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Therapeutic modulation of TREM2-dependent microglial function might provide an additional strategy to slow the progression of Alzheimer's disease. Although studies in animal models suggest that TREM2 is protective against Alzheimer's pathology, its effect on tau pathology and its potential beneficial role in people with Alzheimer's disease is still unclear. Our aim was to study associations between the dynamics of soluble TREM2, as a biomarker of TREM2 signalling, and amyloid β (Aβ) deposition, tau-related pathology, neuroimaging markers, and cognitive decline, during the progression of autosomal dominant Alzheimer's disease.We did a longitudinal analysis of data from the Dominantly Inherited Alzheimer Network (DIAN) observational study, which includes families with a history of autosomal dominant Alzheimer's disease. Participants aged over 18 years who were enrolled in DIAN between Jan 1, 2009, and July 31, 2019, were categorised as either carriers of pathogenic variants in PSEN1, PSEN2, and APP genes (n=155) or non-carriers (n=93). We measured amounts of cleaved soluble TREM2 using a novel immunoassay in CSF samples obtained every 2 years from participants who were asymptomatic (Clinical Dementia Rating [CDR]=0) and annually for those who were symptomatic (CDR>0). CSF concentrations of Aβ40, Aβ42, total tau (t-tau), and tau phosphorylated on threonine 181 (p-tau) were measured by validated immunoassays. Predefined neuroimaging measurements were total cortical uptake of Pittsburgh compound B PET (PiB-PET), cortical thickness in the precuneus ascertained by MRI, and hippocampal volume determined by MRI. Cognition was measured using a validated cognitive composite (including DIAN word list test, logical memory delayed recall, digit symbol coding test [total score], and minimental status examination). We based our statistical analysis on univariate and bivariate linear mixed effects models.In carriers of pathogenic variants, a high amyloid burden at baseline, represented by low CSF Aβ42 (β=-4·28 × 10-2 [SE 0·013], p=0·0012), but not high cortical uptake in PiB-PET (β=-5·51 × 10-3 [0·011], p=0·63), was the only predictor of an augmented annual rate of subsequent increase in soluble TREM2. Augmented annual rates of increase in soluble TREM2 were associated with a diminished rate of decrease in amyloid deposition, as measured by Aβ42 in CSF (r=0·56 [0·22], p=0·011), in presymptomatic carriers of pathogenic variants, and with diminished annual rate of increase in PiB-PET (r=-0·67 [0·25], p=0·0060) in symptomatic carriers of pathogenic variants. Presymptomatic carriers of pathogenic variants with annual rates of increase in soluble TREM2 lower than the median showed a correlation between enhanced annual rates of increase in p-tau in CSF and augmented annual rates of increase in PiB-PET signal (r=0·45 [0·21], p=0·035), that was not observed in those with rates of increase in soluble TREM2 higher than the median. Furthermore, presymptomatic carriers of pathogenic variants with rates of increase in soluble TREM2 above or below the median had opposite associations between Aβ42 in CSF and PiB-PET uptake when assessed longitudinally. Augmented annual rates of increase in soluble TREM2 in presymptomatic carriers of pathogenic variants correlated with decreased cortical shrinkage in the precuneus (r=0·46 [0·22]), p=0·040) and diminished cognitive decline (r=0·67 [0·22], p=0·0020).Our findings in autosomal dominant Alzheimer's disease position the TREM2 response within the amyloid cascade immediately after the first pathological changes in Aβ aggregation and further support the role of TREM2 on Aβ plaque deposition and compaction. Furthermore, these findings underpin a beneficial effect of TREM2 on Aβ deposition, Aβ-dependent tau pathology, cortical shrinkage, and cognitive decline. Soluble TREM2 could, therefore, be a key marker for clinical trial design and interpretation. Efforts to develop TREM2-boosting therapies are ongoing.German Research Foundation, US National Institutes of Health.

The focus of this thesis has been to further advance and develop existing NMR techniques for the study of protein folding. In order to do so, experimental as well as theoretical approaches have been pursued. From the theoretical side, a successful attempt to the development of a general theory for the treatment of residual dipolar couplings in the case of unfolded proteins has been undertaken. Information contained in residual dipolar couplings is especially valuable due to its long-range nature. The dynamic character of unfolded states of proteins, which may be composed of distinct subsets of conformations, renders reliable interpretation of data a non-trivial task. Statistical-coil-based approaches have been shown to be powerful in data interpretation. A consistent theory based on fundamental polymer physics, however, had not been presented so far. The herein presented model addresses this problem building on the original work by Annila and co-workers. In this work, several shortcomings have been identified. These shortcomings have been corrected here leading to a general approach for the treatment of residual dipolar couplings of unfolded proteins. More specifically, it is shown that, in the case of fully unfolded proteins aligned by a steric mechanism, basic dependencies of dipolar couplings such as on chain length and location with in the chain can be analysed in simple analytical terms. The main predictions of the model are compared to experimental data showing reasonable agreement. The presented mathematical framework is principally suited for various improvements which could include the treatment of long-range interactions and of the actual geometry of the given aligment medium. From the experimental side, bovine alpha-lactalbumin has been chosen as a model system for the development of improved time-resolved 1D NMR methods aiming at the observation of conformational transitions by kinetic means. The presented results show that high-quality data can now be obtained at protein concentrations as low as 100uM. Rate constants characterising distinct conformational transitions of up to 8/s have been measured. These are the fastest rate constants which have been reported so far for protein folding events. The NMR data supplemented by complementary biophysical data furthermore demonstrate that the folding of bovine alpha-lactalbumin is more complex than has been anticipated. All data are consistent with a triangular folding mechanism involving parallel pathways of folding for formation of the native state of the protein. Interestingly, such a folding mechanism has also been found for the highly structurally homologous protein lysoyzme from hen egg white. Evidence is presented that the guiding role of long-range interactions in the unfolded state of lysoyzme for mediating intersubdomain interactions during folding is replaced in the case of bovine alpha-lactalbumin by the Ca2+ binding site. Der Schwerpunkt der vorliegenden Arbeit besteht darin, dass bestehende NMR-Techniken zur Charakterisierung der Proteinfaltung weiter entwickelt wurden. Das erste Teil ist rein theoretischer Natur und entwickelt ein mathematisches Gerüst, das die Vorhersage dipolarer Restkopplungen für entfaltete Proteine auf der Grundlage polymerphysikalischer Prinzipien ermöglicht. Auf Grund der Tatsache, dass die im Experiment gemessenen Kopplungen das Ergebnis einer populationsgewichteten Mittelung darstellen, ist ihre Interpretation im strukturellen und dynamischen Sinne schwieriger im Vergleich zu gefalteten Proteinen. Für eine verlässliche Interpretation der Daten ist die Ausarbeitung einer Theorie für die partielle Ausrichtung entfalteter Proteine demzufolge unerlässlich. Als Startpunkt wurde hier der von Annila und Mitarbeitern vorgeschlagene Ansatz gewählt. Eine ausführliche Auseinandersetzung mit der Originalpublikation ergab, dass sie Defizite aufweist. Diese Defizite wurden hier aufgegriffen und korrigiert. Konnte in der Originalpublikation das zweite Legendre-Polynom für ein Kettensegment, das die Ausrichtung relativ zum Magnetfeld wiedergibt, nur numerisch ermittelt werden, so war es hier möglich, eine analytische Lösung zu erarbeiten. Die Vorhersagen auf der Basis des ausgearbeiteten Modells sind, dass (i) dipolare Restkopplungen in der Mitte der Kette größer sind als an den Enden der Kette, (ii) bei einer definierten Konzentration des Ausrichtungsmediums (Barrierendistanz) für kürzere Ketten größer ausfallen als für längere Ketten und (iii) für eine abnehmende Konzentration des Ausrichtungsmediums kleiner werden. Ein ausführlicher Vergleich mit Literaturdaten zeigt gute Übereinstimmung mit den Vorhersagen des Modells. Im zweiten Teil der Arbeit wurden NMR-Experimente zur Untersuchung der Kinetik vorgenommen, mit der sich bovines alpha-Lactalbumin (BLA) vom entfalteten Zustand (U) in den gefalteten Zustand (N) umwandelt. Die Faltung von BLA ist mit verschiedenen biophysikalischen Methoden untersucht worden und das gängige Modell für die Faltung des Proteins, das auf der Basis der Daten erstellt wurde, beschreibt die Bildung eines molten-globule-Intermediates (MG) aus dem entfalteten Zustand und die Umwandlung des Intermediates in den nativen Zustand als einen schrittweisen Prozess: U->MG->N Im Rahmen der vorgenommenen kinetischen Messungen konnten zeitaufgelöste NMR-Methoden derart weiter entwickelt werden, dass (i) die Proteinkonzentration auf 100uM abgesenkt werden konnte, (ii) Faltungsereignisse auf der Millisekundenzeitskala mit Ratenkonstanten von bis zu 8/s detektiert werden konnten und (iii) die kinetischen Daten eindeutig zeigten, dass die Faltung von BLA komplexer abläuft als bisher angenommen wurde. Gestützt durch stopped-flow-Doppelsprung-Fluoreszenzmessungen konnte der Triangular-Faltungsmechanismus für BLA abgeleitet werden. Demnach werden bei der Bildung des gefalteten Zustands von BLA parallele Faltungswege beschritten. I bezeichnet ein Faltungsintermediat, das auf einem der beiden Faltungswege populiert wird und von dem MG-Faltungsintermediat verschieden ist. Besonders interessant hierbei ist, dass für das Hühnereiweißlysozym, das eine hohe Strukturhomologie zu BLA zeigt, ebenfalls dieser sogenannte Triangular-Faltungsmechanismus gefunden wurde. Ein Vergleich der Strukturen des Proteins in Abwesenheit bzw. Anwesenheit des Cofaktors Calcium unter Berücksichtigung der Faltungshomologie legt den Schluss nahe, dass der Calciumbindestelle bei der Faltung von BLA eine tragende Rolle bei der Ausbildung von Kontakten zwischen den Subdomänen zukommt, eine Funktion, die im Falle des Lysozyms durch langreichweitige Wechselwirkungen zwischen den Subdomänen im entfalteten Zustand erfüllt wird.

CSF sTREM2 measurement. Figure S1. Effects of rare TREM2 variants on antibody affinities. Figure S2. CSF sTREM2 in the A/T and in the A/N classifications. Table S1. Demographic and clinical characteristics of the entire sample. Table S2. Associations of CSF sTREM2 with AD CSF core biomarkers including the biomarkers outliers. Table S3. Associations of CSF sTREM2 with AD CSF core biomarkers including subjects carrying a TREM2 rare variant. (DOCX 1992 kb)

CSF sTREM2 measurement. Figure S1. Effects of rare TREM2 variants on antibody affinities. Figure S2. CSF sTREM2 in the A/T and in the A/N classifications. Table S1. Demographic and clinical characteristics of the entire sample. Table S2. Associations of CSF sTREM2 with AD CSF core biomarkers including the biomarkers outliers. Table S3. Associations of CSF sTREM2 with AD CSF core biomarkers including subjects carrying a TREM2 rare variant. (DOCX 1992 kb)

CSF sTREM2 measurement. Figure S1. Effects of rare TREM2 variants on antibody affinities. Figure S2. CSF sTREM2 in the A/T and in the A/N classifications. Table S1. Demographic and clinical characteristics of the entire sample. Table S2. Associations of CSF sTREM2 with AD CSF core biomarkers including the biomarkers outliers. Table S3. Associations of CSF sTREM2 with AD CSF core biomarkers including subjects carrying a TREM2 rare variant. (DOCX 1992 kb)