Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging.

Microglia, the brain's resident macrophages, maintain brain homeostasis and respond to injury and infection. During aging they undergo functional changes, but the underlying mechanisms and their contributions to neuroprotection versus neurodegeneration are unclear. Previous studies suggested that microglia are sex dimorphic, so we compared microglial aging in mice of both sexes. RNA-sequencing of hippocampal microglia revealed more aging-associated changes in female microglia than male microglia, and more sex differences in old microglia than young microglia. Pathway analyses and subsequent validation assays revealed a stronger AKT-mTOR-HIF1α-driven shift to glycolysis among old female microglia and indicated that C3a production and detection was elevated in old microglia, especially in females. Recombinant C3a induced AKT-mTOR-HIF1α signaling and increased the glycolytic and phagocytic activity of young microglia. Single cell analyses attributed the aging-associated sex dimorphism to more abundant disease-associated microglia (DAM) in old female mice than old male mice, and evaluation of an Alzheimer's Disease mouse model revealed that the metabolic and complement changes are also apparent in the context of neurodegenerative disease and are strongest in the neuroprotective DAM2 subset. Collectively, our data implicate autocrine C3a-C3aR signaling in metabolic reprogramming of microglia to neuroprotective DAM during aging, especially in females, and also in Alzheimer's Disease.

Production of MHCII-expressing classical monocytes increases during aging in mice and humans.

Aging is associated with increased monocyte production and altered monocyte function. Classical monocytes are heterogenous and a shift in their subset composition may underlie some of their apparent functional changes during aging. We have previously shown that mouse granulocyte-monocyte progenitors (GMPs) produce "neutrophil-like" monocytes (NeuMo), whereas monocyte-dendritic cell progenitors (MDPs) produce monocyte-derived dendritic cell (moDC)-producing monocytes (DCMo). Here, we demonstrate that classical monocytes from the bone marrow of old male and female mice have higher expression of DCMo signature genes (H2-Aa, H2-Ab1, H2-Eb1, Cd74), and that more classical monocytes express MHCII and CD74 protein. Moreover, we show that bone marrow MDPs and classical monocytes from old mice yield more moDC. We also demonstrate higher expression of Aw112010 in old monocytes and that Aw112010 lncRNA activity regulates MHCII induction in macrophages, which suggests that elevated Aw112010 levels may underlie increased MHCII expression during monocyte aging. Finally, we show that classical monocyte expression of MHCII is also elevated during healthy aging in humans. Thus, aging-associated changes in monocyte production may underlie altered monocyte function and have implications for aging-associated disorders.

SGIP1α, but Not SGIP1, is an Ortholog of FCHo Proteins and Functions as an Endocytic Regulator.

Src homology 3-domain growth factor receptor-bound 2-like interacting protein 1 (SGIP1), originally known as a regulator of energy homeostasis, was later found to be an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins and to function during endocytosis. SGIP1α is a longer splicing variant in mouse brains that contains additional regions in the membrane phospholipid-binding domain (MP) and C-terminal region, but functional consequences with or without additional regions between SGIP1 and SGIP1α remain elusive. Moreover, many previous studies have either inadvertently used SGIP1 instead of SGIP1α or used the different isoforms with or without additional regions indiscriminately, resulting in further confusion. Here, we report that the additional region in the MP is essential for SGIP1α to deform membrane into tubules and for homo-oligomerization, and SGIP1, which lacks this region, fails to perform these functions. Moreover, only SGIP1α rescued endocytic defects caused by FCHo knock-down. Thus, our results indicate that SGIP1α, but not SGIP1, is the functional ortholog of FCHos, and SGIP1 and SGIP1α are not functionally redundant. These findings suggest that caution should be taken in interpreting the role of SGIP1 in endocytosis.

A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease.

Reactive microglia are a major pathological feature of Alzheimer's disease (AD). However, the exact role of microglia in AD pathogenesis is still unclear. Here, using metabolic profiling, we found that exposure to amyloid-ß triggers acute microglial inflammation accompanied by metabolic reprogramming from oxidative phosphorylation to glycolysis. It was dependent on the mTOR-HIF-1α pathway. However, once activated, microglia reached a chronic tolerant phase as a result of broad defects in energy metabolisms and subsequently diminished immune responses, including cytokine secretion and phagocytosis. Using genome-wide RNA sequencing and multiphoton microscopy techniques, we further identified metabolically defective microglia in 5XFAD mice, an AD mouse model. Finally, we showed that metabolic boosting with recombinant interferon-γ treatment reversed the defective glycolytic metabolism and inflammatory functions of microglia, thereby mitigating the AD pathology of 5XFAD mice. Collectively, metabolic reprogramming is crucial for microglial functions in AD, and modulating metabolism might be a new therapeutic strategy for AD.

Autophagy-Mediated Secretory Pathway is Responsible for Both Normal and Pathological Tau in Neurons.

Increased levels of total tau (t-tau) and hyperphosphorylated tau (p-tau) proteins in the cerebrospinal fluid of Alzheimer's disease (AD) patients are well documented and strongly correlate with AD pathology. Recent studies have further shown that human tau can be released into the extracellular space and transferred to nascent neurons. However, because the tau protein has no signal peptide identity, the mechanisms underlying its secretion remain poorly understood. In the present study, we confirmed that tau protein secretion was promoted by autophagy inducers and downregulated by beclin1 knockdown or autophagy inhibitors derived from human wild type tau (wt-tau)-overexpressing SH-SY5Y cells. Moreover, both t-tau and p-tau secretion were increased by autophagy activation. Furthermore, we identified that six isoforms of tau protein are secreted in an autophagy-dependent manner. These results indicate that both normal and pathological tau are secreted via an autophagy-mediated secretory pathway in neurons. Understanding this new pathway for tau secretion may provide critical future insights into tau pathologies, such as AD.

Thrombospondin-1 protects against Aß-induced mitochondrial fragmentation and dysfunction in hippocampal cells.

Alzheimer's disease (AD) is often characterized by the impairment of mitochondrial function caused by excessive mitochondrial fragmentation. Thrombospondin-1 (TSP-1), which is primarily secreted from astrocytes in the central nervous system (CNS), has been suggested to play a role in synaptogenesis, spine morphology, and synaptic density of neurons. In this study, we investigate the protective role of TSP-1 in the recovery of mitochondrial morphology and function in amyloid ß (Aß)-treated mouse hippocampal neuroblastoma cells (HT22). We observe that TSP-1 inhibits Aß-induced mitochondrial fission by maintaining phosphorylated-Drp1 (p-Drp1) levels, which results in reduced Drp1 translocation to the mitochondria. By using gabapentin, a drug that antagonizes the interaction between TSP-1 and its neuronal receptor α2δ1, we observe that α2δ1 acts as one of the target receptors for TSP-1, and blocks the reduction of the p-Drp1 to Drp1 ratio, in the presence of Aß. Taken together, TSP-1 appears to contribute to maintaining the balance in mitochondrial dynamics and mitochondrial functions, which is crucial for neuronal cell viability. These data suggest that TSP-1 may be a potential therapeutic target for AD.

Chemically treated plasma Aß is a potential blood-based biomarker for screening cerebral amyloid deposition.

BACKGROUND: Plasma ß-amyloid (Aß) is a potential candidate for an Alzheimer's disease (AD) biomarker because blood is an easily accessible bio-fluid, which can be collected routinely, and Aß is one of the major hallmarks of AD pathogenesis in the brain. However, the association between plasma Aß levels and AD diagnosis is still unclear due to the instability and inaccurate measurements of plasma Aß levels in the blood of patients with AD. If a consistent value of plasma Aß from the blood can be obtained, this might help determine whether plasma Aß is a potential biomarker for AD diagnosis. METHODS: We predicted the brain amyloid deposit by measuring the plasma Aß levels. This cross-sectional study included 353 participants (215 cognitively normal, 79 with mild cognitive impairment, and 59 with AD dementia) who underwent Pittsburgh-compound B positron emission tomography (PiB-PET) scans. We treated a mixture of protease inhibitors and phosphatase inhibitors (MPP) and detected plasma Aß42 and Aß40 (MPP-Aß42 and MPP-Aß40) in a stable manner using xMAP technology. RESULTS: MPP-Aß40 and MPP-Aß42/40 (MPP-Aßs) were significantly different between subjects with positive amyloid deposition (PiB+) and those with negative amyloid deposition (PiB-) (P < 0.0001). Furthermore, MPP-Aß40 (P < 0.0001, r = 0.23) and MPP-Aß42/40 ratio (P < 0.0001, r = -0.23) showed significant correlation with global PiB deposition (standardized uptake value ratio). In addition, our integrated multivariable (MPP-Aß42/40, gender, age, and apolipoprotein E genotypes) logistic regression model proposes a new standard for the prediction of cerebral amyloid deposition. CONCLUSIONS: MPP-Aß might be one of the potential blood biomarkers for the prediction of PiB-PET positivity in the brain.

Microglia contributes to plaque growth by cell death due to uptake of amyloid ß in the brain of Alzheimer's disease mouse model.

Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid ß (Aß) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain-resident macrophages, are also found surrounding Aß plaques. The study of the brain of AD mouse models revealed that Aß plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aß plaque formation, growth and the mechanisms by which microglia contribute to Aß plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aß plaque formation and their growth in the brain of 5XFAD mice, the Aß-overexpressing AD transgenic mouse model, and performed serial intravital two-photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia-specific GFP-expressing CX3CR1GFP/GFP mice. We found that activated microglia surrounding Aß plaques take up Aß, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aß into the extracellular space, which contributes to Aß plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aß plaques by causing microglial cell death in the brain. GLIA 2016;64:2274-2290.

PiB-PET Imaging-Based Serum Proteome Profiles Predict Mild Cognitive Impairment and Alzheimer's Disease.

Development of a simple, non-invasive early diagnosis platform of Alzheimer's disease (AD) using blood is urgently required. Recently, PiB-PET imaging has been shown to be powerful to quantify amyloid-ß plaque loads leading to pathophysiological alterations in AD brains. Thus, there has been a need for serum biomarkers reflecting PiB-PET imaging data as an early diagnosis platform of AD. Here, using LC-MS/MS analysis coupled with isobaric tagging, we performed comprehensive proteome profiling of serum samples from cognitively normal controls, mild cognitive impairment (MCI), and AD patients, who were selected using PiB-PET imaging. Comparative analysis of the proteomes revealed 79 and 72 differentially expressed proteins in MCI and AD, respectively, compared to controls. Integrated analysis of these proteins with genomic and proteomic data of AD brain tissues, together with network analysis, identified three biomarker candidates representing the altered proteolysis-related process in MCI or AD: proprotein convertase subtilisin/kexin type 9 (PCSK9), coagulation factor XIII, A1 polypeptide (F13A1), and dermcidin (DCD). In independent serum samples of MCI and AD, we confirmed the elevation of the candidates using western blotting and ELISA. Our results suggest that these biomarker candidates can serve as a potential non-invasive early diagnosis platform reflecting PiB-PET imaging for MCI and AD.

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease.

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aß), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aß. Aß increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aß. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aß pathology.

Close Correlation of Monoamine Oxidase Activity with Progress of Alzheimer's Disease in Mice, Observed by in Vivo Two-Photon Imaging.

Monoamine oxidases (MAOs) play an important role in Alzheimer's disease (AD) pathology. We report in vivo comonitoring of MAO activity and amyloid-ß (Aß) plaques dependent on the aging of live mice with AD, using a two-photon fluorescence probe. The probe under the catalytic action of MAO produces a dipolar fluorophore that senses Aß plaques, a general AD biomarker, enabling us to comonitor the enzyme activity and the progress of AD indicated by Aß plaques. The results show that the progress of AD has a close correlation with MAO activity, which can be categorized into three stages: slow initiation stage up to three months, an aggressive stage, and a saturation stage from nine months. Histological analysis also reveals elevation of MAO activity around Aß plaques in aged mice. The close correlation between the MAO activity and AD progress observed by in vivo monitoring for the first time prompts us to investigate the enzyme as a potential biomarker of AD.

Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway.

BACKGROUND: Insulin degrading enzyme (IDE) is a major protease of amyloid beta peptide (Aß), a prominent toxic protein in Alzheimer's disease (AD) pathogenesis. Previous studies suggested that statins promote IDE secretion; however, the underlying mechanism is unknown, as IDE has no signal sequence. RESULTS: In this study, we found that simvastatin (0.2 µM for 12 h) induced the degradation of extracellular Aß40, which depended on IDE secretion from primary astrocytes. In addition, simvastatin increased IDE secretion from astrocytes in a time- and dose-dependent manner. Moreover, simvastatin-mediated IDE secretion was mediated by an autophagy-based unconventional secretory pathway, and autophagic flux regulated simvastatin-mediated IDE secretion. Finally, simvastatin activated autophagy via the LKB1-AMPK-mTOR signaling pathway in astrocytes. CONCLUSIONS: These results demonstrate a novel pathway for statin-mediated IDE secretion from astrocytes. Modulation of this pathway could provide a potential therapeutic target for treatment of Aß pathology by enhancing extracellular clearance of Aß.