Nellie E. Kwang

Lysosomal storage disorders (LSDs) are a large disease class involving lysosomal dysfunction, often resulting in neurodegeneration. Sandhoff disease (SD) is an LSD caused by a deficiency in the β subunit of the β-hexosaminidase enzyme (Hexb). Although Hexb expression in the brain is specific to microglia, SD primarily affects neurons. To investigate how a microglial gene is involved in neuronal homeostasis, here we show that β-hexosaminidase is secreted by microglia and integrated into the lysosomal compartment of neurons. To assess therapeutic relevance, we treat the Hexb ^-/- SD mouse model with bone marrow transplant and colony stimulating factor 1 receptor inhibition, which broadly replaces Hexb ^-/- microglia with Hexb-sufficient cells. Microglial replacement reverses apoptotic gene signatures, improves behavior, restores β-hexosaminidase enzymatic activity and Hexb expression, prevents substrate buildup, and normalizes neuronal lysosomal phenotypes, underscoring the critical role of myeloid-derived β-hexosaminidase in maintaining neuronal health and establishing microglial replacement as a potential LSD therapy.

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair, their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells, derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interact with Aβ uptake. Single dCPEC transcriptomes over time correlate well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition, time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns are then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs, these findings suggest dynamic models of ChP support for the developing human brain.

The function of microglia during progression of Alzheimer's disease (AD) can be investigated using mouse models that enable genetic manipulation of microglial subpopulations in a temporal manner. We developed mouse lines that express either Cre recombinase (Cre) for constitutive targeting, or destabilized‐domain Cre recombinase (DD‐Cre) for inducible targeting from the Cst7 locus (Cst7 ^DD‐Cre) to specifically manipulate disease associated microglia (DAM) and crossed with Ai14 tdTomato cre‐reporter line mice. Cst7^Cre was found to target all brain resident myeloid cells, due to transient developmental expression of Cst7, but no expression was found in the inducible Cst7 ^DD‐Cre mice. Further crossing of this line with 5xFAD mice combined with dietary administration of trimethoprim to induce DD‐Cre activity produces long‐term labeling in DAM without evidence of leakiness, with tdTomato‐expression restricted to cells surrounding plaques. Using this model, we found that DAMs are a subset of plaque‐associated microglia (PAMs) and their transition to DAM increases with age and disease stage. Spatial transcriptomic analysis revealed that tdTomato+ cells show higher expression of disease and inflammatory genes compared to other microglial populations, including non‐labeled PAMs. These models allow either complete cre‐loxP targeting of all brain myeloid cells (Cst7^Cre), or inducible targeting of DAMs, without leakiness (Cst7 ^DD‐Cre).

Background: Apolipoprotein E ε4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer’s disease (LOAD). A recent case report identified a rare variant in APOE, APOE3-R136S (Christchurch), proposed to confer resistance to autosomal dominant Alzheimer’s Disease (AD). However, it remains unclear whether and how this variant exerts its protective effects. Methods: We introduced the R136S variant into mouse Apoe (ApoeCh) and investigated its effect on the development of AD-related pathology using the 5xFAD model of amyloidosis and the PS19 model of tauopathy. We used immunohistochemical and biochemical analysis along with single-cell spatial omics and bulk proteomics to explore the impact of the ApoeCh variant on AD pathological development and the brain’s response to plaques and tau. Results: In 5xFAD mice, ApoeCh enhances a Disease-Associated Microglia (DAM) phenotype in microglia surrounding plaques, and reduces plaque load, dystrophic neurites, and plasma neurofilament light chain. By contrast, in PS19 mice, ApoeCh suppresses the microglial and astrocytic responses to tau-laden neurons and does not reduce tau accumulation or phosphorylation, but partially rescues tau-induced synaptic and myelin loss. We compared how microglia responses differ between the two mouse models to elucidate the distinct DAM signatures induced by ApoeCh. We identified upregulation of antigen presentation-related genes in the DAM response in a PS19 compared to a 5xFAD background, suggesting a differential response to amyloid versus tau pathology that is modulated by the presence of ApoeCh. Bulk proteomics show upregulated mitochondrial protein abundance with ApoeCh in 5xFAD mice, but reductions in mitochondrial and translation associated proteins in PS19 mice. Conclusions: These findings highlight the ability of the ApoeCh variant to modulate microglial responses based on the type of pathology, enhancing DAM reactivity in amyloid models and dampening neuroinflammation to promote protection in tau models. This suggests that the Christchurch variant's protective effects likely involve multiple mechanisms, including changes in receptor binding and microglial programming. Graphical

Lysosomal storage disorders (LSDs) are a large disease class involving lysosomal dysfunction, often resulting in neurodegeneration. Sandhoff disease (SD) is an LSD caused by a deficiency in the β subunit of the β-hexosaminidase enzyme ( Hexb ). Although Hexb expression in the brain is specific to microglia, SD primarily affects neurons. To understand how a microglial gene is involved in maintaining neuronal homeostasis, we demonstrated that β-hexosaminidase is secreted by microglia and integrated into the neuronal lysosomal compartment. To assess therapeutic relevance, we treated SD mice with bone marrow transplant and colony stimulating factor 1 receptor inhibition, which broadly replaced Hexb ^-/- microglia with Hexb -sufficient cells. This intervention reversed apoptotic gene signatures, improved behavior, restored enzymatic activity and Hexb expression, ameliorated substrate accumulation, and normalized neuronal lysosomal phenotypes. These results underscore the critical role of myeloid-derived β- hexosaminidase in neuronal lysosomal function and establish microglial replacement as a potential LSD therapy.

Summary: The function of microglia during progression of Alzheimer’s disease (AD) can be investigated using mouse models that enable genetic manipulation of microglial subpopulations in a temporal manner. We developed a mouse strain that expresses destabilized-domain Cre recombinase (DD-Cre) from the Cst7 locus ( Cst7 ^DD-Cre ) and tested this in 5xFAD amyloidogenic, Ai14 tdTomato cre-reporter line mice. Dietary administration of trimethoprim to induce DD-Cre activity produces long-term labeling in disease associated microglia (DAM) without evidence of leakiness, with tdTomato-expression restricted to cells surrounding plaques. Using this model, we found that DAMs are a subset of plaque-associated microglia (PAMs) and their transition to DAM increases with age and disease stage. Spatial transcriptomic analysis revealed that tdTomato+ cells show higher expression of disease and inflammatory genes compared to other microglial populations, including non-labeled PAMs. This model should allow inducible cre-loxP targeting of DAMs, without leakiness. Highlights: We developed a new mouse strain which specifically enables recombination of loxP sites in disease associated microglia (DAMs) and can be used to manipulate DAM-gene expression. DAMs represent a subset of plaque associated microglia (PAMs), and DAM expression increases with disease progression. Spatial transcriptomic analyses reveal that DAMs have higher expression of disease and inflammatory genes compared to other PAMs.