As the experimental reference set we used a previously published large-scale patient cohort of CSF to serum quotient ratios of immunoglobulins in relation to the respective albumin quotients. We related the inter-individual variances of these quotient relationships to the individual CSF flow time and barrier characteristics. We claim that this new concept allows the diagnosis of inflammatory processes with Reibergrams derived from population-based thresholds to be shifted to individualized judgment, thereby improving diagnostic sensitivity. We further use the source-dependent gradient patterns of proteins in CSF as intrinsic tracers for CSF flow characteristics. We assume that the rostrocaudal gradient of blood-derived proteins is a consequence of CSF bulk flow, whereas the slope of the gradient is a consequence of the unidirectional bulk flow and bidirectional pulsatile flow of CSF. Unlike blood-derived proteins, the influence of CSF flow characteristics on brain-derived proteins in CSF has been insufficiently discussed to date. By critically reviewing existing experimental data and by reassessing their conformity to CSF flow assumptions we conclude that the biomarker potential of brain-derived proteins in CSF can be improved by considering individual subproteomic dynamics of the CSF system.Here we show that the hydrodynamic radii-dependent entry of blood proteins into cerebrospinal fluid (CSF) can best be modeled with a diffusional system of consecutive interdependent steady states between barrier-restricted molecular flux and bulk flow of CSF. The connected steady state model fits precisely to experimental results and provides the theoretical backbone to calculate the  The cerebrospinal fluid is a complex flow system likely involved in many brain-related tasks such as neuronal communication, waste clearance and immune surveillance of the brain Abbott, . Its cloTable 3). This is well explainable with the bulk-flow concept; blood-derived proteins enter the CSF system along the whole flow path of CSF, whereas brain-derived proteins predominantly enter the CSF system in the cranial area. Therefore, the concentration of blood-derived proteins rise with increasing distance from the ventricular system but the concentration of brain-derived proteins remains fairly constant along the spinal flow path (Table 3). With a pulsatile-only CSF flow concept, these subproteomic gradient characteristics are hard to explain. Further, although the pulsatile flow exceeds the bulk flow of CSF up to tenfold  and how their concentration is influenced by CSF dynamics (Section Brain-Derived Proteins in CSF).Another key question is how blood-derived proteins are able to pass the barriers delineating blood from CSF (Section Discussion about the Diffusion Barriers). In the following sections we provide evidence that the passage of blood-borne proteins into CSF is at least predominantly based on diffusion (Section Blood-Derived Proteins in CSF), critically review a preceding diffusion-based model, the molecular flux model (Section The Molecular Flux Theory), and show that the diffusion-based protein exchange between blood and CSF can be precisely modeled with a system of steady states connected by bulk flow (Section The Connected Steady State Model).Q) of blood-derived proteins in CSF (concentration CSF/concentration serum) is dependent on the hydrodynamic radius RH of the protein and is lower for proteins with a higher hydrodynamic radius  and/or the blood-cerebrospinal fluid barrier (BCSFB) remains to be shown on the physiological level, see  can be fitted with a hyperbolic function with the general formula:genhauer  and claiThe Reibergrams Figure  with albAlb values allowed him to draw general conclusions. In short, with increasing QAlb the Q values of the considered immunoglobulins—IgG/-A/-M—also increase. Further, with increasing QAlb the absolute deviation of the mean hyperbolic function to the upper and lower discrimination line increases but the relative variation decreases or remains almost constant (see Table IgX to QAlb was termed population variation coefficient (Table In a follow-up study Reiber repeated the approach but with a considerably higher number of patients (4254 patients) Reiber, . The hignt Table .Alb the specific QIgG/A/M/QAlb ratio is dependent on the difference in RH of the bigger immunoglobulins to the smaller albumin  equates to the respective concentration in CSF, −∞ < x ≤ 0 equates to the semi-infinite media (blood), the boundary is at x = 0 and CSF has values of x > 0, t = time and D is the diffusion constant. Erfc, the error function complement is a standard mathematical function described in Equation 2.11 by Crank (c(x = 0) = 0 for all t > 0. These boundary conditions do not meet the required conditions at the blood surface and this means that the concentration of a blood-derived molecule in CSF can never reach the blood concentration of this molecule. This in turn violates Fick's first law that postulates that a net diffusional flux exists as long as a concentration gradient exists.D is the diffusion coefficient, c the concentration of the diffusing substance and x the position. Another consequence of the selected model is that the molecular flux from blood into CSF increases with increasing c(CSF) as long as c(CSF) <0.5 c(blood). Reiber explained this increase in molecular flux with increased tissue concentration because of increased concentration in CSF  and D is the diffusion coefficient. The concentration gradient dC/dx along L is constant, whereas C decreases linearly over L.The diffusional loss of blood-derived proteins into the CSF system is in steady state with the bulk outflow of the CSF into blood. Furthermore, the blood volume of ~5000 ml is much larger than the CSF volume of ~150 ml. In addition, the fast circulative convection of blood superimposes the low barrier-restricted diffusional protein exchange with CSF. In view of this consideration it is feasible to state that the protein concentration in the blood system is constant. In contrast, the rostrocaudal CSF bulk flow equates to a river-like start-end point system with a rostrocaudal concentration gradient of blood-derived proteins. Both flow systems, CSF and blood, exist continuously; the molecular flux and the CSF bulk flow balance each other out. In conclusion, and as already stated by Reiber , each poD) is proportional to the concentration gradient and therefore inverse to the bulk flow of CSF. In a continuous system like the CSF system, the molecular flux inside the systems is also under steady state conditions. The JD reduces the bulk flow (JBF) induced rostrocaudal gradient of a molecule proportionally to the diffusion constant of that molecule.According to Fick's first law (Equation 4), the molecular flux inside the CSF system  from ventricle vault to lumbar sac is set to LFP = 50 cm. For the volume of CSF, we ignore the cranial SAS volume and for the remaining volume of the CSF system we set VCSF = 100 ml  equals the CSF production rate of 0.3 ml/min along the whole flow path so that For a rough estimate of the inverse influence of JIn conclusion, the molecular bulk flow is far greater than the molecular flux inside the CSF system. This allows diffusional fluxes inside the CSF system to be ignored.H-selective barrier . And analogous for the molecular flux:F is the maximum diffusion rate from blood to CSF, without back-diffusion. Since ͽ is a composite of a general value—D—and an individual value—B—, in the following ͽ is replaced by Mathematically the concentration of diffusing substances at the most upstream position in CSF is zero, and the resulting steady state molecular flux at this position is:Alb:IgX and QAlb is based on diffusion and molecule-dependent barrier properties. A similar approach was used for the molecular flux concept by Reiber  is able to explain the experimental data shown in Figure 0 is ableg QAlb:1QIgX¯ = aAlb, calculated by Equation (10), have to represent physiological values. Figure For Q-values with an increase of the mean CSF flow time (t). With longer CSF flow time (t) the molecular flux (F) from blood to CSF decreases due to an increase in diffusion back from CSF to blood. Since the decay of the net molecular flux (F) is faster for the faster diffusing protein albumin, the ratio ALB values, shown in Figure B = 1, the quotient t and Alb values  we obtain:T have the same influence on all molecules in the same individual. For albumin as well as for the immunoglobulins G/A/M experimentally measured RH values are available. Table H)−1 values to the related Q(ͽ) values derived from Figure In Newtonian fluids the diffusion coefficient is related to the hydrodynamic radius via the Stokes-Einstein relation:Q(ͽ) is slightly increased. This trend is continued when comparing IgA to albumin but not IgM to albumin; the calculated relations are identical. A possibility for the difference in the case of IgA might be that we used the experimentally measured values for the monomeric variant of IgA, but IgA also exists as a dimer. However, in human serum IgA is dominantly monomeric , Q(ͽ) deviates the more from Q(RH)−1 the more the difference in RH of two compared molecules. In the case of IgM relative to albumin, the effect is superimposed by the fact that IgM also exists in variants with a lower RH than used in Table For IgG to albumin, the quotients between ͽ and H)−1 and Q(ͽ) (Table The coincidence of Q(Rͽ) Table  supportsMf) values lower than Q(RH)−1 values (Table H)−1 = Q(D), and therefore Q(D) < Q(RH)−1 is not possible. In the case of barrier-restricted diffusion; Q(ͽ) < Q(RH)−1 is possible since other molecule-specific characteristics included in B, e.g., polarity, may play a superior role to RH, however, as mentioned, this does not seem to be the case for blood proteins and in addition the molecular flux theory describes a scenario for unrestricted diffusion and does not contain any correction factor representing barrier characteristics (like B in the connected steady state model); therefore Q(D) < Q(RH)−1 is not possible and shows again that the diffusion concept used for the molecular flux theory is not applicable for the diffusional exchange between CSF and blood under steady state conditions.In contrast, the molecular flux theory predicts Q. Proteins not adjustable to a reference protein, with remaining high variances, might possess several RH values such as IgM. Care must be taken with proteins in CSF not only derived from blood but at the other site; this fraction can be calculated if their RH is known.Under the assumption that molecule-specific characteristics other than RA direct clinical application is the diagnosis of inflammatory processes in the CNS. Also because of the high variation of the Reibergrams Figure , oligoclH the RH-dependent barrier specificity can be calculated. As an example, if the Q(B) of two molecules with RH of 3 and 4 nm is greater than the Q(B) of two proteins with RH of 4 and 5 nm, then the conclusion is that the barrier is more discriminative between 3 to 4 nm than between 4 and 5 nm. This allows conclusions on the physiological properties of the barrier to be made.Another deduction from the model is that by comparing a set of reference molecules with known RAlb values can be explained by an altered CSF flow rate without the need of assuming a change in barrier permeability. The physiologically normal QAlb in lumbar CSF is around 2–8 [10−3]. This value increases in an age-dependent manner —a turnover reduced by half and therefore assuming a doubled CSF flow time—yields an increase of QAlb e.g., from 2 [10−3] to 20 [10−3] ( change of CSF flow time). Such a high sensitivity of QAlb to CSF flow time is rather unlikely.According to the steady state Equation (10), at this low Qe Figure , so thatAlb > 8 [10−3] is termed as barrier dysfunction. The increase from 2 [10−3] to 20 [10−3] calculated with the connected steady state system would mean a roughly tenfold increase in CSF flow time. With the current physiological understanding of the CSF flow system, a tenfold increase of CSF flow time seems to be a rather large change.A QAlb is mainly dependent on reduced CSF turnover and not based on changed barrier permeability. But pathophysiologically high QAlb values indicate a pathophysiological change of the barrier permeability.In conclusion, the normal age-dependent increase of QAlb and therefore assume that the barrier specificity slightly decreases with increasing QAlb. Regarding barrier permeability and RH-dependent discrimination Felgenhauer speculated: “Holes are formed by random thermal motion of the membrane structure elements and the permeability properties are characterized by formation frequencies and size probability distributions of these holes.”  as constant and therefore he concluded that the barrier specificity remains unchanged which indicates unchanged barrier permeability Reiber, . We inteH-dependent discrimination. A weaker barrier might have a weaker discrimination between molecules with different hydrodynamic radii  at the site of the CP, the blood brain barrier (BBB) which makes up 99% of all brain capillaries .Another possibility is that ventricle CSF is not only “young” CSF produced by the CP but also interstitial fluid from the highly perfused brain parenchyma, which already contains blood-derived proteins, see for instance Section 4.1 in Hladky and Barrand  for a moThe third possibility, an up-mixing of CSF from SAS back into the ventricles, is rather unlikely in normal physiological conditions and the diffusional fluxes are too low. However, most ventricle CSF samples described in the literature (summarized in Table *dl−1 compared to ~46.8 mg*dl−1) , are all intracellular proteins  and the uptake is dependent on cranial and spinal flow characteristics, whereas the uptake of brain-derived proteins predominantly takes place in the cranial space. Further, the reasons why these intracellular proteins are abundant in extracellular fluids is most likely independent of CSF flow characteristics.However, if increased uptake of brain-derived information is counterbalanced by diffusional loss, lumbar CSF-based diagnosis must be highly inaccurate. The extent of diffusion-out for brain proteins can be estimated by observing the diffusion rates of blood-derived proteins. The physiological normal Q Reiber, . TherefoAlb represents not only CSF flow characteristics but also blood to CSF barrier characteristics might explain the lacking correlation of QAlb to the lumbar CSF concentrations of the investigated intracellular brain-derived proteins. Nevertheless, based on the expected common dependency to CSF flow characteristics we expect a certain degree of correlation between QAlb and brain-derived proteins if the sample size is sufficiently large.These aspects and the fact that the QAnother important aspect is that under the assumption of bulk-flow (ventricular CSF mostly upstream and lumbar CSF mostly downstream) and diffusional loss being a very minor factor, the stoichiometry of the lateral ventricular to lumbar CSF protein concentration ratios for the investigated brain-derived proteins presented by Reiber Table .Net fluid introduction into the CSF system past the lateral ventricle CSF and therefore dilution to such an extent that brain-derived proteins are several times higher in lateral ventricle CSF than in lumbar CSF is unlikely considering the gross anatomic relations of the ventricular system; lateral ventricles make up ~80–90% of the ventricular system (Akdogan et al., With an intelligent experimental design Brandner et al. showed that the high levels of brain-derived proteins in ventricular CSF compared to lumbar CSF is an effect of surgical interference (Brandner et al., These findings concur well with the assumption of bulk flow and the relative stable spinal protein concentration of brain-derived proteins along the rostrocaudal axis Table .Again, based on the limited sample and study numbers, the discussed aspects need further experimental elucidation.In our view, the assumed dependency of brain-derived proteins on CSF flow characteristics introducing non-pathological individual variances questions the dominating biomarker discovery strategy based on the comparison of absolute concentration differences. We claim that the diagnostic accuracy can be improved by evaluating relative concentration values with suitable reference proteins similar to the approach used for blood-derived proteins. This aspect is presumed to be of special importance for early diagnosis purposes when subtle concentration changes of diseased to non-diseased individuals can be expected.Alb values (see Section Discussion about the diffusion barriers and Appendix Figure The connected steady state model offers a solution for barrier-restricted diffusion between a continuous regenerating fluid system such as blood, and a river-like start/end system such as CSF. The model might have the potential to be a general solution concept for diffusional exchange of blood to other body fluids such as blood to the urine or lymph. The model fits perfectly to the available experimental data and is able to explain all the experimental findings. For our considerations we used experimental data already interpreted with the hyperbolic function Reiber, . This miThe source-related rostrocaudal gradient of proteins in CSF and the possibility to link the rostrocaudal gradient of blood-derived proteins to pulsatile flow characteristics are highly intriguing and so far underestimated aspects in CSF physiology but worthy of being investigated in more detail in the near future.In spite of the enormous diagnostic potential of brain-derived proteins in CSF, their dependency on CSF flow characteristics is not sufficiently clarified. We assume that a better understanding and a better implementation of CSF dynamics in biomarker discovery approaches is the way toward the development of optimized analyzing strategies of the CSF-Proteome consequently leading to the identification of more sensitive and reliable brain-derived biomarkers.FM designed the study, was involved in all tasks and wrote the paper. DM contributed to the physical aspects of the study and to the discussion of the mathematical aspects of the study Appendix . FS carrThe 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. The reviewer KB declared a shared affiliation, though no other collaboration, with several of the authors FHM, FS to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


Morc2b encodes a functional germ cell-specific member of the MORC protein family. Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution. Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes. Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that Morc2b, a relatively recent gene, has evolved an essential role in meiosis and fertility.The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse  Morc2b. Morc2b belongs to a family of chromatin-remodelling factors. Morc2b is specifically expressed in germ cells. Disruption of Morc2b causes meiotic arrest and infertility in both sexes. Notably, MORC2B regulates the expression of a number of meiosis-specific genes. Interestingly, MORC2B interacts with its sequence homologue MORC2A. These functional studies have uncovered a new protein complex in the regulation of key meiotic processes and suggested the presence of continued selection pressure for evolution of new meiosis-specific factors.In sexually reproducing organisms, meiosis, a process unique to germ cells, produces haploid gametes. Abnormalities in meiosis can lead to infertility, loss of pregnancy, or genetic diseases such as Down syndrome. The meiotic processes are tightly regulated by a large number of genes including many meiosis-specific ones. The majority of meiosis-specific factors are conserved, however, species-specific factors have evolved. Here we report functional studies of a rodent lineage–specific gene named  Arabidopsis to human [Arabidopsis AtMORC1 and AtMORC6 repress transposable elements in a methylation-independent manner and are essential for heterochromatin formation and gene silencing [The microrchidia (MORC) protein family forms a conserved class of chromatin remodeling factors found in diverse species from to human . MORC prilencing . In mammilencing , 4. MORCilencing , 6. MORCilencing . CollectMORC2 cause axonal Charcot-Marie-Tooth disease (CMT) in humans [MORC2 in human diseases.Mutations in n humans . CMT is n humans . The HUSn humans . In addin humans . These sMorc1 and Morc3 in mouse have been reported. Mouse Morc1, the founding member of the MORC family, is specifically expressed in the male germline and its ablation results in male sterility with meiotic arrest [Morc1 deficiency is associated with de-silencing of transposable elements in the male germline [Morc3-/- mice die at birth or within one day after birth [Morc3 mutation reveals a role in bone homeostasis [Morc2 and Morc4 are not known.Genetic requirements of c arrest , 12. Morgermline . Morc3-/er birth . Study oeostasis . HoweverMorc2 are present: Morc2a and Morc2b. Mouse Morc2b was reported to be a transcriptional target of PRDM9, a histone H3 trimethyltransferase required for meiotic progression and involved in speciation [PRDM9 is the only known mammalian speciation gene [Prdm9-deficient testes, Morc2b is not expressed and H3K4me3 at the Morc2b promoter is low, suggesting that PRDM9 normally induces Morc2b expression via H3K4me3 [Morc2b remains unknown. Here, we report that Morc2b is required for chromosomal synapsis and meiotic recombination in both sexes. Inactivation of Morc2b causes mis-expression of a number of genes including meiosis-specific genes. We find that MORC2B interacts with MORC2A. The Morc2b-null mouse mutant exhibits meiotic defects similar to the Prdm9-null mutant, suggesting that MORC2B may be a key downstream effector of PRDM9 in meiosis.In mouse, two paralogues of eciation . PRDM9 iion gene . PRDM9 sion gene . Followiion gene –23. In P H3K4me3 . HoweverMorc2a and Morc2b differs fundamentally: Morc2a contains 26 introns, whereas Morc2b lacks introns in the coding region  is found in more than 100 mammalian species in the NCBI database. In contrast, Morc2b is only present in mouse and rat but not in other non-rodent eutherians, suggesting that the Morc2b retrotransposition event occurred 12–24 million years ago prior to the radiation of mouse and rat   . The fird beyond . This exd beyond . In concMorc2b, we generated a Morc2b-null allele by targeted deletion of exon 2 through homologous recombination in embryonic stem cells (Morc2b coding region. The offspring from intercrosses of heterozygous (Morc2b+/-) mice exhibited a normal Mendelian distribution of genotypes , suggesting that Morc2b is dispensable for embryonic and postnatal development.To assess the function of em cells . Exon 2 Morc2b-/- mice were viable and appeared to be grossly normal. However, both Morc2b-/- males and females were sterile. Adult males of all genotypes were of similar body weight , but Morc2b-/- males had significantly smaller testes than Morc2b+/- control males . Western blot analysis confirmed reduced levels and absence of MORC2B protein in Morc2b+/- and Morc2b-/- testes respectively; MORC1 and MORC2A were present at reduced abundance in Morc2b-/- testes  testes contained germ cells at all stages including pachytene spermatocytes, round and elongating spermatids, whereas Morc2b-deficient tubules contained early meiotic germ cells including pachytene-like spermatocytes but were devoid of any post-meiotic spermatids  [Morc2b-/- mice therefore suggests severe defects in female meiosis. This data is consistent with the expression of Morc2b during meiosis in embryonic ovaries [Morc2b (previously referred to as 4932411A10Rik) transcript is only present in embryonic ovaries at E13.5 and E14.5, but not at E15.5 and beyond including adulthood [Morc2b is essential for meiosis and fertility in both sexes.The ovaries of adult  oocytes . To deteat birth  but disaat birth . TUNEL aat birth . Perinatr Prdm9) , 33, 34. ovaries . The Mordulthood . CollectMorc2b-/- males were at a pachytene-like stage, characterized by apparent chromosome pairing and formation of SC axial elements (SYCP2) but absence of full chromosomal synapsis  axial elements, and SYCP1, a component of SC transverse elements , 36. SC synapsis . We quanke stage .Morc2b-/- oocytes  of unsynapsed chromosomes but was excluded from synapsed regions  and localization of recombination nodules in spread nuclei of Morc2b-/- spermatocytes. During meiosis, following PRDM9-mediated chromatin changes at recombination hotspots, the SPO11 protein catalyses the formation of DSBs, which elicits the DNA damage response [Morc2b-/- leptotene and zygotene spermatocytes  spermatocytes, however, the number of RPA2 foci was sharply higher in Morc2b-/- spermatocytes at both zygotene-like and pachytene-like stages  [Morc2b-/- testes  , retrotr- testes , implyinPrdm9-/- testes  with testicular protein extracts using the MORC2B antibody. Two protein bands were present in immunoprecipitated proteins from wild type testis but not detected in estis IP . The uppestis IP . Mass spestis IP . We veriestis IP . Additioestis IP . Our resMorc2b is required for meiosis and fertility in both males and females. Interestingly, the Morc2b gene evolved in the rodents via retrotransposition from Morc2a. Strikingly, the relatively young Morc2b gene has evolved an essential role in meiosis and fertility, suggesting a strong selection pressure.The MORC family proteins are involved in chromatin remodelling, transcriptional regulation, and transposon silencing. Here we find that Morc gene family, Morc1 and Morc2b are germ cell-specific but exhibit distinct functions. This is evident from differences in the phenotype of the corresponding mouse mutants. The fertility of Morc1 mutant is sexually dimorphic: males are sterile but females are fertile [Morc2b causes sterility in both sexes. The Morc1 mutant phenotype is similar to that of piRNA (Piwi-interacting RNA) pathway mutants: male-only sterility and de-repression of transposable elements (LINE1 and IAP) in male germ cells [Morc1 mutant germ cells, MORC1 most likely protects genome integrity in male germ cells by silencing transposable elements through a different yet unknown mechanism [Among the  fertile , 12, wherm cells , 52. Sinechanism . In contCAIX gene (carbonic anhydrase IX) by decreasing histone H3 acetylation at the CAIX promoter. MORC2 binds to the CAIX promoter and recruits HDAC4 (histone deacetylase 4) to deacetylate histone H3, which is associated with a repressed chromatin state [Human MORC2 plays a critical role in chromatin remodelling in DNA damage response and transcriptional gene silencing –6. MORC2in state , 6. Uponin state . Given tAdam2, Adam3, Clgn, Crisp1, Fmr1, Krt8, Msh5, Piwil1, Rsph1, Tnp1, Tnp2, and Ybx2. The remaining 18 knockout mice exhibit lethality, or no defects, or somatic defects but normal fertility. Several affected genes are known to play critical roles in meiosis: Msh5, Fmr1, and Ccnb3. Msh5 is downregulated in Morc2b-/- testes, whereas Fmr1 and Ccnb3 are upregulated in Morc2b-/- testes. MSH5 forms a heterodimer with MSH4 and functions in meiotic recombination. Inactivation of Msh5 causes a failure in chromosomal synapsis and thus meiotic arrest [CCNB3 transgene in mouse spermatocytes after the zygotene stage disrupts spermatogenesis [Morc2b-/- mice. It is possible that MORC2B regulates the transcription of these genes through chromatin relaxation or epigenetic modifications.Loss of MORC2B results in mis-expression of 71 genes in testes. Out of the 71 genes, 30 genes have been disrupted in mice . Twelve c arrest , 50. FMRc arrest . CCNB3  family of DNA-binding proteins . The resulting working antisera are: anti-MORC2B, UP2419 and UP2420; anti-MORC1, UP2424. Affinity-purified antibodies were used for immunofluorescence analysis and Western blotting. The following additional antibodies were used: MORC2A  and ACTB . The MORC2A antibody (Abnova) was produced against its C-terminal fragment (aa 791–1030), which displays 69% aa sequence identity with MORC2B. The MORC1 antigen (aa 751–950) shows 30% aa sequence identity with MORC2B.The mouse MORC2B C-terminal fragment (aa 823–1022) and MORC1 C-terminal fragment (aa 751–950) were expressed as GST fusion proteins in Morc2b coding exon was replaced with the PGKNeo selection cassette (Morc2b-containing BAC clone (RP24-63E7) by high-fidelity PCR. The HyTK negative selection cassette was cloned after the right arm. V6.5 embryonic stem (ES) cells (on a C57BL/6 x 129S4/SvJae hybrid background) were electroporated with the ClaI-linearized targeting vector. ES cells were cultured in the presence of 350 μg/ml G418 and 2 μM ganciclovir. Out of 96 double-resistant ES cell clones, nine targeted clones were identified by long-distance PCR. Clone 1F3 was injected into blastocysts. Germline transmission of the knockout allele was obtained through breeding of chimera males with C57BL/6 females. Offspring of intercrosses of Morc2b+/- mice were used for all the analyses. Wild-type allele (220 bp) was assayed by PCR with primers TGCACTGAACCCGACACTAC and GGTAGGAGCGGCAGAGATTC. The Morc2b mutant allele (415 bp) was assayed by PCR with primers ATAGCAGGCATGCTGGGGATGCGGT and TGCACCTACACCAGGCAGCTCAGG.In the targeting vector, the 3.2-kb cassette . The twoFor histological analysis, testes and ovaries were fixed in Bouin’s solution, embedded with paraffin, and sectioned. Sections were stained with haematoxylin and eosin. Color histological images were captured on a Leica DM5500B microscope with a DFC450 digital color camera (Leica Microsystems). For immunofluorescence and TUNEL analysis, testes and ovaries were fixed in 4% paraformaldehyde for 3 h or overnight at 4°C, dehydrated, embedded, and sectioned using a cryostat. TUNEL assays were performed with the ApopTag Fluorescein In Situ Apoptosis Detection Kit . Nuclear spread analysis of spermatocytes and oocytes was performed as previously described , 59. TheMorc2b-/-, and Prdm9-/-) were immunostained with H3K4me3 antibody . Prdm9 targeted mice were obtained from Jackson Laboratory (Stock No: 010719) [Sections of postnatal day 14 testes  . Images Morc2b-/-) using TRIzol reagents (Thermo Fisher Scientific). The RNA concentration was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Equal amounts (1 μg) of total RNA from each sample were used to generate RNA-seq libraries using TruSeq Stranded mRNA Library Preparation Kit Set A  according to the manufacturer’s instruction. The concentration of DNA library templates was determined using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The quality of libraries was evaluated using the Agilent 4200 TapeStation (Agilent Technologies). Eight individual libraries (4 wild type and 4 Morc2b-/-) were pooled in equal amounts for sequencing using the Illumina NextSeq 500/550 High Output v2 kit  and the NextSeq 500 system (Illumina). The RNA-seq data are available under GEO accession no: GSE103127.Total RNA was isolated from eight pairs of postnatal day 12 mouse testes  < 0.05 based on Benjamini and Hochberg multiple testing correction. A volcano plot was constructed to illustrate the differentially expressed genes by plotting log2 of the fold change on the X axis and the negative log10 of the p value on the Y axis  using Tophat with default parameters. Mapped reads were subjected to Cufflinks to estimate gene expression levels . The expe Y axis .Morc2b-/- testes at postnatal day 14 was assayed by real-time PCR .The expression of seven differentially expressed genes was analyzed using independent testis samples from postnatal day 10 and 12 mice (3 testis samples per genotype per time point) by real-time PCR. Expression of LINE1 and IAP retrotransposons in wild type and time PCR . Postnat and IAP , 61. ReaCo-immunoprecipitation was performed with postnatal day 20 mouse testes using affinity-purified MORC2B antibodies as previously described . ImmunopMorc2a and Morc2b were cloned into pcDNA3.1/myc-His vector and pcDNA3.1/V5-His-TOPO vector respectively. Plasmids were transfected into HEK 293T cells. Forty-eight hours after transfection, cells were collected and lysed in whole cell lysis buffer . Immunoprecipitation on protein lysate was performed with anti-V5 antibody , followed by Western blotting with anti-Myc antibody .The full-length coding sequences of mouse t-test or χ2 test.Statistical analysis was performed with Student’s S1 Fig(A) Pairwise amino acid sequence identity between mouse MORC proteins. (B) Phylogenetic tree of MORC proteins. A Neighbour Joining tree was built using the BLOSUM62 matrix. MORC protein accession numbers were as follows: MORC1, NP_034946; MORC2A, NP_001152760; MORC2B, NP_808387; MORC3, NP_001038994; MORC4, NP_001180238.(TIF)Click here for additional data file.S2 FigMultiple sequence alignment was performed using CLUSTAL Omega (v1.2.1). Protein sequence accession numbers were as shown in (DOCX)Click here for additional data file.S3 FigMov10l1-/- testes were used as positive controls for LINE1 and IAP de-silencing. *, statistically significant; ns, non-significant.Postnatal day 14 (TIF)Click here for additional data file.S4 Figt-test); ns, non-significant.(A) Testis sections were immunostained with anti-H3K4me3 antibody. DNA was stained with DAPI. Scale bars, 25 μm. (B) Quantification of H3K4me3 fluorescence. The fluorescence in the most advanced spermatocytes  and Sertoli cells were quantified using Image J. The Y axis shows the relative intensity (spermatocyte/Sertoli cell). *, statistically significant Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Huntington disease (HD) is a neurodegenerative disorder phenotypically characterized by progressive chorea, cognitive impairments and emotional disturbances. The crucial pathological hallmark of the disease is the atrophy of the striatum, with a remarkable loss of projecting medium spiny neurons.2A subtype. Thus, several polymorphisms in the A2A receptor gene have been associated to a reduced age of onset of HD [HTT (huntingtin) gene with 51 CAG repeats (Tg51 rats) that an A2A receptor antagonist does not induce any increase in locomotor activity, first suggesting a possible alteration in the receptor density or function [2A receptor agonist produced very similar effects reducing locomotor activity either in the transgenic rats or the wild-type littermate controls and radioligang-binding experiments showed no differences either in the binding properties of striatal A2A receptors [in vivo microdialysis experiments we did observe lower levels of adenosine in the striatum of either heterozygous and homozygous Tg51 rats at 12 month of age. The results were reproduced in a more recently introduced animal model of HD, the zQ175 knock-in mouse, which expresses the entire HTT carrying 188 CAG repeats. Basal levels of striatal adenosine were measured by in vivo microdialysis in 12 month-old zQ175 knock-in mice and found to be significantly decreased as compared to the levels in the wild-type littermates, although the levels of striatal A2A receptors were also substantially reduced in the knock-in mice [Among many disturbances in the homeostasis of the striatum reported in HD, it has been suggested that impairment in adenosine neurotransmission plays an important role in the neurodegenerative process and therefore the progression of the disease . Epidemiet of HD . We havefunction . More reeceptors . All the(SLC29A1) was also significantly increased in human postmortem prefrontal cortex from HD patients with a grade 2 Vonsattel neuropathological severity score and not in more severe stages (Vonsattel severity score 3 and 4) [SLC29A1 serves as a hub of HD-induced gene dysregulation, with ENT1 showing gained of correlations with other genes in the HD group as compared to controls.Adenosine in the brain may be either released or may be the product of degradation of ATP. Adenosine is mainly removed from the extracellular space by uptake through ENT1. We found that ENT1 was up-regulated in the striatum of the zQ175 mice, which clearly points out to this protein as a responsible for the low adenosinergic tone [3 and 4) . We alsoTaking together, all these data show that ENT1 and extracellular adenosine levels can be used as biomarkers of initial stages of neurodegeneration in HD and that ENT1 might represent a therapeutic target, while the use of adenosine receptor agonists is not recommended given the unwanted peripheric effects. On the other hand, ENT1 levels could be measured in the periphery, where they have been described in erythrocytes and leucocytes. The use of this approach presents though a caveat because it is not known if the up-regulation of ENT1 in the brain parallels with an up-regulation in peripheral cells.Given that inhibitors of ENT1 such as dipyridamole, ticagrelor or dilazep have already been used to treat different pathological conditions related to vascular relaxation and platelet aggregation, or the NSAID sulindac sulfide for its anti-inflammatory effects, it would be interesting to use these compounds in clinical studies with people affected by HD in order to see if it is possible to partially reduce the progression of the disease or to delay in a significant way the age of onset.This work was funded by the IRP-NIDA


P<0.05). This was compatible with 3.3- and 1.7-fold reductions in ON vascular permeability after G-CSF treatment compared with PBS treatment (P<0.05). Microglial activation was increased by 3.8- and 3.2-fold in the early (beginning treatment at day 0 or 1) G-CSF-treated group compared with the PBS-treated group (P<0.05). Immediate (within 30 mins of infarct) treatment with G-CSF also induced M2 microglia/macrophage activation. The cytokine levels were lower in the group that received immediate G-CSF treatment compared to those in the later G-CSF treatment group (P<0.05). Early treatment with G-CSF stabilized the blood–ON barrier to reduce macrophage infiltration and induced M2 microglia/macrophage polarization to decrease the expressions of pro-inflammatory cytokines in this rAION model.Granulocyte colony-stimulating factor (G-CSF) was reported to have a neuroprotective effect in a rat model of anterior ischemic optic neuropathy (rAION model). However, the therapeutic window and anti-inflammatory effects of G-CSF in a rAION model have yet to be elucidated. Thus, this study aimed to determine the therapeutic window of G-CSF and investigate the mechanisms of G-CSF via regulation of optic nerve (ON) inflammation in a rAION model. Rats were treated with G-CSF on day 0, 1, 2 or 7 post-rAION induction for 5 consecutive days, and a control group were treated with phosphate-buffered saline (PBS). Visual function was assessed by flash visual evoked potentials at 4 weeks post-rAION induction. The survival rate and apoptosis of retinal ganglion cells were determined by FluoroGold labeling and TUNEL assay, respectively. ON inflammation was evaluated by staining of ED1 and Iba1, and ON vascular permeability was determined by Evans Blue extravasation. The type of macrophage polarization was evaluated using quantitative real-time PCR (qRT-PCR). The protein levels of TNF-α and IL-1β were analyzed by western blotting. A therapeutic window during which G-CSF could rescue visual function and retinal ganglion cell survival was demonstrated at day 0 and day 1 post-infarct. Macrophage infiltration was reduced by 3.1- and 1.6-fold by G-CSF treatment starting on day 0 and 1 post-rAION induction, respectively, compared with the PBS-treated group ( Summary: Early G-CSF treatment (within 24 hours of a rAION infarct) can stabilize optic nerve (ON) vascular permeability to reduce macrophage infiltration into the ON and induce M2 microglia/macrophage polarization at the ON, resulting in decreased inflammation. Non-arteritic anterior ischemic optic neuropathy (NAION) is characterized clinically by acute, painless visual loss with optic disc swelling . NAION iThe ON head (ONH) is sensitive to blood flow changes and susceptible to vascular insufficiencies caused by altered autoregulation, vasospasm and systemic vascular diseases . InflammGranulocyte colony-stimulating factor (G-CSF) is a neuroprotectant that has been used in many ON/retina animal models, including ON axotomy or crush injury, light-induced retinal damage, retinal ischemia and reperfusion, and oxygen-induced retinopathy . We prevIschemic-brain-injury models have demonstrated that G-CSF treatment can reduce neuroinflammation, neutrophil infiltration, infarct volume and blood–brain barrier (BBB) disruption . EvidencP=0.005) and 16.9±4.4 μV (P=0.013) vs 8.1±4.3 μV, respectively]  tests. The amplitudes of the P1-N2 waves in the day 0 (D0)+G-CSF- and D1+G-CSF-treated groups were significantly higher than that of the PBS-treated group  induced the highest level of microglial infiltration in the ONs B. To furP<0.05) (P<0.05) (P<0.05) (P<0.05)  B. In add(P<0.05) B. The IL(P<0.05) B. G-CSF (P<0.05) B. Howeve(P<0.05) B.Fig. 8In the present study, we demonstrated that the time post-infarct of initiating treatment with G-CSF decided the outcome of visual function and morphometry of RGC preservation in rAION. Specifically, early treatment (before 24 h) resulted in better visual function than delayed treatment, as demonstrated by better preservation of amplitudes in FVEPs, and increased survival of RGCs. These findings clearly show that a therapeutic window exists in G-CSF treatment of rAION (before 1 day post-infarct). We further clarified the mechanisms by which G-CSF regulates neuroinflammation. In a rAION model, the blood–ON barrier is disrupted soon after the infarct, allowing for vascular-borne macrophages to cross the barrier and infiltrate into the ON with the massive production of TNF-α and IL-1b, which may then cause further damage to neurons. In this study, immediate subcutaneous G-CSF treatment restored the disrupted blood–ON barrier and prevented extrinsic macrophage infiltration. In addition, it induced M2-type microglia/macrophage activation in the damaged ON, which effectively reduced the production of TNF-α and IL-1β in the damaged ON, and maintained the RGCs in an anti-apoptotic environment.Inflammation is a prominent histological feature of rAION, and both ED1-positive and Iba1-positive cells have been reported to immediately surround anterior ONs 1 day post-infarct . FurtherZhang et al. reported that Iba1-positive cells in the intrascleral and immediate retroscleral portions of the ON largely disappear by 14 days post-rAION induction . As expeAnti-apoptotic effects of G-CSF have been reported in CNS models and RGCs . G-CSF iThis study provides evidence for the mechanism by which early G-CSF treatment can have a neuroprotective effect in a rAION model. Early treatment with G-CSF can stabilize ON vascular permeability to reduce macrophage infiltration into the ON and induce M2 microglia/macrophage polarization at the ON. In addition, attenuation of macrophage infiltration and induction of M2 microglia/macrophage polarization can reduce pro-inflammatory cytokine expressions to prevent cytokine-induced apoptosis in a rAION model. Thus, early treatment with G-CSF (before 1 day post-infarct) can provide neuroprotective effects on RGCs and ONs, as well as preserving visual function.n=18 in each group). Four groups received G-CSF treatment starting at different time points after infarction, including within 30 min after treatment (D0+G-CSF), and 24 h (D1+G-CSF), 48 h (D2+G-CSF) and 7 days (D7+G-CSF) post-rAION induction. One of the other two groups received subcutaneous injections of PBS post-infarct, and the other was a sham group. The treatment groups received a subcutaneous injection of G-CSF  once daily for 5 days . TUNEL assays of the RGC layer were performed. The newly synthesized (extrinsic) macrophages and monocytes (ED1) and microglial cell (Iba1) markers in the ON sections were investigated by immunohistochemistry. We compared the vascular permeability of the ON among groups to assess the intactness of the blood–ON barrier using Evans Blue extravasation and fluorescence post-rAION induction. Levels of the inflammatory cytokines TNFα and IL-1β were measured in the ON samples using western blotting analysis. The type of macrophage polarization was evaluated by real-time reverse-transcriptase (RT)-PCR.In the present study, we investigated the therapeutic window of G-CSF and the role of G-CSF in regulating ON inflammation in a rAION model . After rr 5 days . At the Adult male Wistar rats weighing 150-180 g (7- to 8-weeks old) were used in this study. The rats were obtained from the breeding colony of BioLASCO Co., Taiwan. Animal care and experimental procedures were conducted in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research, and the Institutional Animal Care and Use Committee (IACUC) at Tzu Chi General Hospital approved all of the animal experiments. All experiments were performed with the animals under general anesthesia induced by intramuscular injections of a mixture of ketamine (40 mg/kg body weight) and xylazine , and topical 0.5% Alcaine eye drops  . The ratDetails of the procedures have been reported in our study . Brieflyn=6 rats in each group) to evaluate visual function.FVEPs were recorded 4 weeks after infarction. To avoid VEP responses of albino Wistar rats being contaminated from the contralateral side, we performed the same surgery in both eyes for FVEP examinations. This procedure was approved by the IACUC, and is detailed in our previous reports . We used2 each in the central and mid-peripheral regions of each retina (n=6 per group). The survival rate of the RGCs (presented as a percentage) was calculated as the number of RGCs in each treatment group divided by the number of RGCs in the sham-operated group, and then multiplied by 100.The procedure was performed as described in our previous reports . BrieflyA segment of the ON of about 5-7 mm in length behind the eyeball was collected after the rats had been sacrificed at 4 weeks. The nerves were immediately frozen at −70°C for histological and immunohistochemical (IHC) studies. None of the samples showed massive disc hemorrhage or retinal detachment on histological examination of the ONs .The cornea, lens and vitreous body were removed from the eyeball after the rats had been sacrificed. The remaining eyecups, containing the sclera and the retina, were fixed in 4% paraformaldehyde for 2 h at room temperature. Each retinal cup was cut adjacent to the disc into two pieces. The tissues were then dehydrated in 30% sucrose overnight and kept at −20°C until further processing. Some retinal cups were fixed in 4% paraformaldehyde for paraffin embedding and sectioning .All frozen sections of the retinas were prepared with samples cut at 1-2 mm in distance from the ONH to ensure the use of equivalent fields for comparisons , 2015. TAnti-ED1 and -Iba1 antibodies react against extrinsic macrophages and intrinsic microglia, respectively, and we used monoclonal antibodies of anti-ED1 and -Iba1  in this procedure. Described in a previous report , brieflyg for 45 min. Evans Blue dye in the supernatant was detected spectrophotometrically by absorbance at 620 nm  and 740 nm (subtracted background) and measured in comparison with a standard curve. Blood samples were treated similarly, but without solubilization and with centrifugation for 15 min at 3550 g at 25°C and at 1:1000 dilution prior to spectrometric evaluation. Evans Blue leakage was assessed by measuring ON dye accumulation using the following modified equation . The forward and reverse primers used in this study for amplification of the target genes were as follows (5′-3′): Arg1-F-TCGGAGCGCCTTTCTCTAAG; Arg1-R-ATCCCCGTGGTCTCTCACAT; CD206-F-AACGTTCGCTGATGCAAACC; CD206-R-TGTAAACTGCACCTGCTCGT; Fizz1-F-CAACAGGATGAAGACTGCAACCT; Fizz1-R-GGGACCATCAGCTAAAGAAG; CD86-F-AGACATGTGTAACCTGCACCAT; CD86-R-ACCGACTTTTTCCGGTCCTG; CD32-F-CTGTCGTCCATGTGCTCTCA; CD32-R-CAAGTTTCACCACAGCCTTCG; GypA-F-CACCGTGTTCTTCGACATCAC; GypA-R-CCAGTGCTCAGAGCACGAAAG.Arg1, CD206 and Fizz1 are markers of M2 macrophages, and CD32 and CD86 are markers of M1 macrophages . Tissue The ON samples were collected at day 7 post-rAION induction in each group. Western blotting was used to measure the levels of TNF-α and IL-1β in the ON samples. The ON protein extracts were separated using a 4-12% NuPAGE Bis-Tris gel . The separated proteins were then transferred onto polyvinylidene difluoride membranes and blocked with 5% of nonfat milk in Tris buffer saline/Tween-20 solution containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl and 0.5% Tween-20. The membranes were then reacted with mouse anti-TNF-α antibodies, mouse anti-IL-1β antibodies, and goat anti-mouse immunoglobulin . The blots were then developed using Enhanced Chemiluminescent Substrate , and the relative intensities of the bands were measured using an image analysis system .U-test for un-paired samples was applied. All P-values less than 0.05 were considered to be statistically significant.All statistical analyses were performed using a commercially available statistical software package . The data were presented as mean±s.d. For comparisons of differences among groups, the non-parametric Mann–Whitney


Time integrated ɛHf[t] compositional array of the Hadean xenocrysts indicates a mafic protolith with 176Lu/177Hf ratio of ∼0.019 that was reworked during ∼4.2-4.0 Ga. This also suggests that separation of such an enriched reservoir from chondritic mantle took place at 4.5 ± 0.19 Ga. However, more radiogenic yet subchondritic compositions of ∼3.67 Ga  and ~3.4 Ga zircons  from the same OMTG samples and two other Paleoarchean TTGs dated at ~3.4 Ga and ~3.3 Ga , respectively, corroborate that the enriched Hadean reservoir subsequently underwent mixing with mantle-derived juvenile magma during the Eo-Paleoarchean.Sensitive High-Resolution Ion Microprobe (SHRIMP) U-Pb analyses of zircons from Paleoarchean (~3.4 Ga) tonalite-gneiss called the Older Metamorphic Tonalitic Gneiss (OMTG) from the Champua area of the Singhbhum Craton, India, reveal 4.24-4.03 Ga xenocrystic zircons, suggesting that the OMTG records the hitherto unknown oldest precursor of Hadean age reported in India. Hf isotopic analyses of the Hadean xenocrysts yield unradiogenic  Thus far, the oldest recorded rocks on Earth are the 4.03-3.92 Ga gneisses from the Acasta Gneiss Complex, Slave Province, Canada7. Zircons older than these have been found as detrital grains within metasedimentary rocks from Western Australia13, Western Tibet14, Brazil15 and Southern China16 and as xenocrysts within meta-igneous rocks from Western Australia17 and Central China18. The examination of the Hf isotopic compositions of the oldest zircons, can further contribute to a comprehensive understanding of the differentiation of the early silicate Earth22. Contrasting isotopic signatures and interpretations from such databases have fueled a persistent debate about the nature of the source reservoir of the protolith generating Hadean and Eoarchean zircons and the composition of the earliest crust that developed during the Hadean21. Hf isotopes are tracers that are very widely used to explicate crustal generation processes21. Previously, Lu-Hf isotopic data from the Jack Hill’s zircons of Hadean age revealed both supra-chondritic and subchondritic initial 176Hf/177Hf values23. Highly radiogenic Hadean and Eoarchean zircons reported from the Jack Hill’s metaconglomerate23 implied the presence of depleted reservoirs, which was in contradiction with concurrent Pb-Pb and Lu-Hf isotopic studies yielding predominantly unradiogenic ɛHf[t] values24. The highly positive values observed in previous studies were later described as the artifacts of isotopic mixing between different age domains of zircons with very fine oscillatory zoning25. Here, we report xenocrystic zircons of Hadean (~4.0-4.2 Ga) to Eoarchean age (~3.7 Ga) from the Paleoarchean (~3.4 Ga) Tonalite-Trondhjemite-Granodiorite gneisses (TTG), called the Older Metamorphic Tonalitic Gneiss (OMTG), of the Paleo-Mesoarchean Singhbhum Craton of Eastern India and confirm that the OMTG holds the hitherto oldest precursor rock recorded in India. We also present Lu-Hf isotopic data from these xenocryst cores and their host zircons that add new information to the Hadean zircon isotopic data repository, augmented with interpretations about the nature of their mantle source and the history of crustal formation events in this craton. In addition, the combined U-Pb and Lu-Hf isotopic data of zircons from two other Paleoarchean TTGs (∼3.4 Ga and ∼3.3 Ga) from different locations within the same craton are also presented to further elucidate the characteristics and heterogeneity of the corresponding mantle source reservoir during the Paleoarchean.Zircons, which are the only representatives of the oldest rocks on Earth, preserve robust records of chemical and isotopic characteristics as well as the history of the generation of their parent rocks28  comprises interlayered metabasalt (amphibolite) and metasedimentary rocks 31. The oldest age of the OMG is constrained by a 207Pb/206Pb ion microprobe age of ∼3.5 Ga, obtained from detrital zircon from quartzites from the Champua area31. However, the presence of even older inherited cores of ∼3.55-3.6 Ga within these zircons has led authors to suggest that older crust, with a minimum age of ∼3.55-3.6 Ga, existed in the Singhbhum Craton31. The Sm-Nd isochron age of 3.3 Ga derived from the OMG amphibolites represents their metamorphic age32. The Older Metamorphic Tonalitic Gneiss (OMTG) consists of thinly compositionally layered, medium-grained tonalitic to granodioritic gneisses33. According to Hofmann and Mazumder28, the OMTG represents a suite of TTGs that formed over an extended period between 3.53-3.45 Ga, whereas the OMG represents a supracrustal assemblage that formed as a greenstone succession. The oldest age obtained from the OMTG is a whole-rock Sm-Nd isochron age of 3775 ± 89 Ma34. This age was later questioned and subsequently amended by Moorbath et al.35 to be closer to 3.4 Ga. However, other older ages recently reported from the OMTG include an age of 3664 ± 79 Ma, which was derived from a whole-rock Pb-Pb isochron36, and a xenocrystic zircon core age of ~3.61 Ga (207Pb/206Pb in situ LA-ICP MS dating), which was found within a ~3.4 Ga zircon37. Acharyya et al.38 reported a discordia upper intercept U-Pb zircon age of 3527 ± 17 Ma for the OMTG. Interestingly, the largest population of 207Pb/206Pb zircon ages of the OMTG from previous studies centered around ~3.4 Ga39, reflecting a major felsic magmatic event. However, the ∼3.6 Ga xenocrysts from the OMTG37 and OMG quartzites30 indicate that felsic crustal formation was initiated in the Singhbhum Craton well before the major phase of emplacement of the OMTG.The Paleoarchean Singhbhum Craton, India, consists of an Archean nucleus of voluminous TTG gneisses and intrusive granitoids of ∼3.5-3.2 Ga age, flanked by three Paleoarchean greenstone successions, which are named the Iron Ore Group (IOGs)28 Fig. . The Arc26)-granitoid suite (SG-II and III26), was emplaced in two phases; the older emplacement age (3.45-3.44 Ga37) broadly coincides with the emplacement age of the OMTG. The latter phase of emplacement is constrained at approximately 3.35-3.32 Ga37. The SG batholith is composite in nature and comprises biotite-granodiorite/granite, adamellite-granite, tonalite and trondhjemite27. The SG batholith is encircled by three distinct Archean greenstone successions, namely, the eastern, western and southern Iron Ore Group42 , Singhbhum Granite Phase-I  and Singhbhum Granite Phase-III ; these data are presented in Table 207Pb-206Pb concordant ages of 4031 ± 5, 4036 ± 15, and 4057 ± 8 Ma (Table 43. The oscillatory zoning and higher Th/U ratios (0.44-0.65) of the Hadean zircons suggest an igneous origin44, although exceptions can occur45. Three analyses from the oscillatory zoned rim of an old xenocrystic core with an age of 4241 ± 4 Ga (grain #11) from sample RM-5 yield >10% discordance, thus reflecting Pb loss, which implies hat this rim is probably older than ~3.8-3.9 Ga. In sample RM-5, grain #2 exhibits an inherited core with a concordant age of 3670 ± 7 Ma, which is homogenous in its CL image  and 3399 ± 6 Ma , respectively  of the Singhbhum Granite Phase-I (SBG-I) and another granite TRG-2), collected from the Singhbhum Granite Phase-III (SBG-III), identified from the regional geological map after Saha of the S26 Fig. . Zircons, collect176Hf/177Hf compositions of Hadean xenocrysts (>4.0 Ga) and their host Paleoarchean zircons are summarized in Table 48 ɛHf[t] value of −2.5 ± 1.6 and is similar to the other two younger Hadean xenocrysts of 4031 ± 5 Ma and 4036 ± 15 Ma, which yield εHf[t] values of −4.1 ± 1.3 and −5.2 ± 1.3, respectively  is remarkably close to that of the Hadean core (0.27995 ± 0.00009)  in the OMTG is the least radiogenic  diagram , which intersects the chondritic uniform reservoir (CHUR) line at 4.497 ± 0.19 Ga  calculated from the oldest Jack Hill zircons20. The intersection age (4.497 ± 0.19 Ga) of this array with the CHUR reference line is closer to the CHUR extraction age of 4.46 ± 0.12 Ga as the source reservoir of the Jack Hill zircons21.On the εHf[t] vs ram Fig. , the pre176Hf/177Hfinitial value (0.28041 ± 0.00007) that is higher than those of the older Hadean and Eoarchean age spots. The ~3.3-3.4 Ga age group of zircons from samples RM-1 and RM-5 yields more radiogenic εHf[t] values than Hadean and Eoarchean age spots, ranging from −0.4 ± 1.2 to −3.7 ± 1.8, except for two data points that fall below −4 epsilon units (−4.9 ± 1.5 and −4.3 ± 1.3). Initial Hf ratios of these spots are identical to those of other age spots with lower εHf[t] values, implying that they were derived from the same source. Initial 176Hf/177Hf values of the ∼3.3-3.4 Ga zircons display a relatively small range of values, varying between 0.28047–0.28057, identical with the average of 0.28053 ± 0.00003. This range also includes 176Hf/177Hf initial values of four discordant age spots . However, the oldest Paleoarchean xenocryst, which has a concordant age of 3595 ± 12 Ma, yields an εHf[t] value that is closer to a chondritic value (−1.5 ± 1.3) and a 176Hf/177Hfinitial values that overlap (within uncertainty) the average value  and TRG-2 (0.28060 ± 0.00003) are also comparable Table-The ɛHf[t] values of the 3397 ± 9 Ma zircons from sample TRBG-I (SG-I) and 3286 ± 6 Ma zircons from sample TRG-2 (SG-III) are all subchondritic, yielding ɛHf[t] values ranging from −0.7 ± 1.3 to −4.7 ± 1.3 and from −0.3 ± 1.1 to −3.1 ± 1.2, respectively (Table 176Hf/177Hf ratios of the Bulk Silicate Earth (BSE), i.e., 0.279685 ± 1949 or 0.279781 ± 1822, which are lower than that of the chondritic reservoir, argue against the decades-old paradigm of the chondritic Earth and are explained by the accelerated decay of 176Lu51. However, such accelerated decay is caused by the high rate of irradiation of chondritic or eucritic meteorites by γ and/or galactic cosmic rays, a process whose effectiveness has been questioned for the BSE52 due to the restricted penetration depth of these rays53. Alternatively, it has been assumed that the Earth was developed from chondritic material but was subsequently modified by either collisional erosion during accretion55 or explosive basaltic volcanism in planetesimals56. Hence, we assume that the source reservoir of the Hadean OMTG xenocrysts was initially separated from chondritic material, and we interpret our zircon data considering CHUR48 as a reference frame. The source array of Lu/Hf = 0.019 fitted through these xenocrysts is comparable to ‘mafic protocrust’ with 176Lu/177Hf values proposed by Kemp et al.21 (0.020) and Amelin et al.19 (0.022) calculated from Jack Hill detrital zircon data. The age of separation (∼4.5 Ga) of the enriched reservoir from the chondritic reservoir calculated from the Hadean zircons in this study is also very similar to the CHUR separation age of the source reservoir of the Jack Hill zircons (∼4.5 Ga24 or 4.46 ± 0.12 Ga21). The development of the ~4.49 Ga enriched reservoir recorded in the Singhbhum craton is also in near-agreement with the estimated age of ~4.5 Ga for the separation of the enriched silicate reservoir upon Earth’s solidification, based on recent geodynamic modeling59.Interestingly, the subchondritic Hf composition (ɛHf[t] < 0) of the oldest (4241 ± 4 Ma) xenocryst indicates the presence of a non-chondritic mantle reservoir as early as ∼4.2 Ga. The composition of the earliest mantle reservoirs of Earth has remained controversial. The reported initial 60, the Hadean (~4.2-4 Ga) zircons of the OMTG were presumably generated from minor silicic melts produced as a consequence of the differentiation or re-melting of pre-4.2 Ga juvenile protocrust of mafic composition. The formation and reworking of juvenile crust were either contemporaneous or separated by a short period of ~100-300 My during the Hadean and Archean eons49. To explain the nature of the enriched mantle reservoir that parented the Hadean OMTG zircons, we envisage that such a reservoir may represent an enriched, residual mafic magma generated from a partially solidified magma ocean, analogous to KREEP beneath the lunar anorthositic crust63. This mafic protocrust was presumably reworked and re-melted to generate felsic melt between ~4.2-4.0 Ga without the significant addition of juvenile material from the mantle. The mineral inclusions in the Hadean OMTG xenocrysts, including K-feldspar, titanite and apatite  calculated from the OMTG validates that a mafic-dominated crust prevailed in the Singhbhum Craton during the Hadean, negating the possibility of the significant presence of upper continental (176Lu/177Hf = 0.00866) or ‘TTG-like’ crust (176Lu/177Hf = 0.0120).Assuming that the parental magma of these zircons is likely to be felsic due to the high solubility of zirconium in mafic-ultramafic magmas176Hf/177Hfinitial compositions of these sites , the 3267-3352 Ma zircons in samples TRBG (SG-I) and the 3377-3246 Ma zircons in samples TRG-2 (SG-III) are 0.28053 ± 0.00006, 0.28057 ± 0.00008 and 0.28060 ± 0.00003, respectively. These values are closely comparable except for one spot with an age of 3404 Ma with a slightly less radiogenic 176Hf/177Hfinitial ratio of 0.28041. Clearly, these Paleoarchean zircons were derived from felsic melts with near identical Hf isotopic values, while minor disparity is most likely due to incomplete mixing between the enriched reservoir with depleted juvenile magma. The results of a previous petrological modeling study67 suggested that the protolith of the OMTG was generated by the 40% partial melting of the OMG amphibolites at garnet stability depths32. It is unlikely that the the remnants of the earliest Hadean mafic protocrust survived the constant reworking processes until today. Remnants of the oldest mafic protocrust may have been preserved in the amphibolite enclaves within the OMTG or these enclaves could represent a modified mafic component developed from interactions between the ancient enriched reservoir and mantle-derived, juvenile mafic magma and was preserved as melting residuum of the mafic protolith from which OMTG magma was generated. Interestingly, the zircons from the ~3.5 to ~3.3 Ga TTGs of the Singhbhum Craton, which are located near Keonjhar, exhibit suprachondritic Hf isotopic signatures with average ɛHf[t] values ranging from +2.9 to +2.239, suggesting that they were derived from a depleted source reservoir. However, this implies that a separate depleted reservoir, which was probably complementary with the ancient, enriched reservoir hypothesized in the present study, of Paleoarchean (~3.5 Ga) or even older age, existed under the cratonic lithosphere of the Singhbhum craton and also participated in the generation of TTG magma.Thus, it is necessary to determine the fate of this ancient, enriched Hadean reservoir in the Singhbhum Craton. The Eoarchean (~3.6 Ga) zircon age domains in sample RM-5 record the second-oldest stage of felsic melt generation; these are slightly more radiogenic than the Hadean ones. Therefore, they were probably generated from the modification of the enriched source of Hadean zircons due to its interactions with juvenile (more radiogenic) mantle melt, as is evidenced by the fact that the ɛHf[t] and es Table  in the P69 the condensation of the oldest solids from the solar nebula at 70. Hence, the separation of the enriched reservoir (4.497 ± 0.19 Ga) in the Singhbhum Craton occurred soon after (~40 My) the accretion of the Earth. Evidence from short-lived isotopes, e.g., 146Sm-142Nd74 or 182Hf-182W76, and long-lived isotopic systems (176Lu-176Hf78) suggest that the development of enriched and depleted reservoirs occurred very early in Earth’s history, probably within 100–200 Ma of planetary accretion, and that complementary enriched  and depleted reservoirs  existed during the Hadean79. The nature and fate of this EER remains elusive, although its presence has been deduced from the ~3.4 Ga Ameralik dykes of the Amitsoq complex80. The highly unradiogenic, Hadean zircons of the OMTG most likely represent product of the EER that existed in the Singhbhum cratonic mantle at ~4.2-4 Ga. The presence of ∼3.3-3.4 Ga TTG zircons (OMTG and SG) with unradiogenic Hf signals in this study indicates that this enriched reservoir was sustained until the Paleoarchean. Therefore, the development of the EER from the chondritic mantle at ∼4.5 Ga raises possible questions about the existence of the complementary depleted mantle. Evidence of depleted mantle under the Singhbhum Craton during the Paleoarchean has already been recorded in ∼3.5-3.4 Ga zircon with radiogenic Hf isotope signatures from the TTGs of the Singhbhum Craton39.Based on isotopic constraints, it has been suggested that Earth’s accretion was roughly complete 30 Myr after81. These komatiites may have separated from much older Hadean mantle and may have recorded evidence of early silicate differentiation, i.e., by preserving a record of the Hadean EDR in the highly radiogenic (εHf[t] = up to +8.2) Paleoarchean (~3.5 Ga) Pilbara komatiites82.Is it possible to back-track the oldest depleted Hadean reservoir that is complementary to the enriched one recorded in the Hadean zircons in this study? The most plausible candidate might be the Paleoarchean komatiites (~3.4 Ga) of the Eastern IOG belt, which are the most direct representatives of the Paleoarchean depleted lower mantle (εNd[t] +2 to +4) below the Singhbhum Craton39. Felsic rocks generated before this time have probably now been completely reworked and recycled, as zircons older than this are only found as xenocrysts here and in earlier studies37. A xenocryst with an age of ∼3.38 Ga is found  of their source reservoir indicates the mafic nature of the older crust that originated from an enriched reservoir that separated from the chondritic reservoir at ~4.5 Ga. However, the younger and almost contemporaneous zircons from the OMTG (3.3-3.4 Ga), Singhbhum Granite Phase-I  and Singhbhum Granite-III  yield more radiogenic Hf isotopic signatures, indicating that this enriched reservoir persisted but underwent mixing with juvenile mantle material during the Eo-Paleoarchean.The combined U-Pb SHRIMP and Lu-Hf isotopic data of the ~4.24 and ~4.03 Ga xenocrystic zircons from the ~3.4 Ga TTG of the ‘Older Metamorphic Tonalitic Gneiss (OMTG)’ of the Archean Singhbhum Craton of Eastern India contain records of the oldest crust in India. The essentially subchondritic (ɛHf[t] < 0) isotopic signatures of these Hadean zircons indicate that they originated from the reworking of older crust prior to ~4.2 Ga. The calculated 86. Zircon crystals were obtained using standard crushing and grinding techniques, followed by separation using heavy liquid and magnetic techniques. The hand-picked crystals were cast in epoxy resin discs and polished. The intensity of the primary O2− ion beam was 5 nA and the spot size was 25–30 μm; each site was rastered for 150 s prior to analysis. Five scans through the mass stations were made for each age determination. The standard used for the calibration of elemental abundances was M257, which contains U = 840 ppm87. TEMORA, whose 206Pb/238U age is 417 Ma88, was analyzed for the calibration of 206Pb/238U ratios after every 3 analyses. Detailed CL images of these zircons were captured. All grains were imaged using a CARL-ZEISS MERLIN Compact with GATAN Mono CL4, and the inclusions within Hadean zircons were analyzed using the same scanning electron microscope with OXFORD IE250. The data were processed and assessed using the Squid 1.0289 and Isoplot 3.0090 programs. Common Pb corrections were based on the measured 204Pb contents. The errors given in Table Zircon separation, CL imaging, inclusion analysis and SHRIMP U-Pb dating were carried out at the Beijing SHRIMP Center, Institute of Geology, Chinese Academy of Geological Sciences, using a SHRIMP II following the analytical procedures described by Williamset al.91. Each measurement included an ablation time of ∼26 s for 200 cycles, a repetition rate of 6–8 Hz, a laser power of 100 mJ/pulse and a spot size with a diameter of 44 μm. Helium was used as the carrier gas for the ablated aerosols. The average 176Hf/177Hf ratios of the Mud Tank92 and Plešovice93 standards obtained in this study after repetitive analyses were 0.282500 (n = 25) and 0.282484 (n = 18), respectively. All Lu–Hf isotopic results are reported with 95% confidence limits.The in situ Lu-Hf analyses of zircons from all four TTG samples were conducted on the pits generated during U-Pb dating at the State Key Laboratory of Lithospheric Evolution, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, using a 193 nm UV ArF excimer laser ablation system attached to a Neptune multi-collector ICP MS. The instrumental conditions and analytical procedures were described by Wu 207Pb/206Pb SHRIMP spot analysis ages, chondritic values  and a 176Lu decay constant of 1.865 × 10−11 year−1 94. Selected 207Pb/206Pb concordant and some discordant (10–15% discordant) age data of zircon spots with Hf isotope values consistent with concordant ones are summarized in Tables 207Pb/206Pb age data with >10% U-Pb discordance and Th/U ratios of <0.15 were commonly disregarded. However, some discordant data, such as those with 176Hf/177Hf ratios identical to those of the concordant population, were included because although their U-Pb ratios have been modified, their original Lu-Hf isotopic ratios were preserved.The calculation of εHf[t] values was based on the All data generated or analysed during this study are included in this published article and its Supplementary Tables  and 2.Supplementary Information


No changes were seen in the control group. Studying patients with a short disease duration ( ≤ 5 years) and patients with a long disease duration ( > 5 years) separately, α‐synuclein and tau only increased in the PD group with long disease duration. In the PD group, an increase in phosphorylated tau over 2 years correlated with faster motor progression and faster cognitive decline. An increase in YKL‐40 over 2 years correlated with faster cognitive decline.CSF tau levels correlated strongly with α‐synuclein. The levels of CSF α‐synuclein, tau, phosphorylated tau, neurofilament light, and YKL‐40, but not β‐amyloidCSF biomarkers reflecting Lewy body pathology and neurodegeneration (α‐synuclein), neuronal degeneration , and inflammation (YKL‐40) increase significantly over 2 years in PD. CSF levels of α‐synuclein and tau correlate and remain stable in the early symptomatic phase of PD but increase in the later phase. We hypothesize that CSF α‐synuclein levels might increase as a result of more intense neurodegeneration in PD with long disease duration. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society Still, few longitudinal cerebrospinal fluid (CSF) biomarker studies have yet been performed. Many cross‐sectional studies during the past years have evaluated the diagnostic potential of α‐synuclein (α‐syn), neurofilament light (NFL), tau, phosphorylated tau (P‐tau), and amyloid β (Aβ) for PD‐related disorders. CSF levels of α‐syn have, when compared with controls, been found to be decreased in patients with PD as well as in patients with other synucleinopathies, for example, dementia with Lewy bodies and multiple system atrophy.42 has been shown to predict the subsequent development of cognitive decline in nondemented PD patients.42 positively correlated with faster motor progression measured by UPDRS score, and Stewart and colleagues found a reduction in CSF α‐syn over a follow‐up of approximately 2 years, but this change did not correlate with worsening of motor symptoms or cognitive decline.Some longitudinal studies have investigated the association between baseline CSF biomarkers and subsequent disease progression in PD. Lower CSF Aβ42, NFL, α‐syn, and YKL‐40 both at baseline and after 2 years of follow‐up in neurologically healthy elderly controls and patients with PD. Our aim was to investigate if there are changes over time in these CSF biomarker levels. We also studied if any changes in biomarker levels over time correlated with faster motor progression and/or cognitive decline in patients with PD.In the present prospective and longitudinal study, we investigated the CSF levels of tau, P‐tau, Aβwww.biofinder.se). The study participants are primarily recruited from the southern region of Sweden and are followed with repeated neurologic, psychiatric, and cognitive assessments and collection of CSF and blood samples under standardized conditions. In this study, we included 63 patients with PD (nondemented), of whom 37 had short disease duration ( ≤ 5 years disease duration) and 26 had long disease duration ( > 5 years disease duration) who had had lumbar puncture at baseline and at the 2 year follow‐up. Of the patients with short disease duration, 11 were De Novo. The patients with PD met the National Institute of Neurological Disorders and Stroke Diagnostic Criteria for PD.This prospective and longitudinal study was performed at the Clinic of Neurology, Skåne University Hospital, Sweden, as part of the Swedish BioFINDER Study  among other scales.All individuals gave written informed consent. The study procedure was approved by the local ethics committee at Lund University Sweden and conducted according to the Helsinki Declaration.g for 10 minutes to remove cells and debris and then stored in aliquots at −80°C pending biochemical analysis.CSF samples were obtained by lumbar puncture in the L3/L4 or L4/L5 interspace with patients nonfasting. The samples were collected in polypropylene tubes and gently mixed to avoid gradient effects. All samples were centrifuged within 30 minutes at + 4°C at 200042, tau, and P‐tau were analyzed using AlzBio3 , NFL was analysed using the NF‐light assay , YKL‐40 was analyzed using the Human Chitinase 3‐like 1 Quantikine ELISA Kit , α‐syn was analyzed using the Covance assay , and haemoglobin was analyzed with an assay provided by Bethyl Lab, Inc. . For α‐syn, only samples with haemoglobin <200 ng/ml were used; consequently, 4 samples from PD patients  and 1 sample from the controls were excluded, all baseline samples.CSF AβFor P‐tau there were 8 missing in the control group and 16 missing in the PD group. Baseline and follow‐up CSF samples were always analyzed in the same batch. All analyses were performed using 1 batch of reagents by board‐certified laboratory technicians who were blinded to clinical data. Intra‐assay coefficients of variation were below 10%.U test was used for continuous variables. The Pearson chi‐squared test was used to compare proportions. Univariate associations between 2 continuous variables were analyzed using Pearson's r  or Spearman's rho . Correlations between CSF biomarkers were investigated with linear regression to correct for confounding factors. To investigate changes in CSF biomarker levels over time we used a paired t test, and in the case of nonnormally distributed data we used Wilcoxon signed‐rank test. Correlations between baseline data and changes over 2 years in CSF biomarker levels were investigated with bivariate correlations. Correlations between changes over 2 years in CSF biomarker levels and changes over 2 years in clinical test scores as well as correlations with changes in other CSF biomarkers were then investigated with a linear regression, adjusting for age and Levodopa equivalent (LED).The statistical analyses were accomplished with SPSS for Windows, version 22.0 . To compare demographic and CSF baseline data between groups, the Mann–Whitney P = .028, R = .286), tau , YKL‐40 , and NFL  and negatively with Aβ42 . In the control group, age correlated positively with YKL‐40  and NFL  and negatively with Aβ42 . The levels of NFL, but no other CSF biomarkers, correlated with disease duration . None of the CSF biomarkers correlated with disease severity as measured by the Hoehn &Yahr or UPDRS III. In the PD group, LED correlated with Aβ42 , but not with any of the other CSF biomarkers. In the PD group as a whole and in the PD group with short disease duration, but not in the PD group with long disease duration, there was a significant worsening in the Hoehn & Yahr score  and a significant increase in LED  over 2 years. In the PD group with long disease duration, there was a significant worsening in letter fluency . There were no significant changes in clinical test scores in the control group over 2 years.Demographics and clinical characteristics of study participants at baseline and at 2 years are given in Table P < .001, β > .84), Aβ42 , and YKL‐40 , when adjusting for age and also LED in the case of PD. Tau correlated with YKL‐40  and NFL  when adjusting for age and also LED in the case of PD.In the PD group and in the control group, α‐syn correlated with tau , α‐syn , and YKL‐40 , adjusting for age and LED.In the PD group but not in the control group, P‐tau correlated with tau , P‐tau , α‐syn , NFL , and YKL‐40  when compared with baseline levels  and YKL‐40  after 2 years. There was no significant increase in α‐syn or tau levels in the PD group with short disease duration .In the PD patients with short disease duration , there were significantly increased levels of CSF biomarkers in NFL , tau , NFL , and YKL‐40  after 2 years , there were significantly increased levels of CSF biomarkers in α‐syn , P‐tau , and YKL‐40  over a 2‐year period in PD. Furthermore, increases in tau levels correlated with increases in the levels of P‐tau , YKL‐40 , and NFL . Increases in P‐tau correlated with increases in YKL‐40 , all adjusted for age and LED (Supplementary Table S1).An increase in α‐syn levels correlated with increases in tau . None of the other CSF biomarkers at baseline predicted change in CSF biomarkers over 2 years.Higher levels of P‐tau at baseline correlated with decreases in AβP = .048, β = .292), but not TUG, and with worsening of cognitive executive function as measured by letter fluency , but not MMSE, when adjusting for age and LED. An increase in YKL‐40 over 2 years correlated with worsening of cognitive function as measured by letter fluency , but not MMSE in PD when adjusting for age and LED , the score is a snapshot in time. Finally, the follow‐up period of the present study was relatively short. PD is a disease that develops over many years and in that scope, 2 years is not long enough to draw any definite conclusions regarding long‐term changes in CSF biomarkers. Thus, studies with a longer follow‐up time are needed, and we are currently collecting CSF biomarker data from 4 and 6 years of follow‐up. Also, to give further insight in the role of α‐syn in the pathophysiology of PD, it would be important in future studies to study changes in oligomeric, phosphorylated, and truncated forms of α‐syn as well as total α‐syn.In conclusion, CSF biomarkers reflecting Lewy body pathology (α‐syn), neuronal degeneration , and inflammation (YKL‐40) increase moderately, but significantly, over 2 years in PD. CSF levels of α‐syn and tau remain stable in the early symptomatic phase of PD but increase in the later phase. We hypothesize that CSF α‐syn levels might be a reflection of a more intense neurodegeneration in PD with long disease duration. To our knowledge, these new findings may in the future be of use in both group stratifications as well as monitoring in clinical trials. However, the results need to be validated in future studies.(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique.S.H.: 1B, 1C, 2A, 2B, 3AY.S.: 1C, 2C, 3BA.O.: 1C, 2C, 3BK.B.:1B, 2B, 2C, 3BH.Z.: 1B, 2B, 2C, 3BO.H.: 1A, 1B, 2A, 2C, 3BS.H., Y.S., A.Ö and O.H. report no disclosures relevant to the manuscript. H.Z. and K.B. are cofounders of Brain Biomarker Solutions in Gothenburg AB, a Gothenburg University Holding‐based platform company at the University of Gothenburg. K.B. has served on advisory boards for Immuno‐Biological Laboratories International and Roche Diagnostics.Additional Supporting Information may be found in the online version of this article at the publisher's web‐site.Supplementary InformationClick here for additional data file.