repeat-length determines the impact of DPRs on cellular function and toxicity, with longer repeats causing more severe phenotypes11. As such, it is essential to model DPR pathology at repeat-lengths found in patients wherever technically possible, to ensure relevance to human disease.Recent literature has highlighted defects in nucleocytoplasmic transport as an important feature of C9FTD/ALS, with large-scale genetic screening studies identifying a number of key nucleocytoplasmic transport genes as modifiers of toxicity in fly and yeast models4C2 repeat-RNA, to investigate the specific impact of each dipeptide on the nuclear membrane and nucleocytoplasmic transport machinery. We also investigated the link between C9orf72 dipeptides and TDP-43, which is mislocalised to the cytoplasm in the brain of expansion-carrying patients. Crucially, we used previously characterised constructs8 to express each DPR at long repeat-lengths which are known to be found in patients (> 1000 units), ensuring our findings are relevant to human disease.Here we used alternative-codon sequences for expression of DPRs in the absence of G1020, GR1136, PR1100, AP1024 or empty pEGFP-N1 vector as a GFP-only control. Cells were stained with DAPI to visualise the nucleus and an antibody against lamin-B1, a key component of the lamin matrix which forms the nuclear membrane. A large proportion of cells containing GA, GR or PR inclusions had misshapen nuclei, indicating that DPRs cause structural abnormalities in the nucleus. Rather than forming smooth, rounded structures, nuclei were excessively folded to form “horseshoe” shapes or cause severe creasing in the nuclear membrane .Immunofluorescence imaging was performed on HeLa cells transfected with GAP = 0.0052), and 40.6% for PR (P = 0.0096) compared to 15.7% for GFP-only control (n = 3). Lamin B1 staining of cells containing GA, GR or PR inclusions demonstrated that the lamin matrix had a frayed appearance, suggesting that DPRs caused fragmentation of the nuclear membrane , 62.6% for GR (P = 0.0006), and 50.7% for PR (P = 0. 0071) compared to 22.2% for GFP-only control (n = 3). AP1024 did not cause structural damage to the nucleus, with only 22.5% of AP-transfected cells exhibiting misshapen nuclei (P = 0.6797) and 33.0% exhibiting nuclear membrane fragmentation .43.1% for GR (P = 0.0025), indicating that GR caused mislocalisation of Ran to the cytoplasm. Accumulation of Ran in the cytoplasm was also observed in cells containing GA inclusions, however this occurred less frequently (36.0% of cells) and was not statistically significant , although the severity of this phenotype varied between peptides with arginine-rich dipeptides causing RanGAP accumulation most frequently. The severity of RanGAP mislocalisation was also significantly different between most of the DPR treatments , 37.6% (P = 0.0011) and 29.5% (P = 0.0349) respectively. AP did not cause TDP-43 mislocalisation, with only 18.7% of staining contained within the cytoplasm . Statistical comparisons between each DPR treatment are shown in Table Since DPR expression caused both structural damage to the nucleus and nuclear membrane and disruption to key components of the system that powers nucleocytoplasmic transport, we next investigated whether TDP-43, a nuclear protein known to be mislocalised to the cytoplasm in C9FTD/ALS patient brain, was affected by DPR expression. HeLa cells were co-transfected with an expression construct for mCherry-tagged human TDP-43 and GFP-tagged DPRs or empty pEGFP-N1 control. Immunofluorescence imaging was used to determine whether DPR expression affected the nuclear localisation of TDP-43. Figure P = 0.0044; GR 81.9%, P = 0.0003; PR 71.5%, P = 0.0009), indicating that these two phenotypes occur alongside each other. The vast majority of cells exhibiting TDP-43 mislocalisation also exhibited granular RanGAP staining: 85.6% of cells for GA (P < 0.0001), GR 85.5% (P < 0.0001) and PR 82.1% (P < 0.0001). Therefore, both components of the Ran cycle were mislocalised in cells with DPR-induced TDP-43 mislocalisation to the cytoplasm, highlighting the possibility that defects in the nucleocytoplasmic machinery could be the connection between DPRs and TDP-43 pathology.In order to determine whether the mislocalisation of TDP-43 observed could be linked to DPR-induced disruption to the nucleocytoplasmic transport machinery, we next investigated whether these two things occurred together. Since not all cells containing DPR inclusions exhibited mislocalisation of either Ran, RanGAP or TDP-43, immunofluorescence imaging was performed to determine whether these phenotypes occurred in the same cells. HeLa cells were co-transfected with the mCherry-TDP-43 and DPR-GFP constructs, and a Cy5-conjugated antibody was used to visualise either Ran or RanGAP in these cells , and therefore it was not possible to determine whether this phenotype was caused by DPRs or repeat RNA23. Here, we demonstrate that breakdown of the nuclear membrane is caused by DPRs, specifically GA, GR and PR but not AP. Our finding is supported by a recent report that staining for both lamin-B1 and lamin-A/C was fragmented in a transgenic mouse expressing PR5013. Destabilisation of the lamin matrix which forms the nuclear membrane is likely to have detrimental consequences for cellular function and survival in a number of ways. Firstly, impaired structural integrity of the membrane is likely to impact the transport of proteins and other cargoes across it, leading to nucleocytoplasmic transport defects and perhaps breakdown of the barrier which maintains separation of the nuclear and cytoplasmic compartments. Secondly, lamins have several crucial functions besides supporting the structural integrity of the membrane, including roles in DNA repair, chromatin remodelling, transcription and cell migration25. Furthermore, loss of function mutations in lamins are known to trigger ER stress and activate the unfolded protein response, leading to premature apoptosis27. It is therefore possible that DPR-induced mislocalisation of lamins directly contributes to neurodegeneration in C9FTD/ALS.We have shown that DPRs cause structural damage to the nucleus and fragmentation of the nuclear membrane when expressed at disease-relevant repeat-lengths of over 1000 units. Nuclear membrane fragmentation was previously reported in two 28. Ran and RanGAP mislocalisation have been previously reported to occur in (G4C2)30 flies and human patient iPSC-derived neurons20, however, as with the nuclear membrane fragmentation study, it was not possible to determine the specific cause of this phenotype since these models express G4C2 repeat RNA as well as all five DPRs. We have shown that both phenotypes were caused by DPRs, with all DPRs causing RanGAP to accumulate in the nucleus and primarily GR causing Ran mislocalisation to the cytoplasm. RanGAP puncta have been previously observed in brain tissue from mice expressing GA50, supporting our findings14. Of note, punctate accumulations of RanGAP are also present in post-mortem brain tissue from C9FTD/ALS patients20, demonstrating the relevance of our findings to human disease. Mislocalisation of these key proteins is likely to cause a loss of function phenotype, resulting in nucleocytoplasmic transport impairments. This is supported by genetic screening studies in Drosophila expressing (G4C2)30 which found that overexpression of RanGAP rescued toxicity in this model, while RanGAP knockdown worsened the phenotype20. Deletion of Ran or RanGAP also increased toxicity in separate fly models expressing (G4C2)5823 and PR5021, highlighting the importance of the Ran cycle in cell survival. Combined with our finding that DPRs impair the structural integrity of the nuclear membrane, our data suggests that DPRs are the driving factor causing nucleocytoplasmic transport defects in C9FTD/ALS. Of particular note, the different DPRs had distinct effects on the nucleocytoplasmic machinery, despite their previously demonstrated toxicity in cell culture models 8. This demonstrates that the observed phenotypes were not a by-product of DPR toxicity causing cell death through some other mechanism. Functional assays to investigate of the impact of DPR-induced Ran cycle defects on nucleocytoplasmic transport would be beneficial to fully understand their contribution to disease.We have also shown that DPRs differently impact the localisation of two proteins required for the Ran cycle: the small GTPase, Ran, and its activator protein, RanGAP. The Ran cycle is an essential process which provides the energy required for active transport of proteins across the nuclear membrane via hydrolysis of RanGTP to RanGDP, creating a concentration gradient across the nuclear membraneDrosophila expressing either pure G4C2 repeats or alternative-codon DPR sequences encoding GA64, GR64 or PR6429. GR200 has also recently been found to cause mislocalisation of TDP-43 in mice expressing GR20030, further supporting our findings. GA100 also caused cytoplasmic aggregation of phosphorylated TDP-43 in human neuroblastoma cells31. Interestingly, this study found that neither GR100 nor PR100 caused mislocalisation of TDP-43, indicating that longer repeats may be required to cause a phenotype in this in vitro model. Indeed, the authors note that GA triggered the formation of TDP-43 inclusions in a length-dependent manner, and we have previously shown that DPR repeat-length is strongly correlated with toxicity in human cell culture and zebrafish11.Since the nucleocytoplasmic transport machinery was impacted by DPR expression in our model, we next investigated potential downstream consequences of this by assessing the localisation of TDP-43. TDP-43 is a predominantly nuclear RNA-binding protein which is mislocalised to the cytoplasm in C9FTD/ALS patient brain, where it aggregates to form insoluble inclusions. We found that expression of GA, GR and PR, but not AP, all caused TDP-43 to be mislocalised to the cytoplasm, demonstrating a functional link between these disease proteins. This is supported by a recent report of TDP-43 mislocalisation to the cytoplasm in 4C2)30 fly model and in iPSC-derived neurons from expansion-carrying patients20, and our findings suggest that this was most likely triggered by DPRs. Nuclear depletion of Ran has also been reported to occur alongside TDP-43 mislocalisation to the cytoplasm in a GRN−/− mouse model of non-C9FTD, preceding neuronal loss in the retina32. Impaired nucleocytoplasmic transport may therefore be a common mechanism leading to TDP-43 mislocalisation and neurodegeneration in different subtypes of FTD.We found that DPR-induced TDP-43 mislocalisation occurred alongside DPR-induced Ran and RanGAP mislocalisation, implying that dysfunction of the nucleocytoplasmic transport machinery and TDP-43 pathology could be linked in DPR-expressing cells, although further work would be required to definitively confirm this. Ran and TDP-43 mislocalisation also occurred together in the  was used as a template for PCR using the forward primer (5′ GCTCAAGCTTATATGTCTGAATATATTCGGGTA 3′) and reverse (5′ GAGCGGATCCCTACATTCCCCAGCCAGAAGACT 3′) containing HindIII and BamHI sites respectively. After digestion the product was ligated in pmCherry-C1 (Clontech) in the same restriction sites to create the pmCherry-C1 TDP-43 WT construct. All constructs were verified by DNA sequencing.We previously generated and characterised expression of DPR constructs for expression of GA2 in Dulbecco’s Modified Medium (Sigma) supplemented with 10% foetal calf serum (Gibco), 2 mM l-glutamine (Sigma), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma). Cells were seeded on HCl-treated glass coverslips in 6-well plates overnight and transfected with DPR constructs or empty pEGFP-N1 control vector (800 ng/well) using FuGene HD . Cells were incubated at 37 °C for 48 h with a media change performed at 4 h to reduce DPR toxicity. Co-transfection of DPR and TDP-43 vectors was performed by addition of 500 ng of DPR vector or empty pEGFP-N1 control and 500 ng of TDP-43 vector or empty mCherry control and 9 µl FuGene HD. Cells were incubated at 37 °C for 48 h with a media change 4 h post-transfection.HeLa cells (ECACC #93021013) were maintained at 37 °C with 5% COCells were fixed in 4% paraformaldehyde 48 h post-transfection and permeabilised with 0.05% Triton-X. Primary antibodies were diluted in phosphate buffered saline (PBS) as follows: anti-lamin B1 (Abcam) 1:500, anti-Ran (Abcam) 1:200, anti-RanGAP (Abcam) 1:100. Donkey anti-rabbit AlexaFluor-594, goat anti-mouse AlexaFluor-594 and goat anti-rabbit Cy5 secondary antibodies were used . All antibody incubations were performed at room temperature for 30 min with 3 PBS washes in between. Coverslips were rinsed in MilliQ water and dried overnight before mounting on glass slides using ProLong Diamond Antifade Mounting Media with DAPI (ThermoFisher). All immunofluorescence was performed in triplicate from separate passages of cells.-Apochromat objective and captured using a Coolsnap HQ2 camera (Photometrics) through Micromanager software v1.4.23. Specific band pass filter sets for DAPI, FITC, Texas red and Cy5 were used to prevent bleed through from one channel to the next. Images were processed and analysed using Fiji ImageJ (http://imagej.net/Fiji/Downloads). The relative intensity of antibody staining in the nucleus and cytoplasm was determined by calculating corrected total cell fluorescence (CTCF) values on ImageJ. DAPI staining was used to determine the boundaries of the nucleus.Images were collected on a Zeiss Axioimager.D2 upright microscope using a 63x/1.4 EC PlanP values below 0.05 were considered statistically significant.All experiments were performed in triplicate, with a minimum of 30 cells (chosen from random fields of view based on the presence of GFP-tagged DPR expression) analysed per independent replicate. One-way ANOVAs with Dunnett’s test for multiple comparisons were performed for all experiments using GraphPad Prism. All data generated or analysed during this study are included in this published article.


Tardbp in vivo, we show that oligodendrocyte lineage cells are differentially sensitive to loss of TDP-43. While OPCs depend on TDP-43 for survival, with conditional deletion resulting in cascading cell loss followed by rapid regeneration to restore their density, oligodendrocytes become less sensitive to TDP-43 depletion as they mature. Deletion of TDP-43 early in the maturation process led to eventual oligodendrocyte degeneration, seizures, and premature lethality, while oligodendrocytes that experienced late deletion survived and mice exhibited a normal lifespan. At both stages, TDP-43-deficient oligodendrocytes formed fewer and thinner myelin sheaths and extended new processes that inappropriately wrapped neuronal somata and blood vessels. Transcriptional analysis revealed that in the absence of TDP-43, key proteins involved in oligodendrocyte maturation and myelination were misspliced, leading to aberrant incorporation of cryptic exons. Inducible deletion of TDP-43 from oligodendrocytes in the adult central nervous system (CNS) induced the same progressive morphological changes and mice acquired profound hindlimb weakness, suggesting that loss of TDP-43 function in oligodendrocytes may contribute to neuronal dysfunction in neurodegenerative disease.Generation of oligodendrocytes in the adult brain enables both adaptive changes in neural circuits and regeneration of myelin sheaths destroyed by injury, disease, and normal aging. This transformation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes requires processing of distinct mRNAs at different stages of cell maturation. Although mislocalization and aggregation of the RNA-binding protein, TDP-43, occur in both neurons and glia in neurodegenerative diseases, the consequences of TDP-43 loss within different stages of the oligodendrocyte lineage are not well understood. By performing stage-specific genetic inactivation of  The distribution of myelin in the adult brain is not static, as new oligodendrocytes continue to be generated in response to changes in experience, such as enhanced sensory input and intense motor learning . As a reTARDBP are responsible for both familial and sporadic forms of ALS (TDP-43 (TAR DNA-binding protein 43 kDa) is a DNA/RNA-binding protein that serves as a master regulator of RNA metabolism, controlling transcriptional integrity, post-transcriptional stability, and localization of mRNAs . Within s of ALS , suggests of ALS , suggestTardbp in microglia enhanced expression of NF-κB and NLRP3, leading to a more activated phenotype characterized by enhanced phagocytosis  mice, by administering tamoxifen at P90 and then collecting brain tissue 2 weeks later (P90 + 14) (Rosa-CAG-LSL-EGFP) allows the visualization of the morphology of Cre-recombined cells (n = 4) (panel i) . However, when the interval between tamoxifen administration and sacrifice was increased to 30 days (P90 + 30), OPCs in gray matter were also depleted (panel ii) . Remarkably, OPCs in white matter had repopulated by this time, and highly polarized OPCs were visible migrating out of the corpus callosum into the gray matter   . Inclusied cells . Immunol (n = 4) , resulti (n = 4) , panel i80 + 14) , indicatTardbp from these cells using two constitutive Cre lines which differ in their timing of expression during oligodendrocyte maturation: iCreMog, which has been used extensively to manipulate mature oligodendrocytes   that has been shown to promote oligodendrocyte differentiation  . Single-ng stage . Gene exntiation , suggestfl/fl;RCEMobp-iCre;Tardbp) (Mobp-TDP43 KO) began to exhibit spontaneous seizures at ~P110 , similar to the phenotype exhibited by Cre;Tardbpfl/flCnp mice  (Mog-TDP43 KO) did not exhibit spontaneous seizures or other gross behavioral abnormalities and survived as well as controls (blue lines).Abolishing TDP-43 expression at these different stages resulted in very different outcomes. Mice in which TDP-43 was constitutively removed from premyelinating oligodendrocytes , whereas deletion of TDP-43 from mature oligodendrocytes (Mog-TDP43 KO) was not associated with a significant change in OPC proliferation . Moreover, there was an increase in the number of newly formed oligodendrocytes in Mobp-TDP43 KO, as assessed by the emergence of cells that expressed lncOL1 . Myelinated axons in Mobp-TDP43 KO and Mog-TDP43 KO mice had significantly higher G ratios than controls at both P90 and P180, with thinner sheaths formed , in mice with TDP-43 KO oligodendrocytes, nodes often consisted of Caspr triads and βIV-spectrin doublets (red arrowheads). EM analysis revealed that some axons exhibited double myelination  . HistoloAs the cuff-like structures are reminiscent of astrocyte endfeet , we labeMobp-iCreER mice, which allow selective Cre-dependent recombination in oligodendrocytes in the adult CNS , ASPA+ oligodendrocytes were present in the motor cortex with morphologies comparable to controls  (yellow arrowhead), indicating that TDP-43 is also required to maintain oligodendrocyte structure in the adult CNS. However, the behavioral consequences of TDP-43 deletion in the mature CNS were distinct, as these mice exhibited progressive motor problems, including limp tails, hindlimb clasping  . Howeverclasping , Video 4clasping , but notclasping . Althougclasping , the spiclasping , indicatclasping . Thus, aclasping , triggerclasping .TDP-43 is a highly conserved, multifunctional protein that regulates key aspects of RNA processing. Previous studies have shown that loss of nuclear TDP-43 function, through genetic deletion or disease-induced mislocalization, is sufficient to cause aberrant incorporation of intronic sequences (cryptic exons) into mature mRNA that can alter protein expression and function . Due to Ermn (Ermin), an oligodendrocyte-specific cytoskeletal protein that regulates process branching and sheath extension  Mobp-iCre, (3) iCreMog, and (4) Mobp-iCreER, allowing Tardbp inactivation at discrete stages within the oligodendrocyte lineage in both the developing and mature CNS. Bulk RNA-Seq of purified oligodendrocytes from Mobp-iCre and iCreMog mouse lines showed that this approach was able to target different stages of oligodendrocyte development. In particular, oligodendrocytes labeled by Mobp-iCre showed higher expression of genes enriched in newly formed oligodendrocytes, such as Gpr37 and Mag, whereas those labeled by iCreMog had higher expression of genes enriched in myelin-forming and mature oligodendrocytes, such as Gamt and Opalin  d Opalin . Therefoate OPCs , allowedate OPCs .Tardbp within OPCs resulted in their rapid degeneration, with loss first detected in the white matter of the corpus callosum 2 weeks after tamoxifen exposure, followed closely by OPC depletion in cortical gray matter, demonstrating that these progenitors require TDP-43 for survival. OPCs represent the largest population of actively dividing cells in the brain outside the subventricular and subgranular germinal zones , surviving cells that retain TDP-43 are expected to proliferate to reconstitute the population. Consistent with this hypothesis, all OPCs after 30 days of recovery were EGFP negative  expressed at different stages (early and late) of the maturation of postmitotic oligodendrocytes to determine whether the effects of TDP-43 loss depend on their state of differentiation. In contrast to TDP-43-deficient OPCs, oligodendrocytes were generated, and myelin sheaths were formed in both lines despite inactivation of Tardbp. As the half-life of TDP-43 in vitro was measured to be 12–34 hr (CreCnp-mediated deletion (Mobp-iCre) resulting in oligodendrocyte degeneration, seizures, and premature death by 6 months of age. This phenotype is similar to, but not as severe as that observed in Cre;Tardbpfl/flCnp mice, in which mature oligodendrocytes, OPCs, premyelinating oligodendrocytes, and Schwann cells were genetically manipulated  and enhanced proliferation of OPCs, indicative of oligodendrocyte degeneration, similar to the phenotypic changes observed after inducible deletion of oligodendrocytes in the adult CNS  led to a strikingly different outcome: oligodendrocytes survived, OPC proliferation was not enhanced and there was no gliosis. Correspondingly, these animals also did not exhibit seizures or premature death. Bulk RNA-Seq transcriptional analysis of oligodendrocytes with early and late deletion, even prior to the phenotypic changes in Mobp-iCre mice (P30), revealed that pathways involved in gene expression, cellular localization, cell adhesion, and metabolism were affected. Further, differential gene expression analysis of early, premyelinating and myelinating oligodendrocytes from obtained through single-cell RNA sequencing , change in gene expression as oligodendrocytes mature  led to distinct pathological changes, such as hindlimb weakness, hindlimb clasping, and limp tail, phenotypes not observed with developmental Tardbp inactivation, and these mice did not exhibit seizures or premature death. Although these distinct phenotypes may arise from differences in the proportion of oligodendrocytes affected, they mirror functional differences observed in neurodevelopmental and neurodegenerative diseases. In neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia, abnormal myelination and altered density of oligodendrocytes have been correlated with neural hyperexcitability and circuit dysfunction , including inert nanofibers and fixed axons . HoweverErmn KO mice . Of the other founder lines, the line with highest tamoxifen-dependent recombination efficiency was selected and maintained as a conditional Mobp-iCreERT2 mouse line   .Both female and male mice were used for experiments and randomly assigned to experimental groups. Mice were maintained on a 12 hr light/dark cycle, housed in groups no larger than 5, and food and water were provided ad libitum. All animal experiments were performed in strict accordance with protocols approved by the Animal Care and Use Committee at Johns Hopkins University. All experiments were initially performed with a sample size of 3  for quantification and statistical analyses, and power analysis was then performed to determine the minimum sample size to achieve sufficient statistical power to detect the effect of TDP-43 loss in different transgenic mouse lines at different timepoints. The following transgenic mouse lines were used in this study:Pdgfra-CreER (ra-CreER Tardbpfloxed (bpfloxed Rosa-CAG-LSL-EGFP) (JAX MMRRC stock #32038) (RCE ( #32038) Mobp-iCreMobp-iCreERiCreMog  dissolved in sunflower seed oil (Sigma) and administered by intraperitoneal injection daily for five consecutive days at a dose of 100 mg/kg body weight. Mice were perfused 14, 30, 60, and 90 days from the last day of tamoxifen injection.To induce 2), and their body temperature was maintained at 37°C with a thermostat-controlled heating plate. The skin over the right hemisphere was removed and the skull cleaned. A 3 × 3 mm region of skull over motor cortex was removed using a high-speed dental drill. A piece of cover glass  was placed in the craniotomy and sealed with VetBond (3 M), then dental cement (C&B Metabond) and a custom metal plate with a central hole was attached to the skull for head stabilization. In vivo imaging sessions began 3 weeks after cranial window procedure in order for the surgery-induced inflammation to subside. During imaging sessions, mice were anesthetized with isoflurane and immobilized by attaching the head plate to a custom stage. Images were collected using a Zeiss LSM 710 microscope equipped with a GaAsP detector using a mode-locked Ti:sapphire laser (Coherent Ultra) tuned to 920 nm.Cranial windows were prepared as previously described . Briefly3 and kept in 4°C until use. Free-floating sections were preincubated in blocking solution  for 1 or 2 hr at room temperature, then incubated for 24–48 hr at 4°C or room temperature in primary antibody. Secondary antibody incubation was performed at room temperature for 2–4 hr. Sections were mounted on slides with Aqua Polymount (Polysciences). Images were acquired using a confocal laser-scanning microscope . The following primary antibodies were used in this study: rabbit anti-ASPA , chicken anti-GFP , goat anti-GFP , mouse anti-MBP , chicken anti-MBP , rabbit anti-GFAP , Alexa Fluor 647 isolectin GS-IB4 (Thermo Fisher), rabbit anti-Aquaporin 4 , mouse anti-NeuN , mouse anti-CC1 , mouse anti-Ermin (Gift from E. Peles), guinea pig anti-Caspr (Gift from M. Bhat), rabbit anti-BIV-spectrin (Gift from M. Rasband), goat anti-ChAT , guinea pig anti-NG2 (Generated in D.E. Bergles lab), and rabbit anti-Ki-67 . Appropriate donkey secondary antibodies conjugated to Alexa Fluor 488, Cy3 or Alexa Fluor 546, and Cy5 or Alexa Fluor 647 were used at 1:1000–2000.Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially first with 15–20 ml room temperature PBS (pH 7.4) followed by 10 ml ice-cold 4% paraformaldehyde . CNS tissues were dissected  and postfixed in 4% PFA at 4°C for 4–6 or 12–20 hr depending on the antibodies to be used for immunohistochemistry. The tissues samples were transferred to 30% sucrose solution  for cryoprotection for at least two overnights before sectioning. Tissue samples were frozen in TissueTek and sectioned at 35 μm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Tissue sections were collected into a 24-well plate with PBS with 0.1% NaNG ratio analysis 10 random micrographs covering an area of ~180 µm2 each were taken at a magnification of ×6000 using a LEO912 transmission electron microscope  with an on-axis 2k CCD camera . For random selection of axons the images were overlaid with a grid and axons on grid crossings were analyzed with a minimum of 100 axons per animal. For G ratio determination the area of the axon and the corresponding fiber including the myelin sheath were measured using Fiji/ImageJ  and postfixed at 4°C for two overnights. Subsequent TEM processing was performed according to a previously described protocol . For G ri/ImageJ  and the G ratio quantifications were performed by a blinded observer. Longitudinal image stacks were registered using FIJI plugin ‘Correct 3D Drift’ . Tissue was stored at 4°C for 48+ hr before sectioning. Flatmounted hemicortex were extracted, frozen in TissueTek, and sectioned at 40 µm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Free-floating immunohistochemistry against MBP and EGFP was performed on the first section of the flatmount, which consists of the layer I of the cerebral cortex, as described previously . n represents the number of animals  used in each experiment. Data are reported as mean ± standard error of the mean and p value <0.05 was considered statistically significant.Statistical analyses were performed with GraphPad Prism 7 or Excel (Microsoft). All technical replicates  for each sample were averaged as a single data point. Significance was determined using one-way ANOVA test with Tukey’s correction for multiple comparisons or unpaired, two-tailed Student’s 2+ and Mg2+. Only the cortex was dissected out for downstream processing and mechanically dissociated with a razor blade on ice. The Miltenyi Neural Tissue Dissociation kit was used to perform enzymatic dissociation of the tissue into a single-cell suspension, and Myelin Debris Removal solution (Miltenyi) to remove myelin debris. The cells were resuspended in FACS sorting buffer (5 ml of 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) + 2 ml of Fetal Bovine Serum (FBS) + 800 μl of 0.5 M EDTA (Ethylenediaminetetraacetic acid) in 200 ml HBSS w/o Ca2+, Mg2+) and filtered through 35 μm strainer. The cells were sorted for endogenous GFP fluorescence on BD FACS Aria IIu Cell Sorter using a 100 μm nozzle at the Ross Flow Cytometry Core Facility at Johns Hopkins University School of Medicine.Male and female Mobp-TDP43 and Mog-TDP43 mice aged at P30 were cardiac perfused with ice-cold 1× Hank's Balanced Salt Solution (HBSS) w/o CaRNA was isolated from FACS purified oligodendrocytes (~5000 cells/sample) using QIAGEN RNeasy Micro Kit following the manufacturer’s instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit. Due to low amount of cDNA, Nugen Single-cell RNAseq kit was used to generate cDNA libraries for 100 bp paired-end sequencing on Illumina HiSeq (70–100 million paired reads/sample).Reads were aligned to the latest mouse mm10 reference genome using the STAR spliced read aligner with default parameters. Total counts of read fragments aligned to known gene regions within the mouse mm10 RefSeq reference annotation were used as the basis for quantification of gene expression. Fragment counts were derived using HTS-seq program using mm10 Ensembl transcripts as the model. Various QC analyses were conducted to assess the quality of the data and to identify potential outliers . Principal component analysis correction was performed to correct for batch effects and differences in sample RNA integrity number. Differentially expressed genes were identified using EdgeR Bioconductor package and ranked based on adjusted p values  of <0.05.Ermn and cryptic exon-specific Ermn transcripts were designed using Benchling and Primer3.RNA was isolated from whole mouse brain tissue using ReliaPrep RNA Tissue Miniprep System (Promega) following the manufacturer’s instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit. RT-PCR primers against normal Whole mouse brain tissue was homogenized in 1.5 ml Eppendorf tube with a handheld homogenizer in Pierce RIPA buffer (Thermo Fisher) with Sodium Orthovanadate (Vanadate). After centrifugation at 4°C, supernatant was collected, and the protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher). 50 μg of protein was loaded into each lane of Mini-Protean TGX gels (BioRad) and ran at 120 V for 1.5 hr. Protein was transferred onto Immobilon-FL PVDF Membrane (Millipore) at 100 V for 1.5 hr. Intercept (TBS) Blocking Buffer (Li-Cor) was used for blocking, and the membrane was incubated in primary antibody solution at 4°C overnight with gentle shaking. Mouse-Ermin  and Rabbit-GAPDH  primary antibodies were used. IRDye 680RD Goat anti-Mouse IgG (H + L) (Li-Cor) and IRDye 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor) were used as secondary antibodies for imaging on Odyssey Imaging Workstation (Li-Cor). Heo et al., 2021 investigated the role of the DNA/RNA-binding protein TDP-43 on oligodendrocyte maturation and showed that deletion of this protein induces deleterious effects on oligodendrocyte function in transgenic mouse models. These findings are exciting and support TDP-43 as a key regulator of oligodendrocyte function which may go awry in neurodegenerative disorders. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Maria D Purice (Reviewer #3).Thank you for submitting your article "Stage-specific control of oligodendrocyte survival and morphogenesis by TDP-43" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission with specific attention to the essential revisions.Essential revisions:The majority of the comments are focused on the clarity of the written document, as well as suggestions for making the figures more informative as well as specific recommendations for increasing the clarity of the figures and providing quantification of a few of the figures.– There were a few additional experimental data requested centered on reporter data on recombination efficiency (see reviewer 2 point #3)- and neurofascin splicing (see rev 2 point 5).– Please provide more details and quantification of the immunohistochemistry images shown as noted by reviewer 1 in legends and methods .– Please address reviewer 3 comment regarding potential loss of OPC identity. All reviewers agreed this is important especially since one marker (NG2) was used to define OPCs. Because the absence of TDP-43 alters splicing, it is possible that NG2 is not being expressed. This could also help explain the lack of astrogliosis.Reviewer #1 (Recommendations for the authors):Major Strengths:• Mog-TDP43 and Mobp-TDP43 KO mice represent useful tools for the field to study early and late stages of oligodendrocyte maturation• The stage-dependent role of TDP-43 in oligodendrocyte maturation, morphology, and function is particularly novel.• Transcriptomic analysis  is particularly strong, showing that TDP-43 KO oligodendrocytes exhibit missplicing of cytoskeletal-associated genesMajor Weaknesses:• Very minimal quantification of immunohistochemistry images . Quantification of all images must be included with details in methods/legends• Several figures could be split up into separate figures to enhance clarity. Schematics should also be included in separate figures to provide additional visuals of experiments• No stain of TDP-43 in immunohistochemistry images. Please address.• No quantification of behavior/hindlimb weakness • Narrative is lost through the vast amount of data shown, especially within each figure• Figure 3 axon thinning and density could be shown via A,B,D,E,F and without C and G• Figure 4 does not need examples of abnormal structures (could be put in supplement), and lacks quantification• Both Figures 3 and 4 could be condensed or moved to supplementalReviewer #2 (Recommendations for the authors):The following points are suggestions to improve the overall presentation and impact of the manuscript.1. In the first section of the results the authors mention assessing the efficiency of Cre-mediated recombination in the PDGFRa-TDP43 mice by measuring EGFP expression in NG2-positive OPCs. It is likely that a sentence got deleted that described PDGFRa-CreER mice mated to EGFP Cre reporter (RCE) mice to allow for this assessment. It is mentioned in the figure 1 legend.2. The data presented clearly shows that TDP-43 is required for the survival of OPCs in young and older adult animals. Strikingly, the authors also show that the population of OPCs is quickly restored following their loss. The authors make the reasonable suggestion that the repopulated cells are derived from the minor population of OPCs that did not originally delete TDP-43. Nevertheless, it also seems possible that these cells are derived from a "pre-OPC" progenitor pool that through cell division and maturation are able to replenish the depleted OPC pool. The authors' interpretation is based on their extensive, groundbreaking work on how OPCs replenish lost oligodendrocytes, but it might be possible that distinct mechanisms respond to OPC loss. It might be worth mentioning this alternative possibility in the discussion.3. The authors have done an excellent job demonstrating recombination efficiency and specificity using the PDGFRa and Mobp Cre drivers. Nevertheless, they have not shown similar data for the Mog Cre driver. This data would be helpful when considering the apparent lack of impact on oligodendrocyte viability, in contrast with the other drivers, in the Mog-Cre mice.4. The sentence starting the new section on page 8 begins "Despite the eventual vulnerability of oligodendrocytes to loss of TDP-43, in both Mobp-TDP43 KO and Mog-197 TDP43 KO mice", which is confusing. The authors have clearly shown oligodendrocyte "vulnerability" in the Mobp animals, but up to this point in the manuscript all of the assays presented appear to show unperturbed oligodendrocytes in the Mog animals. Subsequently they demonstrate thinner myelin in these mice, but the introductory statement should be clarified or re-written.5. Chang et al. (2021) recently demonstrated that the loss of TDP-43 in myelinating Schwann cells resulted in aberrant splicing of the paranodal component Neurofascin, which is also expressed by oligodendrocytes. The authors of the current study also demonstrate aberrant splicing in the TDP-43 deficient oligodendrocytes. It would be interesting to know whether Neurofascin RNA is also abnormally processed in the oligodendrocyte mutants. Paranodal abnormalities might contribute to the clinical phenotype of the mutant animals.Reviewer #3 (Recommendations for the authors):Using their mouse models, the authors are able to show the role of TDP-43 in OPC survival, and the role of TDP-43 in early and late stages of postmitotic oligodendrocytes. It would be useful to have a summary figure that shows the role of TDP-43 in OPC/oligodendrocyte development.In lines 164-66, the authors mention that their results are "similar to the phenotype exhibited by Cnp-Cre;Tardbp 165 fl/fl mice , in which recombination occurs in OPCs, early postmitotic oligodendrocytes, and Schwann cells in the PNS ." This statement and citations are confusing. In J. Wang et al., 2018 they confirmed that Cre-mediated recombination occurs for over 95% of mature oligodendrocytes, which are labeled with APC-CC1 and they saw no colocalization of GFP with NG2-positive OPCs, thus suggesting that Cnp-Cre-mediated recombination in the spinal cord is restricted to mature oligodendrocytes. In addition, Lappe-Siefke et al. 2003 does not look at Schwann cells in the PNS. Please clarify these citations.Lines 343-4. The sentence "Thus, death of only a few proliferating OPCs upon removal of TDP-43 is likely to induce a cascading depletion, capturing neighboring cells  as they are induced to proliferate" is confusing. Can the authors clarify what they mean by a cascading-depletion of TDP-43 in other neighboring cells?The methods are easy to follow, however for the RNA isolation section (line 569), it would be helpful to include the number of oligodendrocytes that were sorted and used for RNA extraction. Essential revisions:The majority of the comments are focused on the clarity of the written document, as well as suggestions for making the figures more informative as well as specific recommendations for increasing the clarity of the figures and providing quantification of a few of the figures.– There were a few additional experimental data requested centered on reporter data on recombination efficiency (see reviewer 2 point #3)- and neurofascin splicing ( see rev 2 point 5).Mog-iCre mice was quantified by crossing these mice to a Cre-dependent EGFP reporter mouse line (“RCE”). Analysis of these mice [add experimental details – timing of tam and sac], revealed that 97.3±0.7% of ASPA+ oligodendrocytes were EGFP+ in the cerebral cortex ( = recombination efficiency), whereas 95.9±0.5% of EGFP+ cells were ASPA+ ( = recombination specificity). A small percentage of neurons exhibit Cre activity in this animals . These data have been added as new Figure 2—figure supplement 1.The efficiency of Cre recombination in Nfasc splicing in cortical oligodendrocytes with Tardbp deletion. These data are consistent with observations recently reported by Chang et al. (2021), in which Nfasc missplicing was observed in sciatic nerve, but not in the spinal cord after Cnp-Cre-mediated deletion of Tardbp . These observations have been added to the Discussion section of the manuscript (p. 18).We did not observe any changes in – Please provide more details and quantification of the immunohistochemistry images shown as noted by reviewer 1 in legends and methods .3) of abnormal association of TDP-43 KO oligodendrocytes with non-axonal structures in Figure 4—figure supplement 4. The method of quantification is now described in the Figure 4—figure supplement 4 legend and the Materials and Methods section.We have added quantification of the prevalence  was used to define OPCs. Because the absence of TDP-43 alters splicing, it is possible that NG2 is not being expressed. This could also help explain the lack of astrogliosis.+ TDP-43 KO OPCs disappear, rather than downregulate NG2. We also show that PDGFRα+ cells also disappear upon Cre recombination. These data have been added to Figure 1—figure supplement 1D. Of note, a previous study that used diphtheria toxin to ablate OPCs reported that widespread apoptosis of OPCs does not induce microglial activation or pro-inflammatory changes .We agree this is an important issue. In these experiments, a Cre-dependent EGFP reporter transgene (RCE) was included to follow the fate of Cre-recombined OPCs over time. Immunolabeling for EGFP in these mice revealed that all EGFPReviewer #1 (Recommendations for the authors):Major Strengths:• Mog-TDP43 and Mobp-TDP43 KO mice represent useful tools for the field to study early and late stages of oligodendrocyte maturation• The stage-dependent role of TDP-43 in oligodendrocyte maturation, morphology, and function is particularly novel.• Transcriptomic analysis  is particularly strong, showing that TDP-43 KO oligodendrocytes exhibit missplicing of cytoskeletal-associated genesMajor Weaknesses:• Very minimal quantification of immunohistochemistry images . Quantification of all images must be included with details in methods/legendsQuantification of abnormal association with non-axonal structures has been quantified . The methods of quantification have been added to the Material and Methods section.• Several figures could be split up into separate figures to enhance clarity. Schematics should also be included in separate figures to provide additional visuals of experimentsFigure 3 and 4 have been split and a summary figure has been generated  outlining the distinct role of TDP-43 at different stages of oligodendrocyte development.• No stain of TDP-43 in immunohistochemistry images. Please address.In the mouse brain, TDP-43 protein is most highly expressed by neurons. With available antibodies, immunostaining for TDP-43 only resulted in labeling of neuronal somata. Although TDP-43 is below the limit of detection within oligodendrocyte lineage cells in tissue sections, mRNA for TDP-43 is consistently detected using RNA sequencing approaches and the profound phenotypes we observe indicate its importance in promoting the survival of OPCs, oligodendrocyte maturation, and oligodendrocyte stability.• No quantification of behavior/hindlimb weakness Given the similarity of the motor abnormalities  exhibited by TDP43 cKO mice to that observed in mice subjected to experimental autoimmune encephalomyelitis (EAE), we used the well-established EAE 0-5 severity scoring scale to quantify the behavior of these mice. These data are now included in Figure 5E.• Narrative is lost through the vast amount of data shown, especially within each figure• Figure 3 axon thinning and density could be shown via A,B,D,E,F and without C and G• Figure 4 does not need examples of abnormal structures (could be put in supplement), and lacks quantification• Both Figures 3 and 4 could be condensed or moved to supplementalWe have now separated the main figures  and moved the data indicated into Supplementary Data.Reviewer #2 (Recommendations for the authors):The following points are suggestions to improve the overall presentation and impact of the manuscript.1. In the first section of the results the authors mention assessing the efficiency of Cre-mediated recombination in the PDGFRa-TDP43 mice by measuring EGFP expression in NG2-positive OPCs. It is likely that a sentence got deleted that described PDGFRa-CreER mice mated to EGFP Cre reporter (RCE) mice to allow for this assessment. It is mentioned in the figure 1 legend.Indeed, all of the mice used in this study expressed a Cre-dependent EGFP reporter (RCE) to assess Cre recombination efficiency and to allow visualization of the morphology of TDP-43 KO cells. This information is now included in the Results (p.5).2. The data presented clearly shows that TDP-43 is required for the survival of OPCs in young and older adult animals. Strikingly, the authors also show that the population of OPCs is quickly restored following their loss. The authors make the reasonable suggestion that the repopulated cells are derived from the minor population of OPCs that did not originally delete TDP-43. Nevertheless, it also seems possible that these cells are derived from a "pre-OPC" progenitor pool that through cell division and maturation are able to replenish the depleted OPC pool. The authors' interpretation is based on their extensive, groundbreaking work on how OPCs replenish lost oligodendrocytes, but it might be possible that distinct mechanisms respond to OPC loss. It might be worth mentioning this alternative possibility in the discussion.Thank you for this suggestion. Although we favor the conclusion that repopulation occurs primarily from surviving cells, based on the recombination efficiency and qualitative assessments of proliferation of glial progenitors around the lateral ventricles, we now note this possibility in the Discussion (p. 14).3. The authors have done an excellent job demonstrating recombination efficiency and specificity using the PDGFRa and Mobp Cre drivers. Nevertheless, they have not shown similar data for the Mog Cre driver. This data would be helpful when considering the apparent lack of impact on oligodendrocyte viability, in contrast with the other drivers, in the Mog-Cre mice.We now provide these data in Figure 2—figure supplement 1.4. The sentence starting the new section on page 8 begins "Despite the eventual vulnerability of oligodendrocytes to loss of TDP-43, in both Mobp-TDP43 KO and Mog-197 TDP43 KO mice", which is confusing. The authors have clearly shown oligodendrocyte "vulnerability" in the Mobp animals, but up to this point in the manuscript all of the assays presented appear to show unperturbed oligodendrocytes in the Mog animals. Subsequently they demonstrate thinner myelin in these mice, but the introductory statement should be clarified or re-written.We have removed the text referring to the vulnerability and modified the introductory statement to the following: In both Mobp-TDP43 KO and Mog-TDP43 KO mice, oligodendrocytes were able to mature and form myelin sheaths, global myelin tracts within the brain were established , and there was no evidence of astrogliosis early in development (P30) .5. Chang et al. (2021) recently demonstrated that the loss of TDP-43 in myelinating Schwann cells resulted in aberrant splicing of the paranodal component Neurofascin, which is also expressed by oligodendrocytes. The authors of the current study also demonstrate aberrant splicing in the TDP-43 deficient oligodendrocytes. It would be interesting to know whether Neurofascin RNA is also abnormally processed in the oligodendrocyte mutants. Paranodal abnormalities might contribute to the clinical phenotype of the mutant animals.Nfasc was aberrantly spiced after deletion of Tardbp. Of note, Chang et al. (2021) observed that aberrant splicing of Nfasc occurred in the sciatic nerve but not in the spinal cord in Cnp-Cre cKO mice, which recombine in both the CNS and PNS. This result is now reported in the Discussion (p. 18).We examined our RNAseq datasets and did not observe that Reviewer #3 (Recommendations for the authors):Using their mouse models, the authors are able to show the role of TDP-43 in OPC survival, and the role of TDP-43 in early and late stages of postmitotic oligodendrocytes. It would be useful to have a summary figure that shows the role of TDP-43 in OPC/oligodendrocyte development.We have added a new summary figure to accompany the manuscript .In lines 164-66, the authors mention that their results are "similar to the phenotype exhibited by Cnp-Cre;Tardbp 165 fl/fl mice , in which recombination occurs in OPCs, early postmitotic oligodendrocytes, and Schwann cells in the PNS ." This statement and citations are confusing. In J. Wang et al., 2018 they confirmed that Cre-mediated recombination occurs for over 95% of mature oligodendrocytes, which are labeled with APC-CC1 and they saw no colocalization of GFP with NG2-positive OPCs, thus suggesting that Cnp-Cre-mediated recombination in the spinal cord is restricted to mature oligodendrocytes. In addition, Lappe-Siefke et al. 2003 does not look at Schwann cells in the PNS. Please clarify these citations.We have revised the reference to the following prior studies: Chang et al. 2021 and Tognatta et al. 2017 (p. 7).Lines 343-4. The sentence "Thus, death of only a few proliferating OPCs upon removal of TDP-43 is likely to induce a cascading depletion, capturing neighboring cells  as they are induced to proliferate" is confusing. Can the authors clarify what they mean by a cascading-depletion of TDP-43 in other neighboring cells?We have clarified this statement .The methods are easy to follow, however for the RNA isolation section (line 569), it would be helpful to include the number of oligodendrocytes that were sorted and used for RNA extraction.This method has been clarified.ReferencesEmbo J 30:277–288.Ayala YM, De Conti L, Avendano-Vazquez SE, Dhir A, Romano M, D'Ambrogio A, Tollervey J, Ule J, Baralle M, Buratti E, Baralle FE (2011) TDP-43 regulates its mRNA levels through a negative feedback loop. 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A high performance humidity sensor using tilted fiber Bragg grating (TFBG) and functional graphene oxide (GO)/multi-walled carbon nanotubes (MWCNTs) hybrid nano-materials was proposed. The humidity-sensitive material with three-dimensional (3D) structure was synthesized by the MWCNTs and GOs. Comparing with traditional two dimensional (2D) GOs film, water molecules could be absorbed effectively due to the larger ripples and more holes in GO/MWCNTs layers. The water molecule will fill the entire space in the 3D structure instead of air, which further enhances the absorption efficiency of the hybrid nanomaterial. TFBG as a compact and robust surrounding complex dielectric constant sensing platform was utilized. The mode coupling coefficient or the amplitude of TFBG cladding mode will vary sharply with the imaginary part of permittivity of the hybrid nanomaterial, realizing the high performance RH sensing. In the experiments, we successfully demonstrated that this 3D structural nanomaterial composed by the MWCNTs and GOs has significant advantages for expanding the range of humidity detection (range from 30% to 90%) and enhancing the detection sensitivity (0.377 dB/% RH is twice more than humidity sensor with 2D GO film). The TFBG-based RH sensor also exhibits good repeatability and stability. Our proposed humidity sensor has potential application in environmental and healthy monitoring fields. Humidity monitoring has taken an important role in numerous areas such as corrosion protection, precision electronics manufacturing, food processing, and other industries ,2,3. Tra2 nanoparticles coated S-Taper fiber as an RH sensor achieving the sensitivity of 0.441 dB/% RH and a range of 83.8% RH to 95.2% RH [2 coated side polished fiber (SPF) with a good linear correlation coefficient of 99.39% and a sensitivity of 0.1213 dB/% RH [2 = 0.980) [On the other hand, it is crucial to select a suitable humidity-sensitive material for RH measurement. The humidity-sensitive materials with a lot of hydrophilic functional groups, good swelling degree, and large specific surface area are attractive for measuring the RH. The widely used humidity sensing materials are porous ceramics, semiconducting materials, polymers , and novel carbon materials  ,23,24,2595.2% RH . Y. Luo  dB/% RH . However dB/% RH ,30. For  dB/% RH . K. Prab= 0.980) . The S t= 0.980) . In thosHerein, we proposed a TFBG-based humidity sensor with GO and evenly dispersed multi-walled carbon nanotubes (MWCNTs) hybrid humidity-sensitive nanomaterial. TFBG could couple most of the lights in the core to the cladding and make the lights directly interact with the humidity-sensitive material. The humidity sensing film was synthesized by GO/MWCNTs hybrid nanomaterial with three-dimensional (3D) structure. The MWCNTs play an essential role in supporting and expanding the space between the GO sheets. Via the supporting MWCNTs and GO films, the configuration makes water molecule fill the entire space instead of air which might strengthen the absorption effect of the molecule onto the hybrid nanomaterial. We will creatively combine TFBG with GO/MWCNTs hybrid nanomaterial to measure the relative humidity, and experimentally study the perturbation mechanism of the material’s dielectric constant. The proposed sensor with good performance will show good potential applications for health and environmental monitoring in many fields.rλ meeting the phase matching condition could be expressed as:θ is the tilt angle of the TFBG, Λ is the period of TFBG. rλ, respectively [For a TFBG, the resonance wavelength ectively .R of the TFBG cladding modes are related to the length L of the TFBG and the coupling coefficient κ between the exciting cladding mode and core mode. It can be expressed as follows:The intensity C is a constant depended on the electric field distribution of core mode coreE and cladding mode rE. The coupling coefficient is calculated as follows:e is the unit charge, cμ is the change of the chemical potential, h is Planck’s constant, and Bk is Boltzmann’s constant. T and τ are the environment temperature and vibration frequency [The conductivity σ of GO could be calculated through Equation (4):requency .GO or GO/MWCNTs film will absorb more and more water molecules with the increasing of RH, and the GO layer is filled by the absorbed water molecules. The density of the GO increases with the increasing humidity, which leads to the decrease of the conductivity (Im(nGO)) . For tigIm(nGO)) ,31. WhilIm(nGO)) . For theThe experimental system is shown in The physical precipitation method is selected to coat the GO or GO/MWCNTs on the fiber surface. First, the TFBG sensor is immersed into hydrochloric acid solution (20 min) and then flushed with plenty of deionized water and ethanol. Second, the sensor is immersed in GO aqueous solution or GO/MWCNTs solution to obtain GO or GO/MWCNTs film. At the same time, the sensor with GO aqueous solution or GO/MWCNTs solution is dried in a drying oven for one hour. Finally, to remove unsecured GO or GO/MWCNTs film from the TFBG’s surface, the sensor is washed with ethanol or deionized water and blown dry with nitrogen. The GO/MWCNTs aqueous solution was prepared by simple sonication. CNTs could adhere to the flat layers of GO through strong π-π stacking interaction ,35. TherIn this paper, the humidity response of GO coating TFBG was first performed. 2 of 99.19%. Comparing with the humidity sensor based on TFBG and GO film, the sensitivity under GO/MWCNTs film coating is twice more than that under GO film condition and the sensor detection range of RH is effectively expanded by coating with GO/MWCNTs film as shown in To evaluate the repeatability and reusability of the proposed sensor, three times repeat tests with different relative humidity were performed. It can be seen in Finally, the stability and the response time of the proposed fiber optic humidity sensor were also investigated. First, the TFBG coated with GO/MWCNTs film humidity sensor is placed in a constant temperature and humidity chamber . In this paper, a GO/MWCNTs-based TFBG humidity sensor with high sensitivity and large dynamic range has been demonstrated. In this humidity measurement, the light coupling element is the TFBG which easily excites cladding modes by coupling the core light to the cladding and interacts with the humidity-sensitive material on the fiber surface. The addition of MWCNTs enlarges the distance between the GO sheets and promotes the absorption of water molecules by the enriched surface/ripples/holes of both GO and MWCNTs. Thus, the composite of GO and MWCNTs has obvious advantages for expanding the humidity detection range and improving the detection sensitivity. The experimental results show that the sensitivity of RH detection sensor is 0.377 dB/% RH, which is twice more than that with GO film and the range of humidity detection is 30–90%. Moreover, the proposed humidity sensor possesses excellent repeatability, stability, and fast response characteristics. Therefore, this humidity sensor has potential applications for health and environmental humidity monitoring.