Sustained Trem2 stabilization accelerates microglia heterogeneity and Aß pathology in a mouse model of Alzheimer's disease.

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aß pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

Experimental evidence for temporal uncoupling of brain Aß deposition and neurodegenerative sequelae.

Brain Aß deposition is a key early event in the pathogenesis of Alzheimer´s disease (AD), but the long presymptomatic phase and poor correlation between Aß deposition and clinical symptoms remain puzzling. To elucidate the dependency of downstream pathologies on Aß, we analyzed the trajectories of cerebral Aß accumulation, Aß seeding activity, and neurofilament light chain (NfL) in the CSF (a biomarker of neurodegeneration) in Aß-precursor protein transgenic mice. We find that Aß deposition increases linearly until it reaches an apparent plateau at a late age, while Aß seeding activity increases more rapidly and reaches a plateau earlier, coinciding with the onset of a robust increase of CSF NfL. Short-term inhibition of Aß generation in amyloid-laden mice reduced Aß deposition and associated glial changes, but failed to reduce Aß seeding activity, and CSF NfL continued to increase although at a slower pace. When short-term or long-term inhibition of Aß generation was started at pre-amyloid stages, CSF NfL did not increase despite some Aß deposition, microglial activation, and robust brain Aß seeding activity. A dissociation of Aß load and CSF NfL trajectories was also found in familial AD, consistent with the view that Aß aggregation is not kinetically coupled to neurotoxicity. Rather, neurodegeneration starts when Aß seeding activity is saturated and before Aß deposition reaches critical (half-maximal) levels, a phenomenon reminiscent of the two pathogenic phases in prion disease.

Longitudinal PET Monitoring of Amyloidosis and Microglial Activation in a Second-Generation Amyloid-ß Mouse Model.

Nonphysiologic overexpression of amyloid-ß (Aß) precursor protein in common transgenic Aß mouse models of Alzheimer disease likely hampers their translational potential. The novel AppNL-G-F mouse incorporates a mutated knock-in, potentially presenting an improved model of Alzheimer disease for Aß-targeting treatment trials. We aimed to establish serial small-animal PET of amyloidosis and neuroinflammation in AppNL-G-F mice as a tool for therapy monitoring. Methods:AppNL-G-F mice (20 homozygous and 21 heterogeneous) and 12 age-matched wild-type mice were investigated longitudinally from 2.5 to 10 mo of age with 18F-florbetaben Aß PET and 18F-GE-180 18-kDa translocator protein (TSPO) PET. Voxelwise analysis of SUV ratio images was performed using statistical parametric mapping. All mice underwent a Morris water maze test of spatial learning after their final scan. Quantification of fibrillar Aß and activated microglia by immunohistochemistry and biochemistry served for validation of the PET results. Results: The periaqueductal gray emerged as a suitable pseudo reference tissue for both tracers. Homozygous AppNL-G-F mice had a rising SUV ratio in cortex and hippocampus for Aß (+9.1%, +3.8%) and TSPO (+19.8%, +14.2%) PET from 2.5 to 10 mo of age (all P < 0.05), whereas heterozygous AppNL-G-F mice did not show significant changes with age. Significant voxelwise clusters of Aß deposition and microglial activation in homozygous mice appeared at 5 mo of age. Immunohistochemical and biochemical findings correlated strongly with the PET data. Water maze escape latency was significantly elevated in homozygous AppNL-G-F mice compared with wild-type at 10 mo of age and was associated with high TSPO binding. Conclusion: Longitudinal PET in AppNL-G-F knock-in mice enables monitoring of amyloidogenesis and neuroinflammation in homozygous mice but is insensitive to minor changes in heterozygous animals. The combination of PET with behavioral tasks in AppNL-G-F treatment trials is poised to provide important insights in preclinical drug development.

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157.

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

Longitudinal noninvasive magnetic resonance imaging of brain microhemorrhages in BACE inhibitor-treated APP transgenic mice.

Currently, several immunotherapies and BACE (Beta Site APP Cleaving Enzyme) inhibitor approaches are being tested in the clinic for the treatment of Alzheimer's disease. A crucial mechanism-related safety concern is the exacerbation of microhemorrhages, which are already present in the majority of Alzheimer patients. To investigate potential safety liabilities of long-term BACE inhibitor therapy, we used aged amyloid precursor protein (APP) transgenic mice (APP23), which robustly develop cerebral amyloid angiopathy. T2*-weighted magnetic resonance imaging (MRI), a translational method applicable in preclinical and clinical studies, was used for the detection of microhemorrhages throughout the entire brain, with subsequent histological validation. Three-dimensional reconstruction based on in vivo MRI and serial Perls' stained sections demonstrated a one-to-one matching of the lesions thus allowing for their histopathological characterization. MRI detected small Perls' positive areas with a high spatial resolution. Our data demonstrate that volumetric assessment by noninvasive MRI is well suited to monitor cerebral microhemorrhages in vivo. Furthermore, 3 months treatment of aged APP23 with the potent BACE-inhibitor NB-360 did not exacerbate microhemorrhages in contrast to Aß-antibody ß1. These results substantiate the safe use of BACE inhibitors regarding microhemorrhages in long-term clinical studies for the treatment of Alzheimer's disease.

Pharmacological BACE1 and BACE2 inhibition induces hair depigmentation by inhibiting PMEL17 processing in mice.

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The ß-secretase (BACE1) is responsible for the generation of amyloid-ß (Aß) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer's disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-ß peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2(+/-) and bace2(-/-) mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.