GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.Mutations in  GDAP1 gene cause either an axonapathy or an myelinopathy that can be transmitted recessively or dominantly to offspring. GDAP1 is located in the mitochondrial outer membrane and seems to participate in the mitochondrial network dynamics. To investigate the biological and functional consequences of lack of GDAP1 and to gain insight into the pathophysiology of the GDAP1-related neuropathies we have generated a Gdap1 knockout mouse. Characterization of this model revealed that the absence of GDAP1 induces a peripheral neuropathy with loss of motor neurons and abnormal neuromuscular junctions. We also observed defects in embryonic motor neurons and adult dorsal root ganglia sensory neurons derived from affected animals. Specifically, cultured motor neurons showed large and abnormal mitochondria, dilated perinuclear space and endoplasmic reticulum, changes in acetylation of cytoskeletal α-tubulin and calcium depletion. We propose that pathophysiology of GDAP1-associated recessive CMT neuropathy may be the consequence of abnormal calcium homeostasis and changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interactions. Our findings may be also relevant to understand the role of GDAP1 in relation to other neuropathy-related mitochondrial proteins such as mitofusin 2.Charcot-Marie-Tooth (CMT) disease is an inherited motor and sensory peripheral neuropathy. Mutations in the  Mutations in GDAP1, which maps at human chromosome 8q21.1, are causative for several types of neuropathy and are transmitted through various modes of inheritance including autosomal recessive demyelinating CMT4A with reduced nerve condition velocities (NCVs) , mitochondrial dynamics (MFN1 and MFN2), β-oxidation of fatty acids (HADHA), oxidative stress  and the cytoskeleton  using validated antibodies  transients triggered by emptying of ER-Ca2+ with thapsigargin (TG) in a Ca2+-free medium and the effect of Ca2+ readmission on SOCE activity in 24 hour-cultured motor neurons. Interestingly, neurons lacking GDAP1 showed significant lower [Ca2+]cyt than WT neurons before and after ER-Ca2+ emptying with TG  of GDAP1-related peripheral neuropathy. We observed multiple differences in motor behavior of Gdap1-/- mice. Knockout animals showed significant reduced running time on the rotarod starting at the age of 3 months, suggesting presence of defects in the balance and motor coordination. This difference disappeared at later time-points (>9 months), likely as a consequence of the onset of age-related coordination impairments at control mice or because of the compensatory effects in CNS of the Gdap1 gene paralogue Gdap1l1  changes in motor neurons might be either a compensatory response to the abnormal function of nerves that are not altered enough to show pathological changes, or lack of GDAP1 in mice may be expressed primarily as a motor neuron disease; or (ii) both proximal lesions at neuronal somas and distal changes at NMJs may be the consequence of mitochondrial dysfunction in dendrites and distal synapsis where mitochondria are more present.Reduced CMAPs amplitude in both distal and proximal nerve segments from 5-months-old mice strongly suggested the axonal nature of the neuropathy. Unexpectedly, normal number of axons and axon diameter distribution revealed no evidence of morphological axonopathy as observed in patients ,22,23 orGdap1-/- mice can be caused by various changes in mitochondrial biology: alteration in mitochondrial fission-fusion balance, increase of the total mitochondrial mass or mitochondrial transport defects. Since we observed that in sciatic nerves of Gdap1-/- mice the total mitochondrial mass is not different between WT and knockout mice, we favor the hypothesis of a defective mitochondrial dynamics. Interestingly, an increase in the number of mitochondria in peripheral axons, especially in those with diameters smaller than 3.5 μm, has also been observed in Mfn2 transgenic mice which is a mitochondrial fusion-related protein that is also located in the MOM cyt in both resting status and after pharmacological-mediated calcium depletion with thapsigargin or ionomycin. These findings suggest that the low levels of [Ca2+]cyt associated with the lack of GDAP1 could represent partial depletion of cellular calcium stores and/or release defects. Reduced Ca2+ stores and/or abnormal ER-Ca2+ release may impair proper stored-operated Ca2+ influx as well, which in turn may affect Ca2+ signaling homeostasis. These data in primary mouse MN and those we previously obtained in gene-silenced human neuroblastoma cells [2+]cyt maintenance that can be, at least partially, mediated by SOCE. Thus, GDAP1 might mediate interactions between mitochondria and ER by positioning the mitochondrial network properly. The observation of large, dispersed and non-polarized mitochondria within cultured MNs in electronic microscopy experiments favors our hypothesis. Functional mitochondria are required to maintain SOCE activity [2+ microdomains may reduce the formation of SOCE channels between ER and plasmatic membrane and may reduce the Ca2+ entry operated by ER Ca2+ release. Calcium ions themselves control a wide number of cellular functions underlying cell signaling events. Abnormal calcium signaling has been associated with neurodegeneration [2+] in the ER is observed in neurodegenerative diseases [2+] in the ER is a less known phenomenon. Chronic depletion of cellular Ca2+, either in ER or cytosol or both, affects the fluctuations and signaling of calcium ions and may induce pathological changes in mitochondria and ER and their physiological interaction which in turn may lead to the unfolding protein response (UPR) and activate the ER-stress-induced apoptosis pathway .Cytosolic Caribed by . MNs werp<0.05, with a confidence limit of 95%.Calculations and statistical analysis were performed using Excel , Statgraphic statistical software and Prism software . The specific test applied in each case is indicated in the figure legend or the text. Differences were considered statistically significant when The experimental research performed in animals has been approved by the Bioethics Committee of the Consejo Superior de Investigaciones Científicas (CSIC) and the Animal Experimentation Ethics Committee of the Centro de Investigación Príncipe Felipe (CIPF).S1 Fig(A) Semi-thin cross sections of sciatic nerves from 5-months WT and Gdap1-/-mice. No differences between genotypes were found analysing numbers of fibers in 2 and 5 months old mice (B). Similarly, at proximal and distal sciatic nerves axon size distribution (C) and g-ratio (D) show no differences between 5 months old control and Gdap1-/- mice.(TIF)Click here for additional data file.S2 Fig(A) 20 μg of tissue extracts  were fractionated on 4M urea SDS-PAGE gels, blotted against the indicated antibodies and processed for western blotting. Only antibodies that recognize a single protein band of the expected molecular mass were used in the study. The migration of molecular mass markers is indicated to the left. (B) Shows the details of the scheme of printing of RPPM. One μl samples were spotted in duplicate. Yellow boxed: negative controls of BSA; Green boxed: IgGs spotted as positive control of the secondary antibody used; Blue boxed: standard curves of HCT116 cells; Red boxed: tissue samples. The upper and lower red boxed samples correspond to different tissues. The blue plot illustrates the linear correlation that exists between the fluorescence intensity  and the amount of protein in HCT116 cell lysates. Note the absence of signal in BSA samples (yellow) and the linear increase response of the secondary antibody towards increasing content of spotted IgGs (green). Protein concentrations in the biopsies were calculated according to the fluorescence intensity obtained(TIF)Click here for additional data file.S3 Fig25 μg of tissue extract from peripheral nerves (sciatic and axillar) of two wild type  and two Gdap1-KO  5-months mice were fractionated on SDS-PAGE gels, blotted against the indicated antibodies and processed for western blotting. Note the significant reduction in the expression of LDHA, COXII and MFN2, and the presence of changes in the expression of the mitochondrial SOD2, consistent with the findings reported in RPPM . Represe(TIF)Click here for additional data file.S4 Fig(A) and lumbar spinal cord (B) biopsies of WT and Gdap1-/-mice. Histograms show normalized intensities measured by densitometry and calculated relative to β-actin. A representative blot is shown below each graph. Error bars are SEM. p values were calculated using Student's t test (n = 6. *p<0.05).Western blot analyses of Calreticulin, BIP and IP3R in sciatic nerve (TIF)Click here for additional data file.S5 FigGdap1 paralogue gene Gdap1l1 by semi-quantitative reverse transcriptase PCR. No differences were observed between WT and Gdap1-/- mice tissues.Expression pattern of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Independent HIV-1 replication in the CNS  was detected in 20% of sample pairs analyzed by SGA, or 7% of all sample pairs, and was exclusively observed after four months of infection. In subjects with longitudinal sampling, 30% showed evidence of CNS viral replication or pleocytosis/inflammation in at least one time point, and in approximately 16% of subjects we observed evolving CSF/CNS compartmentalized viral replication and/or a marked CSF inflammatory response at multiple time points suggesting an ongoing or recurrent impact of the infection in the CNS. Two subjects had one of two transmitted lineages (or their recombinant) largely sequestered within the CNS shortly after transmission, indicating an additional mechanism for establishing early CNS replication. Transmitted variants were R5 T cell-tropic. Overall, examination of the relationships between CSF viral populations, blood and CSF HIV-1 RNA concentrations, and inflammatory responses suggested four distinct states of viral population dynamics, with associated mechanisms of local viral replication and the early influx of virus into the CNS. This study considerably enhances the generalizability of our results and greatly expands our knowledge of the early interactions of HIV-1 in the CNS.Compartmentalized HIV-1 replication within the central nervous system (CNS) likely provides a foundation for neurocognitive impairment and a potentially important tissue reservoir. The timing of emergence and character of this local CNS replication has not been defined in a population of subjects. We examined the frequency of elevated cerebrospinal fluid (CSF) HIV-1 RNA concentration, the nature of CSF viral populations compared to the blood, and the presence of a cellular inflammatory response  using paired CSF and blood samples obtained over the first two years of infection from 72 ART-naïve subjects. Using single genome amplification (SGA) and phylodynamics analysis of full-length  Early HIV-1 CNS replication likely provides a foundation for brain injury and a potentially important tissue reservoir. To explore the character and timing of emergence of early HIV-1 CNS replication, we examined paired cerebrospinal fluid (CSF) and blood samples from 72 ART-naïve adults, with one-half having longitudinal samples, during the first two years following HIV-1 subtype B infection. In a cross sectional analysis over the first two years of infection, 10–25% of subjects had evidence of either local viral replication in the CNS, defined by the presence of CSF compartmentalization, or a robust inflammatory response, and in approximately 16% of subjects this CNS involvement persisted over time. In some subjects, one of two transmitted viruses replicated predominantly within the CNS, providing insight into how HIV-1 can establish independently replicating populations early in different parts of the body. Based on their entry phenotype, all viruses were selected for replication in CD4+ T cells, although this phenotype was slightly altered in the compartmentalized virus. Overall, we suggest four states to model the nature of HIV-1 CNS infection, which imply distinct mechanisms of virus/host interaction within the CNS during early infection. While HIV-1 can be detected in both the cerebrospinal fluid (CSF) and brain tissue during the weeks after initial exposure , it is uWhile extensive independent, or compartmentalized, CSF/CNS replication is associated with severe HIV-1 clinical CNS dysfunction –13, geneFor the current study, we used single genome amplification (SGA) and phylogenetic analysis to assess viral populations in the CSF in the presence and absence of cellular inflammation (i.e. pleocytosis) in cross-sectional and often longitudinal paired blood plasma and CSF samples obtained during the first two years of HIV-infection in ART-naïve subjects. We also extended our analytical approach to include Bayesian Evolutionary Analysis by Sampling Trees (BEAST) to assess time to most recent common ancestor (TMRCA) of CSF and blood HIV-1 populations , providiWe analyzed the HIV-1 RNA concentrations in paired blood plasma and CSF samples collected from 72 adult subjects enrolled in an observational neurological study of primary HIV-1 infection, defined as within one year of initial infection. All subjects were infected with HIV-1 subtype B and were ART-naïve at all study intervals, except for one subject, 9018, who was treated with tenofovir, emtricitabine, and atazanavir between the first and second analyzed time points. Paired follow-up samples were assessed for 37 subjects with longitudinal samples available within the initial two years post infection (p.i.). In total, 144 paired samples were available for analysis. Baseline demographic and clinical characteristics at enrollment for the entire cohort (n = 72) and for the subset that had sufficient CSF viral RNA concentrations  to allow adequate sampling of the viral population via SGA (n = 33) are shown in et al. ) and used the stepAIC function to perform stepwise model selection. All correlations employed Spearman’s rank correlation coefficient. For all statistical tests, P values less than 0.05 were considered significant.Compartmentalization was assessed statistically using the Slatkin-Maddison test for gene flow , Wright’st) ,20 and tst) [nn) . Differeenv nucleotide sequences determined in this study have been deposited in GenBank under accession numbers KM353586-KM355197The HIV-1 S1 FigPie chart showing the percent of samples in each phylogenetic state exhibiting minimal to no CSF pleocytosis (CSF WBC <10 cells/μl). States represented include: Not Analyzed by SGA, due to CSF viral load <1,000 copies/ml; Equilibrated (−), CSF WBC <10 cells/μl; and Compartmentalized.(TIFF)Click here for additional data file.S2 FigMDMs were infected with two positive controls that are known to be macophage-tropic (4051_C3  and Ba-L(TIFF)Click here for additional data file.S3 Figenv sequences from the CSF are labeled with solid blue circles and env sequences from the blood plasma are labeled with solid red triangles. Genetic distance is indicated at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between env sequences. Each subject ID and corresponding sampling time point are indicated.Neighbor-joining phylogenetic tree . (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Normalized surface electromyography root mean square was higher in international table tennis players with respect to national ones in all the interactions between muscles and vibration conditions (P < 0.05), with the exception of flexor carpi radialis . The difference in normalized surface electromyography root mean square between international table tennis players and national ones increased in all the muscles with high acceleration load (P < 0.05), with the exception of flexor digitorum superficialis (P > 0.05). The muscle activation during indirect-localized vibration seems to be both skill level and muscle dependent. These results can optimize the training intervention in table tennis players when applying indirect-localized vibration to lower arm muscles. Future investigations should discriminate between middle- and long-term adaptations in response to specific vibration loads.We investigated the electromyographic response to synchronous indirect-localized vibration interventions in international and national table tennis players. Twenty-six male table tennis players, in a standing position, underwent firstly an upper arms maximal voluntary contraction and thereafter two different 30-s vibration interventions in random order: high acceleration load , and low acceleration load . Surface electromyography root mean square from  LL participants' anthropometric data were: age 30.7 ± 3.7 y; mass 76.7 ± 4.3 kg; height 1.75 ± 0.04 m; BMI 25.07 ± 0.82 kg·m−2. The inclusion criteria were: (1) high skill with 15 years of experience in TT training and competitions, and having taken part in international (HL) or national (LL) Championships at the time of the investigation, (2) having regularly competed during the previous competitive season, and (3) possession of a valid medical clearance for competition. The subjects were healthy, without any muscular, neurological, and/or tendinous injury, and were not taking any medications. Each subject was informed of the procedures, methods, benefits, and possible risks of the study. Informed consent for experimentation with human subjects was obtained from all individual participants included in the study. All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments, or with comparable ethical standards.Twenty-six (three left-handed) male TT players participated in the study. All players were divided into two groups according to their best performance and national/international ranking within the Italian Table Tennis Federation. Fourteen players were assigned to the international level (HL) group and the remaining 12 to the national level (LL) group. HL participants' anthropometric data were: age 23.6 ± 3.2 y; mass 63.1 ± 4.5 kg; height 1.72 ± 0.08 m; body mass index (BMI) 21.36 ± 1.54 kg·mstatus. None of the subjects underwent any strenuous endurance activity and/or resistance training outside the usual training protocol.Subjects refrained from drinking alcohol or caffeinated beverages for 24 h before testing, and fasted for at least 4 h prior to visiting the laboratory. Each subject completed all trials at the same time on the testing days to eliminate any influence of circadian variation , from 3 to 6 p.m. to avoid circadian variations , extensor digitorum (ED), flexor carpi radialis (FC), and flexor digitorum superficialis (FS) were recorded during the two vibration interventions. Subjects were asked to contract their muscles against manual resistance to let the belly of the muscle be palpated by the operator. Bipolar  surface electrodes (inter-electrode distance 0.5 cm) were fixed longitudinally over the marked muscle belly and located according to the recommendations of SENIAM  for further analysis. One hour prior to testing, maximal isometric voluntary contractions (MVC) were performed on the vibrating device dumbbell for sEMG normalization  of dominant arm HAL) intervention aPeak was equal to 12.8 g, f = 40 Hz, and D = 4.0 mm, whereas in the low acceleration load (LAL) the aPeak was equal to 7.2 g, f = 30 Hz, and D = 4.0 mm. Each of the two interventions lasted 30 s, separated by 5 min of recovery . In the high acceleration load (norm) values in the BR, ED, FC, and FS were analysed by using a two-way ANOVA with repeated measures and the Bonferroni post-hoc test to adjust the P-value in relation to the number of contrasts that were performed. The within factor was the vibration intervention (2 levels: HAL and LAL), whereas the between factor was the TT level (2 levels: HL and LL). A t-test was used to compare HAL and LAL for each muscle . To assess the variability of the sEMGnorm measures, we calculated their coefficient of variation . The effect size (ES) was calculated with eta squared (η2) and classified as small (0.01 < η2 < 0.06) medium (0.06 < η2 < 0.146) and large (η2 > 0.14) .All the data are reported as the mean ± standard deviation (SD). Assumption of normality was verified by using the Shapiro-Wilk test. Maximal isometric voluntary contractions normalized sEMG (sEMG) Cohen, . Signifinorm values) in BR, ED, FC, and FS during HAL and LAL are summarized in Table F = 20194.99, P < 0.001, η2 = 38.387, ES = large] with a significant interaction between the vibration intervention and TT level . The BR muscle showed significant differences in the sEMG response between the two groups , but the interaction between the vibration intervention and TT level was not significant . In the FC muscle the sEMG response was higher in the LL than the HL group during LAL, whereas during HAL the sEMG response was higher in the HL than the LL group . In the FC muscle the interaction between the vibration intervention and TT level showed the highest effect size among the selected muscle . In the FS muscle the difference in sEMG response between the two groups was the highest during LAL ; the interaction between the vibration intervention and TT level was also significant [F = 11107.07, P < 0.001, η2 = 2.403, ES = large; Table P < 0.001), BR −1.49% (P < 0.001), FC 13.36% (P < 0.0001), and FS −1.74% (P < 0.001). LL revealed a significant change between LAL and HAL in ED −39.61% (P < 0.001), BR −3.54% (P < 0.001), FC −48.47% (P < 00001), and FS 16.48% (P < 0.001).Muscle responses  during LAL; and (2) the vibration acceleration negatively affects muscle activity, particularly in the LL players. In fact, the muscle response differences between HL and LL increased during HAL with the exception of the flexor digitorum superficialis (FS) muscle in LL, in which its response tended to be higher during HAL than during LAL.The main findings of this investigation were that (1) international level (HL) table tennis (TT) players responded with greater muscle activation compared to national level (LL) table tennis players during low acceleration load (LAL) and high acceleration load (HAL) vibration interventions, except for the rms) of the biceps brachii. In addition, Mischi and Cardinale (triceps brachii and biceps brachii during vibration (vibrating dumbells) superimposed on extension-flexion exercises when compared to the non-vibration condition. In contrast, Cochrane and Hawke  in the 50 ms before the collision of the racket with the ball. They showed an extension-flexion-extension sequence of muscle activity that relates to the preparatory “cocking” movements of forearm supination-wrist extension-radial flexion, followed by the action movements of forearm pronation-wrist flexion-ulnar flexion that provide the power at impact. In a comparison of skill among players of different levels, they found that this sequence of muscle activity was well-defined and consistent in skilled players, but insignificant and inconsistent in unskilled players. Their results suggested that the unskilled players were not able to adequately control the final motion of the stroke before impact, and therefore their shot lost power. Previous studies suggest that an increase in sEMG response occurs when the excitation of the alpha motoneurons increases via the muscle spindle system during vibration exposure  was investigated, matching similar biodynamic responses in the kinetic chain system  commonly involved in TT skills. From a biomechanical perspective, TT skills are open kinematic chain movements with a proximal-distal sequence that have the function of accelerating the most distal segment . It seems to be advantageous for TT players to be able to accelerate the racket, because a limited time is allocated for executing the strokes during a match (Ak and Kocak, w et al.  found thrms. However, further investigations will be necessary in order to arrive at a conclusive picture and to state that muscle activation levels of the FC and FS are neurophysiologically- or mechanically-dependent.In the present study, differences in sEMG response between the HAL and LAL interventions emphasize that the muscle activity patterns are modified as a response to changes in the excitation acceleration of input signals. In this context, Siggelkow et al.  have repOur study confirms that indirect-localized vibration applied to the forearm muscles determines acute neuromuscular responses in relation to skill level of TT players. Future investigations should take in account these acute responses that could be used to induce specific adaptations during the training process. Specifically, the acceleration load plays an important role in maximizing the muscle activation in TT players of different skill level. Therefore, these results underline the importance of individualization of the acceleration load by using the sEMG response in the muscles that have the greatest involvement in each task. Particularly, it could be interesting to apply the optimal load and to monitor the vibration effect on the skill level of TT.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


RUNX1/AML1 is among the most commonly mutated genes in human leukemia. Haploinsufficiency of RUNX1 causes familial platelet disorder with predisposition to myeloid malignancies (FPD/MM). However, the molecular mechanism of FPD/MM remains unknown. Here we show that murine Runx1+/− hematopoietic cells are hypersensitive to granulocyte colony-stimulating factor (G-CSF), leading to enhanced expansion and mobilization of stem/progenitor cells and myeloid differentiation block. Upon G-CSF stimulation, Runx1+/− cells exhibited a more pronounced phosphorylation of STAT3 as compared with Runx1+/+ cells, which may be due to reduced expression of Pias3, a key negative regulator of STAT3 signaling, and reduced physical sequestration of STAT3 by RUNX1. Most importantly, blood cells from a FPD patient with RUNX1 mutation exhibited similar G-CSF hypersensitivity. Taken together, Runx1 haploinsufficiency appears to predispose FPD patients to MM by expanding the pool of stem/progenitor cells and blocking myeloid differentiation in response to G-CSF. RUNX1 is one of the most frequently mutated genes in human leukemia. Approximately 30% of acute leukemias carry RUNX1 genetic alterations such as chromosomal translocation, point mutations and deletions.2 A germline mutation of RUNX1 is causally linked to familial platelet disorder with predisposition to myeloid malignancies (FPD/MM), a rare autosomal dominant disorder characterized by modest thrombocytopenia and defective platelet function, with 35% lifetime risk of developing leukemia.6 To date, ~30 families have been reported.7 Most of the RUNX1 mutations identified in these patients concentrate within the Runt domain and disrupt the DNA binding and β heterodimerization capabilities.1 In some cases, mutations are also found in the carboxyl terminus, abrogating the transactivation domain and resulting in formation of dominant negative forms of RUNX1.4Runx1 is well established as a master regulator of hematopoiesis. Runx1−/− murine embryos die at embryonic day 12.5 due to hemorrhage in the central nervous system and inability to generate hematopoietic stem cells (HSCs).9 Inactivation of Runx1 at the adult stage using Runx1 conditional knockout mice results in expansion and subsequent exhaustion of hematopoietic stem and progenitor cells (HSPCs).11Runx1 deficiency is insufficient for leukemogenesis and requires the accumulation of additional mutations for transformation.11Runx1 haploinsufficiency is also insufficient for leukemogenesis, although mild phenotypes such as reduced platelet counts and elevated hematopoietic progenitor counts were observed in Runx1+/− mice, which are genetically identical to FPD patients.13 It remains unclear how RUNX1 haploinsufficiency promotes leukemogenesis in FPD patients.16 Granulocyte colony-stimulating factor (G-CSF) is a potent cytokine that induces HSPC proliferation, mobilization and promotion of granulopoiesis.18 Many infections trigger stressed granulopoiesis through the production of G-CSF to augment granulocyte differentiation. G-CSF is clinically used to mobilize and collect HSCs for peripheral blood stem cell transplantation.19 G-CSF also alleviates severe neutropenia in severe congenital neutropenia patients.20 Recently, there has been growing evidence that suggests an intimate link between RUNX1 and G-CSF signaling. Mutations in RUNX1 and G-CSF receptor (CSF3R) genes were concurrently found in severe congenital neutropenia patients who developed acute myeloid leukemia.21 Treatment with G-CSF and the loss of RUNX1 enhance the detachment of long-term HSCs from the niche and increase the frequency of short-term HSCs.16 We recently reported that deficiency of Runx3, a closely related member of RUNX family, also results in G-CSF hypersensitivity.22HSC behaviors such as self-renewal, proliferation and mobilization are tightly orchestrated by cell intrinsic and extrinsic factors, the latter of which includes secreted factors and cell–cell interactions within the bone marrow (BM) niche.Runx1 haploinsufficiency contributes to leukemogenesis, the steady-state hematopoiesis and cytokine responses of Runx1+/− mice were carefully examined. Runx1+/− mice exhibited G-CSF hypersensitivity as evidenced by HSPC expansion and mobilization to the periphery, as well as myeloid differentiation block. Importantly, FPD patient-derived peripheral blood mononuclear cells harboring a RUNX1 point mutation demonstrated similar G-CSF hypersensitivity when compared with healthy donor cells. These results suggest that Runx1 haploinsufficiency can increase the pool of immature progenitor cells, thereby increasing the probability of acquiring cooperative mutations for leukemic transformation.To better understand how Runx1+/− mice were generated by Okada et al.23 and maintained in the C57BL/6 background. Experiments were carried out using 6 to 8-week-old littermates. For in vivo G-CSF administration, mice were subcutaneously injected with 250 μg/kg/day murine G-CSF or phosphate-buffered saline daily for three consecutive days. Peripheral blood (PB) was obtained via retro-orbital bleeding. Mice were killed at 24 or 72 h after the final injection. BM cells were harvested by flushing femurs and tibias in ice-cold phosphate-buffered saline and incubated with red blood cell lysis buffer. All experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC).PB samples from subjects were collected after obtaining written informed consent. The study was conducted with approval from the internal review board of Keio University School of Medicine, Tokyo, Japan and conformed to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.oC, 5% CO2 and colonies number were scored after 10 days. CFU-C assay for FPD patient was performed as previously described.7Fifty or ten thousand murine whole-BM cells, 100-sorted HSPCs/ myeloid progenitors or 20 μl of PB were seeded into 35 mm dishes in Methocult  supplemented with 10 or 100 ng/ml murine G-CSF, 10 ng/ml granulocyte-macrophage CSF, 10 ng/ml interleukin-3 (IL-3), 500 ng/ml interleukin-6 (IL-6) and 100 ng/ml stem cell factor. All cytokines were purchased from Peprotech . Cell cultures were incubated at 37 Flow cytometric analysis and sorting were performed using LSR II Flow cytometer and FACSAria instrument , respectively. Monoclonal antibodies were purchased from BD Pharmingen  or eBioscience . Antibodies used are listed in 24 pGL3-PIAS3-luc and effector RUNX1 plasmids were transfected into HL-60 cells using FuGENE 6 . Equal amounts of DNA were transfected by supplementing appropriate amounts of the backbone pEF plasmid. Forty-eight hours after transfection, cells were lysed with 1 × passive lysis buffer, and the luciferase assay was performed using the Dual-Luciferase Reporter Assay Kit (Promega).The luciferase reporter plasmid pGL3-CXCR4-luc,Total RNA was extracted using Nucleospin RNA XS kit . Equal amounts of RNA were reverse transcribed into complementary DNA with Superscript III reverse transcriptase . Power SYBR master mix or Taqman gene expression master mix (Life Technologies) were used for real-time PCR analysis. The primers or taqman probes used are listed in t-test. All values are expressed as mean±s.d. unless otherwise stated. P-values<0.05 were considered statistically significant.Differences between samples were evaluated by two-tailed Student's RUNX1 haploinsufficiency on hematopoiesis and leukemogenesis in FPD/MM patients, we used mice that are heterozygous for the Runx1 null mutation (Runx1+/−). Complete blood count analysis of 6–8-week-old Runx1+/− mice showed normal frequency of white blood cells and hemoglobin, but a significant reduction in platelet counts  and cKit+Sca-1-Lin− (KL) fractions were comparable between Runx1+/+ and Runx1+/− mice  was increased, whereas that of CD34-FcγRloKSL megakaryocyte erythrocyte progenitors was reduced in Runx1+/− mice as compared with littermate control , PB cells from Runx1+/− mice formed more colonies than Runx1+/+ mice in methylcellulose culture  compared with Runx1+/+ mice. After 15 and 60 min of G-CSF treatment, p-STAT3 was significantly higher in whole-BM of Runx1+/− mice than Runx1+/+ mice  and suppressor of cytokine signaling (SOCS) families of proteins are well-established negative regulators of STAT signaling. Therefore, we examined if these genes were differentially expressed in the KSL compartment of +/− mice . This re+/− mice . These r+/− mice . It is kRunx1+/− KSL cells prompted us to investigate if Runx1 is a direct regulator of Pias3 expression. Examination of publicly available chromatin immunoprecipitation-sequencing data set showed enrichment in the binding of RUNX1 at the promoter region of PIAS3 in both human CD34+ megakaryocytes26 (O-tetradecanoylphorbol-13-actate-induced murine L8057 cells27 (data not shown). Motif search analysis revealed that 3 RUNX1-binding consensus sequences were found within close proximity in the human PIAS3 promoter. To ascertain the role of RUNX1 in the regulation of PIAS3 expression, the −1.2-Kb promoter region covering these 3 RUNX1-binding sites were cloned upstream of a luciferase reporter. Transfection of RUNX1 and CBFβ strongly induced luciferase expression  were unable to do so , RUNX1Δ189 and RUNX1Δ243 failed to interact with STAT3, whereas Runt domain alone was successfully co-immunoprecipitated with STAT3  expansion and mobilization of HSPCs and (2) differentiation block in G-CSF-induced granulopoiesis. In response to G-CSF, Runx1+/− KSL cells sustained greater activation of STAT3 than Runx1+/+ KSL cells. Our data depicts a model whereby RUNX1 exerts a negative regulation on STAT3 signaling in both transcriptional and non-transcriptional manners  project reported a significant overrepresentation of the enrichment of RUNX1/STAT1/STAT3-binding motifs within the regulatory elements.Runx1+/− mice showed G-CSF hypersensitivity due to insufficient sequestration of STAT3 by RUNX1. Importantly, an intact Runt domain is essential for RUNX1 to inhibit the transcriptional function of STAT3. In previous studies, both Stat1 and Stat3 were reported to interact and sequester Runx2 in the cytoplasm.35 Stat5 was further shown to interact and attenuate the function of all members of RUNX transcription factors.28 Taken together, all members of RUNX and STAT family transcription factors interact and mutually moderate the transcriptional activity of each other. The Runt domain is highly conserved across the different RUNX family members; thus RUNX2 and RUNX3 are likely to use Runt domain to interact with STAT family proteins. Runx3 conditional knockout mice also showed similar G-CSF hypersensitivity.22 Therefore it strongly suggests that RUNX family proteins function as negative regulators of STAT signaling by physical interaction.We report that RUNX1 haploinsufficiency may result in exaggerated cytokine responses during infection, a short period of time when endogenous level of G-CSF and other cytokines can be greatly elevated for emergency granulopoiesis.36 RUNX1 haploinsufficient individuals may experience minor granulocyte differentiation blockage and accumulate HSPCs in the spleen and PB over an extended period of time. Of the many progenitor populations, the most prominently expanded population in Runx1+/− mice, namely GMPs, have been regarded as candidate targets for oncogenic transformation in leukemia.37 Furthermore, GMPs were efficiently transformed into leukemia-initiating cells by retroviral transduction of MLL-AF9.38 Therefore, the sustained expansion of preleukemic GMPs in FPD patients may greatly elevate the likelihood of acquiring cooperating mutations to develop full-blown leukemia. As FPD/MM patients exhibit clinical heterogeneity in both the severity of platelet defects and age of leukemia onset,39 extensive variability in the age of leukemia onset can be explained by the time at which the cooperating mutation was acquired and the severity of these cooperative hits. Mutations in RUNX1 are also considered to be initiating event for onset of various sporadic hematological malignancies such as myelodysplastic syndrome or chronic myelomonocytic leukemia. We speculate that G-CSF-induced preleukemic GMPs expansion may also contribute to the leukemogenesis of these diseases.RUNX1 mutation carriers who do not display distinct clinical features or family history are typically not screened for RUNX1 mutations. This under-diagnosis has repeatedly led to dire consequences: affected siblings with little or no detectable platelet abnormalities acted as donors for HSCT and the recipients eventually developed leukemia due to donor-derived RUNX1 mutation.39 Furthermore, mutations in the RUNX1 coding regions are not the only scenario whereby RUNX1 haploinsufficiency can occur. A significant number of FPD pedigrees did not show mutation in the RUNX1 exons, implying that cis-regulatory elements may also be able to reduce RUNX1 expression.40 Establishment of new accurate diagnosis methods is warranted. Our study shows that G-CSF hypersensitivity is observed in vitro irrespective of the types of RUNX1 alterations. As granulocyte-macrophage CSF hypersensitivity is also used as a criterion to diagnose juvenile myelomonocytic leukemia,41 we propose that G-CSF hypersensitivity would serve as a complementary test to detect affected FPD subjects, including cryptic cases.FPD has been reported to be under-diagnosed due to the large variation in the severity of platelet defects.


Despite decades of intensive research, to date, there is no accepted diagnosis for Parkinson’s disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach.r = -0.37).To explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn . Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer’s disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (Our new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders.The online version of this article (doi:10.1186/s13024-016-0072-9) contains supplementary material, which is available to authorized users. Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. PD is characterized by a large time-gap between the beginning of the neurodegenerative processes and the onset of clinical manifestations . HoweverConsidering the findings presented above, it’s crucial to adopt precise strategies/methods capable to specifically quantify α-syn neurotoxic species in biological samples. In this context, we demonstrated the development of specific, sensitive and reliable ELISA assays using our antibodies to assess different species of α-syn  in different biological samples. The provision of such reliable immunoassays is not only valuable for the discovery of ideal CSF biomarkers but also for a better understanding of the underlying pathophysiological process in PD and related disorders. Next, to explore the potential of our novel assays, we employed them to quantify different species of α-syn in CSF from a cohort of PD patients and age-matched healthy controls (HC). Finally, we investigated the usefulness of combining several biomarkers that reflect different pathogenic pathways, including AD classical biomarkers , phosphorylated-tau (p-tau) and amyloid-beta-42 (Aβ-42)), as candidate CSF biomarkers for PD and related disorders.50 120 pM and KD 9.6 × 10−11 M) [Our mouse monoclonal antibody, Syn-O2, specifically recognizes early “soluble oligomers” and late aggregates “amyloid fibrils” of α-syn Fig. , and has10−11 M) . It exclWe developed, validated and optimized a new sandwich-type ELISA for measuring t-α-syn levels in human CSF. Assay conditions were first optimized using different concentrations of the capture antibody Syn-140 . Based on its sensitivity and background signal, 0.1 μg/ml of the capture antibody was selected for subsequent assays. The detection limit of the assay was as low as 50 pg/ml, which is 20-fold less than the concentration detected in human CSF Fig. . The sta2 of 0.999 was generated , whereas, o-α-syn levels were significantly higher in the PD group (p < 0.0001). CSF p-S129-α-syn levels were elevated in PD compared to HC, with considerable overlap between the groups (p <0.001). However, both p-S129-/t-α-syn and o-/t-α-syn ratios improved the discrimination of PD from HC (p < 0.0001). CSF t-tau, p-tau and Aβ42 levels did not differ significantly between PD and HC groups  within the PD group. The levels of CSF o-α-syn did not correlate with any of AD biomarkers  within the PD group  and negatively correlated with MMSE scores in the PD group , whereas, Aβ42 didn’t correlate significantly with any of the clinical parameters.A positive correlation with t-α-syn was found for both t-tau and p-tau (rrs Table . While Cp = 0.003), o-α-syn (p <0.0001), p-S129-α-syn (p < 0.0001), o-/t-α-syn ratio (p <0.0001) and p-S129/t-α-syn ratio  and it did not recognize the other synuclein proteins (β- or γ-syn). Moreover, Syn-O2 clearly stained LBs and LNs only. Most interestingly, comparing Syn-O2 staining pattern with the other commercial antibodies that are widely used in IHC , Syn-O2 not only showed a lack of cross-reactivity with synaptic α-syn, but also detected pathology not detectable by other pan antibodies confirming its specificity toward α-syn pathology.Both Syn-140 and 11D12 are specific for α-syn, and while Syn-140 recognizes α-syn from different species , 11D12 is specific only for human α-syn. Moreover, PS129 specifically recognizes p-S129-α-syn and does not cross-react with non-phosphorylated α-syn. This diversity in the specifications/characteristics of our antibodies imparts distinctive virtues to our ELISA assays. Therefore, our assays can serve as powerful research tools to investigate the potential of α-syn species in different biological samples. Furthermore, our new ELISA assays provide multiple improvements over other reported immunoassays. First, our ELISA design using 384-well plates is perfectly compatible for accommodating multiple replicates even with limited volume of the sample (50 μl/well). Second, the enhanced sensitivity shown by our assays validates their suitability for the analysis of human CSF specimens. Our assays were shown to be highly target specific based on several methods, including specificity validation of the employed antibodies to their respective antigens, using brain and blood products from genetically modified mice  and monitoring assay precisions. Our ELISAs are also robust since recovery rates from spike experiments were > 90 %.Our group has previously described the development of an ELISA for quantifying o-α-syn in human CSF using the same mouse mAb  for capture and the biotinylated form of 211 for detection , 25. HowAnalyses of the cross sectional cohort revealed that while concentrations of t-α-syn decreased, o-α-syn concentrations significantly increased in PD compared to HC, consistent with other studies , 27–29. PD patients with dementia were not enrolled in our cohort, as reflected in the overall high MMSE scores registered. These high MMSE scores could explain the weakness of the correlation between MMSE scores and t-α-syn levels and the lack of a correlation with Aβ42 levels. Interestingly, CSF o-α-syn levels showed modest negative correlation with disease motor severity (H&Y grade). This inverse correlation, although weak, suggests that o-α-syn increases at the early stage of the disease prior to the manifestation of the motor symptoms. Interestingly, similar finding has been recently reported in sporadic PD patients and PD cases with leucine-rich repeat kinase 2 mutations .An increase in p-S129-α-syn levels was also observed in the PD group compared to HC. Although, approximately 90 % of accumulated α-syn in LBs consists of p-S129-α-syn , it is nNevertheless, including both o-/t-α-syn and p-S129-/t-α-syn ratios improved the ability to discriminate between PD and HC, this was further enhanced by including p-tau, emphasizing the usefulness of combining several CSF biomarkers for diagnostic purposes.Synucleinopathies and tauopathies show significant clinical overlap, making the early diagnosis of PD more challenging. This overlap necessitates a combination of measurements of CSF α-syn species with key AD biomarkers to improve diagnostic accuracy. Moreover, several in vitro and in vivo studies investigated interactions among α-syn and tau proteins, showing that these proteins promote each other’s aggregation, leading to neuronal degeneration and worsening cognitive impairment . The posIn summary, using our novel mAbs, we have developed sensitive and specific ELISAs to measure t-, o- and p-S129-α-syn species in human CSF. Combining measurements of different α-syn species in CSF, we observed marked differential CSF patterns between PD and controls. Our results validate the usefulness of combining multiple CSF biomarkers in improving PD diagnostic accuracy and prognosis. Although the potential use of our assays was confirmed in an independent cohort, we still need to validate the utilization of our assays in large-scale, prospective, longitudinal and well-controlled studies such as the ongoing Parkinson’s Progression Markers Initiative, and by inclusion of patients with other neurological illnesses.Generation and purification of our mouse monoclonal antibodies were performed as previously described . BrieflySyn-140 is a sheep polyclonal antibody raised against full-length human α-syn as previously described , 38. In Monomeric and aggregated forms of recombinant α-, β- and γ-syn or tau proteins and synthetic peptides Aβ-42 , 39; IslSamples of human-, mouse- and rat-brain lysates (15 μg), recombinant α-, β- and γ-syn or human and mouse p-S129-α-syn (50 ng) were mixed with loading buffer  and then separated on 15 % SDS-PAGE gels. The separated proteins were transferred to 0.45 μm nitrocellulose membranes (WhatmanGmbh-Germany) at 100 V for 45 min. The membranes were boiled for 5 min in PBS and then blocked for 1 h with 5 % non-fat milk prepared in PBST prior to incubation with 10 ml of primary antibody (50 ng/ml) for 2 h at RT. The membranes were then washed 4 times with PBST followed by incubation with the secondary antibody. After one hour, the membranes were washed 4 times with PBST, and immunoreactive bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce) according to the manufacturer’s instructions.Expression and purification of recombinant human α-syn was performed as previously described . α-Syn o3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer (PBST containing 2.5 % gelatin) for 2 h at 37 °C. After washing, 50 μl of the CSF samples  was added to each well, and plates were incubated at 37 °C for 2.5 h. 11D12 , diluted in blocking buffer at 1:5 K was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody  diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate . The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was carried out using serial dilutions of recombinant human α-syn in artificial CSF.A 384-well ELISA microplate  was coated by overnight incubation at 4 °C with 0.1 μg/ml Syn-140  in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples  was added to each well, and plates were incubated at 37 °C for 2.5 h. PS129  diluted in blocking buffer (1:1 K) were added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (donkey anti-mouse IgG HRP (Jackson ImmunoResearch)) diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate . The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human p-S129-α-syn in artificial CSF (50 μl/well).A 384-well ELISA microplate  was coated by overnight incubation at 4 °C with 0.5 μg/ml Syn-140  in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples  was added to each well, and plates were incubated at 37 °C for 2.5 h. FL-140 , diluted in blocking buffer at 1:1 K, was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of goat anti-rabbit IgG HRP (Jackson ImmunoResearch) diluted in blocking buffer (1:15 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate . The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human o-α-syn in artificial CSF (50 μl/well).A 384-well ELISA microplate  was coated by overnight incubation at 4 °C with Syn-O2 (0.2 μg/ml) in 200 mM NaHCOConcentrations of t-tau, p-tau and Aβ42 in CSF were determined using the sandwich ELISAs Innotest β-Amyloid (1–42), Innotest h TAU-Ag™ and Innotest Phosphotau (181P)™; (Fujirebio Diagnostics), respectively, as described previously [A description of this cross-sectional cohort has been previously published . We inclU-test was used for comparisons between PD and HC diagnostic groups. Correlations were calculated using Spearman’s Rho (r). Subjects with PD and HC were matched for age (p = 0 · 002), but not for gender since no significant differences between CSF biomarkers in males and females were found in PD or HC groups (p = 0 · 85). The accuracy of the diagnostic value of the biomarkers was assessed based on the area under the curve (AUC) of the receiver operating characteristic (ROC) curve [χ2) tests. Sensitivity and specificity values of the combinations of biomarkers for PD diagnosis and progression values were also calculated.Statistical analyses were performed using using GraphPad Prism (version 5.0) software and IBM SPSS software 21. Continuous variables were described using medians and interquartile ranges. Categorical variables were presented as count or percentages. Mann-Whitney C) curve . Cut-offAdditional file 1:Supplementary Materials and Methods. (PDF 401 kb)


Total tau (T-tau), phosphorylated tau (p-Tau) and Beta-Amyloid 1–42 (AB42) in Cerebrospinal Fluid (CSF) are useful biomarkers in neurodegenerative diseases. The aim of the study was to investigate the role of these and other CSF biomarkers , during hypoxia-reoxygenation in a newborn pig model.2) until Base excess (BE) reached -15 mmol/l. The pigs in the group exposed to severe hypoxia (n = 12) received 8% O2 until BE reached -20 mmol/l or mean Blood Pressure fell below 20 mm Hg, The control group (n = 6) was kept at room air. For all treatments, the CSF was collected at 9.5 hours after the intervention.Thirty newborn pigs were included in a study of moderate or severe hypoxia. The moderate hypoxia group (n = 12) was exposed to global hypoxia pg/ml versus. 1290(SD +/-143) pg/ml (p<0.05), respectively. Further, a non-significant reduction of AB42 was observed in the group exposed to moderate hypoxia T-tau and p-Tau revealed no significant differences between the intervention groups and the control group, however a significantly higher level of S100B was seen in the CSF of pigs receiving hypoxia in comparison to the level in the control group. Further on, there was a moderate negative correlation between the levels of AB42 and S100B in CSF, as well as a moderate negative correlation between Lactate in blood at end of hypoxia and AB42 in CSF.This is the first study to our knowledge that demonstrated a significant drop in AB42 in CSF after neonatal hypoxia. Whether or not this has an etiological basis for adult neurodegenerative disorders needs to be studied with additional experiments and epidemiological studies. AB42 and S100B are significantly changed in neonatal pigs subjected to hypoxia compared to controls and thus may be valuable biomarkers of perinatal asphyxia. Intrapartum events are among the most common causes of neonatal death with more than 800,000 annual cases worldwide .Even though the majority of the children exposed to severe perinatal asphyxia will survive, many of them will suffer from long-term sequelae, such as cerebral palsy and cognitive deficits.In severe cases, perinatal asphyxia may lead to Hypoxic-Ischemic Encephalopathy (HIE), which may cause permanent neurological damage.Proteins, such as S100B and Neuron specific Enolase (NSE), released into the cerebrospinal fluid (CSF) during neuronal injury, might be useful as biomarkers in reflecting disease severity and predicting the clinical outcome after perinatal asphyxia .We addressed the question whether total-tau (T-tau), phospho-Tau (p-Tau) and Beta-Amyloid 1–42 (AB42), biomarkers of adult neurodegenerative disorders, could serve as markers of perinatal asphyxia ,4,5.In pediatric populations altered T-tau levels in CSF have been found in patients with brain tumors  and WestIn two retrospective studies Rondell et al. and Zetterberg et al. showed increased serum levels of Tau-protein and AB42, respectively, after hypoxia due to Cardiac arrest , 10Few, if any, experiments have been conducted to investigate if there is an association between asphyxia in the neonates and the levels of these markers.In addition to T-tau, p-Tau and AB42, we also addressed the question of how NSE and S100B were affected in our model of neonatal hypoxia-reoxygenation.The objective was to determine a possible correlation between the levels of CSF T-tau, p-Tau, AB42, S100B and NSE after hypoxia-reoxygenation in the newborn pig and establish a possible association between perinatal hypoxia-reoxygenation and any of these markers.Thirty newborn pigs, age12–36 hours, Hb > 5g/dl and in good general condition were included in the study.The pigs were given fentanyl 25microg/kg, midazolam 1.0mg/kg and pentobarbitone 20mg/kg intravenously as bolus injections for induction of anaesthesia before they were intubated and placed on their backs and washed for sterile procedures. Anaesthesia was maintained by a continuous infusion of fentanyl (50 microg/kg/h) and midazolam . When necessary, a bolus of fentanyl (10 microg/kg) and midazolam (1 mg/kg) were administered . Pentobarbitone (2.5 mg/kg) was a few times added if there was increased muscular tone that did not respond to fentanyl or midazolam. A continuous iv. infusion, Salidex: saline 0.3% and glucose 3.5%, 10 ml/kg/h was given until start of hypoxia. From 15 min after end of hypoxia the infusion was continued at 5 ml/kg/h.2) 4.5–6.0 kPa) was achieved by adjusting the peak inspiratory pressure or ventilatory rate.The pigs were ventilated with a pressure-controlled ventilator  IMV mode, humidification by Fisher and Paykel MR730, 39°C. Normoventilation , and the right carotic artery was cannulated using a venflon . Both procedures were conducted under sterile conditions, and the cannulas were sutured to the skin. The animals were thereafter placed in a prone position for the rest of the experiment. Rectal temperature was maintained between 38.5 and 39.5°C with a heating blanket and a radiant heating lamp. Mean arterial blood pressure (MABP) was measured continuously in the right carotic artery using BioPac systems MP150-CE.Twelve pigs were included in each experimental group and six pigs were in the control group. After 1 hour of stabilization the pigs in the intervention groups went through global hypoxia and reoxygenation with air.The pigs in the first experimental group were exposed to global hypoxia (8% O2 in Nitrogen) until Base Excess (BE) reached -15 mmol/l (moderate hypoxia). The animals in the second experimental group (severe hypoxia) were exposed to 8% O2 until BE reached -20 mmol/l and/ or mean blood pressure fell below 20 mmHg. During hypoxia, CO2 was added, aiming at a PaCO2 8.0–9.5 kPa, to imitate perinatal asphyxia. After the hypoxic challenge the pigs were reoxygenated with air and observed for 9.5 hours. The pigs in the control group were not exposed to hypoxia. Invasive blood pressure, EEG and ECG were measured continuously. The experiments were performed under Midazolam and Fentanyl anaesthesia, and all efforts were made to minimize suffering. Between 0.5 and 1.0 ml of CSF was collected via lumbar puncture with a 22G spinal needle from each pig at the end of the study and frozen at -70°C for further analysis . Blood fAfter 9.5 hours of reoxygenation the pigs were subjected to euthanasia with an overdose of Pentobarbitone (150mg/kg).The Norwegian Council for Animal Research approved the experimental protocol . The animals were cared for and handled in accordance with the European Guidelines for the use of experimental animals by researchers who have been certified by the Federation of European Laboratory Animals Science Association (FELASA).2 to 1 mL sample. The samples were left to clot for 1 hour and centrifuged at 2500g for 15 minutes prior to assay of S100B. Neuron-specific Enolase was measured using the Kryptor NSE assay , an automated homogenous immunometric assay based on the Time Resolved Amplified Cryptate Emission (TRACE) technology [CSF was sampled from 30 pigs via lumbar puncture. The quality of the CSF of three pigs was insufficient; therefore these were not included in the study. The samples were stored at -70°C until further analyzes. Then they were thawed, mixed and diluted and analyzed according to the manufacturers' instructions. For T-tau, p-Tau and AB42 ELISA kits were applied.  and Innotest Beta-Amyloid; all Innogenetics, Gent, Belgium). S100B was measured using an electrochemiluminescent immunometric assay (ECLIA) on Cobas e 601 immunoassay platform . S100B cannot be assayed in EDTA-plasma, and thus serum was derived from the originally collected EDTA-plasma by adding 20 microL 2M CaClchnology .Brain tissue from hippocampus was homogenized in 5M guanidine HCl buffer with protease inhibitor and PMSF using Omnitip . Homogenate was diluted with sample dilution buffer to 1:50 and 1:200. Final guanidine HCl concentrations were below 0.1 m. Sample duplicates were run on AB42 specific sandwich colorimetric ELISAs following the protocol of the manufacturer . Optical densities at 450 nm of each well were read on a Multiscan Ascent  and sample Aβ42 concentrations were determined by comparison with the AB42 standard curves.Four microm thick sections, which had been stored in 4% formaldehyde, were deparaffinized and rehydrated. The sections kept in citrate buffer, were heated in microwave for 15 min. The sections were then incubated with Formic acid 80% for 10 minutes, prior to blocking in a Peroxidase Block, 3% H2O2 for 10 minutes.Following brief washes with Blocking buffer  for 30 minutes, the sections were incubated with Primary Antibody 6E10 (1:1000), a mouse monoclonal antibody, APP , and incubated over night at -4°C. The next day the slices were incubated with ImmPRESS  for 30 minutes. For visualization DAB plus Peroxidase Substrate  Kit were used.Total RNA was extracted from tissue samples using the EZNA Total RNA Kit II  according to manufacturers’ instructions.Purified RNA was quantified using NanoDrop ND-1000  and 2 microg were reverse transcribed into cDNA with the High capacity cDNA Reverse Transcription kit .Primers were designed using Primer Express 3.0 Software .5-´ CAGATCCGATCCCAGGTTATGA-3´.Primer sequence: Forward: 5-´AGCAGGAACGTTGTAGAGCAGG-3´.Primer sequence: Reverse: A tenfold dilution of each primer showed efficiency of between 90% and 110%.Amplification was performed for both target genes and reference gene P0 in a ViiA 7 Real Time PCR System, universal settings .An amount of 50 ng of each sample was run in duplicate with 400 nmol/l primers and Power SYBR Green Master Mix .Data were analyzed by the comparative Ct method (Delta-delta Ct method).The data were statistically analyzed using the Kruskal-Wallis test and Mann-Whitney U test for variables with non-normal distributions, and independent samples t test when the distribution was normal. Levene´s test for equality of variance was performed before performing the t-test. If Levene´s test documented a significant variance difference between the compared groups, a t-test assuming different variances was performed. Otherwise, a t-test assuming equal variance was performed.The biomarkers S100B and NSE were log-transformed to obtain a normal distribution.Pearson´s correlation was used for calculation of the correlation between the log-values of NSE and S100B and between Lactate andAB42. The statistical analyses were performed by SPSS Statistics 19.0 .Comparing the two treatment groups of either moderate or severe hypoxia and the control group, there were no significant differences in weight, hemoglobin level, age, BE, PaCO2 or glucose level at start . Table 2At the end of hypoxia and 30 minutes after end of hypoxia there were significant differences between both intervention groups and the control group regarding the levels of BE, pH, lactate and glucose (p<0.05). Until 90 minutes after end of hypoxia there were still significant differences in all 4 parameters between the group exposed to severe hypoxia and the control group and BE and lactate remained significantly different until 120 minutes after end of hypoxia (p<0.05). Blood gases taken at later time points of recovery showed no significant differences between the groups.Because of insufficient quality of CSF from 3 pigs, there were 5 pigs in the control group, 10 pigs in the group exposed to moderate hypoxia and 12 pigs in the group exposed to severe hypoxia. There was a slight correlation between AB42 and LogS100B in CSF as well as AB42 in CSF and arterial Lactate .We measured the levels of S100B in CSF and serum and the concentration of NSE in CSF .In spite of no significant difference of NSE in CSF between the hypoxia and control groups, p = 0.11, we found a strong correlation between the levels of NSE and S100B in CSF, . have played a central role in research on AD. However, the function of B-APP and AB in the nervous system is controversial.B-APP-knockout mice show severe behavioral deficits, possibly indicating that B-APP has important physiological functions in the nervous system.In the prodromal and preclinical stages of AD the level of AB42 in CSF is reduced  and we fB, resulting in accumulation of toxic AB aggregates, neuroinflammation and neuronal cell death .AB are known to self-assemble into oligomers, which are thought to be an important source of toxicity by damaging the neurons .Lambert et al.1998) presented in a mouse model that neurotoxins comprising oligomers of AB42 could kill neurons in hippocampus . AB indu98 presenIn accordance to other studies we found that S100B in CSF was significantly higher for those exposed to hypoxia , 22. HowWe did not detect any signs of AB42 in the brain homogenate with ELISA, possibly due to a low concentration of AB42 in the brain of the newborn pigs.Neither did we detect any signs of aggregation of AB42 in the brain tissue with the optical microscope, however a possible presence of small aggregates of AB42, below the detection limit of an optical microscope, cannot be excluded.Taking these points into consideration, it is tempting to speculate that the reduction of AB42 in CSF and the negative correlation with S100B after neonatal hypoxia-reoxygenation, could be a sign of aggregation of AB42, which in turn attacks the neurons, triggering a long-lasting process.In addition, it is an interesting observation that the same cognitive skills which are very often reduced for relatively well-functioning children after perinatal asphyxia are similar to the skills which are influenced in the earliest phases of AD, such as attention and visuospatial skills –25.The moderate negative correlation between AB42 and lactate at end of hypoxia could strengthen the speculation that perinatal asphyxia might inflict neurodegenerative changes, as a study on newborn lambs from 2014 showed a high correlation between lactate 4 hours after asphyxia and histological degeneration of hippocampus 72 hours after asphyxia .Could the neurons injured after neonatal hypoxia-reoxygenation be more prone to increased oxidative stress in late adulthood than their peers and could this make them more prone to neurodegenerative disorders such as AD?It would be worthwhile to study whether AB42 and S100B in CSF could represent useful biomarkers at an early stage of brain damage after perinatal hypoxia-reoxygenation.We are aware that a relatively small number of animals were investigated. The pigs were sacrificed 9.5 hours after end of hypoxia, thus there were no long term follow-up.It would have been highly interesting examining the brain of the pigs with electron microscope and search for possible changes in the neurons. However, to find the regions of interest, when we could not discover any changes in the optical microscope would have been very difficult.As this is an animal study caution should be taken interpreting it on humans.To our knowledge, this study is the first to show an association between AB42 in CSF and perinatal hypoxia. Whether or not the reduction of AB42 in CSF after perinatal hypoxia has an etiological basis for adult neurodegenerative disorders needs to be studied with additional experiments and epidemiological studies.AB42 and S100b are significantly changed in neonatal pigs subjected to severe hypoxia compared to controls, thus they may be valuable biomarkers of perinatal asphyxia.S1 File(PZFX)Click here for additional data file.S2 File(PZFX)Click here for additional data file.


By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs.Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate  CSF-contacting neurons are known to project to locomotor CPGs although their relevance to active locomotion is unclear. Here, the authors show that these cells constitute a mechanosensory organ relying on PKD2L1 channels to detect spinal cord curvature and modulate locomotor frequency in freely moving animals. Xenopus and Danio rerio as KA neuronsIn vitro studies show that in the spinal cord, CSF-cNs selectively express the transient receptor potential channel PKD2L1, a molecular component of the response to pH and osmolarity1415in vivo relay sensory information onto motor circuits during innate locomotion. Here, we combine calcium imaging in moving zebrafish larvae with quantitative behavioural analysis to investigate CSF-cN activity during locomotion and impact on behavior. We show that CSF-cNs respond to both passive and active bending of the spinal cord in vivo. As the PKD2L1 channel is specific to CSF-cNs in the spinal cord, we investigated the role of this channel in the sensory functions of CSF-cNs in vivo. In pkd2l1 mutants, cellular morphology is unaltered but the response to bending is abolished, suggesting that this channel is necessary for CSF-cN mechanosensory response to bending. Furthermore, the loss of CSF-cN sensory function leads to a reduction of tail beat frequency in pkd2l1 mutants as well as in animals with impaired synaptic release from CSF-cNs, confirming the importance of this sensory feedback loop in the regulation of locomotion. Together, our results demonstrate that Pkd2l1 orchestrates a feedback loop that relays information on local curvature of the spinal cord to motor circuits in order to regulate the frequency of locomotion.Behavior has long been known to be modulated by the content of cerebrospinal fluid (CSF)136789pkd2l1 that specifically targets CSF-cNs in the larval spinal cord  triple transgenic larvae  relative to tail movements (<200 ms) to compare calcium signals before and after active tail motion. TagRFP was co-expressed in CSF-cNs with GCaMP5G to track moving cells and to correct for motion artifacts . Moreover, after unilateral tail bends, our results revealed a clear activation of dorsal CSF-cNs ipsilateral to the contracting side . However, the responses of dorsal ipsilateral CSF-cNs to active bending  transgenic larvae  in agreement with European Union legislation. Adults were reared at a maximal density of 8 animals per liter in a 14/10 (light/dark) cycle environment. Fish were fed live artemia twice a day and feeding regime was supplemented with solid extracts matching the fish developmental stage . Larvae were raised at 28.5 °C with a 14/10 day/night light cycle. All experiments were performed at room temperature on 5–6 dpf larvae unless stated otherwise.Tgicm10 transgenic line expressing the Gal4 reporter gene in pkd2l1 expressing cells, we amplified 3.8 kbp of genomic sequence immediately upstream of the predicted ATG site for the zebrafish pkd2l1 gene (ENSDARG00000022503)hsp promoter, while the intronic sequence was placed after the Gal4 stop codon and before the SV40 poly-A site. For establishing the Tg(pkd2l1:GCaMP5G)icm07, the same strategy was used by regular cloning with the PT2 vector containing the GCaMP5G construct. Tg(UAS:GCaMP5G)icm08 was generated by regular cloning in the PT2–14xUAS vector. The transgenic line Tgicm06 was generated by subcloning GCaMP6fTg(pkd2l1:tagRFP)icm17 was generated from pkd2l1:GCaMP5G by swapping the GCaMP cassette for TagRFP using restriction digest/ligation as above. To generate a GFP reporter plasmid that expresses zebrafish optimized Botulinum Toxin Light Chain (BoTxLC) under Gal4/UAS control, we used an expression vector containing the Gal4-target sequence, 5x UAS, followed by an atg-less GFP. A gateway-compatible vector pT2UASMCSGW-R1R2  was used to generate the plasmid pT2UAS:BoTxLC-GFP encoding an in-frame fusion of GFP at the C-terminus of BoTxLC. The generation and validation of the UAS:BoTxLC-GFP line including the electrophysiological characterization that GABA vesicular release is abolished in Tgicm21 transgenic larvae is detailed in a related publicationTgicm23 line was based on the injection of the mnx1 construct previously describedTransgenic lines used in this study are listed in in vitro transcription kit (Invitrogen) and injected into Tg(pkd2l1:GCaMP5G)icm07 embryos at the one-cell stage. Injected embryos were pooled and a crude DNA extract was harvested. A DNA cassette containing the target site was amplified by PCR (primers: 5′-AGGGCAAGAGAATGGCAAGACG-3′ and 5′-TGTGTGCTAGGACTGTGGGG-3′), and the resulting band was digested by SacI (Roche) to confirm disruption of the TALEN site. The mutant alleles were cloned by TOPO cloning (Invitrogen) and sequenced to determine the exact nature of the mutations. The pkd2l1icm02 mutant allele is an 8 bp deletion in exon 2 causing a +1 frameshift that terminates in a stop codon within 35 amino acids of the substitution, upstream of the first transmembrane domain of Pkd2l1. All analysis was performed blind to genotype, which was only assayed at the conclusion of each experiment.RNA coding for each TALEN monomer  was synthesized by the mMessage mMachine Larvae were fixed in 4% PFA +3% sucrose for 2 h at RT followed by 3X 5 min washes in PBS. After removing the skin, larvae were embedded in 4% low melting agarose and cut in 50 μm sagittal sections. Sections were mounted on a slide and blocked for 1 h in blocking solution . Slides were incubated with primary antibody over night at RT or for two days at 4 °C . After washing three times for 5 min in 0.1 M PBS with 0.5% TritonX100 (PBST), slides were incubated in the dark with the secondary antibody in PBST. After washing again three times for 5 min in PBST, mounting medium was added and slices were imaged on a confocal microscope using a 63X objective . Antibodies used here were Anti-GFP , anti-TagRFP , Alexa Fluor 568 goat anti-rabbit IgG , Alexa Fluor 488 goat anti-chicken IgG ; all immunohistochemical samples were counterstained with DAPI .−1 UAS:APEX2-tagRFPTgicm10 embryos at one-cell stage. 2.5 dpf larvae were then selected for CSF-cNs expression and euthanized in 0.2% tricaine methanesulfonate  prior to fixation in 2% glutaraldehyde in 100 mM sodium cacodylate. Briefly, whole embryos were shortly rinsed in Na-cacodylate, to which glycine was added, and rinsed again in Na-cacodylate buffer. The peroxidase was further revealed in DAB (0.5 mg ml−1), to which 10 mM hydrogen peroxide was addedTo label CSF-cNs for electron microscopy, we injected 60 ng μlvivo, Intelligent Imaging Innovations, Inc., Denver) using a 20x objective. Images were acquired at 9 Hz  or 11 Hz (tail-free experiments).5–6 dpf larvae were anesthetized in 0.02% MS-222 diluted in fish facility water and then mounted in glass-bottom dishes  filled with 1.5% low-melting point agarose. Calcium imaging was performed on a two photon microscope Experiments on paralyzed larvae: Larvae were paralyzed by injecting 0.5 nl of 0.5 mM α-Bungarotoxin in the ventral axial musculature  and placed in artificial cerebrospinal fluid solution −F0)/(F0−Fbg) with F0 being the average fluorescence of the 10 first frames and Fbg being the average fluorescence of the 10 first frames in a region with only background signal. To correct for motion artifacts due to cells moving out of the focal plane or incomplete tracking, we calculated the ratio of the GCaMP and the tagRFP signal (ΔR/R) as ΔR/R=(FGCaMP(t) × F0tagRFP/F0GCaMP × FtagRFP(t))−1 with F0 being the average fluorescence of the 10 first frames and both F0 and F(t) background corrected signals post−(ΔR/R)pre with (ΔR/R)pre and (ΔR/R)post being the average five time points before and after stimulation  or the average three time points before and after tail bending (tail free experiments). During motion of the tail, cells can usually not be imaged so between (ΔR/R)pre and (ΔR/R)post there is a gap of 4 frames  or 1 frame (tail free experiment).Regions of interest around CSF-cN cell bodies expressing both GCaMP and tagRFP were manually defined in the software ImageJ. To extract raw fluorescence from the image time series in moving samples , the ROIs were shifted with the moving cell using a custom-made tracking algorithm based on cross correlation. In frames where the normalized correlation coefficient fell underneath a given threshold due to the movement artifact, data were omitted for that given frame. This threshold was manually adjusted to minimize tracking errors for each imaging time series. Any Δnals see . DiffereTg or Tg incrosses were placed in circular swim arenas (Bioptech) atop a plexiglass plate. An Arduino Due software circuit (Arduino) was used to trigger one second-long recordings on a high speed camera (Vieworks) operating at 650 fps. To trigger escape behaviours, a 500 Hz, 10 ms sine wave stimulus was played through a 20 W amplifier (Adafruit) over two speakers (Monacor) at maximum volume 200 ms into the recording. Larvae were allowed to acclimate to the chamber for 5 min prior to the experiment and were allowed to rest 2 min between each stimulus. Each larva was subjected to five trials, the properties of which were averaged within subjects. Larvae were genotyped after data acquisition. Sample sizes were selected by performing a Lehr analysis on preliminary data sets with a threshold of 5% difference in means.To assess fish behavior, a video recording chamber was constructed. 6 dpf larvae from α was calculated by identifying the midpoint of the two eye points, then calculating a head direction vector based on this point and the center of the swim bladder. A tail direction vector based on the last point on the tail minus the swim bladder was then calculated, and α was calculated as the inverse cosine of these two vector lengths. To remove noise due to tracking error, the raw trace of α was median-filtered, linearly interpolated, and finally smoothed using a smoothing spline method in MATLAB's Curve Fitting Toolbox. Simultaneously, the unprocessed tail angle trace was differentiated to estimate angular velocity, which was smoothed as above. The angular velocity trace was used to define frames of movement—any frame with angular velocity >1°/frame is defined as ‘in motion'. These frames were then connected into movement bouts using morphological closing with a block size of 10 frames. Detected bouts were then assigned priority weights by their distance from stimulus onset to identify the escape. Non-escape bouts were discarded and not analyzed. Next, local maxima and minima corresponding to bend amplitudes were identified within the escape bout by calculation of regional differences (see http://www.billauer.co.il/peakdet.html). These peaks were then marked as sequential  or non-sequential. A hemicycle for each peak was calculated as the number of frames from each peak to the next in sequence, regardless of direction. Each peak therefore receives a maximum angle, hemicycle duration, and is flagged as being either positive (in the direction of the C-bend) or negative (in the direction of the counter bend), and either sequential or non-sequential. To improve accuracy, detection of the bout onset was modified from the initial approach, which used a single threshold of the angular velocity. A straight line segment was fit to the ascending phase of the first bend, and a single-term exponential fit was calculated between this segment and a straight horizontal segment at y=0 between the stimulus onset and the intercept of the rising phase segment with the y-axis. Escape onset was then defined as the frame at which the resulting exponential fit is greater than a 2° threshold. This method reliably identified a movement onset that was independent of the amplitude of the first bend as well as resistant to noise in the angle trace. From these data, escapes were defined as those movement bouts occurring within 30 ms of the stimulus and in which the first bend is >60° . Movement bouts failing these inclusion criteria are not analyzed. From the inputs described above we are able to derive latency (escape onset-time of stimulus presentation), bout duration (escape offset-escape onset), bout distance (travel distance of point corresponding to the swim bladder), bout speed (distance/duration), number of oscillations (sum of detected positive and negative peaks/2), C-bend amplitude (amplitude of first detected peak). Mean tail beat frequency was calculated as the inverse of two times the mean hemicycle of sequential peaks. MATLAB package is available on request; the tail-tracking algorithm is property of R&D Vision.Two software packages were used to extract kinematic parameters from high-speed videos of fish during escapes. The first executable, developed in collaboration with R&D Vision, fits a series of eleven points to the fish body—two for each of the eyes, one for the swim bladder, and the remaining eight to the tail. The program identifies these 11 points for each of the 650 frames of video. The second executable consists of a series of MATLAB scripts that derives kinetic parameters from these points. First, the tail angle Tg(pkd2l1:GCaMP5G) and Tg(pkd2l1:tagRFP) larvae were plated acutely in zebrafish ACSF (see Ca2+ imaging section) on plastic petri dishes based on a published protocolDissociated cells from 4 dpf All values are mean±s.e.m. unless otherwise noted. To account for the fact that all our data are intrinsically nested , we used linear mixed models for all our hypothesis testingHow to cite this article: Böhm, U. L. et al. CSF-contacting neurons regulate locomotion by relaying mechanical stimuli to spinal circuits. Nat. Commun. 7:10866 doi: 10.1038/ncomms10866 (2016).Supplementary Figures 1-5, Supplementary Table 1 and Supplementary ReferencesA CSF-cN in vivo showing a beating cilium in a 4 dpf larvae.A CSF-cN in vitro on a cover slip showing a beating cilium three hours after plating.Calcium imaging, cell position and simultaneous recording of locomotor behavior in Tg.


Experimental evidence has cast doubt on the classical model of river-like cerebrospinal fluid (CSF) flow from the choroid plexus to the arachnoid granulations. We propose a novel model of water transport through the parenchyma from the microcirculation as driven by Starling forces. This model investigates the effect of osmotic pressure on water transport between the cerebral vasculature, the extracellular space (ECS), the perivascular space (PVS), and the CSF. A rigorous literature search was conducted focusing on experiments which alter the osmolarity of blood or ventricles and measure the rate of CSF production. Investigations into the effect of osmotic pressure on the volume of ventricles and the flux of ions in the blood, choroid plexus epithelium, and CSF are reviewed. Increasing the osmolarity of the serum via a bolus injection completely inhibits nascent fluid flow production in the ventricles. A continuous injection of a hyperosmolar solution into the ventricles can increase the volume of the ventricle by up to 125%. CSF production is altered by 0.231 µL per mOsm in the ventricle and by 0.835 µL per mOsm in the serum. Water flux from the ECS to the CSF is identified as a key feature of intracranial dynamics. A complete mathematical model with all equations and scenarios is fully described, as well as a guide to constructing a computational model of intracranial water balance dynamics. The model proposed in this article predicts the effects the osmolarity of ECS, blood, and CSF on water flux in the brain, establishing a link between osmotic imbalances and pathological conditions such as hydrocephalus and edema. The total volume of CSF is 150 mL for the average adult where 125 mL is confined to the subarachnoid spaces and 25 mL to the ventricles  and basolateral (blood facing) surface of the choroid plexus epithelium create a concentration gradient. This gradient drives water filtration from the fenestrated arterials feeding the choroid plexus into the CSF in the ventricles  alone when compared to the cerebral capillaries (140 000 cm2). In vitro experiments  into the SSS, the large vein that drains the cerebral vasculature. These AG are located at the interface between the subarachnoid space and the SSS . EndotheRecent experimental evidence suggests that there exists a pathway for the movement of water and solute in the perivascular space (PVS) surrounding the blood vessels in the brain -38. ThisIn vivo measurements are obtained using ventriculo-cisternal perfusion (VCP) to measure the effect of osmolarity on the bulk flow of nascent fluid, which constitutes CSF production by the choroid plexus and any bulk that is drawn into the ventricle as the result of an osmotic gradient.Studies linking the production and reabsorption of CSF with the osmolarity of the blood and ventricles -30 sugge3H20 into either the lateral ventricle or the bloodstream and compared the radioactivity of the blood and the CSF in the cisterna magna.Radiolabeled water experiments were also conducted that utilized an injection of These experiments suggest that water is not confined to CSF space or blood lumen, but is constantly interchanged between the parenchyma and the blood throughout the brain. These observations form the basis of the microvessel hypothesis in which water transport between the blood, ECS, and ventricles is driven by Starling forces. The microvessel hypothesis -49 has bAmple experimental evidence suggests that the transport of water between the blood, ECS, and ventricles is driven by osmotic pressure gradients -30,52,53- across the choroid plexus epithelium. Additionally, the effect of osmolarity on the rate of CSF production and water transport within the brain is described. The discussion section introduces a novel computational model that synthesizes the classical hypothesis with the microvessel hypothesis. The conclusion outlines how the computational model can be used to examine the effect of osmolarity on intracranial pathology including hydrocephalus and edema.The goal of this investigation is to present a phenomenological description of CSF production and reabsorption, describe the experimental methods used to study the physiology of CSF, and gather relevant data from those experiments. These observations were then compiled into a novel computational model that can be used to interrogate the effects of osmolarity gradients on water accumulation in the ECS and CSF. The methods section of this article describes experimental techniques used to study the physiology of CSF formation and the effect of osmolarity on the flow rate of CSF and ventricular volume. The results section presents empirical evidence for the active transport of ionsThe following section summarizes the methods used to study CSF physiology. Protocols for measuring the effect of osmolarity of the serum and ventricle on bulk flow rates of nascent fluid and ventricular volume are described. The measurement of ionic concentrations in the blood, CSF, and choroid plexus epithelium is also explained. Finally, methods for determining the flux of ions across the choroid plexus epithelium are presented.inj is the bulk flow rate of the perfusion fluid, Qout is the bulk flow rate of the collection fluid, QSAS is the bulk flow rate of the fluid in the SAS, Cinj is the tracer concentration in the perfusion fluid, and Cout is the tracer concentration in the collection fluid.Ventriculo-cisternal perfusion (VCP) measures the effect of osmolarity on the formation of CSF . An infu[1][2]SAS and Qf are unknown. The effect of osmolarity on the bulk flow of nascent fluid in the ventricles is examined by altering the osmolarity of either the blood  1 and 2 altered the osmolarity of the ventricles, while experiments 3-5 altered the osmolarity of the serum. The data collected in these experiments are described in the Results section.E1. Ventricular osmolarity and the bulk flow of nascent fluid. Wald et al . According to classical hypothesis of CSF physiology, the active transport of Na+, K+, Cl-, and HCO3- between the fenestrated arterioles feeding the choroid plexus, the choroid plexus epithelium, and the CSF in the ventricles creates an osmotic gradient that drives CSF production. The Na+ and K+ concentration in the blood, CSF and choroid plexus epithelium were determined via flame atomic emission spectrometry , and Kh is the Henry's law constant for the solubility of carbon dioxide in blood (0.03 mmol/mL/mmHg).trometry -16, whertrometry . Concentequation  as shown[3]+, K+, Cl-, and HCO3- in the choroid plexus epithelium was determined by a method devised by Johanson et al  into the femoral vein of a canine model . The fluxes and concentrations of Na+, K+, Cl-, and HCO3- in the serum, choroid plexus epithelium, and CSF [21][22][23][24])[25]Albumin transport. The concentration of albumin in each compartment, Calb, is determined by constructing molar conservation balances for each compartment. Albumin convects freely through the vasculature and in between the ventricles and the SAS. Due to its large size, the protein will be limited in its transport through membranes by the albumin reflection coefficient, σAlb, as shown in the following mass conservation equations eq. 26-33. The numerical values in the concentration subscripts are the same as for the osmotic pressure as shown in [26][27][28][29][30][31][32][33]Ion transport. The transport of ions concentration in each compartment, Cion, is determined by constructing molar conservation balances as shown in eq. 34-41. For each ionic compound, this mass conservation must be solved, specifically the K+ and Na+ as they provide a significant contribution to the osmotic pressure in each compartment. Ions convect freely through the vasculature and in between the ventricles and the SAS. Though they are small enough to fit through the various junctions that connect these compartments, charge barriers exist as represented by the ionic reflection coefficient, σion. The concentration of ions in the arterial blood is a boundary.[34][35][36][37][38][39][40][41]Glucose transport. Glucose transport throughout the intracranial system is computed by molar conservation balances as shown in eq. 42-49. Glucose concentration, Cglu, provides a significant contribution to osmotic pressure, as well as to metabolic water production in the ECS determined by the first order destruction constant for glucose in the parenchyma, kgluc, the concentration of glucose at the ECS node, Cgluc,6, and the volume of the ECS node, V6. Glucose transports freely in the vasculature and the aqueducts. However, its transport through membranes is mediated by active transport represented by the reflection coefficient, σGlu. The concentration of glucose in the arterial blood is a boundary.[42][43][44][45][46][47][48][49]Osmotic pressure determination. The osmolarity of a solution can be calculated by equation 50, where the osmotic coefficient, φ, is related to the number of particles into which the molecule dissociates, n, and the molar concentration, C, for each solute in the solution, i. The osmotic coefficient accounts for the degree of non-ideal solute dissociation.[50]The determination of the osmotic pressure, π, is complicated by a number of factors that are derived from the discrepancy between ideal theoretical osmotic coefficients and measured osmotic coefficients ,60. The The presented empirical data show the effect of osmolarity on the nascent fluid bulk flow per mOsm. Perfusion of sucrose solution into the lateral ventricle was shown to alter nascent fluid flow by 0.231 µL per mOsm , while b3H2O can be computed for ventricles, cisterna magna, and plasma. These data can be compared directly with the empirical data obtained in experiment E2 performed by Klarica et al (Further validation of the model can be accomplished by computing the transport of radiolabeled water between the compartments in the brain. The time dependent concentration of ca et al , illustrHydrocephalus, edema, and normal water drainage critically depend on osmotic pressure gradients between the compartments of the brain creating an abnormal accumulation of water. Hydrocephalus is an undesirable accumulation of water in the ventricles, while edema is associated with an accumulation of water in the ECS. It is possible to explain hydrocephalus by a pathologically high osmolarity in the ventricles drawing water from the blood or ECS, in fact, it has been shown ,53,61 th