Monoclonal antibodies against N-Brev and Brev-A were used to develop two ELISAs detecting each epitope. A comparison of brevican fragments in serum from individuals with AD (n = 28), other dementia (OD) (n = 41), and non-dementia-related memory complaints (NDCs) (n = 48) was conducted. Anti-N-Brev and anti-Brev-A antibodies selectively recognized their targets and dilution and spike recoveries were within limits of ±20%. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. For the N-Brev biomarker, serum from patients with OD showed significantly lower levels than those with AD (p = 0.05) and NDCs (p < 0.01). The opposite pattern was evident for Brev-A: serum levels in patients with OD were significantly higher than for AD (p = 0.04) and NDCs (p = 0.01). For both N-Brev and Brev-A, levels did not differ between AD and NDCs. The ratio of N-Brev/Brev-A resulted in increased significant differences between OD and AD (p < 0.01) and between OD and NDCs (p < 0.0001). The ratio discriminated between NDCs and OD  and between OD and AD . In conclusion, we developed the first assays detecting the N-terminal of brevican as well as an ADAMTS4-cleaved fragment of brevican in blood. Differential levels of N-Brev and Brev-A between AD and OD allow for these biomarkers to possibly distinguish between different forms of dementias.Evidence indicate that the brain-specific protein, brevican, is proteolytically cleaved during neurodegeneration, hence positioning fragments of brevican as potential blood biomarkers of neurodegenerative diseases, such as dementia. We aimed to develop two assays capable of detecting the brevican N-terminal (N-Brev) and the ADAMTS4-generated fragment (Brev-A), cleaved at Ser Current research diagnostic criteria are based solely on CSF and imaging biomarkers of amyloid and tau pathology [1–42 and P-tau. Targeting such neo-epitopes as biomarkers of dementia has gained much attention, as the protein fingerprint on key proteins is likely to provide insight into central disease mechanisms [To date, several symptomatic treatments are available for AD but no disease-modifying therapy exist. Efforts to develop such therapies are in part hampered by an inability to diagnose patients early and accurately. Early and accurate diagnosis is difficult due to the initiation of AD pathology years before the first clinical manifestations ,2 as welathology . Markersathology –8 and haathology ,10. Althathology ,12. Consathology ,13. The athology ,13. Plasathology –16 althoathology ,18. The athology . One poschanisms . The strchanisms –25. Takechanisms .Brevican is suggested to be an important component of highly organized extracellular matrix (ECM) structures named perineuronal nets (PNNs) . These n646 [401 (corresponding to Ser396 in rat) for a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member [in vitro Aβ stimulation of astrocytes [Two isoforms of brevican exist. The major form is secreted into the extracellular space. The minor form, lacking the C-terminal part of the full protein, is attached to the cell membrane via a glycosylphosphatidylinositol (GPI)-anchor at Ala646 . The >14y member ,48. ADAMy member . Rodent trocytes ,51. The trocytes ,52. Furttrocytes .In summary, brevican is an important component of the PNNs suggested to be implicated in synaptic plasticity and neuroprotection. There is evidence of altered expression and cleavage of brevican in relation to AD and neurodegeneration in general. This places brevican and specifically the fragments resulting from ADAMTS4-cleavage as potential biomarkers of processes related to AD. Therefore, we set out to develop highly specific serum-based enzyme-linked immunosorbent assays (ELISAs) for detection of two distinct sites on brevican, the N-terminal (N-Brev) and the ADAMTS4-cleaved neo-epitope (Brev-A), with the potential to reflect pathological processes in the AD brain. We assessed the potential of these protein sites to provide information for differential diagnosis of dementia in a cross-sectional study encompassing non-demented subjects and patients diagnosed with AD or other forms of dementia.GDVLEGDSSED. For Brev-A, synthetic peptides include: a) Immunogenic peptide SRGAIYSIPI-GGC-KLH (Keyhole-Limpet-Hemocyanin), b) screening peptide SRGAIYSIPI-biotin, c) selection peptide SRGAIYSIPI and d) elongated peptide GSRGAIYSIPI.All chemicals were standard high-quality chemicals from Merck  and Sigma-Aldrich . Monoclonal antibodies (mAb) were produced and validated using synthetic peptides purchased from the Chinese Peptide Company . Peptide conjugation reagents were produced by Pierce . For N-Brev, synthetic peptides include: a) Immunogenic peptide DVLEGDSSED-GGC-KLH (Keyhole-Limpet-Hemocyanin), b) screening peptide DVLEGDSSED-biotin, c) selection peptide DVLEGDSSED and d) elongated peptide 23DVLEGDSSED32 corresponding to the N-terminal sequence of mature brevican (present in both isoforms). The Anti-Brev-A antibody was raised against the decamer peptide 401SRGAIYSIPI410 corresponding to the neo-epitope sequence generated by ADAMTS4 cleavage of brevican at Ser401 (present in both isoforms). Both peptide epitopes were blasted for homology to other proteins using the NPS@:network protein sequence analysis with the UniProt/Swiss-Prot database [The Anti-N-Brev antibody was raised against the decamer peptide database .™ Protein G Sepharose column . Furthermore, the anti-N-Brev antibody was labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit  according to manufacturer instructions.The methods for producing these antibodies in mice were as previously described . All theUsing our monoclonal antibodies, competitive ELISAs were developed for selective detection of N-Brev and Brev-A. Preliminary experiments evaluating different buffers, incubation time and temperature as well as antibody and antigen concentrations, were performed to identify the optimal ELISA conditions. ELISA-plates used for the assay development were Streptavidin-coated (Roche cat. no.11940279). All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, .2SO4) and the plate was measured at 450 nm with 650 nm as the reference. The screening peptide was diluted in coating buffer . Standards, samples and the anti-N-Brev mAb were diluted in assay buffer A .The final settings for the N-Brev ELISA were as follows: A 96-well ELISA plate pre-coated with streptavidin was further coated with 1.4 ng/mL of the screening peptide for 30 min at 4°C, 300 rpm. The plate was washed five times in wash buffer . Hereafter, 20 μL/well of standard or sample was added followed by 100 μL/well of the peroxidase conjugated anti-N-Brev mAb diluted in assay buffer. The plate was incubated for 1 h at 4°C, 300 rpm and washed 5 times in wash buffer. Finally, 100 μL/well tetramethylbenzidine (TMB) (Kem-En-Tec cat. no. 4380) was added and the plate was incubated for 15 min at 20°C, 300 rpm. The colorimetric reaction was stopped by adding 100 μL/well of stopping solution .The final settings for the Brev-A ELISA were as follows: A 96-well ELISA plate pre-coated with streptavidin was further coated with 1.5 ng/mL of the screening peptide for 30 min at 20°C, 300 rpm. The plate was washed five times in wash buffer. Hereafter, 20 μL/well of standard or sample was added followed by 100 μL/well of the anti-Brev-A mAb diluted in assay buffer. The plate was incubated for one h at 20°C, 300 rpm and washed five times in wash buffer. Then, 100 μL/well of peroxidase conjugated rabbit anti-mouse Ab (Jackson Immunoresearch cat. no. 315-035-045) was added and the plate incubated for one h at 20°C, 300 rpm followed by five times wash with wash buffer. Finally, 100 μL/well TMB was added to the wells and the plate was incubated for 15 min at 20°C, 300 rpm. The colorimetric reaction was stopped by adding 100 μL/well of stopping solution and the plate measured at 450 nm with 650 nm as the reference. The screening peptide was diluted in coating buffer. Standards, samples, anti-Brev-A mAb and peroxidase conjugated rabbit anti-mouse Ab were diluted in assay buffer B  and the Upper Limit of Detection (ULOD). In this explorative setting, samples determined in duplicates, with CVs < 20% were accepted as reliable measurements if levels were above the LLOD, while levels below the LLOD were not considered reliable and thus given the value of the LLOD. The LLOD was established in an analytical run with 21 determinations of the lowest standard (i.e. assay buffer). The mean OD - 3x standard deviations (SD) equals LLOD. The ULOD was established based on 10 independent analytical runs determining the mean concentration of the highest possible standard concentration giving rise to an intra-assay CV < 10%. This mean concentration - 3x SD equals the ULOD.The selectivity of the assays for their intended targets was assessed by performing an analytical run with serial dilutions of selection- and elongated peptides within the assay range. The degree to which a single amino acid (elongated peptide) prevented the binding of antibodies, represented the selectivity of the assays. Furthermore, the ability of the Brev-A ELISA to selectively detect ADAMTS4-cleaved rh-brevican, not full rh-brevican, was assessed as a supporting measure of selectivity.Inter- and intra-assay variation was assessed by performing 10 independent analytical runs measuring duplicates of at least four different test samples consisting of serum as well as assay buffer spiked with selection peptide and calculating the mean CV%.To assess potential differences in analyte detection between standard diluent and sample matrix, spiking recoveries were performed. For spiking recovery, two-four different serum samples were back-calculated from various amounts of spiked recombinant human brevican (rh-brevican) to estimate the recovery percentage. Linearity of dilution was assessed by performing dilution recoveries. Four different serum samples were back-calculated from 1:3, 1:4 and 1:5 dilutions to 1:2 diluted samples to estimate the recovery percentage. Serum samples were diluted in assay buffer as this was also the diluent for patient samples.In vitro cleavage of rh-brevican with rh-ADAMTS4 was performed by dissolving 15 μg of rh-brevican in cleavage buffer  in a ratio of 1:2. 0.15μg of Rh-ADAMTS4 was added to the dissolved protein in a ratio of 1:100 and incubated at 37°C for 2, 24 and 72h. Cleavage was ended by placing the solutions at -80°C. Solutions were tested in the Brev-A ELISA.2O2 in 99% ethanol) for 30 min at RT. Then, tissues were sequentially hydrated in 96% and 70% ethanol and dH2O. To break formalin-induced methylene bridges, heat-mediated antigen retrieval was performed in citrate buffer  ON at 37°C. Slides were cooled to RT and washed in wash buffer before blocking with blocking buffer (0.5% casein in TBST) for 20 min. at RT. Tissues were incubated ON at 4°C with commercial available anti-brevican mAb (Merck Millipore cat no. MABN491), pre-neutralised anti-N-Brev mAb incubated ON at 4°C with selection peptide at a ratio of 1:50 moles) or non-neutralised anti-N-Brev mAb. Slides were then washed in wash buffer and incubated for 30 min at RT in HRP-labeled anti-mouse secondary antibody . Visualization was performed by incubation with 3,3’-diaminobenzidine (DAB)  for 10 min at RT and counterstaining was done using Mayer’s hematoxylin 2, 12 H2O, 1 mM NaIO3) for 1–2 minutes until staining of nuclei was observed. Finally, the slides were sequentially dehydrated in H2O, 70%, 96%, 99% ethanol and toluene and visualized using an Olympus IX-70 microscope and Olympus DP71 camera.To investigate the spatial localisation of brevican, histology was performed on formalin fixed paraffin embedded (FFPE) human hippocampal tissue from a patient diagnosed post-mortem with Alzheimer’s Disease (BioChain cat. no. T2236052Alz). Tissue slides were incubated for 1h at 60°C, deparaffinized in toluene and rinsed in 99% ethanol. Endogenous peroxidase activity was blocked using hydrogen peroxide . To aid lysis, samples were treated with a homogenizer. After homogenization, samples were spun down at 4°C for 10 min at 10.000 g before collection of supernatant and storage at -80°C. Later, proteins in the lysates were separated on an SDS-PAGE gel under denaturing conditions. An equal amount of protein from each tissue was loaded on the gel by first determining the protein concentration of each sample, using a Bio-Rad protein assay kit (Bio-Rad), and then equalizing the protein concentrations with MilliQ water. Following separation on the gel, proteins were transferred to a membrane and the membrane was blocked in 5% milk-TBST for two h. Then, the membrane was incubated with anti-brevican mAb (Merck Millipore cat no. MABN491) and anti-p38 MAPK Ab  as a loading control ON at 4°C. Finally, membranes were washed in TBST and the primary antibodies were visualized using secondary peroxidase-conjugated mouse and rabbit antibodies followed by membrane development using ECL Western Blotting detection reagents .To investigate the CNS specificity of brevican, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting (WB) on 10 different tissues of rat: kidney, spleen, heart, liver, lung, muscle, hippocampus, frontal cortex, brainstem and cerebellum. First, protein extraction from snap frozen tissue was performed. Tissues were pulverised in a Bessman Pulveriser. Then, the powder was dissolved in extraction buffer , Other Dementia (OD)  (n = 41) and patients with memory complaints originating from another causes than dementia (n = 48), (NDCs) . All samKruskal–Wallis test by ranks. Dunn’s pairwise tests were carried out for the three pairs of groups. Results are shown as Scatter plots with the median and 95% CI indicated. Correlation analysis were performed using the Spearman’s Rank correlation Coefficient. The significance level was set at 0.05. Graphs and statistical analyses were performed using GraphPad Prism version 7  and MedCalc Statistical Software version 14.8.1 .Differences between groups were tested using the 23DVLEGDSSED32 and 401SRGAIYSIPI410, respectively, as the addition of a single amino acid at the N-terminal of both epitopes reduced the binding of antibody almost completely  and NDCs (p < 0.01) with mean ranks of 47.5, 63.5 and 66.2, respectively (p = 0.04) and NDCs (p = 0.01) with mean ranks of 67.9, 54.5 and 54.1, respectively. For both markers, levels did not differ between AD and NDCs (p = 0.74 for N-Brev and p = 0.95 for Brev-A). Looking into the ability of serum brevican to reflect pathophysiology related to AD, we performed correlation analyses on serological levels of N-Brev and CSF levels of core biomarkers of AD. Brev-A levels were not investigated here, as the large percentage of samples with the value of LLOD would prevent reliable interpretation of the correlation matrix. Levels of brevican showed modest correlation with CSF P-Tau (A summary of cohort characteristics is given in ectively . The oppSF P-Tau . There wp < 0.01) and NDCs (p < 0.0001) with mean ranks of 40.8, 66.5 and 70.2, respectively. No difference in ratios was observed between AD and NDCs (p = 0.65).Given the variation in serum levels of N-Brev in all subjects of this cohort, we speculated that, besides the possibility of disease-related alterations in total brevican expression, basal expression levels of total brevican may vary between individuals. In this case, normalisation via ratios could allow for a more detailed picture of pathology possibly reflected by ADAMTS4 cleavage of brevican. Ratios of N-Brev/Brev-A serum levels were significantly lower in OD compared to both AD (p < 0.0001) as well as OD and AD . No separation was obtained between AD and NDCs   . Optimal = 0.66) . AD and  = 0.66) .In this study, we present two competitive ELISAs detecting distinct sites on the CNS-specific ECM protein, brevican. To our knowledge, there are no commercially available assays specifically detecting these distinct sites, thus highlighting the novelty of the data presented here. These sites are quantified in serum of AD, OD and NDCs using our competitive ELISAs, suggesting that 1) brevican and/or fragments of brevican are transferred from the CNS into the peripheral blood compartment and 2) our assays are capable of quantifying the level of these potential markers. Overall, this allows the possibility that brevican has potential as a blood-based biomarker of CNS disease and, given that the sensitivity of measurements is improved, our assays have potential for clinical utility.401, Brev-A. The in-house ELISAs showed low serum matrix interference as well as high technical reliability with inter- and intra-assay variations well below the acceptable limits. Both ELISAs showed little cross-reactvity towards an elongated peptide suggesting high selectivity towards the intended target. Selectivity of the Brev-A assay was further supported by specific detection of in vitro ADAMTS4-cleaved rh-brevican and not full-length rh-brevican which, unlike the screening and elongated peptides, displayed a natural tertiary structure better resembling a true biological condition. We note that the N-Brev assay also detected rh-brevican as evident by the spiking recovery tests , as alteration in brevican levels, as well as modification of brevican, has been shown in brain lesion models ,50,52 asThere are obvious advantages of the use of blood biomarkers. However, the CNS is separated from the peripheral blood by the blood-brain barrier (BBB) possibly affecting the peripheral composition of CNS-derived protein species. Therefore, an interesting next step in our investigation on the biology underlying the release of N-Brev and Brev-A from the brain parenchyma is the measurement of these markers in CSF. In the present study, we did not have access to patient CSF.The presence of the BBB raises the concern that only small fractions of brain proteins enter the blood. Indeed, this study shows that sensitivity of measurements is a major challenge when targeting CNS-specific proteins in blood. One limitation of this study was that only 24% of subjects had Brev-A levels higher than the LLOD. The remaining subjects were given the value of the LLOD. Biologically, it makes sense that the pool of brevican fragments cleaved by a specific protease at a specific site (Brev-A) would amount to less than the pool of full-length brevican including all possible fragments of brevican (N-Brev). However, from a technical point of view, the low concentration of Brev-A in serum necessitates highly sensitivity detection methods in order to separate low Brev-A values of one subject from that of another. Correct quantification would provide a more detailed picture of Brev-A levels and possibly improve separation of different subject groups. From this study it is clear that sensitivity of the assay needs to be improved going forward.Another limitation of this study was the heterogeneity of the group “other dementia.” The heterogeneity makes it difficult to set forth solid conclusions based on the observed differences in biomarker-levels between this group, AD and NDCs. It is possible that the variation of N-Brev and Brev-A within the group of OD merely reflects the heterogeneity of pathology. On the other hand, an equivalent variation in biomarker-level is observed in the two other groups. The difference in mean age between all demented and NDCs is also a limitation of this cohort as we cannot exclude the effect of age on the level of biomarkers.In summary, we developed the first assays detecting the N-terminal of brevican as well as an ADAMTS4-cleaved fragment of this protein in blood. In rat, we showed specific expression of brevican in the CNS and this finding is in line with the scientific consensus on brevican as a protein of the CNS. The most important and straightforward finding of this study is the detection and quantification of this CNS protein in blood of both demented and non-demented subjects. This finding allows for the possibility of gaining information on the pathology underlying CNS diseases in a non-invasive and inexpensive manner.Investigations on the diagnostic potential of N-Brev and Brev-A are positive as levels of both markers showed differential expression between groups. However, it is not clear from this study whether our markers reflect an AD-specific profile. Investigations on the potential of N-Brev and Brev-A within prognosis and monitoring of efficacy of treatment is of great interest as is the case for the potential of the markers within other types of neurodegeneration.S1 FigShown are the three sequences producing significant alignments to the epitope sequence of N-Brev. The sequence was blasted for homology to other proteins using the “NPS@: Network Protein Sequence Analysis with the UniprotKB/Swiss-prot database” software online.(TIF)Click here for additional data file.S2 FigShown are the five sequences producing significant alignments to the neo-epitope sequence of Brev-A. The sequence was blasted for homology to other proteins using the “NPS@: Network Protein Sequence Analysis with the UniprotKB/Swiss-prot database” software online.(TIF)Click here for additional data file.S1 TableGoing left to right, the columns contain information on: the extent of dilution, concentrations of N-Brev or Brev-A in diluted serum samples, percent recovery of N-Brev or Brev-A in serum diluted from 1:2 to 1:5. Optimal dilution for each assay is indicated by black square.(DOCX)Click here for additional data file.S2 TableGoing left to right, the columns contain information on: measured concentrations of N-Brev and Brev-A in serum samples, measured concentration of cleaved or full length rh-brevican for spiking, expected concentration of spiked serum samples, measured concentration of spiked serum samples, percent recovery of cleaved or full length rh-brevican and mean percent recovery in all samples.(DOCX)Click here for additional data file.S3 TableListed are the Spearman’s rho correlation coefficients (r) with the 95% confidence interval. Aβ, amyloid-β; T-tau, total tau; P-tau, phosphorylated tau; n, number of patients.(DOCX)Click here for additional data file.S4 TableFor ROC curve analysis performed on serum levels of N-Brev and Brev-A, reported are the AUC, optimal cut-off values calculated by the Youden index and the corresponding measures of sensitivity and specificity for each comparison.(DOCX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its pathophysiological cascade is still enigmatic. More than 90% of ALS patients suffer from sporadic ALS, which makes it specifically demanding to generate appropriate model systems. One interesting aspect considering the seeding, spreading and further disease development of ALS is the cerebrospinal fluid (CSF). We therefore asked whether CSF from sporadic ALS patients is capable of causing disease typical changes in human patient-derived spinal MN cultures and thus could represent a novel model system for sporadic ALS. By using induced pluripotent stem cell (iPSC)-derived MNs from healthy controls and monogenetic forms of ALS we could demonstrate a harmful effect of ALS-CSF on healthy donor-derived human MNs. Golgi fragmentation—a typical finding in lower organism models and human postmortem tissue—was induced solely by addition of ALS-CSF, but not control-CSF. No other neurodegenerative hallmarks—including pathological protein aggregation—were found, underpinning Golgi fragmentation as early event in the neurodegenerative cascade. Of note, these changes occurred predominantly in MNs, the cell type primarily affected in ALS. We thus present a novel way to model early features of sporadic ALS. Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its initial cause and the mechanisms of further progression are still enigmatic nowadays. The cerebrospinal fluid (CSF) seems to play a major role within the deathly cascade, which is particularly true concerning pathological protein aggregates and their spreading across the central nervous system ,2.Several studies have demonstrated diverse pathological effects of ALS-CSF in different in vivo and in vitro models ,4,5,6,7.Golgi fragmentation in MNs themselves has been reported in different in vitro and in vivo ALS models ,17; reviThe main goal of our study was to establish a new model system for sporadic ALS by investigating the ability of CSF derived from sporadic ALS patients to induce motor neuronal degeneration in the current gold standard cell culture model of ALS, namely patient specific iPSC-derived MNs.We used human iPSC-derived neural progenitor cells (NPC) from healthy subjects ,36,37,38−, Vimentin+) numbers, while not affecting neuronal morphology. Prolonged incubation time additionally increased non neuronal cell numbers. This effect was still obvious, albeit to a lesser extent, when cells were allowed to differentiate for 10 days prior to being incubated with CSF. Therefore, all further experiments were performed under the latter condition . Furthermore, the control cell line showed neither significant difference in the number of MNs relative to total cells  in control cultures, but no ALS-CSF specific effect. In detail, the amount of neuronal cells treated with either ALS- or control-CSF was significantly decreased compared to non-treated cells  (H(2) = 7.636, p < 0.05; z = 2.763, p < 0.05 with Dunn-Bonferroni post-hoc test), but no difference between ALS- and control-CSF exposition .When analyzing a monogenetic ALS cell line, namely the SOD-1-mutant cell line (p.R115G), we saw a significant decrease in the number of MNs relative to total cells (MN/Hoechst) when comparing ALS- or control-CSF-treatment to non-treated cells  b. Of notThere was no overt visible difference in the neuronal network comparing cells treated with control-CSF or ALS-CSF . We therIntrathecal infusion of ALS-CSF in an in vivo rat model caused pathological abnormalities similar to those found in the postmortem sections of ALS patients, namely accumulation of cytoplasmic TDP-43, indicating a spread of the disease via CSF . We therp < 0.001 by one-way ANOVA with Tukey post-hoc test) or without CSF . Control-CSF had no effect when compared to the non-treated condition .Golgi fragmentation has been reported in different ALS-models and postmortem sections of ALS patients ,19,22,23p < 0.001 by two-way ANOVA with Bonferroni post-hoc test), indicating a higher vulnerability of MNs to ALS-CSF.Similar results could be seen in the Golgi fragmentation rates of non-MN-neurons (SMI32−/ TuJ-1+), albeit to a much smaller extent compared to MNs  CSF derivation and (ii) cell derivation.Add (i): CSF was taken from 11 ALS patients and 8 control subjects  under clinical routine conditions and independent of this study. The use of CSF was approved by the local Ethics committee . CSF was immediately centrifuged (2100 rpm at room temperature for 10 min) and supernatant was stored at −80 °C until use. We included patients with definite, probable or possible amyotrophic lateral sclerosis, according to the revised El Escorial criteria [Add (ii): We included cell lines carrying FUS (p.R521C), TDP-43 (p.S393L), SOD1 (p.R115G) mutations and systematically compared them to four control iPSC lines from healthy volunteers . All proThese procedures were performed as previously described by Nauman et al., Japtok et al., and Kreiter et al. ,35,37.The basal medium (N2B27) for the final MN differentiation consisted of 48.75% DMEM/F12 , 48.75% Neurobasal medium , 1% penicillin/streptomycin/glutamine , 1% B27 supplement without vitamin A  and 0.5% N2 supplement . NPCs were taken from the liquid-nitrogen-storage (−196 °C), then seeded and expanded using N2B27 medium containing additional 150 µM ascorbic acid , 3 µM CHIR99021  and 0.5 µM purmorphamin . After at least two more passages during NPC expansion, patterning was started after reseeding, using N2B27 medium with 1 µM purmorphamin and 1 µM retinoic acid . After 9 days cells were re-seeded with a density of 50,000 cells per well (26.316 cells/cm²) on PLO/laminin-coated coverslips and differentiation was started using N2B27 medium additionally containing 10 ng/mL rhBDNF , 500 µM dbcAMP  and 10 ng/ mL rhGDNF . Following this protocol all SMI-32+ cells correspond to MNs and were positively co-stained for Hb9 (Homeobox gene 9), Cholinacetyltransferase (ChAt) and ISLET1 (ISL LH4 homeobox 1) ,37. All Since CSF caused a significant proliferation of NPCs in our cell cultures , we had 2+/Mg2+  and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Paraformaldehyde was aspirated and cells were washed three times with PBS. Fixed cells were first permeabilized for 10 min in 0.2% Triton X solution and subsequently incubated for 1 h at room temperature in blocking solution  and 0.02% Triton X-100  in PBS). Following blocking, primary antibodies were diluted in blocking solution and cells were incubated with primary antibody solution for 12 h at 4 °C. The following primary antibodies were used: chicken anti-SMI-32 , mouse anti-FUS , rabbit anti-beta-III-Tubulin , mouse anti-GM-130 , mouse anti-SOD1 , rabbit anti-TDP-43 , rabbit anti-GFAP , mouse anti-vimentin . The nuclei were counterstained using Hoechst 33342 .For immunofluorescence staining, cells were washed twice with phosphate-buffered-saline (PBS) without CaAfter immunofluorescence staining, cells were counted, and relative amounts were calculated. We took at least 15 pictures of each condition at random fields of view across the respective well. This resulted in from 350 to up to 3000 cells counted in each condition for one individual experiment, which we always performed at least three times. Furthermore, the morphology of the Golgi apparatuses was analyzed using a Zeiss Axio Observer Z1 microscope  and classified in different categories . One catWe performed the analysis as previously described by Glaß et al. . After i


Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b+Gr1+ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b+Gr1+ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient’s endogenous MDSCs.Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2K Nevertheless, number of challenges remain for organ recipients after the transplantation, as they suffer from acute and chronic rejection as well as complications of immunosuppression9. One of the more critical issues pertains to the current state of standard immunosuppressive protocols. Calcineurin inhibitor (CNI) based immunosuppressive protocols are not designed to promote antigen-specific tolerance, and may exacerbate chronic allograft injury 10. Therefore, there is an urgent need to establish safer anti-rejection strategies by targeting antigen-specific alloimmunity.Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. The Scientific Registry of Transplant Recipients (SRTR) reported that 14,313 kidney14, regulatory T cells (Tregs)15 and regulatory B cells (Bregs)16, have shown the potential application in solid organ transplantation. In particular, Thompson and colleagues conducted a first-in-human study to demonstrate that adoptive transfer of DCreg allows complete withdrawal of immunosuppression regimen in low risk patients20. Myeloid derived suppressor cells (MDSCs) comprise a heterogeneous cell population including immature myeloid cells, which thereafter differentiate into monocytes, dendritic cells and neutrophils21. While sharing similar functional features, MDSCs are shown to offer much powerful immunosuppression compared to DCregs. MDSCs inhibit T cell responses by inducing the apoptosis of antigen-primed and activated T cells23 while inducing FoxP3+ Treg24. In vivo models have shown MDSCs attenuate GvHD25 as well as autoimmunity27. Finally, higher frequency of MDSCs were detected in blood samples from stable renal transplant patients24.Cell therapies, including regulatory dendritic cells (DCregs)28 by culturing donor bone marrow cells within 6 days in the presence of GM-CSF29 , TGFβ30 and IL1032. In addition, we added IFNγ on day 5 to promote the suppressive function of MDSCs33. Finally, we demonstrated that the administration of donor-derived MDSCs prolong allogeneic cardiac graft survival in a donor-specific manner through induction of recipients’ endogenous MDSCs.Based on the above clinical and preclinical studies, we hypothesized that donor-derived MDSCs may induce donor-specific immune suppression in allogeneic organ transplantation. Of note, we modified standard in vitro MDSCs generation protocolb, I-Ab) with BALB/c  derived conventional DCs (cDCs) stimulates an alloreactive T cell proliferation. We observed that the addition of BALB/c MDSCs to this alloMLR system significantly inhibited the proliferation of CD4+ and CD8+ T cells compared to conventional myeloid derived cells (cMDCs)  was performed to examine the regulatory function of donor-derived MDSCs. Co-culturing naive T cells isolated from C57BL/6 mice (H2KCs) Fig. A,B.Figur6 BALB/c MDSCs or BALB/c cMDCs 7 days prior to the cardiac transplantation with BALB/c donors  7 for hematoxylin and eosin (H&E) staining and immune fluorescence staining. H&E staining revealed the attenuated myocardial lesion as well as the decreased lymphocyte infiltration in the donor MDSCs treated group compared to the cMDC group Fig. A. Immuneups Fig. B.Figure +FoxP3- helper T cells, the proportion of effector T cells (Teff) defined as CD44+CD62Llo population, was significantly decreased in the MDSCs treated group compared to the cMDCs treated group  were isolated from allografts (POD7) for flow cytometry analysis. Gating on the CD4Finally, qRT-PCR of allografts  showed that the treatment with donor-derived MDSCs markedly downregulated the mRNA expression of proinflammatory cytokine: IL1α, IL2, IL4, IL13, NLRP3, IFNγ, IRF1, and TNF Fig. .+ and CD8+ Teff induction was analyzed by flow cytometry. Cells were gated on CD4+ Teff (CD4+FoxP3-CD44+CD62lo) and CD8+ Teff (CD8+FoxP3-CD44+CD62lo). We found that the proportion of Ki67+ Teff (Ki67+CD44+CD62lo) in CD4+FoxP3- as well as CD8+FoxP3- population were decreased in the MDSCs group in comparison to the cMDCs group  at the ratio of 20:1 or 10:1 for 4 days Fig. A. CD4+ a+Gr1+34, defined broadly as MDSCs34, was significantly increased in the donor-derived MDSCs treated group  is from the clonal expansion of the transferred donor-derived MDSCs or from the induction of recipients’ endogenous MDSCs, we traced the transferred donor-derived MDSCs using H2Kd antibody. 1 × 106 BALB/c MDSCs were intravenously injected to C57BL/6 recipients, and their splenocytes were examined at 3, 6 and 24 h post-injection. We found that the transferred donor-derived MDSCs peaked 3 h post-injection (PIH3) and then disappeared at PIH24  and Ly6C+Ly6G+ MDSC   Fig. . Taken t+Gr1+ cells were isolated by fluorescence activated cell sorting (FACS) from recipients’ splenocytes on POD7 and were used as modulators. As the transferred donor-derived MDSCs disappeared 24 h after injection, these CD11b+Gr1+ MDSCs are endogenous MDSCs  anti-Gr1 (RB6-8C5) post-transplantation and observed the allograft survivalb, I-Ab) mice received a single-dose intravenous injection of 1 × 106 BALB/c  or C3H  MDSCs 7 days prior to the transplantation. C3H or BALB/c mice were used as cardiac allograft donors  cells in recipients play a key role in allogeneic immune suppression  and compared the SE between the primary donor type cDCs and the third-party cDCs stimulated group   Fig. B. In abosay Fig. A.Figure +Gr1+ from donor-derived MDSCs achieved the similar allograft survival with the whole MDSCs treatment group. However, C57BL/6 recipients treated with CD11b+Gr1- or CD11b- cells from donor-derived MDSCs did not show significant graft survival benefit , Ly6G+Ly6C-  and Ly6C+Ly6G+ MDSC. Our in vitro generated MDSCs are composed with approximately 47.14% of M-MDSC, 2.74% of G-MDSC and 34.43% of Ly6C+Ly6G+ MDSC  4.71 5 of M-MDSC; (2) 3.44 5 of Ly6C+Ly6G+ MDSC; (3) 2.74 4 of G-MDSC. Surprisingly, none of the single population treatment group   Fig. C, but on+Gr1+ cells, while generated donor-derived cMDCs (control) contained only 40% CD11b+Gr1+ cells   based therapies have been shown to protect allografts and attenuate GvHD29, TGFβ30 and IL1032. As IFNγ is essential for the suppression capability of MDSCs via STAT1 signaling activation55 and iNOS and NO production56, we added IFNγ on day 5 to promote the suppressive function of MDSCs33 , we found that MDSCs phenotypically separated from cMDCs, suggesting distinct transcriptional programs 70. Study of transplant patients has demonstrated that high expression of S100A9 predicts better graft outcomes71. In line with these positive clinical outcomes, DCs treated with S100A8 or S100A9 maintain their immature phenotype, and show significant reduction in their capacity to induce T cell proliferation or to produce IFNγ71.Several conclusions are drawn from the current study. First, the in vitro generated MDSCs showed immune suppressive phenotype at the level of both protein and mRNA. Our generated MDSCs not only indicate the ≥ 85% purity of CD11bype Fig. B. In addams Fig. E. Differams Fig.  showed t+Gr1+ is observed in donor MDSCs treated recipients’ spleen as well as in allografts. Depletion of this population in vivo abolished the donor MDSCs induced allograft protection. Furthermore, the addition of these recipients’ endogenous MDSCs showed a powerful immune suppression in primary donor type APC (cDCs) stimulated alloMLR system in comparison to the third-party APC stimulated alloreaction, that indicated the immune suppression function of the recipients’ endogenous MDSCs is donor-specific. While the exact mechanism requires more investigation, this observation may be explained in a two-step process. First, pretreatment with donor-derived MDSCs conditions recipients by inhibiting donor-reactive Teff. Pretreatment with donor-type MDSCs acts as an immune suppressive vaccine, which leads to a primary host versus donor antigen response. Recipient’s cognate T cells engage with donor MDSCs presenting allo-antigen with poor co-stimulatory signal leading to the suboptimal activation72 which in turn results in the generation of donor- specific Tregs73 and anergy of donor-specific Teffs74. Our data supports this by showing increase in activated Treg , BALB/cByJ , C3H/HeJ  mice were from The Jackson Laboratory.Female mice 6–12 weeks of age were used for all experiments. Wide-type C57BL/6 J .Femoral and tibial BMCs were obtained from female BALB/c and C3H mice. To generate MDSCs, BMCs were cultured in RPMI-1640 medium  supplemented with 10% FBS , 20 ng/mL GM-CSF, IL10 and TGFβ  and 50 mM 2-Mercaptoethanol  for 6 days in 24-well TC-treated plates . 10 ng/mL IFNγ  was added on day 5.To generate cMDCs, BMCs were cultured in RPMI-1640 supplemented with 10% FCS, 10 ng/mL GM-CSF, IL4  and 50 mM 2-Mercaptoethanol for six days in 24-well cell culture plates. 10 ng/mL IFNγ  was added on day 5.The graphic protocol is shown in Fig. Cells were stained with antibodies from Biolegend , eBioscience . For intracellular staining, FoxP3/Transcription Factor Staining Buffer Set was used . Flow cytometry was determined by Canto-II instrument (BD) and analyzed by FlowJo v10.+ cells were pre-enriched with anti-CD11b microbeads . Enriched CD11b+ cells were stained with anti-CD11b and anti-Gr-1 for FACS by using an MoFlo Astrios system (Beckman Coulter).For cell sorting, CD11bDetailed reagents information is listed in Table 75. Briefly, samples were lysed with RLT Buffer (Qiagen) and RNA was isolated using MyOne Silane Dynabeads (Thermo Fisher Scientific). RNA was fragmented and barcoded with 8 bp barcodes. Primers were removed using Agencourt AMPure XP bead cleanup (Beckman Coulter/Agencourt). Samples were amplified with 14 PCR cycles. Libraries were gel purified and quantified using a Qubit high sensitivity DNA kit (Invitrogen) and library quality was assessed using Tapestation high sensitivity DNA tapes (Agilent Technologies). RNA was sequenced on an Illumina NextSeq sequencer (Illumina) according to manufacturer’s instructions, sequencing 50 bp single end reads. Analysis was performed using the CLC Genomics Workbench version 8.0.1 RNAseq analysis software package (Qiagen). Briefly, reads were aligned  to the mouse genome and differential expression analysis was performed . Results were normalized to reads per million.RNAseq library preparations were performed as previously described57. Graft survival was considered complete at the time of cessation of a palpable heart beating and confirmed visually by laparotomy.All transplant procedures were performed under anesthesia with isoflurane. Fully vascularized heterotopic hearts from BALB/c or C3H were transplanted into C57BL/6 recipients using a microsurgical techniqueg for 5 min to pellet the cells. The pellet was suspended in 5 ml PBS, loaded onto 5 ml of Lympholyte-M  and centrifuged at 1500 g for 25 min at room temperature. Cells were isolated from the Lympholyte-M interface and washed twice in PBS at 300 g for 5 min and prepared for flow cytometry analysis.Tissues were disrupted mechanically in 10 ml digestion solution, which include 0.5 mg/ml collagenase IV , 50 U/ml DNaseI  in RPMI-1640 medium and incubated at 37 °C for 30 min. After that, 10 ml of iced RPMI 1640 with 5% FBS was added. The suspension was filtered through a nylon mesh (70 μm) to remove aggregates. The resulted cell suspension was centrifuged at 8005/well) were labeled with CellTrace violet  and used as responders. Cultures were maintained in the complete medium for 3–4 days at 37 °C in 5% CO2 in air. The reaction system and other details are shown in the associated figures.Allogeneic MLR was performed in duplicate in 96-well, round-bottom plates . Nylon wool-eluted spleen T cells  and then embedded in paraffin. Sections of 4 µm were made for hematoxylin and eosin (H&E) staining.76. Antibodies used in this experiment is listed in Table Grafts harvested on POD7 were embedded in OCT compound  and preserved in − 80 °C freezer. Cryo sections of 4 µm were made for immuno fluorescent staininglater stabilization for freezing . Total RNA was extracted from frozen tissue samples using TRIzol method . Then, RNA was reverse transcribed to cDNA using iScript cDNA Synthesis Kit . Quantitative RT-PCR was performed using a SsoAdvance Universal SYBR Green system  on the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). The normalized threshold cycle (Ct) value of each gene was obtained by subtracting the Ct value of 18S rRNA. The sequences used in our study are shown in Table Cardiac grafts were harvested on POD3 and POD7 and submerged in RNA44 antibody  or a rat IgG2β isotype control  at POD 0, 2, 4, 6.Recipients received the intraperitoneal injection of 250 μg RB6-8C5The data were analyzed using GraphPad Prism, version 5.0 (GraphPad Software). One-way ANOVA and Tukey's test were used to compare the means of more than two groups. Student's t test was used to compare the means of two groups. A statistical evaluation of graft survival was performed using Kaplan–Meier curves and compared using log-rank tests. RNA-seq data was analyzed by using extraction and analysis of differential gene expression (EDGE) test. All in vitro experimental data were representative of at least three independent experiments. p values of less than 0.05 were considered statistically significant.Supplementary file 1Supplementary file 2Supplementary file 3Supplementary file 4Supplementary file 5


Serial cerebrospinal fluid (CSF) analysis might be required in clinical neurologic disease. The effect of lumbosacral (LS) or cervical (C1‐C2) centesis on subsequent CSF cytologic analyses has not been investigated in horses.To evaluate the effect of thecal puncture on subsequent CSF analysesTen healthy adult horses.P < .05.Prospective study. Horses were randomly assigned to undergo CSF collection twice, 14 days apart, from either the C1‐C2 or LS space. After a 4‐month washout period, CSF collection was repeated from the alternate site. Continuous data were analyzed using linear mixed‐effects models and count data using mixed‐effects negative binomial regression. Statistical significance was set at P = .12; LS: 64 [95% CI: 41‐100] mg/dL day 0 vs 83 [95% CI: 53‐129] mg/dL day 14, P = .37), or nucleated cell count . There was no significant difference in EPM titer or EPM serum : CSF ratio between days 0 and 14.There was no significant effect of collection day (day 0 or day 14) for any CSF analytes, including protein concentration (C1‐C2: 45 [95% CI: 33‐57] mg/dL day 0 vs 49 [95% CI: 39‐62] mg/dL day 14, Repeat thecal puncture from the LS or C1‐C2 space 2 weeks apart does not appear to impact CSF analytes. AIantibody indexCSFcerebrospinal fluidEPMequine protozoal myeloencephalitisLSlumbosacralRBCred blood cellWBCwhite blood cell1Complete neurologic system evaluation and diagnosis of neurologic disease requires evaluation of cerebrospinal fluid (CSF). In many cases of equine neurologic disease, a single spinal centesis is performed, however there are occasions where repeat CSF analysis is warranted. Clinical scenarios in which this becomes relevant include when (a) clinical signs have worsened despite treatment, (b) repeat diagnostics are needed to determine duration of treatment or confirm initial results, or (c) for research purposes. Repeat CSF collection is performed regularly in human medicine, particularly in cases of bacterial meningitis.The objective of our study was to evaluate the effect of prior thecal puncture on subsequent CSF analyses in horses. The authors' hypothesized that there would be no clinically relevant difference in analyses of CSF collected 14 days after initial thecal puncture.22.1All experimental procedures were approved by the University of Georgia Institutional Animal Care and Use Committee. The study population consisted of 10 adult Quarter Horses, aged 3‐22 years. There were 4 mares, 4 geldings, and 2 stallions. All horses belonged to the University of Georgia research herd. Before enrollment, horses were determined to be healthy based on normal physical and neurologic examinations. The horses were housed in individual box stalls for 12 hours before and 48 hours after CSF collection. The horses were otherwise housed on pasture with access to shelter. Ad libitum water and coastal Bermuda hay were available throughout the study period.2.2https://www.randomizer.org/) was used to determine which site was sampled first. CSF collection from the same site was repeated 14 days later. After a 4‐month washout period, physical and neurological examinations were repeated, and the groups were switched to undergo CSF collection from the alternative site. CSF collection from that same site was then repeated 14 days later.Horses were randomly assigned to undergo CSF collection from either the C1‐C2 or LS space. An online random number generator  within 30 minutes of collection. If 3 aliquots could not be collected the second 1 mL aliquot was submitted. Manual TNCC and RBC count were performed on each sample using a standard Neubauer hemocytometer. Total protein concentration was quantitated via the biuret method . Cytologic evaluation was performed in batches by a clinical pathology resident (Bridgette Wells) and board‐certified clinical pathologist (Melinda Camus).2.4Sarcocystis neurona  was performed.Serum and CSF from each horse was stored at −80°C and shipped to Equine Diagnostic Solutions Laboratory  for batched analysis within 6 months of collection. An enzyme linked immunosorbent assay to detect antibodies to 2.5α = .05. Normality of the data was assessed based on examination of histograms and normal Q‐Q plots of the residuals. Homogeneity of variance was assessed by plotting residuals against predicted values. Data were transformed to the natural logarithm when necessary. Protein concentration, RBC count, EPM titer in CSF, and serum : CSF titer ratios were assessed using linear mixed‐effects models with anatomic site and collection day modeled as fixed nominal effects and horse included as a random effect to account for repeated measurements. EPM titers reported as <2.5 in CSF samples or as <250 in serum samples were arbitrarily assigned values of 1.25 and 125, respectively, for statistical analysis. Samples with a CSF EPM titer <2.5 were considered to have a negative serum : CSF ratio, and were arbitrarily assigned a serum : CSF ratio of 400 for statistical comparisons. Counts of TNCC were fitted to a mixed‐effects negative binomial model. Percentages of neutrophils, lymphocytes and large mononuclear cells were analyzed using fractional probit regression with robust standard errors clustered on horse to account for the correlated structure of the data. Results of statistical models were reported using estimated marginal means to ensure that the effect of each variable was adjusted for other variables included in the model. For all analyses, model fit was assessed using Akaike's information criterion values. Values of P < .05 were considered statistically significant. Analyses were performed using commercially available statistical software .Sample size calculation using published nucleated cell counts and protein concentrations measured from CSF collected from the LS space in horses3After randomization, 7 horses were assigned to undergo LS centesis first and 3 horses to undergo C1‐C2 centesis. After a 4‐month washout period, groups were switched, and horses underwent CSF collection from the second site. One horse was euthanized, for reasons unrelated to the study, during the washout period after undergoing LS centesis. Therefore, 10 horses underwent 2 LS centeses, and 9 horses underwent repeat cervical centesis, resulting in comparison of 19 CSF samples for day 0 and day 14.CSF analytes are summarized by collection day and anatomic site in Table The median number of collection attempts required to obtain 38 CSF samples was 2 (range 1‐10). For cervical centesis (18 samples), a median of 1 attempt was required (range 1‐5). For LS centesis (20 samples), a median of 3 attempts were required (range 1‐10). In horses with substantial blood contamination (≥9000 RBC/μL), an average of 2 collection attempts (range 1‐3) were required for both cervical and LS centesis.For samples collected from the C1‐C2 space, 1/9 horses had a higher CSF EPM titer on day 14 compared to day 0, and 8/9 horses had the same titer on both days Table . All horFor samples collected from the LS space, 5/10 horses had a higher CSF EPM titer on day 14 compared to day 0, 1/10 horses had a lower titer, and 4/10 horses had the same titer on both days. No horses had a serum : CSF titer ratio ≤ 50 on day 0. On day 14, 2/10 horses transitioned from a negative to a positive result (serum : CSF titer ratio of 50). One horse (EPM CSF titer of 5) had substantial blood contamination of CSF (51 900 RBC/uL) at day 14. The other horse (CSF EPM titer of 10) had minimal blood contamination of CSF at either day 0 or day 14, however CSF protein concentration was markedly increased at day 14 (257 mg/dL) compared to day 0 (53 mg/dL). Antibody indices for both horses were below 1 , therefore they were classified as EPM negative despite the serum : CSF titer ratio.After CSF collection, no abnormalities in physical or neurologic examinations were noted. No gross inflammation was observed at either the LS or C1‐C2 centesis sites after CSF collection on day 0 or day 14.4Based on the results of our study, recent thecal puncture does not appear to affect subsequent CSF analysis to a degree that would interfere with clinical interpretation of results.In human medicine, a small increase in TNCC, neutrophil count, and mononuclear cell count was identified in healthy adults undergoing repeat lumbar puncture, and 56% of these patients met the definition for CSF pleocytosis on day 14.Historically, formulas have been used to correct TNCC and protein concentration in CSF with blood contamination, however this has now been shown to be inaccurate.S neurona antibody titer was of extraneural origin. The horses were therefore considered EPM negative.A primary reason for repeat thecal puncture in horses is in cases of suspected EPM with acute onset of clinical signs where initial diagnostic testing is negative. Neurologic signs can be observed before seroconversion in recently infected horses, yielding false negative results.The most common reason for repeat thecal puncture in human medicine is in cases of bacterial meningitis that fail to improve with antimicrobial treatment. Bacterial meningitis is rare in adult horses but occurs more commonly in foals. Other indications in human medicine include reducing intracranial pressure, monitoring response to treatment, or investigating patients with persistent or relapsing fever.A 14 day time period between CSF collection was chosen. This time period is (a) sufficient to allow seroconversion in horses recently infected with EPMOne limitation of our study is the small sample size. The number of horses enrolled was based on power calculations to detect a statistical difference based on TNCC and protein concentrations, and was limited by availability of horses from the University of Georgia research herd. Because of the extended washout period between CSF collection from the C1‐C2 and LS sites, only university‐owned animals were used to avoid cost‐prohibitive fees associated with boarding donation horses.In conclusion, this study suggests that repeat thecal puncture from the LS or C1‐C2 space 2 weeks apart does not result in clinically important changes in CSF analytes including total protein concentration, TNCC and RBC count. The difference in EPM serum : CSF titer ratio between day 0 and day 14 observed in 2 horses was unexpected, however AI was not consistent with intrathecal antibody production, and would not have led to a false positive diagnostic test result for EPM. In practice, a clinically important difference in CSF analytes is not expected from thecal puncture performed 14 days earlier.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.This study was approved by the University of Georgia IACUC.Authors declare human ethics approval was not needed for this study.Table S1 Cerebrospinal fluid red blood cell (RBC) count, white blood cell (WBC) count, and total protein concentration from individual horses from both the cervical (C1‐2) and lumbosacral (LS) space on day 0 and day 14.Click here for additional data file.Table S2 Serum and cerebrospinal fluid equine protozoal myeloencephalitis (EPM) titers and serum : CSF titer ratios from individual horses from both the cervical (C1‐2) and lumbosacral (LS) space on day 0 and day 14.Click here for additional data file.


Despite advances in systemic therapies for solid tumors, the development of brain metastases remains a significant contributor to overall cancer mortality and requires improved methods for diagnosing and treating these lesions. Similarly, the prognosis for malignant primary brain tumors remains poor with little improvement in overall survival over the last several decades. In both primary and metastatic central nervous system (CNS) tumors, the challenge from a clinical perspective centers on detecting CNS dissemination early and understanding how CNS lesions differ from the primary tumor, in order to determine potential treatment strategies. Acquiring tissue from CNS tumors has historically been accomplished through invasive neurosurgical procedures, which restricts the number of patients to those who can safely undergo a surgical procedure, and for which such interventions will add meaningful value to the care of the patient. In this review we discuss the potential of analyzing cell free DNA shed from tumor cells that is contained within the cerebrospinal fluid (CSF) as a sensitive and minimally invasive method to detect and characterize primary and metastatic tumors in the CNS. Comprehensive genomic profiling (CGP) of formalin-fixed paraffin-embedded (FFPE) tumor tissues has allowed many cancer patients to benefit from precision medicine by identifying genomic alterations for which targeted therapy or immunotherapy may be preferentially utilized. While the clinical value of tissue based CGP is well-established, this approach requires patients to undergo a tissue acquisition procedure such as a fine need aspiration, core biopsy, or surgical resection. As nearly all patients will be biopsied at presentation to establish a tissue diagnosis of malignancy, there is often tumor tissue available for CGP.Despite advancements in therapeutic strategies for solid tumors, progression frequently occurs, manifesting as metastatic disease or recurrence. At this point, the ability to acquire a second biopsy can be challenging for several reasons including, the patient's general health status, the ability and/or willingness to undergo another invasive procedure, and the patient's safety associated with the location of the lesion to be biopsied. For patients with primary brain tumors , most patients will undergo a tissue sampling procedure at the time of presentation to establish the diagnosis, while only a subset 10–20%) of patients will ever have a second procedure to acquire tissue at tumor recurrence  from the plasma of peripheral blood samples has provided a novel mechanism for interrogating the genomic landscape of tumors without invasive surgical procedures. This type of assay is frequently referred to as a “liquid biopsy” and encompasses a broad category of minimally invasive tests that can effectively isolate DNA shed from cancer cells that are circulating in the blood or other relevant fluids. In contrast to circulating tumor cells (CTCs), which are intact tumor cells in the peripheral circulation, ctDNA is a component of the total circulating “cell-free” or cfDNA that includes both ctDNA and DNA fragments in plasma contributed by white blood cells and apoptotic normal non-hematologic cells. ctDNA testing platforms range from variant-specific and “hot-spot” assays to broad next generation sequencing panels that can assess for variants in hundreds of genes simultaneously. For some solid tumors, such as breast, lung, prostate, melanoma and colorectal cancers, ctDNA assays have demonstrated strong concordance of genomic profiles compared to tissue-based assays , 4. MoreWhile advanced-stage tumors demonstrate increasing yields of ctDNA in the plasma, detecting ctDNA in the plasma of primary brain tumor patients is limited, with only a minority yielding detectable levels. Across published reports, the rate of ctDNA detection in plasma for primary brain tumors varies from 10 to 50% of samples with higher detection rates associated with glioblastoma; however, all studies consistently report that even when detected, the ctDNA concentrations are much lower compared to other advanced stage tumors , 5–7. AlSeveral studies have identified cerebrospinal fluid (CSF) as a rich and reliable source of ctDNA (ctDNA-CSF) in patients with primary and metastatic brain tumors. Secreted by the choroid plexus, CSF is a clear body fluid in the subarachnoid space of the brain and spinal cord that provides specialized immunologic protections and serves as a cushion for the brain . Given iIn this review we will explore CSF testing for primary and metastatic brain tumors and discuss the clinical utility of testing for management of patients affected by brain cancer.For tumors that arise outside of the CNS , the presence of tumor cells in the CSF correlates with poor response to therapy and overall poor prognosis , 11. To CSF is typically collected through a minimally invasive lumbar puncture but can also be accessed via existing shunts or during intracranial surgical procedures, with a typical volume of 2–10 mLs of fluid with each collection. Current methodologies for evaluating CSF focus on determining the presence of cancer cells with cytology, immunohistochemistry, or flow cytometry; however, while these techniques can be highly specific, they lack the sensitivity that is needed for early detection of malignancy. As an alternative, ctDNA from the CSF (ctDNA-CSF) can be extracted and sequenced, which increases sensitivity and provides additional information about tumor heterogeneity and progression of disease ; Figures, 2. In tHER2 amplification in the ctDNA-CSF sample but barely detectable levels in the pelleted fraction. In all, known variants were detected in 100% of ctDNA-CSF samples but only 63% of CSF cell pellets from their cohort  of ctDNA-CSF samples, however only 84.6% (22/26) and 73.1% (19/26) of pelleted CSF and plasma ctDNA samples, respectively, identified the mutation  compared to plasma ctDNA (21%). In another study of patients with leptomeningeal carcinomatosis, ctDNA-CSF analysis detected the presence of disease in 100% of cases while neuroimaging and CSF cytology identified 63 and 71% of cases, respectively. In another institution's experience, ctDNA-CSF genomic profiling from patients with malignant CNS disease detected clinically actionable mutations in 40% (11/27) of patients. After consultation with an interdisciplinary molecular tumor board, seven patients were recommended to receive a targeted therapy based on the ctDNA-CSF results, with four patients being clinically stable to proceed with the new treatment regimen. These patients included two metastatic NSCLC patients with EGFR mutations who received erlotinib or afatinib, a NSCLC patient with an EZR/ROS1 fusion who was treated with crizotinib and a HER2 positive metastatic breast cancer patient with FGFR amplification who received everolimus  only represent 1% of primary brain tumors in pediatric and young adult patients, the CNS is the second most common location for extragonadal tumors. These are a heterogeneous group of tumors that can be difficult to diagnose because of their non-specific clinical presentations and subsequently predict which tumors are more likely to metastasize to the brain. The propensity to spread throughout the CNS increases the need for highly specific and sensitive markers that can be detected in the CSF of patients. Currently monitoring certain protein biomarkers such as alpha-fetoprotein and human chorionic gonadotropin within the CSF only provides information for certain GCT subtypes and has limited sensitivity in terms of diagnosing CNS dissemination . There iPrimary CNS lymphoma (PCNSL) patients may also benefit from CSF liquid biopsies as these tumors often disseminate via CSF. Notably, surgical resection is not standard of care, and if PCNSL is diagnosed during an intraoperative frozen section, the surgical procedure is routinely stopped as this disease is best managed with chemotherapy. While this is considered best practice, it does limit the amount of tissue available for genomic testing and further reduces the likelihood of additional tissue sampling at the time of tumor recurrence. Detection of mutational profiles and immunoglobulin gene rearrangements in CSF has the potential to improve diagnostic sensitivity, especially in setting of steroid administration, which can make tissue sampling of PCNSL challenging because of tumor cell lysis. In contrast, one can speculate that the use of steroids for PCNSL prior to ctDNA-CSF liquid biopsy may enhance the amount of ctDNA available in the CSF compartment. In one study 19 patients with B-cell lymphomas were evaluated to compare the utility of ctDNA-CSF or plasma in diagnosing CNS involvement and explore whether the analysis of ctDNA could monitor treatment response in CNS lymphomas . GenomicFurther utility for CSF liquid biopsy in PCNSL includes identifying tumor recurrence, particularly in cases associated with extensive leptomeningeal involvement in which diagnosing recurrent tumor in the post-treatment setting solely by neuro-imaging studies can be very difficult, often when cytologic analysis is unrevealing. Indeed, sequential ctDNA-CSF testing in patients undergoing treatment demonstrated that despite a decrease in tumor cells by flow cytometry or in some cases no detectable disease by conventional techniques, patients still harbored CNS disease when assessed with a more sensitive method .MYD88, CARD11, TNFAIP3, and CD79B. Therefore, in the recurrent PCNSL setting, should CSF liquid biopsy identify mutations in these key genes, it would likely guide therapy away from BTKi  ibrutinib as monotherapy in salvage treatment of PCNSL, this highlights the value of mapping the genomic landscape of recurrent PCNSL. While BTKi may become an important option for salvage therapy, it is worth noting that several mechanisms of resistance have been identified including mutations in rom BTKi –33.Liquid biopsies are increasingly demonstrating clinical utility for a variety of cancers. Guidelines have evolved most rapidly in non-CNS cancers such as lung cancer where CAP guidelines suggest that liquid biopsy may be acceptable at the time of diagnosis should tissue not be available for testing.Additionally, this methodology can help clarify radiologic findings and differentiate between radiation-induced necrosis and tumor progression. Pseudoprogression, defined as post-radiotherapy changes that typically resolve spontaneously, occurs in 10–30% of GBM patients typically within the first 12 weeks of treatment . The abiH3F3A (K27M) mutation, it would suggest a molecular diagnosis of diffuse midline glioma, H3F3A K27M mutant, WHO Grade IV; alternatively, if the tumor harbored a KIAA1549-BRAF fusion, that would suggest a lower grade glioma such as pilocytic astrocytoma WHO Grade I. A retrospective study of 20 glioma patients demonstrated combined digital droplet PCR and targeted sequencing of known glioma associated mutations on ctDNA-CSF samples could correctly classify gliomas in 85% of cases; the only samples that failed were low-grade gliomas or tumors where ctDNA-CSF yields were low , given the anatomic location, it is often not possible to biopsy these patients and therefore they are routinely treated clinically based on imaging studies. If however ctDNA-CSF testing was available and showed a were low . ProspecThere is significant potential for ctDNA-CSF testing to disrupt the way neuro-oncology is currently practiced by providing neurosurgeons, oncologists and patients with pre-surgical, pre-radiation and pre-systemic therapy genomic diagnoses that can inform clinical management strategies. The value of total and near-total resection for high grade gliomas remains an important therapeutic and prognostic indicator. ctDNA-CSF molecular analysis has the potential to provide more information to neurosurgeons for planning surgical procedures based on genomic profiles. In instances where the tumor is based in an eloquent area of the brain or deep gray structures where resection is not possible and the purpose of surgical intervention is solely to sample tissue for diagnosis, CSF ctDNA testing may be an alternative to biopsy procedures.ERBB2 levels and other tumor-specific mutations during treatment with trastuzumab emtansine, CSF ctDNA revealed continued high levels of ERBB2, MYC copy numbers, PIK3CA, and TP53 mutations, which were identified prior to the start of treatment (H3F3A K27M levels in CSF ctDNA correlated with the presence of contrast-enhancing areas on MRI in pediatric DIPGs (As several studies have demonstrated, ctDNA-CSF sequencing can also provide opportunities for early detection of metastatic disease, which may have significant implications for therapeutic intervention. Retrospective studies have demonstrated that in the context of metastatic disease ctDNA-CSF mutations reflect CNS specific alterations, which are not identified in plasma ctDNA or extracranial metastases . Incorporeatment . This hiic DIPGs .Importantly, to fully understand the potential of ctDNA-CSF testing and incorporate the methodology into routine clinical care, prospective clinical trials are needed. Larger studies should be performed that evaluate the correlations between ctDNA-CSF, plasma ctDNA and tumor tissue samples. Future prospective studies that incorporate ctDNA-CSF testing in parallel with standard of care diagnostic and management procedures will help refine and implement the utility of ctDNA-CSF testing for patients with brain metastases and primary tumors.All of the authors contributed to the preparation and writing manuscript as each individual has specific expertise in different areas, including neuro-oncology, metastatic CNS disease, circulating tumor DNA, genomic profiling, neurosurgery, and integration of molecular data into direct clinical care.Some authors are employed by Foundation Medicine, Inc. 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.


In Huntington’s disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities. The precise pathogenic roles of the mHTT aggregates remain elusive. Certain studies suggest that they may be protective2 or neutral4, while others show that they are pathogenic7 and possibly contribute to the selective vulnerability of striatal medium spiny neurons (MSNs) in HD8.Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by CAG repeat expansion encoding a polyglutamine (polyQ) stretch near the N-terminus of mutant Huntingtin (mHTT). The mutant polyQ repeat increases the propensity of mHTT to misfold and form aggregates in the striatum and cortex of HD patients10. The Singulex single-molecule counting immunoassay (SMC) can detect a significant increase of HTT in cerebrospinal fluids (CSF) and plasma from premanifest to manifest HD stages10. Importantly, this assay has been successfully employed in the antisense oligonucleotide (ASO) trial in HD patients to show the reduction of mHTT in the CSF of HD patients11. The ELISA-based meso-scale discovery (MSD) electrochemiluminescence assay is a platform that sensitively quantifies and differentiates soluble and aggregated mHTT in brain tissues and blood cells14. The microbead-based immunoprecipitation and flow cytometry assay (IP-FCM) has been shown to detect mHTT in the brain of HD mouse models and CSF of patients15. The study further showed that ASO against HTT could reduce CSF mHTT in BACHD, a mouse model of HD16. These sensitive antibody-based methods demonstrated the presence of mHTT in the more accessible patient biofluids. However, since HTT is a large protein (350 kDa) with extensive post-translational modifications and aggregates to form higher order structures18, it is likely that these antibody-based assays may capture a subset (e.g. specific conformations) of mHTT species.Several antibody-based platforms have been developed to quantitatively detect HTT species as a biomarker in biosamples20. This is postulated to be a key mechanism in disease progression20. Previous studies reported that the aggregation of recombinant mHTT fragments in vitro could be greatly accelerated by the addition of preformed mHTT aggregates21, a phenomenon often referred to as mHTT “seeding activities”. Moreover, extracellular application of preformed mHTT aggregates or crude extracts of aggregates from postmortem HD brains or mouse models of HD could also seed intracellular aggregation of otherwise soluble forms of HTT25. One later study demonstrated that extracts from modified PC12 cells expressing inducible GFP-tagged mHTT-exon 1 fragment with 103Q showed increased aggregation with the addition of CSF from HD patients26. It sheds the light of using seed-induced aggregation for detecting mHTT seeding species in the biosamples. However, this assay is limited by its sensitivity and scalability since the measurements of induced aggregation rely on cell counting and filter retardation assays, and the cell line used in this study spontaneously forms reporter protein aggregates without the addition of extracellular mHTT seeds26.Emerging studies show that pathogenic proteins in a variety of neurodegenerative disorders may exhibit “prion-like” properties, leading to the spreading and template-based amplification of misfolded disease proteins (e.g. aggregated α-synuclein in Parkinson’s disease and hyperphosphorylated Tau in Tauopathy and Alzheimer’s disease)In this study, we developed cell lines stably expressing wildtype and engineered mHTT-exon 1 reporters. We found that deleting the N17 domain of mHTT-exon 1 greatly augments the sensitivity of the reporter cells for detection mHTT seeding species. Importantly, with CSF samples from postmortem brains and living HD patients, we showed that the seedable mHTT species in the patient CSF increased with the progression of clinical disease presentation and neuropathology. Moreover, we showed the CSF mHTT seeding activities can be abolished by overexpressing the chaperone DNAJB6 and by antibodies against the polyproline domain of HTT. These results suggest the presence and level of seeding-competent mHTT species in the patient CSF may correlate with neuropathological progression, and substantiate the use of this highly sensitive and scalable cell-based assay for studying mHTT seeding activities in HD clinical progression and response to therapy.26. However, these cell lines fall short of sensitivity, specificity and scalability in detecting the presence of mHTT seeds in HD patient biosamples, such as CSF. To develop a reporter cell line that fulfill these requirements, we reasoned that the cells should stably express a mHTT N-terminal fragment with a moderately expanded polyQ repeat, which thus could be nucleated to form aggregates while not spontaneously aggregates at the baseline. We first generated a stable HEK293 cell line expressing mHTT-exon 1 fragment with 46Q and in-frame C-terminal green fluorescent protein fusion  that leads to increased oligomer formation, nuclear mHTT aggregation and HD-like disease phenotypes in vivo34. These two cell lines are termed mHTT-SD-GFP and mHTT-ΔN17-GFP, respectively. Similar to the mHTT-46Q-GFP cell line, both mHTT-SD-GFP and mHTT-ΔN17-GFP cell lines have neglectable aggregation at baseline and when treated with mHTT-51Q monomers  of mHTT is critical in regulating the subcellular localization, oligomerization and aggregation, and toxicities of mHTT in vitro and in vivolls Fig. . Togethe38. Among the three reporter cell lines, only the mHTT-ΔN17-GFP line is sensitive enough to show induced aggregation with sonicated striatal extracts from old (14 months of age) but not young (4 months of age) Q175 mice  knock-in mouse model carrying an expanded CAG repeat and showing early and progressive mHtt aggregation, transcriptional dysregulation, and behavioral impairment Fig. 35–38. Amice Fig. o,p, nor ice Fig. n. Importnes Fig. . These r+ aggregates over time. We noticed that the smaller aggregates (< 0.9 μm) were not detected reliably by the automated image analysis, possibly due to the optical limitation and background heat noise from the instrument. Thus, for optimal signal detection over the background, we chose to only quantify the GFP+ aggregates at the size between 1 and 3 µm. As a result, the 384-well format and the high-content imaging system were capable of capturing the time-dependent progression of mHTT-ΔN17-GFP (referred to as mHTT-GFP thereafter) aggregation  or Parkinson’s disease (PD) patients  . We further test the all combination of these covariates plus CAG length. HD grades regressed on the aggregation index remains the best model, since addition of other covariates was more likely deteriorate the fit  Fig. a. Imported) Fig. . We foun40. In PREDICT-HD, the enrolled individuals were evaluated annually for motor and cognitive functions, performed brain imaging and collected biosamples. They are categorized by the diagnostic confidence level (DCL), where DCL0 means indistinguishable from healthy individuals, DCL2 and DCL3 are prodromal stages with increasing likelihood of HD diagnosis, and DCL4 is most likely to be clinically diagnosed HD. To prevent experimental bias, we were provided 60 PREDICT-CSF samples by NINDS/BioSEND and performed the seeding assays while blinded to any identification or clinical information. Samples were unblinded only after the results were submitted back to NINDS BioSEND. We found that the 60 CSF samples were actually duplicates of 30 samples collected from 2 visits of 15 individuals . When the covariates are added individually, the association between aggregation score and DCL4 remained significant  associated with aggregation index in any of the models. There are combinations of covariates  with which the association with DCL becomes non-significant, likely because these combinations of covariates explain a large fraction (mostly over 50%) of the DCL score variation. As a diagnostic check, especially for the small PREDICT-HD sample size, we tested normality of the residuals of aggregation index using Shapiro–Wilks test and found non-significant results , a longitudinal phenotypic and biomarker study of prodromal HD see “40. In PRr = 0.3539; Fig. 41, and CAG repeat length  score  in individuals diagnosed with HD but not in non-carriers or HD gene carriers in the DCL0 to DCL3 categories Fig. . These pr = 0.5849, p = 0.0054; Fig. r = − 0.7476, p < 0.0001; Supplemental Fig. 41. We again used mixed effect models to test these associations while accounting for repeat visits and found that the associations of aggregation index and TFC remain significant even after Bonferroni correction for the 69 tested clinical variables  were used to immunodeplete mHTT seeding species from the HD CSF samples. The immunodepleted CSF samples were then applied to the mHTT-ΔN17-GFP cells. We found that HD patient CSF, immunodepleted by both anti-polyP antibodies, significantly diminished its seeding ability in the assay  for minimal aggregation at the baseline and deletion of the N17 domain, which showed remarkable enhancement in sensitivity for detecting mHTT seeding species, including those in HD patient biosamples. Furthermore, by adapting the new cell line into a 384-well, high-content imaging assay platform, we are able to reproducibly detect and quantify mHTT seeding activities in the HD patient CSF. Our preliminary study of clinical samples reveals a significant increase in the mHTT seeding activities in the CSF of HD gene carriers upon their initial diagnosis of HD (i.e. DCL4), and a continuing increase of such seeding activities with progression of neuropathological grades in the postmortem samples. Finally, our assay reveals the mHTT seeding activities could be inhibited by overexpression of the chaperone DNAJB6 and by immunodepletion using antibodies against the polyP domain of mHTT. Further studies of larger premanifest and manifest HD patient cohort are needed to fully evaluate the utility of longitudinal CSF mHTT seeding activities, as detected by the highly sensitive cell-based assay, in tracking the clinical onset and progression of HD. Finally, our study also demonstrated the utility of this cell-based assay to evaluate molecules that can suppress the mHTT seeding activities from HD patient CSF, which in turn could be further investigated for their roles in disease modifications.46. However, changes in the levels of mHTT seeding species have not been described over the course of the disease and with the concurrence of symptoms. In this study, we demonstrate the neuropathological and clinical stage-dependent increase  in mHTT CSF seeding activities. Despite the limited availability of multi-visit samples in PREDICT-HD, our study also showed that the seeding propensity of CSF is also augmented in subsequent visits of HD patients but not in those of premanifest mutant carriers. Moreover, our preliminary findings showed the CSF seeding propensity is positively correlated with the disease severity, including TFC and total motor score, similar to the currently clinically used SMC assay11. The SMC assay is significantly correlated with the CAP score41. One important question is whether the CSF mHTT seeding activities are tightly correlated with the CAG length and/or age of the patients. We found that the CSF mHTT seeding propensity does not appear to correlate with the CAP score of the patients. Thus, our assay does not seem to probe the same mHTT species in the patient CSF as the SMC assay. We acknowledge that a limitation of our study is the relatively small number of CSF samples from premanifest and manifest HD patients used. Therefore, our findings related to the relationships of CSF seeding activities and clinical disease stages should be considered preliminary. Future large-scale studies with longitudinal CSF samples and clinical data from both HD gene carriers and patients is needed to rigorously evaluate the clinical utility of this high-throughput, cell-based mHTT seeding assay in monitoring HD onset and progression and in evaluating efficacies of HD therapeutics.One important finding of this study is that the propensity of the mHTT seeding is relatively minimal in HD gene carriers prior to the symptom onset, and is significantly elevated upon disease diagnosis and appears to continuously increase with neuropathological progression. Previous studies have reported increased mHTT species in the CSF before and after the disease onset43 and other chaperones47, could be a meaningful approach to identify molecular targets and candidate therapeutic reagents to block mHTT seeding in the patients.We demonstrated that overexpression of DNAJB6 in the reporter cell line can block the seeding activities from preformed seeds and patient CSF. These findings confirmed that our assay is detecting template-based, aggregate-inducing activities of mHTT species in the CSF. It further suggests that studying chaperone-mediated mHTT proteostasis, such as those mediated by DNAJB6 and DNAJB850. It is worth noting that a commonly used anti-HTT antibody, EM48, of which epitope is mapped to the polyP region, could not elicit the same blocking effect. This might be attributed to the intrinsic difference of antibodies in epitope recognition and affinity. Recent structure studies suggest a “bottle brush” model for HTT exon 1 structure, with the polyP domain forms bristled helix-rich structures projecting away from the fibril core of N17 and polyQ52. Aggregation of mHTT is a stepwise event, initiated by N17 interaction, followed by β-sheet formation of polyQ and, lastly, crowding of the polyP bristles. Given the slowest step lies on the final structure maturation as polyP crowding52, presence of antibodies against the polyP domain might reduce mHTT-GFP aggregation through interference with polyP bristles compaction. Future studies are needed to test whether extracellular delivery of anti-HTT-polyP antibodies could be an effective measure to block the propagation of mHTT aggregation and HD pathogenesis in vivo.A key advance in our study is to provide strong evidence that the CSF seeding activities from HD patients are likely to be mediated by specific mHTT species, of which the polyP region is exposed and might play a key role in the seeding process. We showed that antibody-blockade and immunodepletion with three different anti-polyP antibodies could significantly reduce mHTT-GFP aggregation induced by recombinant seed and CSF from HD patients, while anti-N17 or anti-polyQ antibodies could not elicit similar effects. This result is consistent with prior studies showing that co-expressing polyP-specific intrabodies inhibited aggregation of mHTT proteins in HD transgenic miceFinally, our scalable, highly sensitive cell-based mHTT seeding assay can be used in large scale perturbation studies, e.g. anti-HTT antibodies, intracellular gene-based screenings  and small molecule screenings to systematically identify molecules that could modulate mHTT seeding activities. A good example is the antibody screening reported in this paper. This is made possible as our assay requires only very small amount of CSF (i.e. 10–20 μl) per sample. Thus, future large-scale, phenotype-driven screening with our assay could identify genetic targets or small molecules to curb such pathogenic activities in the patients.In conclusion, we reported the development of a novel high-throughput cell-based system that could sensitively and specifically detect disease-related mHTT seeding activities in HD patient CSF. By using this platform, we identified molecules (i.e. DNAJB6 expression or anti-HTT polyP antibodies) that could block such seeding activities. Our study will enable future systematic analyses of the pathogenic mHTT seeding activities in HD patient CSF to evaluate its potential utility as disease-progression biomarkers, and for large-scale screening of molecules to ameliorate pathogenic seeding activities in HD.40. The PREDICT-HD participants visits the centers annually for biosample collection , brain imaging and cognitive and other neurological evaluation54. CSF were collected through lumbar puncture (LP) after fasting. Basic CSF quality analyses were conducted for cell count, erythrocytes, total protein, and glucose54. All paired multi-visit CSF samples from the same individuals were collected 1 year apart.Prior to the seeding assays, samples were randomized and blinded to investigators. Autopsy brain tissues of subjects with HD were obtained from UCLA Human Brain and Spinal Fluid Resource Center  fragment C-terminally tagged with EGFP were cloned into a mammalian expression vector, pcDNA3.1, to generate the mHTT-46Q-GFP plasmid. mHTT-SD-GFP and mHTT-ΔN17-GFP plasmids were generated by direct point mutation of S13/S16 residues to D13/D16 and deletion of 2–16 a.a. residues of mHTT on the mHTT-46Q-GFP plasmid using a QuikChange site-directed mutagenesis kit (Agilent), respectively. Resulting constructs were validated by Sanger’s sequencing. For generation of stable reporter cell lines, HEK293 cells  were transfected with one of aforementioned plasmids using FuGENE HD (Promega) as described by the manufacturer's protocol. Two days following transfection, the culture media was replenished and G418 (500 ng/ml) was administered, allowing the selective propagation of transfected cells in culture. Neomycin resistant, fluorescent-labeled colonies were identified by a fluorescent microscope on day 14. Single transgenic colonies were picked by a micropipette, and transferred into a 24-well culture dish. The cells continuously proliferated in the presence of G418 (reduced to 200 ng/ml), and the expression of transgenes was monitored by a fluorescent microscope. Cellular expression of mHTT-GFP was confirmed by immunostaining using an anti-HTT antibody, EM48 (Millipore). Stable cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and gentamycin (25 ng/ml) at 37 °C.30. In brief, Rosetta 2 (DE3) pLysS competent cells expressing pGEX-mHtt-Ex1-Q51 plasmid were induced with 1 mM IPTG for 2.5 h at 16 °C. Cell pellets were lysed using an Emulsiflex  and incubated with GSH-Sepharose resin  and washed with 0.1% Triton, 500 mM NaCl, and 5 mM Mg-ATP before eluting protein with 15 mM Glutathione. Protein was concentrated and buffer exchanged with 50 mM Tris-HCl, pH 8.0; 100 mM NaCl; 5% glycerol. Concentrated mHTT-51Q protein was 0.2 μm PVDF filtered. Aggregation reaction was conducted by incubating 20 μM of mHTT-51Q in TEV reaction buffer (ThermoFisher) at 30 °C for 60 h. Aggregates were centrifuged at 16,000×g for 1 h at room temperature to pellet fibers, which were resuspended in TEV buffer and then sonicated 1 s-on/1 s-off pulsing using a Fisher Scientific 120W Sonic Dismembrator (ThermoFisher). The size of the sonicated fibers was measured by DLS using a Zetasizer Nano ZS .Recombinant mHTT-51Q proteins were produced as previously describedAβ peptides corresponding to the human Aβ amino acids 1–42 were purchased from American Peptide Co.  and dissolved to a final concentration of 1 mg/ml in DMSO. To generate fAβ seeds, lyophilized Aβ peptides were reconstituted in sterile distilled water and incubated at 37 °C for 1 week to allow fibrillization. fAβ was then sonicated for two 10 s-on/10 s-off cycles to generate fAβ seeds.d-lysine coated coverslips or 384-well plates and cultured with DMEM plus 10% FBS. mHTT-51Q (2 μg/ml or designated concentrations), Aβ (10 μg/ml), Tau (10 μg/ml), brain extract  or CSF  were directly applied to the cells. For DNAJB6 experiments, cells were transfected with a DNAJB6 expressing plasmid pcDNA5/FRT/TO DNAJB6b43 a day prior to seeding. The media were then replenished and recombinant mHTT seeds (2 μg/ml) or CSF  were added. For evaluating the role of the mHTT domains in seed-induced aggregation, two methods were used to apply mHTT domain-specific antibodies: (1) immunoblocking—the recombinant seeds were preincubated with antibodies (2 μg/ml) for 10 min before applying to the cells; (2) immunodepletion—recombinant mHTT seeds or CSF  were incubated with 2 μg of antibodies and 10 μl of Protein A/G agarose beads in 100 μl of PBS overnight. The mixture were then centrifuged at 13,000×g for 2 min. The resulting supernatant were applied to the cells at the concentration as if they had not been immunodepleted (seeds at 2 μg/ml and CSF at 1:20). The cells were then incubated for 3 or 5 days to allow aggregate formation for low throughput (i.e. coverslips) or high throughput (i.e. 384-wells) experiments, respectively. Confocal images were taken using a Zeiss LSM 500 confocal or ImageXpress Micro Confocal (Molecular Devices) and analyzed by ImageJ or MetaXpress (Molecular Devices), respectively. The particle measurement function in ImageJ were used to quantify the aggregates in the cells. High-content image analysis is described below.One day prior to seeding, cells were plated on poly-High-content images were acquired using automated the ImageXpress Micro Confocal system with a 10 × objective at the confocal slit mode. Z-stacked images were taken to ensure capturing all aggregates within the cells. Four sites per well were imaged to avoid in-well variation. Maximum intensity projected images were analyzed by MetaXpress with Granularity module at Standard analysis mode. Raw readouts were normalized to the internal controls within each plate. The normalized values are labeled as “aggregation index” in the figures. For all experiments, 6–8 wells were used as the technical replica and averaged to generate one data point for a biological replicate.g for 2 min. The supernatants were then immediately applied to the cells and incubated for designated time for the seeding assay.HD patient CSF (1:10) were mixed with 2 μg of an anti-HTT antibody and Protein A/G-conjugated agarose beads  in DMEM and incubated with rocking for 24 h at 4 °C. The mixture were centrifuged at 13,000×Group comparisons without covariates were performed using one-way ANOVA with Tukey post-hoc test. To assess possible confounders, linear models were used for post-mortem CSF data, while linear mixed effect models were used for PREDICT-HD data to account for the fact that there are two data points (visits 1 year apart) for each subject. Bonferroni correction was used to account for testing of 82 clinical scores. Shapiro–Wilks test was used to test for normality of residuals. Pearson correlation and the corresponding significance estimate were used to quantify association between continuous variables.Supplementary Information.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.