Recurrence is a common feature of craniopharyngiomas, benign tumors that origin from squamous epithelial remnants of Rathke’s pouch- arising at any segment of its whole course. There are two histotypes, showing different morphology and clinical behavior: adamantinomatous(adaCP) and papillary (papCP). An univocal strategy of management has not yet been defined, being considered the combination of surgery and radiotherapy the most effective, especially in case of incomplete resection. Therefore, the identification of factors influencing the biological and clinical behaviour is of paramount importance.β-catenin is a cell-cell adhesion protein, whose nuclear localization has been linked to the pathogenesis of adaCP: its nuclear accumulation is associated to the presence of a tumor stem cell subpopulation. The latter is made of cells capable of self-renewal, hence believed to be responsible of recurrence, metastases and resistance to therapy in all tumors.ATM is a kinase activated by autophosphorylation (p-ATM) upon DNA double-strand breaks. It is involved not only in DNA repair, but also in tumor migration and invasiveness. Its expression may have prognostic implications in many neoplastic diseases.In this study, we measured the immunohistochemical expression of β-catenin, stem cell markers , Ki67 and pATMin 45 craniopharyngiomas and correlated it with clinicopathologic features.p = 0.0039), Ki67  and CD166 . A slight tendency to a higher expression of β-catenin was recorded for adaCP rather than papCP (p = 0.0895).Fisher’s exact test showed that CD166 was significantlyrelated with recurrence (p = 0.0040). Furthermore, cytoplasmic pATM was more expressed in adaCPs (p = 0.0470), compared to papCPs that displayed a more evident nuclear signal (p = 0.0313) instead.Statistical analysis revealed strong correlation of β-catenin with recurrence (Backing upon these data, we could weigh in on the need of identifying β-catenin and CD166 as prognostic markersthat could be useful in predicting thebiologicalbehavior, as recurrence risk incraniopharyngiomas. The final goal is to drew up a prognostic algorithm to be of aid in the planning of an appropriate treatment strategy. Furthermore, our findings demonstrate that pATM could be used as additional distinction-marker between the two histotypes. Craniopharyngiomas (CPs) are rare benign tumors of the sellar and parasellar region, classified as low histologic grade (WHO grade I) that account for 2–4% of all intracranial tumors . Two hisThus far, the possibility of identifying inner tumor factors influencing the biological and clinical behavior of this “chronic disease” is paramount.Pathogenesis of CP accounts on an embryogenic theory for adaCP and a so-called metaplastic for papCP: the adamantinous type arises from remnant cells of the Rathke’s pouch, while the latter from adenohypophyseal cells, which underwent to squamous metaplastic changes , 3. DysrIn case of exon3 deletion of CTNNB1 gene, an active form of β-catenin protein is translated but it lacks the amino acids that allow its phosphorylation and then its degradation, thus resulting in an over-activation of the WNT/β-catenin pathway .Studies in mice showed that WNT/β-catenin pathway is required for normal pituitary morphogenesis and differentiation. A constitutive activation of this pathway in Rathke’s pouch undifferentiated precursor cells is at the base of childhood-onset adaCP, while CTNNB1 mutations in β-catenin accumulating cell clusters are the cause of adulthood-onset adaCP . GrowingComplete removal at first surgical attempt has been suggested as the most effective treatment, although itcould eventually determine visual impairment, endocrinological disturbances, and/or hypothalamic disturbances, resulting in impairment of social and behavioral disturbances. However, craniopharyngiomas can recur even after radical resection with a rate of 23% –16. The When analyzing the issue of recurrence, the need of defining morphologic , histologic  and/or molecular factors (Ki67 and p53) is crucial to rule out the predictive role , but relStem cells represent a small proportion of tumor cells capable of self-renewal and differentiation towards mature cells, hence believed to be responsible of cancer evolution, recurrence, metastases and resistance to chemotherapy and radiation in all tumors –21. CD13ATM is a kinase activated by autophosphorylation (p-ATM) upon DNA double-strand breaks arising from errors during replication, byproducts of metabolism, chemotherapy or ionizing radiations . ATM expThe aim of our study was to assess the expression of β-catenin and compare it with stem cell markers, as CD133 and CD166, with the proliferative index (Ki67 Labeling index) in a quite conspicuous number of craniopharyngiomas. Furthermore, we measured pATM expression in all those cases. The results were then compared with the recurrence rate, in order to evaluate the potential prognostic role of all these factors.Forty five samples of craniopharyngioma were analyzed. They were all obtained as formalin fixed tissue from the archive of the Department of Anatomic Pathology of the Federico II University Hospital of Naples. Patients underwent surgery, with endoscopic endonasaltransphenoidalapproach, at the Department of Neurosurgery of the same hospital. The cases were identified between 1998 and 2015 and the diagnoses were made in accordance with the World Health Organization (WHO) classification (1). Only specimens containing sufficient amountsof the respective CP subtype were taken into account. Of each specimen, samples exclusively made of fibro-inflammatory reaction, that is common in this type of pathology, were excluded from this analysis.For each patient, follow-up data were available and we had a written informed consent to use part of the specimen for scientific and/or research scopes.Four μm sections were used for immunohistochemistry. Sections were dewaxed in xylene, hydrated in graded series of alcohol and subjected to heat-induced antigen retrieval. After blocking endogenous peroxidase activity, the tissue was incubated with monoclonal antibodies for anti β-catenin , CD166 , p-ATM (phosphoS1981) and Ki67  and with the polyclonal antibody CD133 , all for 90 min. Subsequently, the slices were rinsed and incubated with the biotinylated secondary antibody, at room temperature, for 30 min. The bound antibody complexes were stained for 3–5 min or until appropriate for microscopic examination with diaminobenzidine, and they were then counterstained with hematoxylin (30s) and mounted. Appropriate positive controls were chosen: mammary fibromatosis for β-catenin, normal skin for CD166 and CD133 and normal pancreas for p-ATM. Negative control was obtained by omitting the primary antibody.All the slices were reviewed by two experienced pathologists , using light microscopy. In discordant cases the slides were re-evaluated on a multi-headed microscope to achieve consensus.0 = 0%, 1 < 10%, 2 = 10–50%, 3 = 51–80%, 4 > 80%) and an Intensity score . The same IS was used also for CD133 and CD166 membrane signal. The 3 markers were then divided into two scoring groups, on the base of the IS: absent/low expression (IS = 0–2) and moderate/high expression (IS = 3–12).β-catenin was considered as positive only in case of nuclear staining and a total immunostaining score (IS) was calculated as the product of a Proportion score : For the evaluation of the proliferating index Ki67 , “hot spot” areas were chosen and an average of the values on 5 adjacent fields (at least 500 neoplastic cells) was calculated: normally highly proliferating areas were excluded, namely basal cells. A cut-off of 5% was identified because the median value was of 6%.In some cases not all the markers could be tested because of tissue exhaustion.The study of association between the scoring group of each marker  and clinico-pathological features  was carried out by Fisher’s exact test. For pATM the population was divided into positive and negative cases.Spearman correlation test was used to examine the correlation between β-catenin and, respectively, CD133, CD166 and Ki67 and between CD166 and Ki67.p value ≤ 0.05 was considered statistically significant. All tests were two sided and carried out with GraphPad Prism 5 software .A Upon 45 patients Table  30 were All 45 cases were examined with β-catenin antibody. β-catenin immunoreactivity was detected at the cell membrane in most cases and in the nucleus in 28 cases. We focused on the nuclear signal that was located in neoplastic cells forming “whirl-like” structures and in palisading basal cells arranged around the stellate reticulum. The signal was variable in intensity and proportion of distribution Fig. .Fig. 145β-catenin immunostaining score was negative in 17 cases and in positive cases it ranged from 1 to 9, being 1, 2, 3, 4, 6 and 9 in 4, 11, 2, 7, 3 and 1 cases, respectively. Based on the calculated IS, all specimens were subsequently divided into two different scoring groups: 32 were absent/low (IS = 0–2) and 13 were moderate/high (IS > 2).p = 0.0039). No other correlations with clinical variables  were found out. A slight tendency to a higher expression of β-catenin was recorded for the adamantinomatoushistotype, rather than the papillary (p = 0.0895). In all 7 papCPs that were analyzed, β-catenin-IS was ≤2 (6 cases were 0 and one was 2).Out of 32 cases with absent/low β-catenin IS, 5 were recurrent, while out of 13 with moderate/high β-catenin IS, 8 were recurrent. Statistical analysis (Table p = 0.0385) association between high Ki67 L.I. (>5%) and recurrences was found but not as strong as with β-catenin IS. Cells forming “whirl-like” clusters did not show high Ki67 L.I . A statistically significant  scoring group, while 14 to the moderate/high (IS > 2).The signal was mainly located at the cell membrane Fig.  with difp = 0.0040).Out of 13 recurrent cases, 3 showed a CD166-IS ≤ 2, while in 10 cases the immunoscore was > 2. Fisher’s exact test showed that CD166-IS was significantlyrelated with recurrence risk  and CD133-IS were observed.p = 0.0011, r = 0.4903), β-catenin and CD166  and CD166 and Ki67 ; no significant relation was disclosed between β-catenin and CD133 .The Spearman test showed direct correlation between β-catenin and Ki67 , while CpATM was more present in adaCPs (p = 0.0470)  Fig. . No diffFinally, we analyzed the likelihood of recurrence for papCPs, on the base of NpATM, and adaCP, on the base of CpATM; no statistically significant correlation was observed Table .Table 3EThe unpredictable features and biological behavior, along with anatomical relationships that craniopharyngiomas establish, represent a key aspect to be considered to rule out prognosis and recurrence risk of these lesions.β-catenin is a protein with a dual function, regulating cell-cell adhesion and gene transcription. It plays a key role in the Wnt signaling that is involved in morphogenesis and in the process of motility in adaCPs. It is known that exon3 deletion of CTNNB1 gene causes β-catenin intranuclear accumulation that can be highlighted by immunohistochemistry and, though it can represent a diagnostic hallmark.In our study we examined the nuclear expression of β-catenin in adaCP specimens from 45 patients and investigated the association between the signal and recurrence risk. We found out that a moderate/high signal was more often detected in recurrent tumors.Probably this could be related to the role of co-transcription factor: indeed, when inside the nucleus, β-catenin binds transcriptional activators, switching on target genes .Furthermore, Wnt signaling and the elevated level of β-catenin are involved in the maintenance of pluripotency in many cell types . Few stuCD166 is an activated leukocyte cell adhesion molecule (ALCAM), with a controversial role in cancer: immunohistochemical studies on melanocytic lesions showed that CD166 overexpression was associated with tumor progression , as wellIn our series, we observed that CD166 was expressed in CPs and it showed a membrane signal whose immunoscore correlated with β-catenin, Ki67 and recurrence risk. It remains unclear whether it behaves as a stem cell marker in CPs: though, it seems to spare the whirl-like cell clusters in adaCPs, where there should be the stem cell niches. However, regardless of its role in stemness, it can be of help, together with β-catenin, to predict recurrences in CPs. In our cohort CD133 was expressed in both histotypes, while it was described to be almost undetectable in papCPs (11).Many other markers of stemness  have been studied in pituitary lesions and, curiously, in adaCP, SOX9 nuclear immunostaining was observed not only in the palisaded basal cell layer of the epithelium, but also in the stellate stromal cells under the epithelium . The samThe potential of Ki67 as prognostic marker was confirmed. A cut-off of 5% was established as a valid tool to effectively distinguish highly proliferative forms from those with a low rate of proliferation. The Labeling Index was calculated on cells other than those of the basal layer where a high proliferative activity was expected.Our results could weigh in on the need of a prognostic algorithm for the management of craniopharyngiomas: the identification of a batch of prognostic factors, indeed, could be helpful to determine a more appropriate integrated therapeutic modality.As well, pATM expression was assessed in the attempt of identifying potential factors that can affect radiosensitivity in CPs. This kinase has a specific role in the DNA double-strand breaks repair, that is exercised in the nucleus, and a cytoplasmic function that is related to autophagy , a proceIn our series, we observed mainly a nuclear signal in papCPs and a cytoplasmic in adaCs: this allowed us to consider that pATM different expression patterns in CP subtypes might be used as additional diagnostic and prognostic tool. No predictive role of recurrence was observed for pATM, neither cytoplasmic nor nuclear.The prevalent cytoplasmic localization of pATM in adaCPs may be the effect of a different pathogenetic role of this kinase in the two histotypes. Further studies are needed to clear this aspect.In modern histopathology there is a strong need to identify prognostic markers able to predict at best the biology of tumors. In craniopharyngiomas recurrence is an event that should be managed and prevented with multidisciplinary approach, in order to define the most appropriate treatment strategy. Overall, our results demonstrate how β-catenin, CD166 and Ki67 could be a valid supplementary tool. They can be also regarded as potentially useful antitumor targets.Furthermore, our findings could suggest to introduce pATM as additional distinction-marker between adamantinomatous and papillary histotypes.


Cognitive performance in attention-deficit/hyperactivity disorder (ADHD) is characterised, in part, by frequent fluctuations in response speed, resulting in high reaction time variability (RTV). RTV captures a large proportion of the genetic risk in ADHD but, importantly, is malleable, improving significantly in a fast-paced, rewarded task condition. Using the temporal precision offered by event-related potentials (ERPs), we aimed to examine the neurophysiological measures of attention allocation (P3 amplitudes) and preparation , and their associations with the fluctuating RT performance and its improvement in ADHD. 93 participants with ADHD and 174 controls completed the baseline and fast-incentive conditions of a four-choice reaction time task, while EEG was simultaneously recorded. Compared to controls, individuals with ADHD showed both increased RTV and reduced P3 amplitudes during performance on the RT task. In the participants with ADHD, attenuated P3 amplitudes were significantly associated with high RTV, and the increase in P3 amplitudes from a slow baseline to a fast-paced, rewarded condition was significantly associated with the RTV decrease. Yet, the individuals with ADHD did not show the same increase in CNV from baseline to fast-incentive condition as observed in controls. ADHD is associated both with a neurophysiological impairment of attention allocation (P3 amplitudes) and an inability to adjust the preparatory state (CNV) in a changed context. Our findings suggest that both neurophysiological and cognitive performance measures of attention are malleable in ADHD, which are potential targets for non-pharmacological interventions.The online version of this article (doi:10.1007/s10548-017-0554-2) contains supplementary material, which is available to authorized users. Inconsistent performance on reaction time tasks is one of the most prominent features of cognitive performance in ADHD   studies have reported that ADHD and healthy controls show differential patterns of brain activation related to RTV. In adolescents with ADHD, increased RTV was associated with decreased basal ganglia and thalamus activity , RTV has previously been linked to the P3 component in a neurotypical population  is observed also for the attentional P3 and the CNV. Third, we aimed to examine the relationship between RTV, P3 and CNV within and across task conditions. As lower IQ is associated with ADHD , but a significant difference in IQ was observed  .Seven ADHD participants were excluded from the analyses: two became very drowsy and could not complete the task, in two cases there was EEG equipment failure, and in three cases there were less than 20 acceptable segments available as required for averaging of EEG data. Two control participants were excluded, as they met ADHD criteria based on parent report. The final follow-up sample consisted of 93 ADHD participants  and 174 controls . The two groups did not differ in age  or gender  Wechsler  were admThe baseline (slow-unrewarded) condition followed a standard warned four-choice RT task. A warning signal  first appeared on the screen. At the end of the fore-period , the circle designated as the target signal for that trial was filled (coloured) in. The participant was asked to make a compatible choice by pressing the response key that directly corresponded in position to the location of the target stimulus. Following a response, the stimuli disappeared from the screen and a fixed inter-trial interval of 2.5 s followed. Speed and accuracy were emphasised equally. If the participant did not respond within 10 s, the trial terminated. First, a practice session was administered, during which the participant had to respond correctly to five consecutive trials. The baseline condition, with a fore-period of 8 s and consisting of 72 trials, then followed.To investigate the extent to which a response style characterised by slow and variable speed of responding can be maximally reduced, the task includes a comparison condition that uses a fast event rate (fore-period of 1 s) and incentives. This condition started immediately after the baseline condition and consisted of 80 trials, with a fixed inter-trial interval of 2.5 s following the response. If the participant did not respond within 10 s, the trial terminated. Speed and accuracy were emphasised equally. The participants were told to respond really quickly one after another, to win smiley faces and earn real prizes in the end. Participants won a smiley face for responding faster than their own MRT during the baseline (first) condition consecutively for three trials. The smiley faces are continuously updated and remain on the screen throughout this condition until the end of the task. The baseline MRT was calculated here based on the middle 94% of responses , therefore excluding extremely fast and extremely slow responses. The smiley faces appeared below the circles in the middle of the screen and were updated continuously. The fast-incentive condition is always administered after the baseline condition and, as such, does not involve a similar learning phase. Participants earned £5 in cash after the task battery.Due to the longer fore-period in the baseline condition, the two conditions were not matched on task length, but were matched on the number of trials. We analysed RTV performance, defined as within-subject standard deviation of mean RT on all correct trials, in both the full baseline condition and separately on the length-matched segment , with a 500 Hz sampling-rate, impedances kept under 10kΩ, and FCz as the recording reference. The electro-oculograms (EOGs) were recorded from two additional electrodes above and below the left eye and at the outer canthi.The EEG data were analysed using Brain Vision Analyzer 2.0) . After down-sampling the data to 256 Hz, the EEG data were re-referenced to the average and filtered offline with digital band-pass  Butterworth filters. Ocular artifacts were identified from the data using Independent Component Analysis [ICA,  command in Stata . As these were sibling data, the data were analysed using random intercept models and logistic regression in Stata. The random intercept model is a multilevel regression model that can be used as an alternative to ANCOVA to control for genetic relatedness in a repeated-measures design, using a “robust cluster” command to estimate standard errors  and control (n = 81) groups. Singletons were randomly drawn from complete sibling pairs using the “sample” command in Stata. The robust cluster command was not available in the correlational analysis, so for these analyses only the singletons (n = 194) were included. As the CNV was maximal at Cz, we computed the correlations with the CNV at Cz only, to reduce the number of statistical comparisons.We first computed the group differences in both the baseline and the fast-incentive condition, followed by a post-hoc group analysis of the difference score between conditions. As the RTV difference scores could not be successfully transformed, non-parametric Kruskal–Wallis tests were used on the singletons, by removing the siblings from the ADHD  = 18.29, p < 0.01). We obtained comparable results using the length-matched segment of the baseline condition  = 12.68, p < 0.01), compared to the controls.A random intercept model indicated significant main effects of group  and condition  for RTV, and a significant group-by-condition interaction   and condition  for P3 amplitudes, but the group-by-condition interaction did not reach significance   but not at Pz . The main effect of condition was significant at both Cz and Pz . The group by condition interaction was also significant at Cz  and Pz . Post-hoc comparisons revealed no group differences on the CNV in the baseline condition at Cz Figs. a or Pz  and condition  for P3 amplitudes, with the group-by-condition interaction just reaching significance  . Post-hoc comparison revealed significant group differences in the baseline  but not in the fast-incentive  condition.To confirm that the P3 group by condition effect observed following CSD transformation was due to the CNV, we re-ran the analysis on P3 including CNV amplitude at Pz as a covariate, both with CSD transformation. This covariate approach allows us to take into account the group effects during the baseline intervals, similar to using a technical zero baseline approach for the P3 in which a significant group by condition interaction for P3 was observed . With CNV amplitudes included in the same model, a group by condition interaction now emerged for the P3 , with the main effects of group and condition remaining significant . Post-hoc analyses indicated that the ADHD group showed significantly reduced P3 amplitudes compared to the control group in the baseline condition  but not in the fast-incentive condition .All analyses were re-run without IQ as a covariate, and the pattern of results remained the same with two exceptions. First, the main effect of group for P3 amplitudes diminished . Further examination of the effects of IQ on P3 amplitudes was, therefore, carried out on the ADHD and control groups separately. A negative association between IQ and P3 amplitudes was observed in the control group in the baseline condition , but not in the fast-incentive condition . In the ADHD group, IQ and P3 amplitudes were not significantly correlated in the baseline condition  but were associated at trend level in the fast-incentive condition . Second, the magnitude of group difference in the CNV at Pz in the fast-incentive condition changed from a trend (p = 0.06) to statistical significance  when IQ was not controlled for. The latter finding suggests that IQ differences between ADHD and control group may account for the group differences in the CNV at Pz.As age correlated significantly with P3 in both groups (Table S2), and RTV varied as a function of gender in the ADHD group, they were included as covariates in the within-group correlational analyses, in addition to IQ. In the ADHD group, a significant association was observed between RTV and P3 amplitudes and between CNV and P3 amplitudes in both the baseline and fast-incentive conditions, while a significant association between RTV and CNV was only observed in the fast-incentive condition Table . In the Correlations of the difference scores across conditions indicate that a reduction in RTV from the baseline to fast-incentive condition was significantly associated with an increase in P3 amplitudes in the ADHD group, whereas this pattern was not observed in the control group, with the difference in the correlation coefficients reaching significance between groups  is linked to the cognitive performance measure of attentional fluctuation (RTV) in individuals with ADHD, with both measures showing malleability: attenuated P3 amplitudes were significantly associated with high RTV, and the increase in P3 amplitudes from a baseline to a fast-paced, rewarded condition was significantly associated with the decrease in RTV observed between conditions. Yet, third, the individuals with ADHD did not show the same increase in CNV from baseline to fast-incentive condition as observed in controls, indicating that they were unable to adequately adjust the preparatory state in a changed context.Our findings on RTV replicate those reported in the previous study on a partially overlapping group of children performing the identical Fast task 6 years earlier  suggests that the lack of group by condition interaction effect in P3 may be partially accounted for by subtracting the more prominent increase in CNV in controls than in the ADHD group during the fast-incentive condition as the baseline. Thus, the preparatory brain processes in individuals with ADHD were not comparable to controls in the fast-rewarded condition. As RTV improves under the fast-incentive condition, we observed a significantly greater magnitude of change in preparatory CNV activity from the baseline to the fast-incentive condition among controls compared to individuals with ADHD. The overall pattern of findings suggests that the inability to adjust the preparatory state in a changed context may explain why RTV does not fully normalise in ADHD. These findings also highlight the importance of considering the effects of pre-stimulus state measures, such as the CNV, when interpreting the findings on post-stimulus ERPs, particularly in individuals with ADHD. Studies that subtract the pre-stimulus baseline ERP activity from the post-stimulus ERPs without testing for systematic effects during this pre-stimulus period may risk overlooking crucial neurophysiological processes that underlie behavioural states and their influence on subsequent components, which could lead to misinterpretations.Little is known about how attentional processes on a neural level relates to attentional fluctuation on a cognitive performance level, even amongst the general population. The results from our control group indicate that, under a slow and unrewarded baseline condition, RTV relates more strongly with preparation (CNV) than with allocation of attention (P3 amplitudes). By comparing the associations between these ERP markers with RTV across conditions, we further demonstrate that for individuals with and without ADHD, both attentional resource allocation (P3 amplitudes) and response preparation (CNV) are necessary for optimal cognitive performance in the fast-incentive condition. As the main effects of condition were significant for both P3 and CNV amplitudes, it suggests that regardless of ADHD status, attentional allocation and response preparation are both contributing factors for optimal cognitive performance in the fast-incentive condition. This finding is consistent with a recent study that the prospect of gaining monetary reward increased CNV amplitudes and reduced reaction time in healthy adults , we obtained identical findings whether or not length-matched segments were used. The design of the Fast Task paradigm is not ideal for measuring a conventional CNV, as the interval between the cue is very long in the baseline condition and relatively short for the fast-incentive condition. In spite of the short time interval in the latter condition, we observed a typical CNV distribution in the fast condition, and a prominent group difference in this preparatory activity, suggesting that CNV is a sensitive marker of ADHD also at short intervals. Future studies could replicate these findings regarding anticipatory activity using other ERP tasks. Due to the long inter-stimulus interval of our task design, we were unable to obtain an accurate latency measure. However, as previous studies suggest, both amplitude and latency of the stimulus-aligned P3 are dependent on RT distribution


FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)–mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma  leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition. In most cases, ALS is a sporadic disease although ~10% of patients have a clear family history. Mutations in the superoxide dismutase 1 (SOD1), the TAR DNA-binding protein (TARDBP), the fused in sarcoma (FUS), and the chromosome 9 open reading frame 72 (C9orf72) genes are the most prevalent ones1. No effective treatment is available for ALS. Riluzole, an FDA-approved drug which has anti-excitotoxic properties, prolongs life by only a few months2. Although intensive basic research has been done based on different ALS animal models, none of the discovered strategies has been successfully translated into a therapy3. As a consequence, more reliable ALS models are urgently needed. MNs differentiated from patient-derived-induced pluripotent stem cells (iPSCs) provide a new opportunity to model human disease starting from patient material and avoiding effects of species differences and/or overexpression. It can mimic the developmental process in vitro, which could contribute to understand basic disease mechanisms, especially early pathological changes that are potentially amenable to drug screening4.Amyotrophic lateral sclerosis (ALS) is the most common degenerative disorder of motor neurons (MNs) in adults and is characterized by the selective death of both upper and lower MNs. This wasting of MNs leads to progressive paralysis and death of the patient due to respiratory failure usually within 2 to 5 years after symptom onsetFUS was first identified as an oncogene and was reported as an ALS-causing gene in 20097. It contains a glycine-rich region, an RNA recognition motif and a nuclear localization signal (NLS). Many point mutations in FUS have been discovered since then and a large number of these are situated in the C-terminal NLS region8. FUS can also cause a rare and very aggressive juvenile onset ALS9. FUS functions as a DNA/RNA-binding protein and is involved in multiple aspects of DNA/RNA metabolism8. The most significant pathological change in post mortem tissue is the cytoplasmic mislocalization of FUS. In iPSC models, cytoplasmic mislocalization of mutant FUS was reported by three independent groups in their patient-derived MNs12. Cytoplasmic FUS aggregates were observed by Liu et al.10 for the P525L mutation. In addition, electrophysiological changes have been frequently observed in ALS patient-derived MNs15. Both hyperexcitability and hypoexcitability have been proposed as pathophysiological defects in these models15. A recent study based on iPSC-derived MNs carrying mutations in C9orf72 or in TARDBP suggested a switch from hyperexcitability to hypoexcitability, which could result in MN death12. For FUS, intrinsic membrane hyperexcitability was presented by Wainger et al.14, whereas hypoexcitability  was reported by Naujock et al.1316. In this dying-back theory, MNs lose their function at the distal axon and retract back to the MN soma. The longest and largest axons with the highest metabolic demand seem to be the most vulnerable ones, which suggest that defects in axonal transport could be involved in this neurodegenerative process17. Most of the energy comes from mitochondria transported to the distal site where they are most needed17. In addition, multiple cargos such as proteins, mRNAs, lipids, and organelles are mostly synthesized in the cell body and are transported to the distal part of the axon to maintain their function18. About 5–20% of the mitochondria are in very close proximity to the endoplasmic reticulum (ER), which seems to have an important role in many neurodegenerative diseases, including ALS20. This region is called the mitochondria-associated ER membrane (MAM) and is linked to intracellular trafficking of mitochondria and ER, Ca2+ and phospholipid exchange, energy metabolism, mitochondrial biogenesis, ER stress responses, and autophagy20. MAMs were reported to be regulated by TDP-43, which is the protein encoded by TARDBP and by FUS in transfected cells and in mouse models through activation of glycogen synthase kinase-3β (GSK-3β)22.There is no clear explanation why MNs selectively degenerate in ALS. One hypothesis is based on the observation that MNs characterized by long axons degenerate first and that altered functions of the most distal sites occurs at initial stages of disease23. In contrast to the other family members of the HDAC family that mainly deacetylate histones in the nucleus, HDAC6 is localized in the cytoplasm and is the major deacetylating enzyme of α-tubulin24. Acetylation of α-tubulin is important for the binding of molecular motor proteins to the microtubules26. The importance of HDAC6 in ALS is illustrated by the fact that genetic deletion of HDAC6 significantly slowed disease progression and prolonged survival of the mutant SOD1G93A mouse model27. FUS and TDP-43 have also been reported to regulate HDAC6 expression30. As a consequence, one of our aims was to investigate the therapeutic potential of HDAC6 inhibition.We previously discovered that histone deacetylase 6 (HDAC6) inhibitors could rescue axonal transport defects in dorsal root ganglion (DRG) neurons from a transgenic mouse model of the axonal form of Charcot–Marie–Tooth disease (CMT2)FUS mutations, as well as from family members without mutations. In addition, we generated isogenic control lines using the CRISPR-Cas9 technology and overexpressed wildtype and mutant FUS in human embryonic stem cells (hESCs). Highly pure MN cultures were obtained by differentiating these iPSCs or hESCs. The cells expressing mutant FUS showed cytoplasmic accumulation of FUS, hypoexcitability, and axonal transport defects. We could successfully rescue the axonal transport defects by treating the MNs with HDAC6 inhibitors and by silencing HDAC6 using antisense oligonucleotides (ASOs). Our results indicate that axonal transport could have an important role in ALS pathology, and that HDAC6 inhibition could become a promising therapeutic strategy for FUS-induced ALS.To further investigate the pathological mechanism and to identify potential therapeutic strategies, we generated iPSCs from fibroblasts of ALS patients carrying different 31. In addition, we established two control iPSC lines  from two healthy family members of one of our patients. We also included another two published healthy controls  for patch clamp measurements13. Reprogramming of fibroblasts was performed using Sendai viral vectors expressing 4 reprogramming factors . This led to an integration-free conversion of fibroblasts into iPSCs. The absence of Sendai virus was confirmed by qPCR analysis  to assess for the integrity of the mitochondria. However, we could not detect any obvious morphological changes in the mitochondria in the soma or axons of patient-derived MNs  vesicles using ER-tracker Fig. . PatientTo confirm that the observed phenotypes were due to the mutation in FUS and to eliminate possible effects of the genetic background on these phenotypes, we established an isogenic control iPSC line for one of the patient-derived iPSC lines carrying the R521H mutation using the CRISPR-Cas9 technology. Sequencing confirmed that the point mutation was corrected Fig. . As indiAAVS1 locus35 via recombinase-mediated cassette exchange , whereas mitochondria were labeled using translocase of the outer mitochondrial membrane protein-20 (TOM20). Pearson’s correlation coefficient was used for the analysis. ALS patient-derived MNs all showed a significantly decreased overlap of ER and mitochondrial staining especially in neurites  conversion to PC than wildtype cells37. Therefore, production of PC can be used to assess MAM disturbances. We harvested culture medium at different time points during the in vitro differentiation process  and measured the level of released PC. We observed a reduction of PC in mutant FUS MNs  responsible for the axonal transport defects in the iPSC-derived MNs-containing mutant FUS. As indicated before, we first assessed the mitochondrial integrity in mutant FUS and control-derived MNs by using EM. We could not identify abnormal mitochondrial morphology in the FUS mutant MNs Fig. . In addites Fig. . MAMs al23. In a previous study, we discovered that HDAC6 inhibitors restore mitochondrial axonal transport defects in DRG neurons from a CMT disease mouse model, which correlated with increased α-tubulin acetylation23. In this study, we assessed the acetylation level of α-tubulin in MNs differentiated from a mutant FUS line and its isogenic control. Using western blot analysis, we detected a slight decrease of acetylated α-tubulin in patient-derived MNs compared with the isogenic control  in the NLS region of FUS, as well as from two healthy controls. Furthermore, we used CRISPR-Cas9 technology to correct the point mutation in one of the ALS patient-derived iPSC lines as an isogenic control line, and we overexpressed wildtype and mutant FUS in hESCs. In this way, we investigated the causative relationship between FUS mutations and the observed defects in MNs derived from these iPSCs/hESCs. In addition, we developed a highly efficient MN differentiation protocol, which generated more than 80% of cells with MN morphology, expressing typical MN markers and exhibiting typical electrophysiological characteristics. The MNs derived from these iPSCs of ALS patients exhibited typical FUS mislocalization and hypoexcitability. In addition, progressive mitochondrial and ER vesicle transport defects developed in these iPSC-derived MNs. These axonal transport defects as well as the MAM reduction could be rescued by HDAC6 inhibition and by genetic knockdown of HDAC6 using ASOs.12, we observed cytoplasmic FUS mislocalization in patient cells including fibroblasts, iPSCs, and MNs. The degree of cytoplasmic mislocalization of FUS was correlated before with the mutation type and was associated with the severity of the clinical presentation and the age of disease onset8. This indicates that cytoplasmic FUS localization is a very early pathological change, which occurs in different cell types and of which the severity depends on the type of FUS mutation. Unlike other studies, we did not observe FUS aggregates in our mutant lines41. As aggregates were reported to colocalize with stress-granule markers, we may not have detected FUS aggregates because we did not apply extra stress to the MNs42. It has been suggested that the mislocalization of FUS serves as a first hit in the pathophysiological cascade that makes the cell more vulnerable6. A second hit, such as cellular stress and/or defects in protein degradation, is proposed to assemble cytoplasmic FUS into stress granules and could trigger neurodegeneration6.Consistent with previous reports using iPSC models or post-mortem patient tissue33 reported hyperexcitability in iPSC-derived MNs carrying SOD1, C9orf72, and FUS mutations based on increased spontaneous firing and reduced delayed rectifier K+ currents by multielectrode array recordings. Devlin et al.15 proposed a switch from hyperexcitation to hypoexcitation of MNs, which was dependent on the differentiation time of human iPSCs obtained from patients harboring TARDBP or C9orf72 ALS mutations. Recently, Naujock et al.13 showed hypoexcitability in MNs derived from mutant SOD1 and FUS iPSCs differentiated for 7 weeks. Although the possible change from hyperexcitability to hypoexcitability was evaluated, only increased synaptic activity but not enhanced AP firing frequencies was found in cells differentiated in culture for 3–4 weeks. The hypoexcitability was confirmed in our study as patient-derived MNs presented with a reduced frequency of APs and postsynaptic currents at week 7 of differentiation. Hypoexcitation could be a late phenotype, which can eventually cause cell death. However, we did not observe obvious cell death in the MNs derived from patient iPSCs. As a consequence, we consider the hypoexcitability as an early pathological change rather than a direct cause of cell death.Several studies demonstrated a number of physiological changes in different iPSC-derived ALS models. Kiskinis et al.44. SOD1 mutations can affect neurofilaments, mitochondria, and vesicle transport44. Only one study based on an ALS iPSC-based model has previously identified mitochondrial transport defects in MNs derived from ALS patients carrying an A4V mutation in SOD133. In that study, mitochondrial damage was observed based on EM and it was hypothesized to be the cause of the mitochondrial transport defects33. In the current study, we did not observe obvious structural changes in the mitochondria of mutant FUS MNs. In addition, ER vesicle transport along the axons was also decreased indicating that the disturbance in axonal transport was a more general problem. These axonal transport defects developed as a function of time in culture. They were significant at the third week after plating and became progressively worse when cells were maintained in culture. Defective axonal transport defects were also observed as a common phenotype in Drosophila models of TARDBP, FUS, and C9orf7217. These axonal transport deficits are considered as an early pathological change during the development of ALS, which might cause dysfunction of the neuromuscular junctions, which highly rely on energy supplied by mitochondria and on efficient synaptic cargo delivery to control proper muscle contraction45. If axonal transport is hampered in MNs, this leads to the loss of neuromuscular junctions, axon degeneration, and finally death of the soma. This sequence of events has led to the concept of a dying back mechanism in ALS16. As axonal transport defects were rescued in the isogenic genetically corrected R521R iPSC-derived MNs, the mutation itself is sufficient to induce this phenotype. Moreover, the same mitochondrial transport defects were detected in MNs generated from hESC in which two different mutant FUS genes were inducibly overexpressed from within the AAVS1 locus, whereas wildtype FUS overexpression had no effect. This is comparable to a previous study detecting axonal branching and growth defects in primary neuronal cultures of mice overexpressing mutant FUS (P525L and R521H), whereas no defects were observed after overexpression of wildtype FUS46. Overexpression of caz, the Drosophila orthologue of human FUS in flies, did also not induce axonal transport defects, whereas caz variants mimicking mutant FUS also induced mitochondrial transport defects17. Both gain-of-function and loss-of-function mechanisms have been proposed as the basic mechanism underlying the pathogenic effect of mutations in FUS. Our data strongly suggest that at least the defects in axonal transport are due to a gain of function of mutant FUS-related toxicity, which is also consistent with recent studies in a mouse model47. In a recent study, inhibition of the mitochondrial localization of TDP-43 can prevent mitochondrial dysfunction and neuronal loss in a TDP-43M337V mouse model. This indicates that there is a link between TDP-43 localization and mitochondrial movement, which may also be true for FUS48.In addition, we identified axonal transport defects in MNs derived of mutant FUS iPSCs. Axonal transport abnormalities have also been reported in several animal models of ALS, especially in SOD1 models49, we did not observe any significant changes in the staining for NFL in both patient and control cells. Next, we investigated whether MAMs were affected by FUS mutations. MAMs have multiple functions including lipid synthesis, mitochondrial dynamics, Ca2+ homeostasis, ER stress, intracellular trafficking, and autophagy19. Overexpression of TDP-43 was reported to cause MAM disruption in vivo and in vitro21. In a recent report, FUS was suggested to activate GSK-3β, which disrupts MAMs through a VAPB–PTPIP51 interaction22. By staining for both mitochondria and ER, we found that overlap of ER and mitochondrial markers was significantly lower in FUS compared with the control MNs. A limitation of our study is that we could only estimate the MAM reduction using stainings. As MAMs represent a very small region where the distance between mitochondria and ER is limited to 10–30 nm, and where only 5–20% of the mitochondrial surface is closely connected to the ER membranes19, it is difficult to accurately quantify the percentage of MAMs. As another readout for MAM integrity, phospholipid release by FUS-mutant MNs was assessed. This demonstrated significantly decreased PC release by FUS-mutant MNs, which was reversed following genetic correction.To gain insights into the mechanism(s) underlying the axonal transport defects in mutant FUS MNs, we first assessed cytoskeletal changes. Although a previous study demonstrated that NFL was decreased in mutant SOD1 iPSC MNs50. All these motor proteins are involved in axonal transport of mitochondria and vesicles. KIF5C belong to the Kinesin1 family, which drives anterograde (=synapse directed) transport of mitochondria along axons51. An outer mitochondrial protein, Miro1, connects mitochondria with Kinesin151. Miro1 preferentially localizes in MAM sites and part of the ER will be transported together with mitochondria based on this association51. It could be that if MAMs decrease, axonal transports will decrease because of this reduced Miro1 linkage.FUS can bind and regulate mRNAs of several motor proteins including KIF5C, KIF1B, and KIF3A28. However, we did not observe any downregulation of HDAC6 expression in MNs derived of mutant FUS iPSCs. In line with the rescue of axonal transport deficits in DRG neurons from a mouse model of CMT2 and in a Drosophila model of Parkinson disease52, we believe that the therapeutic effect of HDAC6 inhibitors could be related to a more general effect due to the increase of the acetylation level of α-tubulin in the microtubules, rather than a (mutant) FUS-specific effect. This is also in line with the observation that genetic deletion of HDAC6 in an ALS mouse model with mutant SOD1G93A extended the lifespan of these mice by maintaining the integrity of the motor axon, whereas there was no effect on the disease onset27. Our hypothesis illustrated in Fig. We could rescue both mitochondrial and ER vesicle transport defects by using two different HDAC6 inhibitors. HDAC6 inhibition also increased the total number of ER vesicles. These compounds had no effect on healthy control-derived MNs indicating that axonal transport is already maximal and cannot be increased by inhibiting deacetylation in these cells. Knockdown of HDAC6 using ASOs also resulted in the same rescue effects on the axonal transport. This did not only confirm that HDAC6 has an important role in this rescue effect, it also confirms the specificity of the pharmacological HDAC6 inhibitors, used in our study. It was reported before that the mRNA expression of HDAC6 was regulated by a TDP-43 and FUS complex53. As a consequence, pharmacological HDAC6 inhibition seems to be an interesting strategy to counteract general axonal transport defects, which correlates with an increased acetylation of α-tubulin.Both Tubastatin A and ACY-738 are considered as being specific HDAC6 inhibitors. Although Tubastatin A has a higher selectivity, the advantage of ACY-738 is that it has a better capability to cross the blood–brain–barrier and that a related compound  is in clinical trials without reported side effects until nowFUS result in clear defects in MNs derived from FUS-ALS patients, which is confirmed by gene editing and overexpression approaches. Furthermore, we can rescue the defects pharmacologically by HDAC6 inhibition and by genetic silencing of HDAC6, which suggests that this could become a new therapeutic strategy for ALS.In conclusion, our study showed that point mutations in 13.Primary human fibroblasts were obtained from skin biopsies of ALS patients and controls with the approval of the ethical committee of the University Hospitals Leuven (S50354). The 1/1 and 2/2 samples were obtained from a 33–year-old and a 71-year-old female patient, respectively. Both of these patients carry a R521H mutation in FUS. The 3/1 sample is from a 17-year-old male patient carrying a de novo P525L FUS mutation. The 3/2 and 3/3 samples are from the unaffected parents of patient 3/1. The 2/1 sample is from a presymptomatic patient carrying a R521H mutation. Human iPSC lines were generated by using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Reprogramming factors  were transfected by using non-integrating Sendai virus vectors from the kit. The clearance of these Sendai-virus vectors was confirmed by using TaqMan® iPSC Sendai Detection Kit (Invitrogen). Control BJ1 iPSC were generated from BJ fibroblasts (ATCC® CRL-2522™). The 4/1 and 4/2 are published healthy control iPSCsCreation of isogenic controls of iPSC carrying the R521H mutation in FUS was performed by CellSystems . iPSCs were transfected with gRNA vector, Cas9 vector, and donor DNA. Transfected cells were selected with puromycin for 2 days. Single clones were genotyped with genomic DNA PCR and subsequently sequenced. The absence of the FUS mutation was confirmed by this sequencing.AgeI and MluI sites of the pZ:F3‐P–Tet-On-3f-tdT-F plasmid (constructed by the lab35) to replace the tdT cassette to allow inducible FUS expression. The RMCE protocol used was described before35. Puromycin (100–200 ng/ml) and 0.5 μM 1‐(2‐deoxy‐2‐fluoro‐beta‐d‐arabinofuranosyl)‐5‐iodouracil (FIAU) were used for colony selection. All PCR primers are listed in Supplementay Table The FRT-containing hESC line H9 (WA09) was purchased from WiCell Research Institute  and used for recombinase-mediated cassette exchange to establish the FUS hESC-overexpressing FUS. Wildtype FUS cDNA sequences were purchased from OriGene Technologies . Point mutations were introduced by PCR and the cDNA was inserted into the ycin 100–00 ng/ml TM) in EssentialTM 8 medium  with 1000 U/ml penicillin–streptomycin. Colonies were routinely passaged with 0.5 mM EDTA  in Dulbecco’s phosphate-buffered saline (DPBS). Cultures were routinely analyzed by PCR for mycoplasma contamination.hESCs and human iPSCs were maintained on GeltrexR LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix  and injected with 100 μl matrigel (Becton Dickinson) subcutaneously in the back of immunodeficient RAG2−/− γc−/− mice. Tumors generally developed within 4–8 weeks. Animals were killed for dissection. Teratomas were fixed overnight in 4% paraformaldehyde, washed in 70% ethanol and subsequently embedded in paraffin. After sectioning, the presence of cells from the three germ layers was assessed following hematoxylin and eosin staining. Hematoxylin–eosin staining was performed by incubating sections for 2.5 min in Harris’ hematoxylin, washing with water for 10 min, and incubation for 10 s in 0.01 g/ml eosin. These experiments were approved by the Ethical Committee for animal experiments of the KU Leuven.iPSCs were collected through enzymatic dissociation, 5–10 × 1032. iPSC clones were treated with collagenase type IV to form small clusters and resuspended in EssentialTM 8 medium. During the first 2 days, medium was changed every day with Neuronal basic medium  supplemented with 40 μM SB431542 (Tocris Bioscience), 0.2 μM LDN-193189 (Stemgent), 3 µM CHIR99021 (Tocris Bioscience), and 5 µM Y-27632 (Merck Millipore). From day 3 on, 0.1 µM retinoic acid (Sigma) and 500 nM SAG (Merck Millipore) was added. From day 8 on, BDNF  and GDNF  were added. DAPT  was added on day 9. Floating clusters were dissociated into single cells for plating on day 11 by using 0.05% trypsin (GibcoTM). Motor neuron progenitors were subsequently plated on laminin (20 μg/ml)-coated 12-well plates at 0.5–2 × 105 cells per well. From day 17 on, the cells were switched to motor neuron maturation medium supplemented with BDNF, GDNF, and CNTF  to keep long term cultures. Media were changed every other day by replacing half of the medium. For rescue experiments, motor neurons were treated overnight with either 1 µM Tubastatin A (Sigma), 1 µM ACY-738  or an equivalent amount of DMSO.Motor neuron differentiation was performed as described before, with some modifications5 cells per well (12 well plate) at day 10 of motor neuron differentiation. At day 25, oligonucleotides dissolved in sterilized water were added and mixed. The LNA oligonucleotides were used at a final concentration of 50 nM. The total incubation time before cell lysis or axonal transport analysis was 1 week.Locked Nucleic Acid (LNA™) oligonucleotides for FUS and HDAC6 were purchased from Exiqon , Motor neuron progenitors were seeded at low plating density of 0.5 × 1013. Whole-cell recordings were low-pass filtered and digitized at 2.9 and 10 kHz, respectively. Patch Master software (HEKA) was used for recording and for the final evaluation of the data. Borosilicate glass pipettes (Science Products) were pulled and polished to yield a resistance of 3–4 MOhm when filled with the internal solution . Pharmacological compounds were applied to the external bath solution . NBQX  or bicuculline  were applied via gravity through the modified SF-77B perfusion fast-step system (Warner Instruments).Patch-clamp recordings of iPSC-derived MNs were performed at room temperature with an inverted microscope (Zeiss) and an EPC-10 amplifier (HEKA)TM). All samples were run in triplicate and relative quantification was done using the ΔΔCt method with normalization to GAPDH. A list of primers can be found in Supplementary Table RNA was isolated using an RNeasy kit (Qiagen) and reverse transcription was performed using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative RT-PCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems™) on the 7500 Step OnePlus™ Real-Time PCR System .Cells were manually collected on ice and fresh-frozen cell samples were maintained at −80 °C until further processing. Samples were hydrolyzed in RIPA buffer  NP40, 0.5% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol) complemented with protease inhibitors . Protein concentrations were determined using the microBCA kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Western blotting was performed as described beforeFor ELISA, the Phosphatidylcholine Colorimetric/Fluorometric Assay Kit (BioVision) was used to detect phosphatidylcholine level in motor neuron culture medium according to the manufacturer’s instructions. The medium was collected after 2 days incubation with cells. No dilution was needed for the measurements. Absorbance was measured at 540 nm. Because of the large variation in absolute values between different batches, representative experiments are shown.Cells plated on coverslips were fixed in 4% paraformaldehyde for 20 min at room temperature and were washed with PBS. Permeabilization and blocking was done for 30 min using PBS containing 0.2% Triton X-100 (Acros Organics) and 5% donkey serum (Sigma) for 1 h. Cells were incubated overnight at 4 °C in blocking buffer (2% donkey serum) containing the different primary antibodies (Abs). After washing with PBS, cells were incubated with secondary Abs (Invitrogen) for 1 h at room temperature. Primary Abs and dilutions are listed in Supplementary Table Fluorescent and bright field micrographs were captured using a Zeiss Axio Imager M1 microscope (Carl Zeiss) equipped with an AxioCam MRc5  or a monochrome AxioCam Mrm camera . Images were analyzed using ImageJ with Coloc2 plug-in. Pearson’s correlation coefficients were used for expressing the intensity correlation for colocalization.2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES). Measurements were performed on an inverted Zeiss Axiovert 200 M microscope (Carl Zeiss) with a ×40 water immersion lens.Motor neurons  from patients and controls were loaded with MitoTracker-Red  or ER Tracker-Red , washed and left to equilibrate (20 min) in motor neuron maturation medium, before transferring them to a HEPES buffered salt solution  based on typical morphology consisting of a soma and long-extended neurites. Both MitoTracker-RED and ER Tracker-Red were excited at 580 nm, using a TILL Poly V light source (TILL Photonics) and image sequences were recorded (200 images at 1 Hz) onto a cooled CCD camera (PCO Sensicam-QE) using TillVisION (TILL Photonics) software. A heated gravity-fed perfusion system was used to keep cells at 36 ± 0.5 °C during the recordings.54. In brief, kymographs or spatio-temporal maps were constructed for each of the neuronal processes. In these maps, stationary mitochondria appear as vertical lines and moving mitochondria generate tilted lines. Proportions of moving and stationary mitochondria were extracted from the maps by marking and analyzing the properties of each of the mitochondrial trajectories.All image analysis was performed in Igor Pro (Wavemetrics) using custom-written routines based on a previously described analysis algorithm55. MNs were identified by their morphology and were analyzed by using a JEOL JEM1400 transmission electron microscope . Digital acquisitions were taken by a numeric camera .MNs were plated on plastic coverslips on the tenth day of differentiation. On day 38, cells were fixed and processed for embedding and ultrathin sectioning. Subsequently, the samples were contrasted with uranyl acetate and lead citrate as described beforeA minimum of three independent experiments based on three different differentiation batches was always performed. Statistical analysis was performed using Graphpad Prism version 5.0b.P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 were considered significant. Data values represent mean ± SEM, unless indicated otherwise.Mann–Whitney test were used as statistical analysis for patch clamp data. One-way ANOVA was used for the other experiments with post-hoc Tukey’s test to determine statistical differences between groups.*The data that support the findings of this study are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


In this review, we summarize the different limitations of currently available culture models, and discuss the challenges that have to be overcome for developing more reliable and translational platforms for the in vitro study of spinal MN degeneration.Pathogenic conditions involving degeneration of spinal motor neurons (MNs), such as amyotrophic lateral sclerosis, sarcopenia, and spinal cord injury, mostly occur in individuals whose spinal MNs are fully mature. There is currently no effective treatment to prevent death or promote axonal regeneration of the spinal MNs affected in these patients. To increase our understanding and find a cure for such conditions, easily controllable and monitorable cell culture models allow for a better dissection of certain molecular and cellular events that cannot be teased apart in whole organism models. To date, various types of spinal MN cultures have been described. Yet these models are all based on the use of immature neurons or neurons uncharacterized for their degree of maturity after being isolated and cultured. Additionally, studying only MNs cannot give a comprehensive and complete view of the neurodegenerative processes usually involving other cell types. To date, there is no confirmed  However, these neurons are literally in their immature state  and their peripheral axons occurs in neurodegenerative disorders  , as wellre state , raisingre state . While tre state . This fiin vivo? At embryonic day (E) 9.5, gradient signals of two morphogens (retinoic acid and sonic hedgehog) created in the neural tube organize topographical positioning of neural precursor cells and induce their differentiation into spinal MNs  to efficiently and promptly execute their energy-consuming tasks, such as neurotransmitter release and vesicle recycling (in vitro manifest a hand-like morphology (growth cones) and are devoted to active regeneration, lengthening in response to neurotrophic factors supplemented in the culture media  the original cause or trigger of the disease (degeneration); (ii) the molecular and cellular cascade responsible for the degenerative process; and (iii) the regenerative mechanisms after the disease onset. An exception may be spinal cord injuries, for which (i) is already known. Studying the process of axonal regeneration in a simple MN mono-culture without muscle cells may sound reasonable, as this is a process prior to spinal MN settlement. However, careful attention still needs to be paid when utilizing embryonic neurons for understanding adult diseases, as their regenerative potential may not be the same as that of adult spinal MNs. The best would be to harvest primary spinal MNs from adult mice. In fact, several protocols detailing the methods for this are available ; howeverin vivo receive all these glial benefits in a spatiotemporally regulated manner based on local biological requirements. This situation is quite different from that of dissociated or differentiated MNs harvested “naked” in a plate and forced to regenerate in culture media supplemented with fixed and spatially homogenous concentrations of trophic factors.In the ventral horn, spinal MNs are supported by glial cells at the structural, trophic, and metabolic levels . Chondroin vitro, the naked neuron mono-culture is still useful for the investigation of certain biological aspects, such as the capture of disease-related cell-autonomous events. A high-throughput drug screening study that has recently been conducted by utilizing the survival of ALS patient iMNs as readout has identified bosutinib, the Src/c-Abl inhibitor approved by FDA for treating patients with chronic myelogenous leukemia, as a potential therapeutic candidate for ALS . The most utilized stem cells for studying MN biology are mouse ES cells and human iPS cells. The main advantages of these cell lines compared to primary neurons are that they can unlimitedly be expanded and stored as homogeneous populations. They are thus suited for studies requiring a large quantity of neurons for repetitive tasks, such as drug screening, proteomics, and biochemistry. On the other hand, primary MNs, which have fully differentiated in vivo before being dissociated from the spinal cord, should possess a more genuine identity of spinal MNs compared with MNs differentiated in vitro from stem cells. In terms of purity and quantity, however, primary cultures often face variable contamination with other cell types, and the number of viable primary MNs obtainable per embryo is much more limited. Of prime importance, the iPS cells are the only source enabling us to study patient-derived MNs in vitro . With this method, freshly dissected thin cross sections of the spinal cord are directly explanted in a dish. Thus, spinal MNs “embedded” in the explant do not undergo harsh procedures of chemical digestion and mechanical dissociation, but regenerate in vitro within a micro-environment resembling the in vivo condition. This culture system would also allow us to minimize the supplemental control of trophic factors as glial cells integrated in the system may supply them upon local requirements. Noteworthy, the freshly prepared spinal cord culture can serve as a ready-to-use platform for investigating neural networks by electrophysiological recording  for establishing healthy mature and fully functional NMJs in compartmented systems are still under development.Knowing the significant applications that an bjective . In addibjective . What ha growing . By allo growing . Another growing . Despite growing , no studin vitro? Difficulty in culturing these “grown-up” neurons is thought to be associated with their phenotypic changes. As animals grow, MN dendrites in the ventral horn become longer and form a more complex structure, which increases the chance of MNs to receive more injuries during the isolation, leading to their excitotoxicity-associated cell death  we will be able to compare and study, for the first time in vitro, spinal MNs from different stages of maturity  or diseases ; and (3) it will serve as a novel cell source for drug screening. Yet, all these exciting applications are based on an assumption that adult spinal MNs plated in vitro continue to retain their “mature” properties previously acquired in vivo. Therefore, following successful isolation, it is important to verify whether these adult neurons remain as expected or retrieve “immature” properties during regeneration in an artificial in vitro environment.What if we could achieve robust regeneration of adult mouse-derived mature spinal MNs ll death . Anotherll death . As an ain vitro models of many other cell types. Each culture model has different advantages and disadvantages; it is not a matter of which one is superior to the other. What is important is to select the one most fitted for individual objectives. The good news is that the range of possible models is widening, thanks to recent advance in technologies. Nonetheless, there is certainly still quite some room for progress, and further challenges have to be addressed toward establishing more reliable and translational in vitro platforms.Spinal MNs cannot truly mature and age alone. They need good supports and influences from multiple neighbors, or say “coworkers,” like CNS glia and peripheral residents, such as Schwann cells and muscle cells. All these cells constituting the neuromuscular system benefit the MNs either directly or indirectly. Incidents or diseases sabotaging their good relationships may disrupt the healthy natural maturation and/or aging of spinal MNs, and often result in motor disorders of various severities. The different limitations that we have raised and discussed here apply not only to spinal MN cultures but also to MB, SJM, DBR, and SK contributed to devising, editing, and revising of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gadus morhua) in the Baltic Sea has dramatically decreased during the past two decades, with large implications for the fishery relying on this resource. Here, we statistically investigated the potential drivers of the Baltic cod condition during the past 40 years using newly compiled fishery-independent biological data and hydrological observations. We evidenced a combination of different factors operating before and after the ecological regime shift that occurred in the Baltic Sea in the early 1990s. The changes in cod condition related to feeding opportunities, driven either by density-dependence or food limitation, along the whole period investigated and to the fivefold increase in the extent of hypoxic areas in the most recent 20 years. Hypoxic areas can act on cod condition through different mechanisms related directly to species physiology, or indirectly to behaviour and trophic interactions. Our analyses found statistical evidence for an effect of the hypoxia-induced habitat compression on cod condition possibly operating via crowding and density-dependent processes. These results furnish novel insights into the population dynamics of Baltic Sea cod that can aid the management of this currently threatened population.Investigating the factors regulating fish condition is crucial in ecology and the management of exploited fish populations. The body condition of cod ( Variations of condition in wild fish populations have been attributed to several factors, such as food availability , densityClupea harengus and sprat Sprattus sprattus [Gadus morhua), the main piscivore and the most commercially important species of the system [In the Central Baltic Sea, a large focus has been addressed to investigate the factors driving the body condition of pelagic clupeid species, i.e. herring sprattus –14. On te system ,16. The e system . Concomie system ,18. The e system . Analysee system . Howevere system , but theOne of the most prominent abiotic changes that occurred in the Baltic Sea ecosystem during the past two decades has been the fivefold increase in the extent of hypoxic and anoxic areas ,23, whicPseudoterranova decipiens (cod worm) and Contracaecum osculatum (liver worm) [Halichoerus grypus) [Another factor that previously has been related to Baltic cod condition is the intensity of infection with the parasites er worm) . The inf grypus) –36, thatHere we used for the first time a newly compiled time-series of fishery-independent biological data to investigate the changes of Baltic cod condition during the past 40 years. We firstly characterized the variations in condition by investigating the potential differences between areas and size-classes. Thereafter, the changes in condition were put in relation to both the biological context  and hydrological circumstances (extent of hypoxic areas). These analyses, by discerning the main factors relating to cod condition, are essentials for the management of this ecologically and commercially key species in the Baltic Sea.2.L), total weight (W), age, sex and maturity stage and were retrieved from the ICES DATRAS database (www.ices.dk). Further data collected during bottom trawl surveys performed in 1976–1990 were retrieved from the national databases of the former Swedish Board of Fisheries  and the former Baltic Research Institute of Fishery of Latvia (currently the Latvian Institute of Food Safety BIOR). Cod individual body condition (Fulton's K) was estimated as K = W/L3 × 100, where W is the total weight (g) and L the total length (cm) of the fish. Condition was then averaged per 10 cm length-class  for each SD, year and country. Thereafter, a generalized linear model (GLZM) was used to predict the year effect on cod condition for each SD after scaling out the country effect. Typically, during the surveys each country covers a specific area within the same SD. Therefore, this correction allowed accounting for potential spatial differences in condition within an SD when its area was not entirely covered by the survey. Condition data were normally distributed, and thus the normal distribution with an identity function was used in the GLZMs. This procedure has been used before to estimate SD-specific condition of Baltic Sea sprat [n < 50 for some SDs, countries or years). We focused on the cod condition in autumn (i.e. quarter 4), corresponding to the cod main feeding season [Biological data on Eastern Baltic cod individuals were collected during the Baltic International Trawl Survey  betweenea sprat . Mean cog season . MoreoveIndices of cod abundance  from the BITS survey in SDs 25–28 were retrieved from ICES DATRAS database. The indices are based on bottom trawl hauls typically swept between 20 and 100 m depth, whereas hauls shallower than 20 m and deeper than 100 m are very rare . The timTime-series of the biomass of the main pelagic prey of cod (the clupeids herring and sprat), by age and 0.5 cm length-class, in SDs 25–28 were from the autumn Baltic International Acoustic Survey  and hisThe grey seal data used in this study is based on counts of grey seals hauling out during moult. Monitoring of the grey seal moult has been carried out in the Baltic Sea since the mid-1970s and Swedish surveys have been coordinated by the Swedish Museum of Natural History ever since. In 1989, population trends of Baltic grey seals became part of the Swedish marine monitoring programme with the aim to provide data for analyses of population trends for the Baltic population. Grey seal surveys are carried out during peak moulting time (late May early June during a pre-defined two week period). Even if the surveys are geared to provide a good estimate of population trends for the entire Baltic population, studies of grey seal site fidelity ,43 have 2) of hypoxic bottoms  by SD (SDs 25–28) were obtained from the Swedish Meteorological and Hydrological Institute . Time-series of the depth of hypoxic waters (i.e. mean depth at which hypoxia was encountered) by SD were also from SMHI. We used 1 ml l−1 as threshold for hypoxia because the Baltic cod has been shown to avoid oxygen concentrations below this value [−1 (the latter more commonly used in the literature as threshold for hypoxia) are highly correlated (r = 0.94). We estimated the suitable areas for cod as those with an oxygen concentration > 1 ml l−1, excluding the areas shallower than 20 m and deeper than 100 m, as these are not well and consistently sampled during the BITS. Moreover, at depths > 100 m cod is very rare [−1 within the 20–100 m depth interval. Hypoxic areas were also used in our study as an indicator of benthic productivity [Time-series of total areas . The fofunction , Vi … Vna priori. All variables were expressed as standardized anomalies prior to analysis (X′ = X − mean/s.d.).As predictors for cod condition (response) the following variables were employed in the full models: cod abundance (i.e. density-dependence), biomass of herring and sprat  included both separately or taken together as clupeids (i.e. food availability), seal abundance  and extent of hypoxic areas . These predictors were selected based on acknowledged ecological and physiological mechanisms potentially affecting cod condition ,21,25,33p-value was excluded at each step and the model was run again, until the GCV reached a minimum. If excluding a predictor increased the GCV, the predictor was retained and the procedure ended. We limited the maximum degrees of freedom acceptable for each term to k = 4. A normal distribution with an identity function was used in the GAMs. We calculated the deviance explained by the final models, and the deviance contribution of each predictor based on the percentage difference in explained deviance of the final models after deletion of one predictor at a time while keeping the others (i.e. with replacement) (Difference Dev. Expl. %) [To find the best possible set of predictors, we ran a model selection based on statistical significance and generalized cross validation (GCV) using a backward stepwise procedure. The GCV criterion allows an optimal trade-off between the amount of deviance explained by the model and the model complexity measured through the equivalent degrees of freedom. From the full model, the predictor with the lowest Expl. %) . ResiduaExpl. %) . We modeExpl. %) ,54 and fExpl. %)  of the Cwww.r-project.org) and Statistica v. 6.1. The significance level was set to α = 0.05 for all tests.The statistical analyses were performed using the mgcv library of R v. 3.0.2 . The drop in cod condition after the mid-1990s was strongest for the intermediate and larger cod  compared with the 10–19 cm length-class (11% decrease) and the 20–29 cm length-class (15% decrease) . However, the temporal variations in condition were synchronous for all the length-classes, as shown by pairwise correlations at different time lags  increased between 1976 and 1995, whereas it dropped from the mid-1990s to 2010, and stabilized at a low level in the last few years in all the SDs and size-classes (figures 5–28 incr3ecrease) a,b. Howeime lags . The con3.2.a). The abundance of cod ≥ 30 cm was used as predictor in the GAM modelling because they constitute the spawning fraction of the Baltic cod population [b). Seal abundance was low up to the early 1990s, a little higher between 1993 and 2003, and since the mid-2000s it has increased strongly (c).Cod adult abundance  increased from the mid-1970s to the mid-1980s, followed by a drop that culminated in the early 1990s. Cod abundance has been low from 1991 to the mid-2000s. Afterwards, it increased up to the late 2000s, followed by a slight decrease a. The abpulation  and comppulation . In the strongly c.Figure3.3.−1 oxygen concentration) between 1993 (lowest level in the time period considered) and 2011 (highest level in the time period considered) are shown as an example in the maps of figure 6a. Hypoxic areas in SDs 25–28 decreased from the late 1970s to the lowest levels in mid-1990s (approx. 10 × 103 km2), increased strongly up to the mid-2000s, and remained constant afterwards at around 50 × 103 km2 (figure 6b). Hypoxic areas within the main depth interval of cod (i.e. down to 100 m depth) showed very similar patterns (figure 6b). Suitable areas for cod  increased from the late 1970s to the mid-1990s , decreased until the late 2000s and remained thereafter stable at around 90 × 103 km2  (figure 6c). In percentage, the decrease in suitable areas between the early 1990s and the late 2000s has been approximately 30%.The changes in the extent of hypoxic areas . Cod abundance, sprat and herring biomass and seal abundance were discarded by the model selection procedure (their stepwise elimination decreased the GCV of the model). The residuals did not violate the normality and homogeneity assumptions, and were not autocorrelated . The use of ‘cod density’ instead of ‘cod abundance’ improved the overall performance of the model .The final model of cod condition for the period 1976–1993 explained 36.4% of the total deviance . Cod abufigure 7b). Cod abundance, herring biomass and seal abundance were discarded by the model selection procedure (their stepwise elimination decreased the GCV of the model). The residuals did not violate the normality and homogeneity assumptions and were not autocorrelated . The use of the ‘biomass of the most suitable prey size’ instead of ‘prey biomass’ increased the deviance explained by the model (87.2%) but also increased its GCV .The final model for the period 1994–2014 explained 82.9% of the total deviance . Sprat bfigure 7We also modelled the temporal changes in cod condition in the whole time period (1976–2014). The final model explained 85.9% of the total deviance . Cod abu4.Our study suggests that the temporal variations in the condition of the Eastern Baltic cod have been caused by multiple biotic and abiotic factors. Our analyses, in fact, show a strong negative correlation between cod condition and the extent of hypoxic areas and a positive correlation between cod condition and the biomass of sprat after the mid-1990s. On the other hand, a negative relation between cod condition and cod abundance, as indication of intra-specific competition, was evident before the mid-1990s in correspondence of large variations in cod population abundance.The factors affecting the condition of other gadoid populations, including cod, have been intensively investigated worldwide. These factors range from physical forcing and prey availability to selective fishing and internal population control such as intra-specific density-dependence ,21,56,574.1.et al. [per capita food availability. Our analyses performed separately for the two periods, 1976–1993 and 1994–2014, showed evidence for density-dependence in the first period and food limitation in the second period. However, while in the second period with low cod abundance the sprat dynamics have been mainly driven by recruitment variations, in the first period it was driven by cod predation [p = 0.03). These results suggest that feeding opportunities on pelagic prey, either driven by density-dependence or food limitation, have been important to shape cod condition in the past 40 years.One of the main biotic changes in the Baltic Sea ecosystem during the past four decades has been the large increase in the total population size (SDs 22-23) of sprat (i.e. the main pelagic fish prey for cod) starting in the early 1990s. After having reached a peak in the mid-1990s, the sprat population size somewhat decreased again but has remained at relatively high levels since the early 2000s . Beside et al.  who founredation ,60. Ther4.2.Although feeding opportunities on pelagic fish prey were important to explain cod condition, another strong factor in our model was represented by the extent of low-oxygen bottoms. The effect of hypoxic areas was especially strong in our models in the second time period, i.e. 1994–2014. Hypoxic areas increased dramatically during the 1990s, levelling off since the mid-2000s, which depicts an inverse pattern to that shown by cod condition. Hypoxic areas can affect Baltic cod through several mechanisms, which are not mutually exclusive and whose effects may, therefore, sum up to lead to the observed changes in condition .Figure Firstly, adverse oxygen circumstances can result in physiological and behavioural stress in fish. Experimental studies have shown a decline in fish growth (in terms of increase in body size or in body condition) when fish were exposed to hypoxia  at a temperature around 5–10°C [−1 used in our study as avoidance level  that can be advocated to lay behind the strong relation between cod condition and hypoxic areas found in our statistical models presupposes that cod do not completely avoid low-oxygen areas. In , using t3 ml l−1  which co3 ml l−1 . Moreoved 5–10°C , which irding to ) could, Saduria entomon [Saduria entomon has diminished in the past two decades due to the increasing extent of hypoxic areas, constituting an additional factor explaining the decrease in cod condition . Previous analyses on Baltic cod stomach data for the period 2007–2014 showed that the proportion of cod with benthic food in their stomachs decreased in regions with prevalence of hypoxic bottoms [A second mechanism (benthos productivity mechanism) we propose to explain the negative relation between cod condition and hypoxic areas is the availability of the benthos, an important component of the diet across all sizes of cod . Benthic entomon . This sp entomon ,72, and  bottoms . This ca bottoms  probably−1 [−1) has decreased by around 30% from the early 1990s. The spatial compression of the suitable habitat and the consequent crowding of the population may trigger density-dependent processes, such as a decline in condition. In the case of the Baltic cod, the habitat compression has been concomitant with an increase in cod population abundance since the mid-2000s, potentially worsening the density-dependent response. Our analyses showed that cod density  was a better explanatory variable than cod abundance, suggesting that crowding, mediated by the shrinkage of suitable habitats, has been an important factor in the decrease of cod condition in the past 20 years (see [A third mechanism (habitat compression mechanism) we propose mediating the link between hypoxia and cod condition is represented by the habitat compression that hypoxic areas may cause on aquatic populations, with several examples spanning worldwide from open oceans, coral reefs and estuaries ,29,73–75−1  and our ars (see , for an ars (see , and the4.3.Seal parasites have been advocated as a factor that could have contributed to the decrease in cod condition during the past three decades ,19. Duri5.The decrease in the condition of Baltic cod that started in the mid-1990s has been a matter heavily debated in the past few years both in the scientific forums and media, as it has large economic and ecological implications ,18,19. OFrom a management perspective, our results highlight the importance of regulating anthropogenic nutrient inputs, to dampen eutrophication and thus combat oxygen depletion  also for


The Wnt signaling pathway is essential in the development and homeostasis of blood and immune cells, but its exact role is still controversial and is the subject of intense research. The malignant counterpart of normal hematopoietic cells, leukemic (stem) cells, have hijacked the Wnt pathway for their self-renewal and proliferation. Here we review the multiple ways dysregulated Wnt signaling can contribute to leukemogenesis, both cell autonomously as well as by changes in the microenvironment. Blood cell formation, or hematopoiesis, is sustained by a rare population of multipotent cells that can be found in the adult bone marrow (BM) and that are responsible for the continuous production of blood cells throughout life . These cHSCs are functionally defined by their ability to mediate long-term (LT) multi-lineage repopulation after transplantation. The most stringent version of this operational definition requires that HSCs have to be serially transplantable in recipient animals while retaining both self-renewal and multi-lineage differentiation capacities . As in sAs Wnt signaling plays an important role in normal hematopoiesis ,9, it isIn short, the Wnt signaling cascade is often discerned into canonical or Wnt/β-catenin pathways and the non-canonical pathways. In the absence of Wnt ligands, cytoplasmic levels of β-catenin are kept very low through the action of a protein complex  that actively targets β-catenin for degradation. This complex is composed of two negative regulatory kinases, including Glycogen Synthase Kinase 3β (GSK-3β) and at least two anchor proteins that also function as tumor suppressor proteins, namely Axin1 or Axin2 and APC (adenomatous polyposis coli). APC and Axin function as negative regulators of the pathway by sequestering β-catenin in the cytoplasm. Activation of the pathway by Wnt leads to inactivation of the destruction complex, allowing build-up of the dephosphorylated form of β-catenin and its migration to the nucleus. In the nucleus, β-catenin binds to members of the TCF/LEF transcription factor family, thereby converting them from transcriptional repressors into transcriptional activators.Researchers in the field of hematologic malignancies currently aim to elucidate the exact role of aberrant Wnt signaling during pathogenesis. Before discussing the potential role of Wnt signals in leukemias, it is important to note that different hematologic malignancies arise at different anatomical locations, e.g., the bone marrow where all acute and chronic leukemias are thought to originate versus the thymus where T-cell acute lymphoblastic leukemia (T-ALL) originates, and that the various malignancies originate from different progenitor cells or from HSCs see . Both thFinally, a note on nomenclature in mice vs. man. In animal models of leukemia, certain acute leukemias, i.e., those of B and T progenitors in the BM and thymus, are referred to as lymphomas, whereas in patients such malignancies are termed (acute) leukemias. In man, only malignancies in peripheral (secondary) lymphoid organs and other organs, i.e., skin, are termed lymphomas. For the sake of clarity we will use the latter terminology in this review.Acute myeloid leukemia (AML) has many other names, including acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia. “Acute” means that this leukemia can progress quickly if not treated, and would probably be fatal within a few months. “Myeloid” refers to the cell type that is transformed, i.e., myeloid cells of the monocytic/granulocytic lineages. It is now well-established that AML is a clonal malignancy that originates in hematopoietic stem cells (HSCs) or very immature myeloid progenitor cells. It is the type of leukemia that is most closely related to normal HSCs. In AML, chromosomal translocations resulting in abnormal fusion proteins are frequently identified. Also, mutations that lead to FLT3 internal tandem duplications (ITDs), deletions of certain chromosomes, or abnormal karyotypes are all found, making the molecular causes of this disease highly heterogeneous.The first studies describing a role for Wnt in AML showed that AML fusion proteins, such as AML1-ETO, PML-RARα, and PLZF-RARα specifically activated γ-catenin , the homologue of β-catenin, and lead to enhanced plakoglobin-LEF complexes, hence higher active Wnt signaling ,17. AlsoMany studies demonstrate that epigenetic inactivation of Wnt pathway inhibitors by CpG island methylation provides an additional mechanism for the observed Wnt-pathway activity in AML leukemic cells. The methylation status of Wnt antagonists, such as SFRP-1, 3, 4, and DKK1, was shown to be responsible for the activation of the Wnt pathway in AML cells and correlated with poor prognosis ,23,24,25Important insights into the involvement of Wnt signaling in initiation and progression of AML blasts has come from mouse models for this disease. Both in a Mixed Lineage Leukemia (MLL) fusion protein induced AML mouse model (MLL-ENL)  and in aIn clinical samples it was shown that activating mutations in FLT3 (FLT3 amplifications occur in about 30% of AML patients) were associated with high β-catenin levels ,31. The Another interesting notion is that not only cell autonomous changes in the hematopoietic or leukemic stem cells are responsible for malignant transformation, but also that disruption of niche regulation by transformed hematopoietic cells, which may overexpress Wnt signaling or intrinsic stromal defects, is a collaborative factor in leukemogenesis. For instance, in AML, deregulation of the expression of certain Wnt ligands  has been shown ,36.In conclusion, active Wnt signaling appears to play an important role in the propagation/acceleration of AML and has been shown to be an important secondary oncogenic event in mouse models of AML to transform pre-LSCs into LSCs. Based on these insights, new therapeutic opportunities have been explored by investigators who show that small-molecule WNT-pathway inhibitors, which inhibit the interaction between β-catenin and LEF1, selectively induce cell death in AML cell lines and primary AML blasts . Recent An at first sight disparate result is the favorable outcome of cytogenetically normal AML patients that were associated with high LEF1 expression, reported by Metzeler and co-authors . Other iChronic myeloid leukemia is a myeloproliferative disorder characterized by the disease-causing translocation t, which gives rise to a shortened chromosome 22, the Philadelphia chromosome (Ph) ,46 The tExpression of the BCR-ABL fusion protein starts early in disease progression and was shown to correlate with high levels of active β-catenin during the blast crisis of CML . BCR-ABL2+/NFAT pathway  in resistant CML cells and in particular the role of Fzd8. Resistant CML cells appear to be dependent on their Fzd8 expression for survival. This effect was suggested to be mediated via cytokine-induced Wnt5a production. Another explanation for the resistance of CML tumor cells towards TKIs is the selective survival of leukemic stem cells (LSCs) during treatment [However, another possibility is the involvement of the non-canonical Wnt-pathway in the pathogenesis of CML and ALL as has recently been demonstrated by Gregory et al. . In theireatment . The surreatment . They shIn conclusion, as the fusion protein BCR-ABL can actively modulate β-catenin levels in the cells, the most severely affected Wnt-pathway in CML is the canonical Wnt pathway. However, as recent studies show, in CML cells resistant to tyrosine kinase inhibitors the non-canonical Wnt-pathway might interfere when the BCR-ABL-mediated mechanism is inhibited. Therefore, novel therapeutics should not only be aimed at affecting the canonical Wnt-pathway but should also take into account the redundant effects of the non-canonical pathway, all in combination with tyrosine kinase inhibitors targeting BCR-ABL.Acute lymphoblastic leukemia (ALL) is a rapidly progressive disease that involves immature lymphocytes, most often B-cell progenitors (80%) but also T-cell progenitors (20%) as malignant counterparts of thymocytes. The importance of the Wnt-pathway in normal B-cell development has been shown in two different settings; mice lacking Lef1 or Fzd9 show disrupted B-cell development with severely reduced B cell numbers ,58. The 5+CD19+ B cells. Although it is a slowly developing disease, eventually malignant cells take over and cause reduced platelets and erythrocyte numbers, and lower numbers of T cells with associated clinical manifestations such as internal bleeding and infections. It is thought that the aberrant survival of these cells is mediated by mechanisms that evade apoptosis. Strikingly, WNT3 was highly and selectively expressed in CLL cells compared to other B cell malignancies (Diffuse Large B Cell Lymphoma (DLBCL) and follicular lymphoma) and normal T and B cells [Chronic lymphocytic leukemia is characterized by extended survival and accumulation of mature dysfunctional CD B cells ,64. As W B cells ,64. Besi B cells . Secondl B cells ,66,67. AThus, active WNT signaling in CLL seems to be mediated via at least two mechanisms. First, the production and secretion of WNT proteins concomitant with the expression of WNT-receptors (such as FZD3 and LRP5/6), provides an autocrine loop. Second, the methylation status of the WNT-antagonists, explained by the high methylation status of antagonists as found in primary CLL samples, will contribute to higher WNT-activity in CLL cells.An intriguing concept was recently explored by Gutierrez and co-workers. They reasoned that if the detrimentally high levels of LEF1 in CLL cells was closely correlated to the differentiation status of the cells, modulation of the CLL cells could affect disease progression . Indeed,Leukemic cells in T-cell Acute Lymphoblastic Leukemia are characterized by an immature phenotype of developing T lymphocytes in the thymus. Hence, pathways involved in the development of T cells ,71 are oOf special interest are two recent studies that show a clear tumor suppressor role for Tcf1 in T-ALL development ,82. BothIn contrast to AML, leukemic stem cells in ALL have long been controversial, although based on deregulated Wnt signaling such stem cells have long been predicted . Recent For all hematologic malignancies discussed in this review, Wnt signaling  appears to be involved at a certain phase during pathogenesis. There seem to be at least two different stages at which dysregulation of Wnt signaling plays a role in leukemogenesis. First, at the initiation phase of the disease, elevated Wnt signaling could be the crucial factor to progress from a pre-LSC into an LSC (as for AML) and as has been recently discovered for T-ALL where the absence of the Wnt-nuclear factor Tcf1 appears to predispose to T-ALL development. However, also in progression of established disease, as described for CML, ectopically activated Wnt signaling is important. Hence, depending on the differentiation status of the cell and or the location of the leukemia-initiating cells in its microenvironment (niche), Wnt signaling plays different roles in leukemogenesis.There seem to be at least five different mechanisms involved in the observed deregulation of Wnt signaling in leukemias. First, aberrant levels of Wnt proteins (and/or Wnt antagonists) can be secreted by tumor cells themselves and/or the microenvironment, frequently reported as an autocrine feedback loop of the tumor cells. Second, the sensitivity of the tumor cells towards the Wnt proteins (and antagonists) might be altered . As a third mechanism, epigenetic changes are repeatedly reported in most types of leukemias. Both methylation of Wnt antagonists (thereby interfering with their suppressive function) and of the Wnt5a promoter  have been found. Fourth, activating mutations in β-catenin or inactivating mutations in APC or Axin have been described in ALL, akin to well-known activating mutations in colon carcinomas. And fifth, the balance of the Tcf/Lef factors within a tumor cell appears to be an important determining factor whether Wnt signaling derails during development (see also a summary in Needless to say, these alterations in Wnt signaling offer possibilities for therapeutic intervention, especially because the levels of Wnt signaling in hematological malignancies are significantly higher than their normal counterparts ,87. In a