Funktionelle Charakterisierung von BACE, einer für die Alzheimer Krankheit relevanten Protease

Die Alzheimer Krankheit ist die häufigste Altersdemenz. Ein spezifisches pathologisches Merkmal der Alzheimer Krankheit ist die Amyloid-Ablagerung im Gehirn. Die Hauptkomponente der so genannten Amyloid-Plaques ist das Amyloid beta-Peptid (A-beta). A-beta entsteht durch sequenzielle proteolytische Spaltung aus einem membrangebundenen Vorläuferprotein, dem beta-APP (betaamyloid precursor protein). Die kürzlich identifizierte beta-Sekretase (BACE, beta-site APPcleaving enzyme) generiert den Schnitt am N-Terminus von A-beta. Es entsteht ein C-terminales, membrangebundenes beta-APP-Fragment, das beta-APP-CTF. Beta-APP-CTF ist das direkte Substrat für die gamma-Sekretase, die innerhalb der Membrandomäne schneidet, wodurch A-beta freigesetzt wird. In der vorliegenden Arbeit kann erstmalig gezeigt werden, dass BACE auf dem sekretorischen Transportweg aus dem Endoplasmatischen Retikulum (ER), über den Golgi-Apparat zur Zelloberfläche transportiert wird. Auf dem Transport wird BACE durch N-Glycosylierung und Propeptidabspaltung posttranslational modifiziert. BACE wird im ER N-glycosyliert und die mannosereichen Zucker werden auf dem Transport durch den Golgi-Apparat in Endoglycosidase H resistente Zucker des komplexen Typs modifiziert. Die Propeptidabspaltung, durch Furin oder furinähnliche Propeptidkonvertasen, findet unmittelbar vor dem Aufbau der komplexen Zucker statt. Ferner konnte gezeigt werden, dass der Transport von BACE die A-beta-Entstehung limitieren kann. In polarisierten Madin-Darby canine kidney (MDCK) Zellen wird BACE überwiegend zur apikalen Plasmamembran transportiert und damit entgegengesetzt zu seinem Substrat beta-APP. Der gegensätzliche Transport von BACE und beta-APP begrenzt die A-beta Entstehung. Wird der apikale Transport von beta-APP durch Deletion seines basolateralen Sortierungssignals erhöht, entsteht vermehrt A-beta. Der differenzielle Transport von BACE und beta-APP könnte ein Hinweis darauf sein, dass beta-APP nicht das physiologische Substrat von BACE ist.Alzheimer`s disease is the most common cause of progressive cognitive decline in the aged population. Pathologically Alzheimer`s disease is characterized by the invariant accumulation of senile plaques. Senile plaques are predominantly composed of the amyloid beta-peptide (A-beta), which is derived from the membrane bound beta-amyloid precursor protein (beta-APP) by sequential proteolytic cleavage. The recently identified beta-secretase (BACE) is responsible for the cleavage at the N-terminus of the A-beta domain. This cleavage generates membrane-bound beta-APP-Cterminal fragments (beta-APP-CTF) which are the immediate precursor for gamma-secretase cleavage and therefore for liberation of A-beta. The present work shows that BACE moves along the secretory pathway, while it undergoes post-translational modifications, which can be monitored by a significant increase in the molecular mass and cleavage of its pro-peptide. BACE becomes N-glycosylated within the ER and the increase in molecular mass is caused by complex N-glycosylation. The mature form of BACE is resistant to endoglycosidase H treatment; this indicates that BACE traffics through the Golgi. Furthermore the mature form of BACE does not contain the pro-peptide anymore. Pro-BACE is predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurs immediately before full maturation by furin or a furin-like proprotein convertase. Moreover traffic of BACE can limit A-beta generation. In the well established model system of polarized Madin-Darby canine kidney (MDCK) cells, the majority of BACE is sorted to the apical domain. Interestingly it has been shown previously that the substrate of BACE, beta-APP is transported to the basolateral surface of MCDK cells. Therefore, substantial amounts of BACE are targeted away from beta-APP to a non-amyloidogenic compartment, a cellular mechanism that limits A-beta generation. Upon deletion of the basolateral sorting signal of beta-APP, apically missorted beta-APP is processed by BACE. The differential targeting of BACE and its substrate beta-APP suggest that beta-APP might not be the major physiological substrate of BACE

Presenilin-1 affects trafficking and processing of βAPP and is targeted in a complex with nicastrin to the plasma membrane

Amyloid β-peptide (Aβ) is generated by the consecutive cleavages of β- and γ-secretase. The intramembraneous γ-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with γ-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Aβ production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the γ-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with γ-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by γ-secretase inhibitors results in delayed reinternalization of the β-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in γ-secretase processing and in trafficking

Loss of TMEM106B potentiates lysosomal and FTLD-like pathology in progranulin-deficient mice

Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP-43 pathology in single and double knockout animals. Grn-/- /Tmem106b-/- mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease-associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn-/- knockouts only occasionally show TDP-43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP-43. Thus, a loss of function of TMEM106B may enhance the risk for GRN-associated FTLD by reduced protein turnover in the lysosomal/autophagic system

Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

Background: Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear. Methods: mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins. . Results: We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN. Conclusions: The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells

Early increase of CSF sTREM2 in Alzheimer's disease is associated with tau related-neurodegeneration but not with amyloid- pathology

BackgroundTREM2 is a transmembrane receptor that is predominantly expressed by microglia in the central nervous system. Rare variants in the TREM2 gene increase the risk for late-onset Alzheimer's disease (AD). Soluble TREM2 (sTREM2) resulting from shedding of the TREM2 ectodomain can be detected in the cerebrospinal fluid (CSF) and is a surrogate measure of TREM2-mediated microglia function. CSF sTREM2 has been previously reported to increase at different clinical stages of AD, however, alterations in relation to Amyloid -peptide (A) deposition or additional pathological processes in the amyloid cascade (such as tau pathology or neurodegeneration) remain unclear. In the current cross-sectional study, we employed the biomarker-based classification framework recently proposed by the NIA-AA consensus guidelines, in combination with clinical staging, in order to examine the CSF sTREM2 alterations at early asymptomatic and symptomatic stages of AD.MethodsA cross-sectional study of 1027 participants of the Alzheimer's Disease Imaging Initiative (ADNI) cohort, including 43 subjects carrying TREM2 rare genetic variants, was conducted to measure CSF sTREM2 using a previously validated enzyme-linked immunosorbent assay (ELISA). ADNI participants were classified following the A/T/N framework, which we implemented based on the CSF levels of A(1-42) (A), phosphorylated tau (T) and total tau as a marker of neurodegeneration (N), at different clinical stages defined by the clinical dementia rating (CDR) score.ResultsCSF sTREM2 differed between TREM2 variants, whereas the p.R47H variant had higher CSF sTREM2, p.L211P had lower CSF sTREM2 than non-carriers. We found that CSF sTREM2 increased in early symptomatic stages of late-onset AD but, unexpectedly, we observed decreased CSF sTREM2 levels at the earliest asymptomatic phase when only abnormal A pathology (A+) but no tau pathology or neurodegeneration (TN-), is present.ConclusionsA pathology (A) and tau pathology/neurodegeneration (TN) have differing associations with CSF sTREM2. While tau-related neurodegeneration is associated with an increase in CSF sTREM2, A pathology in the absence of downstream tau-related neurodegeneration is associated with a decrease in CSF sTREM2

Depletion and activation of microglia impact metabolic connectivity of the mouse brain

AimWe aimed to investigate the impact of microglial activity and microglial FDG uptake on metabolic connectivity, since microglial activation states determine FDG-PET alterations. Metabolic connectivity refers to a concept of interacting metabolic brain regions and receives growing interest in approaching complex cerebral metabolic networks in neurodegenerative diseases. However, underlying sources of metabolic connectivity remain to be elucidated.Materials and methodsWe analyzed metabolic networks measured by interregional correlation coefficients (ICCs) of FDG-PET scans in WT mice and in mice with mutations in progranulin (Grn) or triggering receptor expressed on myeloid cells 2 (Trem2) knockouts ((-/-)) as well as in double mutant Grn(-/-)/Trem2(-/-) mice. We selected those rodent models as they represent opposite microglial signatures with disease associated microglia in Grn(-/-) mice and microglia locked in a homeostatic state in Trem2(-/-) mice;however, both resulting in lower glucose uptake of the brain. The direct influence of microglia on metabolic networks was further determined by microglia depletion using a CSF1R inhibitor in WT mice at two different ages. Within maps of global mean scaled regional FDG uptake, 24 pre-established volumes of interest were applied and assigned to either cortical or subcortical networks. ICCs of all region pairs were calculated and z-transformed prior to group comparisons. FDG uptake of neurons, microglia, and astrocytes was determined in Grn(-/-) and WT mice via assessment of single cell tracer uptake (scRadiotracing).ResultsMicroglia depletion by CSF1R inhibition resulted in a strong decrease of metabolic connectivity defined by decrease of mean cortical ICCs in WT mice at both ages studied (6-7 m;p = 0.0148, 9-10 m;p = 0.0191), when compared to vehicle-treated age-matched WT mice. Grn(-/-), Trem2(-/-) and Grn(-/-)/Trem2(-/-) mice all displayed reduced FDG-PET signals when compared to WT mice. However, when analyzing metabolic networks, a distinct increase of ICCs was observed in Grn(-/-) mice when compared to WT mice in cortical (p < 0.0001) and hippocampal (p < 0.0001) networks. In contrast, Trem2(-/-) mice did not show significant alterations in metabolic connectivity when compared to WT. Furthermore, the increased metabolic connectivity in Grn(-/-) mice was completely suppressed in Grn(-/-)/Trem2(-/-) mice. Grn(-/-) mice exhibited a severe loss of neuronal FDG uptake (- 61%, p < 0.0001) which shifted allocation of cellular brain FDG uptake to microglia (42% in Grn(-/-) vs. 22% in WT).ConclusionsPresence, absence, and activation of microglia have a strong impact on metabolic connectivity of the mouse brain. Enhanced metabolic connectivity is associated with increased microglial FDG allocation

A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer's disease models

van Lengerich et al. developed a human TREM2 antibody with a transport vehicle (ATV) that improves brain exposure and biodistribution in mouse models. ATV:TREM2 promotes microglial energetic capacity and metabolism via mitochondrial pathways. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD