Background: Genetic mutations underlying familial Alzheimer’s disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. Methods: We engineered a novel App knock-in mouse model (App^SAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-β pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous App^SAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aβ content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. Results: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aβ content. The App^SAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. Discussion: Our findings demonstrate that fibrillar Aβ in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and loss-of-function coding variants are major risk factors for late onset Alzheimer’s disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling in microglia, we generated a TREM2 reporter mouse. In APP transgenic animals, bulk RNA-sequencing of isolated microglia sorted based on reporter expression highlighted TREM2 level-related changes in major immunometabolic pathways, and enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. Metabolic and lipidomic profiling of sorted microglia showed that, independent of Aβ pathology, TREM2 expression correlated with signatures consistent with increased cellular redox, energetics, and cholesterol homoeostasis. In accordance, metabolic activity correlated with phagocytic capacity. Finally, we performed chronic treatment with a TREM2 agonist antibody and identified a window of TREM2 expression where microglia are most responsive, thereby informing clinical applications of TREM2 agonists.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD- GRN ). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD- GRN , namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD- GRN –associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)–derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD- GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD- GRN and potentially other CNS disorders.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1 gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration. Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1 in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we report severe alterations in microglial lipidomic profiles, including the enrichment of bis(monoacylglycero)phosphate, increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hyperactivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a pathological cascade resembling NPC-like phenotypes, including a shortened life span, motor impairments, astrogliosis, neuroaxonal pathology, and increased neurofilament light chain (NF-L), a neuronal injury biomarker. As observed in the mouse model, patients with NPC showed increased NF-L in the blood and microglial hyperactivity, as visualized by TSPO-PET imaging. Reduced TSPO expression in blood-derived macrophages of patients with NPC was measured after N -acetyl- l -leucine treatment, which has been recently shown to have beneficial effects in patients with NPC, suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further investigated for therapeutic targeting and disease monitoring.

Soda consumption in adolescents has been linked to poorer metabolic outcomes. We tested whether replacing soda with reduced fat milk would improve features of atherogenic dyslipidemia and other cardiometabolic risk factors. Thirty overweight and obese adolescent boys who were habitual consumers of sugar-sweetened beverages were randomly assigned to consume 24 oz/day of sugar-sweetened soda or an energy equivalent of reduced fat (2%) milk for 3 weeks with crossover to the alternate beverage after a ≥ 2 weeks washout. Plasma lipids and lipoproteins and other laboratory measures were assessed after each beverage period. Lipid and lipoprotein measurements, C-reactive protein, and serum transaminases did not differ significantly between the soda and milk phases of the study. Systolic blood pressure z-score and uric acid concentration were significantly lower after consuming milk compared to soda. Milk consumption also significantly decreased plasma glucosyl ceramide (d18:1/C16:0) and lactosylceramides (d18:1/C16:0 and d18:1/C18:0). While no effects of replacing soda with milk on lipid and lipoprotein measurements were observed in these normolipidemic weight-stable adolescent boys, decreases in systolic blood pressure, uric acid, and glycosphingolipids suggest that an overall favorable effect on cardiometabolic risk can be achieved following a short-term dietary intervention.

The Alzheimer’s disease (AD) genetic landscape identified microglia as a key disease-modifying cell type. Paired immunoglobulin-like type 2 receptor alpha (PILRA) is an immunoreceptor tyrosine–based inhibitory motif domain–containing inhibitory receptor, expressed by myeloid cells such as microglia. The known protective PILRA G78R gene variant reduces AD risk in apolipoprotein E4 ( APOE4 ) carriers and is enriched in a cohort of healthy centenarians. However, mechanisms underlying protective effects in microglia are undefined. Here, we identified biological functions of PILRA in human induced pluripotent stem cell–derived microglia (iMG) and chimeric AD mice. PILRA knockout (KO) in iMG rescued ApoE4-mediated immunometabolic deficits and prevented lipotoxicity through increased lipid storage, improved mitochondrial bioenergetics, and antioxidant activity. PILRA KO also enhanced microglial chemotaxis and attenuated inflammation. With pharmacological inhibitor studies, we showed that peroxisome proliferator–activated receptor and signal transducer and activator of transcription 1/3 mediated PILRA -dependent microglial functions. AD mice transplanted with human PILRA KO microglia exhibited reduced amyloid pathology and rescued synaptic markers. A high-affinity ligand blocking PILRA antibody phenocopied PILRA KO iMG. These findings suggest that PILRA is a pharmacologically tractable therapeutic target for AD.