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Abstract Microglial activation occurs early in Alzheimer's disease (AD) and previous studies reported both detrimental and protective effects of microglia on AD progression. Here, we used CSF sTREM2 to investigate disease stage‐dependent drivers of microglial activation and to determine downstream consequences on AD progression. We included 402 patients with measures of earliest beta‐amyloid (CSF Aβ1‐42) and late‐stage fibrillary Aβ pathology (amyloid‐PET centiloid), as well as sTREM2, p‐tau181, and FDG‐PET. To determine disease stage, we stratified participants into early Aβ‐accumulators (Aβ CSF+/PET−; n = 70) or late Aβ‐accumulators (Aβ CSF+/PET+; n = 201) plus 131 controls. In early Aβ‐accumulators, higher centiloid was associated with cross‐sectional/longitudinal sTREM2 and p‐tau181 increases. Further, higher sTREM2 mediated the association between centiloid and cross‐sectional/longitudinal p‐tau181 increases and higher sTREM2 was associated with FDG‐PET hypermetabolism. In late Aβ‐accumulators, we found no association between centiloid and sTREM2 but a cross‐sectional association between higher sTREM2, higher p‐tau181 and glucose hypometabolism. Our findings suggest that a TREM2‐related microglial response follows earliest Aβ fibrillization, manifests in inflammatory glucose hypermetabolism and may facilitate subsequent p‐tau181 increases in earliest AD.

Abstract Microglia activation is the brain's major immune response to amyloid plaques in Alzheimer's disease (AD). Both cerebrospinal fluid (CSF) levels of soluble TREM2 (sTREM2), a biomarker of microglia activation, and microglia PET are increased in AD; however, whether an increase in these biomarkers is associated with reduced amyloid‐beta (Aβ) accumulation remains unclear. To address this question, we pursued a two‐pronged translational approach. Firstly, in non‐demented and demented individuals, we tested CSF sTREM2 at baseline to predict (i) amyloid PET changes over ∼2 years and (ii) tau PET cross‐sectionally assessed in a subset of patients. We found higher CSF sTREM2 associated with attenuated amyloid PET increase and lower tau PET. Secondly, in the AppNL‐G-F mouse model of amyloidosis, we studied baseline 18F‐GE180 microglia PET and longitudinal amyloid PET to test the microglia vs. Aβ association, without any confounding co‐pathologies often present in AD patients. Higher microglia PET at age 5 months was associated with a slower amyloid PET increase between ages 5‐to‐10 months. In conclusion, higher microglia activation as determined by CSF sTREM2 or microglia PET shows protective effects on subsequent amyloid accumulation.

Abstract Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease‐associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full‐length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α‐secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α‐secretase‐mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho‐SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid β‐peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9‐bound. Moreover, in a mouse model for Alzheimer's disease‐related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease‐associated state.

Abstract The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non‐canonical translation of the expanded repeat results in abundant poly‐GA inclusion pathology throughout the CNS. (GA)149‐CFP expression in mice triggers motor deficits and neuroinflammation. Since poly‐GA is transmitted between cells, we investigated the therapeutic potential of anti‐GA antibodies by vaccinating (GA)149‐CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin‐(GA)10 conjugates and pre‐aggregated carrier‐free (GA)15. Only ovalbumin‐(GA)10 immunization induced a strong anti‐GA response. The resulting antisera detected poly‐GA aggregates in cell culture and patient tissue. Ovalbumin‐(GA)10 immunization largely rescued the motor function in (GA)149‐CFP transgenic mice and reduced poly‐GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin‐(GA)10‐immunized poly‐GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP‐43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.

Abstract Microglia adopt numerous fates with homeostatic microglia (HM) and a microglial neurodegenerative phenotype (MGnD) representing two opposite ends. A number of variants in genes selectively expressed in microglia are associated with an increased risk for neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Among these genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). Both cause neurodegeneration by mechanisms involving loss of function. We have now isolated microglia from Grn−/− mice and compared their transcriptomes to those of Trem2−/−mice. Surprisingly, while loss of Trem2 enhances the expression of genes associated with a homeostatic state, microglia derived from Grn−/− mice showed a reciprocal activation of the MGnD molecular signature and suppression of gene characteristic for HM. The opposite mRNA expression profiles are associated with divergent functional phenotypes. Although loss of TREM2 and progranulin resulted in opposite activation states and functional phenotypes of microglia, FDG (fluoro‐2‐deoxy‐d‐glucose)‐μPET of brain revealed reduced glucose metabolism in both conditions, suggesting that opposite microglial phenotypes result in similar wide spread brain dysfunction.

Abstract Progranulin (PGRN) is predominantly expressed by microglia in the brain, and genetic and experimental evidence suggests a critical role in Alzheimer's disease (AD). We asked whether PGRN expression is changed in a disease severity‐specific manner in AD. We measured PGRN in cerebrospinal fluid (CSF) in two of the best‐characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). In carriers of AD causing dominant mutations, cross‐sectionally assessed CSF PGRN increased over the course of the disease and significantly differed from non‐carriers 10 years before the expected symptom onset. In late‐onset AD, higher CSF PGRN was associated with more advanced disease stages and cognitive impairment. Higher CSF PGRN was associated with higher CSF soluble TREM2 (triggering receptor expressed on myeloid cells 2) only when there was underlying pathology, but not in controls. In conclusion, we demonstrate that, although CSF PGRN is not a diagnostic biomarker for AD, it may together with sTREM2 reflect microglial activation during the disease.

Abstract Sequence variations occurring in the gene encoding the triggering receptor expressed on myeloid cells 2 (TREM2) support an essential function of microglia and innate immunity in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. TREM2 matures within the secretory pathway, and its ectodomain is shed on the plasma membrane. Missense mutations in the immunoglobulin (Ig)‐like domain such as p.T66M and p.Y38C retain TREM2 within the endoplasmic reticulum and reduce shedding as well as TREM2‐dependent phagocytosis. Using mass spectrometry, we have now determined the cleavage site of TREM2. TREM2 is shed by proteases of the ADAM (a disintegrin and metalloproteinase domain containing protein) family C‐terminal to histidine 157, a position where an AD‐associated coding variant has been discovered (p.H157Y) in the Han Chinese population. Opposite to the characterized mutations within the Ig‐like domain, such as p.T66M and p.Y38C, the p.H157Y variant within the stalk region leads to enhanced shedding of TREM2. Elevated ectodomain shedding reduces cell surface full‐length TREM2 and lowers TREM2‐dependent phagocytosis. Therefore, two seemingly opposite cellular effects of TREM2 variants, namely reduced versus enhanced shedding, result in similar phenotypic outcomes by reducing cell surface TREM2.

Abstract Cell‐to‐cell transmission of protein aggregates is an emerging theme in neurodegenerative disease. Here, we analyze the dipeptide repeat (DPR) proteins that form neuronal inclusions in patients with hexanucleotide repeat expansion C9orf72, the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Sense and antisense transcripts of the (G4C2)n repeat are translated by repeat‐associated non‐ATG (RAN) translation in all reading frames into five aggregating DPR proteins. We show that the hydrophobic DPR proteins poly‐GA, poly‐GP, and poly‐PA are transmitted between cells using co‐culture assays and cell extracts. Moreover, uptake or expression of poly‐GA induces nuclear RNA foci in (G4C2)80‐expressing cells and patient fibroblasts, suggesting an unexpected positive feedback loop. Exposure to recombinant poly‐GA and cerebellar extracts of C9orf72 patients increases repeat RNA levels and seeds aggregation of all DPR proteins in receiver cells expressing (G4C2)80. Treatment with anti‐GA antibodies inhibits intracellular poly‐GA aggregation and blocks the seeding activity of C9orf72 brain extracts. Poly‐GA‐directed immunotherapy may thus reduce DPR aggregation and disease progression in C9orf72 ALS/FTD.

Abstract Immunotherapeutic approaches are currently the most advanced treatments for Alzheimer's disease (AD). Antibodies against amyloid β‐peptide (Aβ) bind to amyloid plaques and induce their clearance by microglia via Fc receptor‐mediated phagocytosis. Dysfunctions of microglia may play a pivotal role in AD pathogenesis and could result in reduced efficacy of antibody‐mediated Aβ clearance. Recently, heterozygous mutations in the triggering receptor expressed on myeloid cells 2 (TREM2), a microglial gene involved in phagocytosis, were genetically linked to late onset AD. Loss of TREM2 reduces the ability of microglia to engulf Aβ. We have now investigated whether loss of TREM2 affects the efficacy of immunotherapeutic approaches. We show that anti‐Aβ antibodies stimulate Aβ uptake and amyloid plaque clearance in a dose‐dependent manner in the presence or absence of TREM2. However, TREM2‐deficient N9 microglial cell lines, macrophages as well as primary microglia showed significantly reduced uptake of antibody‐bound Aβ and as a consequence reduced clearance of amyloid plaques. Titration experiments revealed that reduced efficacy of amyloid plaque clearance by Trem2 knockout cells can be compensated by elevating the concentration of therapeutic antibodies.

Abstract TREM2 is an innate immune receptor expressed on the surface of microglia. Loss‐of‐function mutations of TREM2 are associated with increased risk of Alzheimer's disease (AD). TREM2 is a type‐1 protein with an ectodomain that is proteolytically cleaved and released into the extracellular space as a soluble variant (sTREM2), which can be measured in the cerebrospinal fluid (CSF). In this cross‐sectional multicenter study, we investigated whether CSF levels of sTREM2 are changed during the clinical course of AD, and in cognitively normal individuals with suspected non‐AD pathology (SNAP). CSF sTREM2 levels were higher in mild cognitive impairment due to AD than in all other AD groups and controls. SNAP individuals also had significantly increased CSF sTREM2 compared to controls. Moreover, increased CSF sTREM2 levels were associated with higher CSF total tau and phospho‐tau181P, which are markers of neuronal degeneration and tau pathology. Our data demonstrate that CSF sTREM2 levels are increased in the early symptomatic phase of AD, probably reflecting a corresponding change of the microglia activation status in response to neuronal degeneration.

Abstract As stated by the prevailing amyloid cascade hypothesis, Alzheimer's disease (AD) is caused by the aggregation and cerebral deposition of long amyloid‐β peptide (Aβ) species, which are released from a C‐terminal amyloid precursor protein fragment by γ‐secretase. Mutations in its catalytic subunit presenilin‐1 (PS1) increase the Aβ42 to Aβ40 ratio and are the major cause of familial AD (FAD). An opposing hypothesis states that loss of essential presenilin functions underlies the disease. A major argument for this hypothesis is the observation that the nearly inactive PS1 L435F mutant, paradoxically, causes FAD. We now show that the very little Aβ generated by PS1 L435F consists primarily of Aβ43, a highly amyloidogenic species which was overlooked in previous studies of this mutant. We further demonstrate that the generation of Aβ43 is not due to a trans‐dominant effect of this mutant on WT presenilin. Furthermore, we found Aβ43‐containing plaques in brains of patients with this mutation. The aberrant generation of Aβ43 by this particular mutant provides a direct objection against the presenilin hypothesis.