Samira Parhizkar

Cerebral amyloid angiopathy (CAA), characterized by cerebrovascular amyloid deposition, is strongly associated with Alzheimer’s disease, ischemic injury, and lobar hemorrhages. APOE4 is the strongest known genetic risk factor for CAA, yet the cellular sources of APOE4 that drive pathogenic vascular amyloid deposition remain poorly defined. Here, we identify vascular-associated fibroblasts (VAFs) as a key source of APOE4 that mediates CAA and cerebrovascular dysfunction. In post-mortem brain tissue from APOE4/E4 patients with confirmed CAA, APOE colocalizes with COL6⁺ VAFs around Aβ-laden vessels. Therefore, to study the contribution of APOE4 derived from VAFs in CAA, independently of parenchymal sources, we used 5xFAD mice in which the endogenous mouse Apoe coding exons are replaced with floxed human APOE4 sequences. This enables selective suppression of APOE4 expression in VAFs upon tamoxifen induction of Col1a2-CreERT ⁺ . APOE4 reduction in VAFs markedly reduced CAA, with the strongest effects observed in leptomeningeal vessels. Additionally, Aβ40, which preferentially associates with vascular amyloid, was significantly reduced in the vascular fraction. VAF APOE4 depletion resulted in fewer small microbleeds and an approximately 50% reduction in parenchymal border macrophages within the leptomeningeal compartment, whereas astrocytic reactivity remained unchanged. Functional assessment using in vivo multi-parametric photoacoustic microscopy revealed improved arterial dilation and blood flow in amyloid-laden leptomeningeal arteries. Together, these findings establish VAF-derived APOE4 as a compartment-specific driver of CAA and cerebrovascular dysfunction. One Sentence Summary: Vascular-associated fibroblasts are a major source of pathogenic APOE4 that drives vascular dysfunction in cerebral amyloid angiopathy.

Sleep disturbances are associated with the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and primary tauopathies. Here we demonstrate that administration of the dual orexin receptor antagonist lemborexant in the P301S/E4 mouse model of tauopathy improves tau-associated impairments in sleep–wake behavior. It also protects against chronic reactive microgliosis and brain atrophy in male P301S/E4 mice by preventing abnormal phosphorylation of tau. These neuroprotective effects in males were not observed after administration of the nonorexinergic drug zolpidem that similarly promoted nonrapid eye movement sleep. Furthermore, both genetic ablation of orexin receptor 2 and lemborexant treatment reduced wakefulness and decreased seeding and spreading of phosphorylated tau in the brain of wild-type mice. These findings raise the therapeutic potential of targeting sleep by orexin receptor antagonism to prevent abnormal tau phosphorylation and limit tau-induced damage.

Background: Sleep disturbances are associated with the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD) and primary tauopathies. We have previously shown that APOE4, the strongest genetic risk factor for AD, directly influences the severity of key pathological hallmarks of neurodegeneration including tau deposition, microglial reactivity and brain atrophy. Sleep loss influences tau accumulation and microglial reactivity in both mice and humans, suggesting that sleep loss may contribute to neurodegeneration not only by influencing protein aggregation, but also through an immune mechanism. Therefore, we aimed to investigate whether promoting sleep as a therapeutic strategy could mitigate the damaging effects of chronic microglial reactivity that contribute to tau‐mediated neurodegeneration.Method: We used lemborexant, a dual orexin receptor antagonist that promotes sleep in both mice and humans. We orally gavaged P301S/APOE4 mice, a model of tauopathy with brain atrophy, and non‐tau depositing APOE4 knock‐in mice daily with 30mg/kg lemborexant or vehicle (n = 16‐20/genotype and treatment group) at one‐hour post‐dark onset. Mice were treated from 7.5 months (M), when tau‐mediated neuroinflammation is observed without overt neuronal loss in P301S/APOE4 mice, until 9.5M.Result: In P301S/APOE4 mice, lemborexant not only improved tau‐associated sleep loss, specifically non‐rapid eye movement sleep, but also dramatically reduced pathological tau deposition. Antagonizing orexin receptor signaling improved tau‐mediated neurodegeneration noted by a decrease in plasma neurofilament light chain levels, as well as brain atrophy compared to vehicle‐treated P301S/APOE4 controls. In support of these findings, lemborexant‐treated P301S/APOE4 mice displayed reduced microglial reactivity of disease‐associated microglia including immunostaining for CD68 and Clec7a compared to controls. Both astroglial and microglial APOE co‐localization were significantly reduced in lemborexant‐treated P301S/APOE4 mice, the latter of which is more commonly observed during elevated inflammatory and damaging conditions. Unbiased transcriptome profiling provided potential mechanistic insights into functional pathways influenced by lemborexant in P301S/APOE4 mice, including those regulating synaptic activity such as Slc17a7, Shank1, Shank2, which was further accompanied by reduced pre‐ and post‐synaptic loss.Conclusion: Our study provides novel therapeutic evidence that antagonizing the orexin signaling pathway using lemborexant is neuroprotective by restoring sleep deficits as well as limiting tau‐mediated neuronal and synaptic damage, potentially by suppressing chronic neuroinflammation.

Alzheimer’s disease (AD) is the most common cause of dementia. The APOE-ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset AD. The APOE genotype modulates the effect of sleep disruption on AD risk, suggesting a possible link between apoE and sleep in AD pathogenesis, which is relatively unexplored. We hypothesized that apoE modifies Aβ deposition and Aβ plaque–associated tau seeding and spreading in the form of neuritic plaque–tau (NP-tau) pathology in response to chronic sleep deprivation (SD) in an apoE isoform–dependent fashion. To test this hypothesis, we used APPPS1 mice expressing human APOE-ε3 or -ε4 with or without AD-tau injection. We found that SD in APPPS1 mice significantly increased Aβ deposition and peri-plaque NP-tau pathology in the presence of APOE4 but not APOE3. SD in APPPS1 mice significantly decreased microglial clustering around plaques and aquaporin-4 (AQP4) polarization around blood vessels in the presence of APOE4 but not APOE3. We also found that sleep-deprived APPPS1:E4 mice injected with AD-tau had significantly altered sleep behaviors compared with APPPS1:E3 mice. These findings suggest that the APOE-ε4 genotype is a critical modifier in the development of AD pathology in response to SD.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer’s disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-β (Aβ) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aβ plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aβ plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aβ plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aβ pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aβ deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach.

Background: β‐amyloid PET (Aβ‐PET) is an important tool for quantification of amyloidosis in the brain of suspected Alzheimer’s disease (AD) patients and transgenic AD mouse models. Despite the excellent correlation of Aβ‐PET with gold standard immunohistochemical assessments, the relative contributions of fibrillar and non‐fibrillar Aβ components to the in vivo Aβ‐PET signal remain unclear. Thus, we obtained two murine cerebral amyloidosis models that present with distinct Aβ plaque compositions and performed regression analysis between immunohistochemistry and Aβ PET to determine the biochemical contributions to Aβ‐PET signal in vivo.Method: We investigated groups of App^NL‐G‐F and APPPS1 mice three, six and 12 months of age by longitudinal [¹⁸F]‐florbetaben Aβ‐PET and with immunohistochemical analysis of the fibrillar and total Aβ burdens. We then applied group level inter‐modality regression models using age and genotype matched sets of fibrillar/ non‐fibrillar Aβ data (predictors) and Aβ‐PET results (outcome) for both transgenic models. An independent group of double‐hit APPPS1 mice with dysfunctional microglia due to knock‐out of triggering receptor expression on myeloid cells 2 (Trem2^‐/‐) served for validation and evaluation of translational impact.Result: Neither fibrillar nor non‐fibrillar Aβ content alone sufficed to explain the Aβ‐PET findings in either transgenic AD model (Figure 1). A regression model compiling fibrillar and non‐fibrillar Aβ together with the estimate of individual heterogeneity and age at scanning could explain a 93% of variance of the Aβ‐PET signal (pin vivo Aβ‐PET signal in App^NL‐G‐F mice, and 25±12% in the APPPS1 mice. Corresponding results in groups of APPPS1/Trem2^‐/‐ and APPPS1/Trem2^+/+ mice validated the calculated regression factors and revealed that the altered fibrillarity due to Trem2 knockout impacts the Aβ‐PET signal (Figure 3).Conclusion: Taken together, the in vivo Aβ‐PET signal derives from the composite of fibrillar and non‐fibrillar Aβ plaque components. While fibrillar Aβ has inherently higher PET tracer binding, the greater abundance of non‐fibrillar Aβ plaque in AD model mice contributes importantly to the PET signal.

Progressive accumulation of Amyloid-β (Aβ) deposits in the brain is a characteristic neuropathological hallmark of Alzheimer’s disease (AD). During disease progression, extracellular Aβ plaques undergo specific changes in their composition by the sequential deposition of different modified Aβ species. Microglia are implicated in the restriction of amyloid deposits and play a major role in internalization and degradation of Aβ. Recent studies showed that rare variants of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are associated with an increased risk for AD. Post-translational modifications of Aβ could modulate the interaction with TREM2, and the uptake by microglia. Here, we demonstrate that genetic deletion of TREM2 or expression of a disease associated TREM2 variant in mice lead to differential accumulation of modified and non-modified Aβ species in extracellular plaques and intraneuronal deposits. Human brains with rare TREM2 AD risk variants also showed altered deposition of modified Aβ species in the different brain lesions as compared to cases with the common variant of TREM2. These findings indicate that TREM2 plays a critical role in the development and the composition of Aβ deposits, not only in extracellular plaques, but also intraneuronally, that both could contribute to the pathogenesis of AD.

Rare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid β peptides (Aβ). Aβ peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post‐translational modifications result in the production of several distinct Aβ variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post‐translational modifications of Aβ affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aβ, and that phosphorylation of Aβ increases this interaction. Phosphorylation of Aβ also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aβ variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aβ species in preclinical models of Alzheimer's disease.

In Alzheimer’s disease (AD) models, AD risk variants in the microglial-expressed TREM2 gene decrease Aβ plaque–associated microgliosis and increase neuritic dystrophy as well as plaque-associated seeding and spreading of tau aggregates. Whether this Aβ-enhanced tau seeding/spreading is due to loss of microglial function or a toxic gain of function in TREM2-deficient microglia is unclear. Depletion of microglia in mice with established brain amyloid has no effect on amyloid but results in less spine and neuronal loss. Microglial repopulation in aged mice improved cognitive and neuronal deficits. In the context of AD pathology, we asked whether microglial removal and repopulation decreased Aβ-driven tau seeding and spreading. We show that both TREM2KO and microglial ablation dramatically enhance tau seeding and spreading around plaques. Interestingly, although repopulated microglia clustered around plaques, they had a reduction in disease-associated microglia (DAM) gene expression and elevated tau seeding/spreading. Together, these data suggest that TREM2-dependent activation of the DAM phenotype is essential in delaying Aβ-induced pathological tau propagation.

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.