Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders

Implicating particular genes in the generation of complex brain and behavior phenotypes requires multiple lines of evidence. The rarity of most high impact genetic variants typically precludes the possibility of accruing statistical evidence that they are associated with a given trait. We show here that the enrichment of a rare Chromosome 22q11.22 deletion in a recently expanded Northern Finnish sub-isolate enables the detection of association between TOP3β and both schizophrenia and cognitive impairment. Biochemical analysis of TOP3β revealed that this topoisomerase is a component of cytosolic messenger ribonucleoproteins (mRNPs) and is catalytically active on RNA. The recruitment of TOP3β to mRNPs was independent of RNA cis-elements and was coupled to the co-recruitment of FMRP, the disease gene product in fragile X mental retardation syndrome (FXS). Thus, we uncover a novel role for TOP3β in mRNA metabolism and provide several lines of evidence implicating it in neurodevelopmental disorders

Funktionelle Charakterisierung des TTF-Komplexes und seine Rolle in neurologischen Entwicklungsstörungen

The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific “mRNP code” which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs. The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level. Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with “early” mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression. Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work. The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation.Die eukaryotische Genexpression bedarf einer umfassenden Regulation um die Homöostase der Zelle zu gewährleisten und um dynamische Reaktionen auf externe Einflüsse zu ermöglichen. Obwohl viele der regulatorischen Mechanismen die Transkription als ersten Schritt der Genexpression betreffen, findet auch eine intensive Regulierung auf der Ebene des post-transkriptionellen mRNA-Metabolismus statt. Dabei spielt die jeweilige Zusammensetzung der mRNPs eine zentrale Rolle, da je nachdem, mit welchen Faktoren eine mRNA assoziiert ist, ein sog. „mRNP-Code“ entsteht, der das Schicksal der mRNA bestimmt. Viele der an der Regulierung und dem mRNA-Metabolismus beteiligten Proteine sind in Krankheiten betroffen und gerade neurologische Erkrankungen resultieren häufig von einem fehlerhaften mRNP-Code, der zu Veränderungen in der Regulation und Expression von mRNPs führt. Im Zentrum dieser Arbeit stand ein trimerer Proteinkomplex, der aufgrund seiner Untereinheiten TDRD3, TOP3β und FMRP als TTF-Komplex bezeichnet wird. Biochemische Daten haben gezeigt, dass die drei Komponenten des TTF-Komplexes nucleo-cytoplasmatische „Shuttle“-Proteine sind, die sich im „steady-state“ hauptsächlich im Cytoplasma befinden, mit mRNPs assoziieren und vermutlich mit der Translation in Verbindung stehen. Unter zellulären Stressbedingungen konzentrieren sich die TTF-Komponenten in Stress Granula. Der TTF-Komplex ist damit Teil des mRNP-Codes, dessen zelluläre Ziel-RNAs und Funktion bislang aber völlig unbekannt sind. Da der Verlust von funktionellem FMRP zu der Ausprägung des fragilen X Syndroms (FXS) führt und TOP3β mit Schizophrenie und geistiger Retardation in Verbindung steht, verbindet der TTF-Komplex phänotypisch verwandte neuro-psychiatrische Krankheiten auf molekularer Ebene miteinander. Das Ziel dieser Arbeit war es daher, den TTF-Komplex biochemisch zu charakterisieren und seine Funktion im mRNA-Metabolismus zu definieren. Im Zuge dieser Arbeit gelang der Nachweis, dass TDRD3 als zentrale Einheit des TTF-Komplexes agiert und sowohl FMRP als auch TOP3β direkt bindet. Die Interaktion von TDRD3 und TOP3β ist hierbei sehr stabil, FMRP ist hingegen eine dynamische Komponente. Interessanterweise wird der TTF-Komplex nicht direkt an mRNA gebunden, sondern über den Exon-Junction-Komplex (EJC) an mRNPs rekrutiert. Diese Interaktion wird durch ein spezifisches Bindungsmodul in TDRD3, dem sog. EBM vermittelt. In einer Reihe von biochemischen und systembiologischen Studien konnte das Interaktom des TTF-Komplexes bestimmt und seine Rolle im mRNA-Metabolismus definiert werden. Die Daten offenbarten, dass der TTF-Komplex primär mit „frühen“ mRNPs assoziiert ist und sehr wahrscheinlich an der „pioneer round of translation“ beteiligt ist. Weiterhin zeigte sich, dass TOP3β das Ribosom direkt bindet und somit eine Verbindung des EJC und der Translationsmaschinerie etabliert. Die Reduktion von Komponenten des TTF-Komplexes in kultivierten Zellen führte zu selektiven Änderungen im Proteom, wobei einzelne Proteinteilgruppen, jedoch nicht die globale Expression durch den TTF-Komplex reguliert zu sein scheinen. Die enzymatische Analyse von TOP3β hat darüber hinaus gezeigt, dass es sich um eine Topoisomerase vom Typ IA handelt, die nicht nur DNA sondern auch RNA angreifen kann. Dieser Aspekt ist besonders interessant im Zusammenhang der in dieser Arbeit aufgedeckten Verbindung von frühen mRNPs mit der Translation. Die im Rahmen dieser Arbeit erhaltenen Daten legen nahe, dass der TTF-Komplex eine Rolle bei der Regulation des Metabolismus „früher“ mRNP-Teilgruppen möglicherweise im Zuge der „Pionierrunde“ der Translation spielt. Dabei ist die Verbindung einer RNA-Topoisomerase mit dem mRNA-Metabolismus bisher einzigartig und eröffnet so eine ganz neue Sichtweise auf die post-transkriptionellen Schritte der Genexpression und ihre Regulation

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

<div><p>Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.</p></div

Determination of K^b/B_22–29-tetramer⁺ CD8 T-cells in diabetic RIP-B7.1 tg mice.

<p>(<b>A</b>) TAP-deficient RMA-S cells were either not pulsed (−/−) or pulsed for 6 h with high doses (100 µg/ml) of K^b/A_12-N21A or K^b/B_22–29 peptides, followed by surface staining of trimeric K^b-molecules and FCM. (<b>B</b>) RIP-B7.1 tg mice were immunized with pCI, pCI/ppins or pCI/ppinsΔA_12–21. CD8 T-cells were prepared from pancreata of early diabetic (pCI/ppins, pCI/ppinsΔA_12–21) or non-diabetic (pCI) mice and directly stained with K^b/B_22–29-tetramers. Primary FACS data are shown for representative mice. The actual percentage of K^b/B_22–29-tetramer⁺ CD8 T-cells within the pancreas-infiltrating CD8 T-cell population is shown in brackets. (<b>C</b>) The numbers of K^b/B_22–29-tetramer⁺ CD8 T-cells were determined during the course of pCI/ppinsΔA_12–21-mediated EAD: group 1, health mice (n = 3) with blood glucose levels <200 mg/dl; group 2, early diabetic mice (n = 3) with blood glucose levels between 250–350 mg/dl; group 3, diabetic mice (n = 3) with severe diabetes (i.e., blood glucose levels between 400–550 mg/dl). Pancreata of representative mice out of groups 1 to 3 were analyzed histologically for CD8 T-cell influx (CD8+) or stained with hematoxylin-eosin (H&E).</p

Characterization of autoreactive CD8 T-cell responses in PD-L1^−/− mice.

<p>(<b>A</b>) PD-L1^−/− mice were immunized with pCI (group 1, n = 3), pCI/ppins (group 2, n = 10) or pCI/ppinsΔA_12–21 (group 3, n = 20) and cumulative diabetes incidences (%) were determined (left panel). CD8 T-cells were prepared from pancreata of pCI/ppins-immune and diabetic PD-L1^−/− mice. Pancreatic cell preparations from eight ppins-immune mice were pooled and restimulated <i>ex vivo</i> for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFNγ⁺ CD8 T-cells were determined by FCM. The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown (middle panel). Pancreatic cell preparations from ppins-immune and diabetic (group 2, n = 3), and from control (pCI) or ppinsΔA_12–21-immune and healthy PD-L1^−/− mice (groups 1 and 3, n = 3) were directly stained with K^b/B_22–29-tetramers. The percentage of K^b/B_22–29-tetramer⁺ CD8 T-cells (±SD) within the pancreas-infiltrating CD8 T-cell population is shown (right panel). (<b>B</b>) RIP-B7.1⁺/PD-L1^−/− mice were immunized with pCI (group 1, n = 3) or pCI/ppinsΔA_12–21 (group 2, n = 4) and cumulative diabetes incidences (%) (left panel) and K^b/B_22–29-tetramer⁺ CD8 T-cells in the pancreata (right panel) were determined as described above. (<b>C</b>) PD-L1^−/− mice were immunized with pCI (group 1, n = 2) or both, pCI/ppins+pCI/ppinsΔA_12–21 vectors (group 2, n = 4) into the right and the left tibialis anterior muscles, respectively and cumulative diabetes incidences (%) (left panel) and K^b/B_22–29-tetramer⁺ CD8 T-cells in the pancreata (right panel) were determined as described above. The statistical significance of diabetes induction in immunized mice (<b>A–C</b>) was determined using the log-rank test. Values of P<0.05 were considered significant.</p

The RIP-B7.1 diabetes model.

<p>(<b>A</b>) Map of ppins antigens. The expression vectors encoding the ppins and the mutant ppinsΔA_12–21 are shown. The signal peptide (SP), the insulin B- and A- chains, the C-peptide and the position and sequences of the K^b/A_12–21 epitope (•), its K^b/A_12-N21A variant and of the newly identified K^b/B_22–29 epitope (○) are indicated. (<b>B,C</b>) RIP-B7.1 tg mice were immunized with pCI (groups 1, n = 6), pCI/ppins (groups 2, n = 6) or pCI/ppinsΔA_12–21 (groups 3, n = 6). At indicated times after immunization, blood glucose levels (<b>B</b>) and cumulative diabetes incidences (<b>C</b>) were determined. The statistical significance of diabetes induction in immunized mice was determined using the log-rank test. Values of P<0.05 were considered significant. (<b>D</b>) CD8 T-cells were prepared from pancreata of pCI/ppinsΔA_12–21-immune and diabetic RIP-B7.1 tg mice. Pancreatic cell preparations from ten mice were pooled and restimulated <i>ex vivo</i> for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFNγ⁺ CD8 T-cells were determined by by flow cytometry (FCM). The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown. CD8 T-cell frequencies <0.05% are defined negative. (<b>E</b>) RIP-B7.1 tg mice were immunized with pCI (group 1), pCI/ppins (group 2), pCI/ppinsΔA_12–21 (group 3) or pCI/ppins and pCI/ppinsΔA_12–21 (group 4). In group 4, the indicated plasmids were injected into the right and the left tibialis anterior muscles, respectively. CD8 T-cells were prepared from pancreata of diabetic (groups 2–4) or non-diabetic (group 1) mice and restimulated <i>ex vivo</i> with A_12-N21A or B_22–29 peptides. Specific IFNγ⁺ CD8 T-cell frequencies were determined by FCM. The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (±SD) of a representative experiment (n = 3 mice per group) is shown. The statistical significance of differences between A_12-N21A- (groups 2 and 4) and K^b/B_22–29-specific CD8 T-cell frequencies (groups 3 and 4) was determined by the unpaired Student’s t-test (ns, not significant).</p

Recruitment of different ‘bystander’ cell populations into the pancreatic target tissue.

<p>PD-L1^−/− mice were immunized with both, pCI/ppins+pCI/ppinsΔA_12–21 vectors into the right and the left tibialis anterior muscles, respectively. Pancreata of representative healthy (at 3 days post immunization) (A) or early diabetic mice (at 15–20 days post immunization) (B) were analyzed histologically for insulin expression (insulin) and influx of CD4⁺ T-cells (CD4⁺), macrophages (F4/80⁺) or DCs (CD11c⁺).</p

Priming of K^b/B_22–29-specific CD8 T-cell responses and EAD by mutant ppins antigens.

<p>(<b>A</b>) Map of the expression vectors pCI/ppinsΔA_12–21, pCI/SP-B (encoding the ER-targeting signal peptide and the insulin B-chain) and pCI/SP-B-C (encoding the ER-targeting signal peptide up to the C-peptide). The position of the K^b/B_22–29 epitope (○) is indicated. (<b>B</b>) RIP-B7.1 tg mice were immunized with pCI/ppinsΔA_12–21 (group 1, n = 4), pCI/SP-B (group 2, n = 8) or pCI/SP-B-C DNA (group 3, n = 8) and cumulative diabetes incidences were determined. The statistical significance of diabetes induction in immunized mice was determined using the log-rank test. Values of P<0.05 were considered significant. (<b>C</b>) HEK-293 cells were transiently transfected with pCI (lane 1), pCI/ppins (lane 2) or pCI/ppinsΔA_12–21DNA (lane 3). Cells were labeled with ³⁵S-methionine/cysteine, lysed and immunoprecipitated with an anti-insulin (H86) Ab and protein G sepharose. Immunoprecipitates were processed for SDS-PAGE, followed by fluorography of the gels. The position of pins is indicated (<b>D</b>) HEK-293 cells were transiently transfected with pCI/ppins (lane 1) or pCI/ppinsΔA_12–21 (lanes 2–4). At 28 h after transfection, cells were either non-treated (lanes 1 and 2), or incubated for 6 h with the proteasome-inhibitors expoxymycin (ep; lane 3) or lactacystein (lac; lane 4) and subsequently lysed. Total cell extracts were subjected to high resolution tricine-urea-SDS-PAGE (16%) followed by anti-insulin (H86) specific western blotting.</p

Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes.

Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high- versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.peerReviewe