The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells

AbstractSRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells

Serum Thioredoxin-80 is associated with age, ApoE4, and neuropathological biomarkers in Alzheimer’s disease: a potential early sign of AD

[EN] Background: Thioredoxin-80 (Trx80) is a cleavage product from the redox-active protein Thioredoxin-1 and has been previously described as a pro-inflammatory cytokine secreted by immune cells. Previous studies in our group reported that Trx80 levels are depleted in Alzheimer's disease (AD) brains. However, no studies so far have investigated peripheral Trx80 levels in the context of AD pathology and whether could be associated with the main known AD risk factors and biomarkers. Methods: Trx80 was measured in serum samples from participants from two different cohorts: the observational memory clinic biobank (GEDOC) (N = 99) with AD CSF biomarker data was available and the population-based lifestyle multidomain intervention trial Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) (N = 47), with neuroimaging data and blood markers of inflammation available. The GEDOC cohort consists of participants diagnosed with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), and AD, whereas the FINGER participants are older adults at-risk of dementia, but without substantial cognitive impairment. One-way ANOVA and multiple comparison tests were used to assess the levels of Trx80 between groups. Linear regression models were used to explore associations of Trx80 with cognition, AD CSF biomarkers (A beta 42, t-tau, p-tau and p-tau/t-tau ratio), inflammatory cytokines, and neuroimaging markers. Results: In the GEDOC cohort, Trx80 was associated to p-tau/t-tau ratio in the MCI group. In the FINGER cohort, serum Trx80 levels correlated with lower hippocampal volume and higher pro-inflammatory cytokine levels. In both GEDOC and FINGER cohorts, ApoE4 carriers had significantly higher serum Trx80 levels compared to non-ApoE4 carriers. However, Trx80 levels in the brain were further decreased in AD patients with ApoE4 genotype. Conclusion: We report that serum Trx80 levels are associated to AD disease stage as well as to several risk factors for AD such as age and ApoE4 genotype, which suggests that Trx80 could have potential as serum AD biomarker. Increased serum Trx80 and decreased brain Trx80 levels was particularly seen in ApoE4 carriers. Whether this could contribute to the mechanism by which ApoE4 show increased vulnerability to develop AD would need to be further investigated.Open access funding provided by Karolinska Institute. This research was supported by the Margaretha af Ugglas Foundation, the Karolinska institutet KID funding, Gun och Bertil Stohnes Stiftelse, Stiftelsen Syskonen Svenssons, the Karolinska Institutet fund for geriatric research Stiftelsen Gamla Tjanarinnor, and the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet

Genetic predisposition to increased blood cholesterol and triglyceride lipid levels and risk of Alzheimer disease: a Mendelian randomization analysis.

BACKGROUND: Although altered lipid metabolism has been extensively implicated in the pathogenesis of Alzheimer disease (AD) through cell biological, epidemiological, and genetic studies, the molecular mechanisms linking cholesterol and AD pathology are still not well understood and contradictory results have been reported. We have used a Mendelian randomization approach to dissect the causal nature of the association between circulating lipid levels and late onset AD (LOAD) and test the hypothesis that genetically raised lipid levels increase the risk of LOAD. METHODS AND FINDINGS: We included 3,914 patients with LOAD, 1,675 older individuals without LOAD, and 4,989 individuals from the general population from six genome wide studies drawn from a white population (total n=10,578). We constructed weighted genotype risk scores (GRSs) for four blood lipid phenotypes (high-density lipoprotein cholesterol [HDL-c], low-density lipoprotein cholesterol [LDL-c], triglycerides, and total cholesterol) using well-established SNPs in 157 loci for blood lipids reported by Willer and colleagues (2013). Both full GRSs using all SNPs associated with each trait at p<5×10-8 and trait specific scores using SNPs associated exclusively with each trait at p<5 × 10-8 were developed. We used logistic regression to investigate whether the GRSs were associated with LOAD in each study and results were combined together by meta-analysis. We found no association between any of the full GRSs and LOAD (meta-analysis results: odds ratio [OR]=1.005, 95% CI 0.82-1.24, p = 0.962 per 1 unit increase in HDL-c; OR=0.901, 95% CI 0.65-1.25, p=0.530 per 1 unit increase in LDL-c; OR=1.104, 95% CI 0.89-1.37, p=0.362 per 1 unit increase in triglycerides; and OR=0.954, 95% CI 0.76-1.21, p=0.688 per 1 unit increase in total cholesterol). Results for the trait specific scores were similar; however, the trait specific scores explained much smaller phenotypic variance. CONCLUSIONS: Genetic predisposition to increased blood cholesterol and triglyceride lipid levels is not associated with elevated LOAD risk. The observed epidemiological associations between abnormal lipid levels and LOAD risk could therefore be attributed to the result of biological pleiotropy or could be secondary to LOAD. Limitations of this study include the small proportion of lipid variance explained by the GRS, biases in case-control ascertainment, and the limitations implicit to Mendelian randomization studies. Future studies should focus on larger LOAD datasets with longitudinal sampled peripheral lipid measures and other markers of lipid metabolism, which have been shown to be altered in LOAD. Please see later in the article for the Editors' Summary

Alzheimer's disease biomarker profiling in a memory clinic cohort without common comorbidities

Alzheimer's disease is a multifactorial disorder with large heterogeneity. Comorbidities such as hypertension, hypercholesterolaemia and diabetes are known contributors to disease progression. However, less is known about their mechanistic contribution to Alzheimer's pathology and neurodegeneration. The aim of this study was to investigate the relationship of several biomarkers related to risk mechanisms in Alzheimer's disease with the well-established Alzheimer's disease markers in a memory clinic population without common comorbidities. We investigated 13 molecular markers representing key mechanisms underlying Alzheimer's disease pathogenesis in CSF from memory clinic patients without diagnosed hypertension, hypercholesterolaemia or diabetes nor other neurodegenerative disorders. An analysis of covariance was used to compare biomarker levels between clinical groups. Associations were analysed by linear regression. Two-step cluster analysis was used to determine patient clusters. Two key markers were analysed by immunofluorescence staining in the hippocampus of non-demented control and Alzheimer's disease individuals. CSF samples from a total of 90 participants were included in this study: 30 from patients with subjective cognitive decline (age 62.4 ± 4.38, female 60%), 30 with mild cognitive impairment (age 65.6 ± 7.48, female 50%) and 30 with Alzheimer's disease (age 68.2 ± 7.86, female 50%). Angiotensinogen, thioredoxin-1 and interleukin-15 had the most prominent associations with Alzheimer's disease pathology, synaptic and axonal damage markers. Synaptosomal-associated protein 25 kDa and neurofilament light chain were increased in mild cognitive impairment and Alzheimer's disease patients. Grouping biomarkers by biological function showed that inflammatory and survival components were associated with Alzheimer's disease pathology, synaptic dysfunction and axonal damage. Moreover, a vascular/metabolic component was associated with synaptic dysfunction. In the data-driven analysis, two patient clusters were identified: Cluster 1 had increased CSF markers of oxidative stress, vascular pathology and neuroinflammation and was characterized by elevated synaptic and axonal damage, compared with Cluster 2. Clinical groups were evenly distributed between the clusters. An analysis of post-mortem hippocampal tissue showed that compared with non-demented controls, angiotensinogen staining was higher in Alzheimer's disease and co-localized with phosphorylated-tau. The identification of biomarker-driven endophenotypes in cognitive disorder patients further highlights the biological heterogeneity of Alzheimer's disease and the importance of tailored prevention and treatment strategies

Association study of cholesterol-related genes in Alzheimer's disease

Alzheimer's disease (AD) is a genetically complex disorder, and several genes related to cholesterol metabolism have been reported to contribute to AD risk. To identify further AD susceptibility genes, we have screened genes that map to chromosomal regions with high logarithm of the odds scores for AD in full genome scans and are related to cholesterol metabolism. In a European screening sample of 115 sporadic AD patients and 191 healthy control subjects, we analyzed single nucleotide polymorphisms in 28 cholesterol-related genes for association with AD. The genes HMGCS2, FDPS, RAFTLIN, ACAD8, NPC2, and ABCG1 were associated with AD at a significance level of P ≤ 0.05 in this sample. Replication trials in five independent European samples detected associations of variants within HMGCS2, FDPS, NPC2, or ABCG1 with AD in some samples (P = 0.05 to P = 0.005). We did not identify a marker that was significantly associated with AD in the pooled sample (n = 2864). Stratification of this sample revealed an APOE-dependent association of HMGCS2 with AD (P = 0.004). We conclude that genetic variants investigated in this study may be associated with a moderate modification of the risk for AD in some sample

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

A novel Alzheimer disease locus located near the gene encoding tau protein

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordAPOE ε4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ε4+ (10 352 cases and 9207 controls) and APOE ε4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ε4 status. Suggestive associations (P<1 × 10-4) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ε4+: 1250 cases and 536 controls; APOE ε4-: 718 cases and 1699 controls). Among APOE ε4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10-9). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ε4+ subjects (CR1 and CLU) or APOE ε4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10-7) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P≤1.3 × 10-8), frontal cortex (P≤1.3 × 10-9) and temporal cortex (P≤1.2 × 10-11). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10-6) and temporal cortex (P=2.6 × 10-6). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ε4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted

Biological functions of SRPKs in K562 erythroleukamic cells: detection of elevated autoantibodies against SRPK1 in Alzheimer’s disease patients

SPRK1 represents the most studied member of the serine/arginine family of kinases, which specifically phosphorylate serine residues within serine-arginine⁄arginine-serine domains. During the first part of this study, we investigated the possible impact of SRPK1 down-regulation, by applying RNAi technology, in K562 cells. Down-regulation of SRPK1 expression levels in K562 cells induces erythroid differentiation. A dominant cytoplasmic distribution of SRPK1 was observed in these cells, whereas a minor fraction of the kinase was found in the nucleus. Interestingly, a fraction of the cytoplasmic kinase, was found to be associated with polysomes.SRPK1 post-transcriptional down-regulation results in decreased phosphorylation of a certain number of proteins associated with the polysomal fraction. Following LC-MS/MS analysis, we identified a set of 28 proteins, participating in various aspects of mRNA maturation, transport and translation. TAF15 was one of the identified proteins. We present evidence that TAF15 is not phosphorylated by SRPK1. However, these two proteins were found to interact in vitro via SRPK1’s first catalytic domain and most importantly TAF15 was shown to negatively regulate SRPK1 activity. TAF15 localizes almost exclusively in the nucleus of K562 and HeLa cells. Taking into account the prevalent cytoplasmic localization of SRPK1, the two proteins do not seem to interact under normal cellular conditions. However, SRPK1 translocates to the nucleus, following genotoxic stress of HeLa cells, where it co-localizes with TAF15 in the periphery of particular subnuclear structures. This interaction is functional, as TAF15 could inhibit SRPK1-mediated splicing of a reporter gene, in an in vivo splicing assay. In the second part of this study, we investigated the potential implication of SRPK1 in the immunopathobiological mechanisms leading to Alzheimer’s disease (AD) development, by detecting specific autoantibodies against SRPK1 in the sera and cerebrospinal fluid of AD patients. In particular, a significant difference in anti-SRPK1 serum autoantibodies between Alzheimer’s disease patients and control individuals was observed (p=0.031). Furthermore, the epitopes mainly recognized by the antibodies in AD patients were mapped within the first catalytic domain of the kinase (p= 0.029). Elevated levels of SRPK1 autoantibodies were also detected in cerebrospinal fluid samples of the patients.Η SRPK1 αποτελεί το πιο καλά μελετημένο μέλος της οικογένειας κινασών πρωτεϊνών σερίνης/αργινίνης, φωσφορυλιώνοντας σερίνες σε επαναλαμβανόμενα μοτίβα σερίνης/αργινίνης. Στο πρώτο μέρος της παρούσας διατριβής, διερευνήσαμε τις επιπτώσεις της καταστολής της SRPK1, με την τεχνική της RNA παρεμβολής, στα κύτταρα Κ562. Παρατηρήθηκε ότι η καταστολή της έκφρασης της κινάσης στα κύτταρα Κ562 προκαλεί την διαφοροποίησή τους προς ερυθροκύτταρα. Η κινάση εντοπίστηκε κυρίως στο κυτταρόπλασμα και ένα μέρος της ανιχνεύθηκε στα πολυσώματα. Η καταστολή της SRPK1, επιφέρει μείωση του επιπέδου φωσφορυλίωσης συγκεκριμένων πρωτεϊνών που απομονώθηκαν από το εκχύλισμα των πολυσωμάτων. Ύστερα από ανάλυση LC-MS/MS, ταυτοποιήθηκαν 28 πρωτεΐνες, οι οποίες σχετίζονται με τις διαδικασίες ωρίμανσης, μεταφοράς και μετάφρασης του mRNA. Μία από τις πρωτεΐνες που ανιχνεύθηκαν ήταν η TAF15. Στην παρούσα διατριβή δεν παρατηρήθηκε φωσφορυλίωση της TAF15 από την SRPK1. Ωστόσο, δείχτηκε ότι οι δύο πρωτεΐνες αλληλεπιδρούν in vitro, μέσω της πρώτης καταλυτικής περιοχής της SRPK1 και μάλιστα βρέθηκε ότι η TAF15 αναστέλλει τη δράση της SRPK1. Η TAF15 κατανέμεται κυρίαρχα στο πυρήνα κυττάρων Κ562 και HeLa, ενώ ένα μέρος της εντοπίζεται στο κυτταρόπλασμα. Δεδομένης της κυτταροπλασματικής κατανομής της SPRK1, οι δύο πρωτεΐνες υπό φυσιολογικές συνθήκες δεν φαίνεται να συνεντοπίζονται. Ωστόσο, υποβάλλοντας τα κύτταρα σε γενοτοξικό stress, παρατηρήθηκε μετατόπιση της SRPK1 στον πυρήνα, όπου και συνεντοπίζεται με την TAF15, γύρω από συγκεκριμένες υποπυρηνικές δομές. Η συνεντόπιση αυτή φαίνεται να έχει και λειτουργική σημασία, αφού σε δοκιμασία in vivo ματίσματος διαπιστώθηκε καταστολή του ματίσματος ενός γονιδίου αναφοράς από την TAF15. Στο δεύτερο μέρος της διατριβής, μελετήσαμε την ενδεχόμενη συμμετοχή της SRPK1 σε ανοσοβιολογικούς παθογενετικούς μηχανισμούς της νόσου Alzheimer, με την ανίχνευση ειδικών αυτοαντισωμάτων που στοχεύουν στην SRPK1 στον ορό και εγκεφαλονωτιαίο υγρό ασθενών με νόσο Alzheimer. Συγκεκριμένα, παρατηρήθηκε σημαντική διαφορά στον τίτλο αυτοαντισωμάτων έναντι της SRPK1 στον ορό των ασθενών με νόσο Alzheimer, σε σχέση με τους υγιείς μάρτυρες (p=0.031). Μάλιστα, διαπιστώθηκε ότι η πρώτη καταλυτική περιοχή της κινάσης αποτελεί την κύρια αντιγονική περιοχή που αναγνωρίζεται από τα αντι-SPRK1 αυτοαντισώματα στους ασθενείς (p=0.029). Παράλληλα, διαπιστώσαμε την ύπαρξη των συγκεκριμένων αυτοαντισωμάτων και στο εγκεφαλονωτιαίο υγρό, όπου παρατηρήθηκε αυξημένη παρουσία τους στους ασθενείς

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases&rsquo; partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins