Selective targeting and modulation of plaque associated microglia via systemic hydroxyl dendrimer administration in an Alzheimer's disease mouse model.

BACKGROUND: In Alzheimer's disease (AD), microglia surround extracellular plaques and mount a sustained inflammatory response, contributing to the pathogenesis of the disease. Identifying approaches to specifically target plaque-associated microglia (PAMs) without interfering in the homeostatic functions of non-plaque associated microglia would afford a powerful tool and potential therapeutic avenue. METHODS: Here, we demonstrated that a systemically administered nanomedicine, hydroxyl dendrimers (HDs), can cross the blood brain barrier and are preferentially taken up by PAMs in a mouse model of AD. As proof of principle, to demonstrate biological effects in PAM function, we treated the 5xFAD mouse model of amyloidosis for 4 weeks via systemic administration (ip, 2x weekly) of HDs conjugated to a colony stimulating factor-1 receptor (CSF1R) inhibitor (D-45113). RESULTS: Treatment resulted in significant reductions in amyloid-beta (Aß) and a stark reduction in the number of microglia and microglia-plaque association in the subiculum and somatosensory cortex, as well as a downregulation in microglial, inflammatory, and synaptic gene expression compared to vehicle treated 5xFAD mice. CONCLUSIONS: This study demonstrates that systemic administration of a dendranib may be utilized to target and modulate PAMs.

Effects of SPI1-mediated transcriptome remodeling on Alzheimer's disease-related phenotypes in mouse models of Aß amyloidosis.

SPI1 was recently reported as a genetic risk factor for Alzheimer's disease (AD) in large-scale genome-wide association studies. However, it is unknown whether SPI1 should be downregulated or increased to have therapeutic benefits. To investigate the effect of modulating SPI1 levels on AD pathogenesis, we performed extensive biochemical, histological, and transcriptomic analyses using both Spi1-knockdown and Spi1-overexpression mouse models. Here, we show that the knockdown of Spi1 expression significantly exacerbates insoluble amyloid-ß (Aß) levels, amyloid plaque deposition, and gliosis. Conversely, overexpression of Spi1 significantly ameliorates these phenotypes and dystrophic neurites. Further mechanistic studies using targeted and single-cell transcriptomics approaches demonstrate that altered Spi1 expression modulates several pathways, such as immune response pathways and complement system. Our data suggest that transcriptional reprogramming by targeting transcription factors, like Spi1, might hold promise as a therapeutic strategy. This approach could potentially expand the current landscape of druggable targets for AD.

Loss of Cholinergic and Monoaminergic Afferents in APPswe/PS1ΔE9 Transgenic Mouse Model of Cerebral Amyloidosis Preferentially Occurs Near Amyloid Plaques.

Alzheimer's disease (AD) is characterized by a loss of neurons in the cortex and subcortical regions. Previously, we showed that the progressive degeneration of subcortical monoaminergic (MAergic) neurons seen in human AD is recapitulated in the APPswe/PS1ΔE9 (APP/PS) transgenic mouse model. Because degeneration of cholinergic (Ach) neurons is also a prominent feature of AD, we examined the integrity of the Ach system in the APP/PS model. The overall density of Ach fibers is reduced in APP/PS1 mice at 12 and 18 months of age but not at 4 months of age. Analysis of basal forebrain Ach neurons shows no loss of Ach neurons in the APP/PS model. Thus, since MAergic systems show overt cell loss at 18 months of age, the Ach system is less vulnerable to neurodegeneration in the APP/PS1 model. We also examined whether the proximity to Aß deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aß deposits exhibit a more severe loss of afferents than the areas that are more distal to Aß deposits. Collectively, the results indicate that the APP/PS model recapitulates the degeneration of multiple subcortical neurotransmitter systems, including the Ach system. In addition, the results indicate that Aß deposits cause global as well as local toxicity to subcortical afferents.

Transcranial Magneto-Acoustic Stimulation Protects Synaptic Rehabilitation from Amyloid-Beta Plaques via Regulation of Microglial Functions.

Transcranial magneto-acoustic stimulation (TMAS), which is characterized by high spatiotemporal resolution and high penetrability, is a non-invasive neuromodulation technology based on the magnetic-acoustic coupling effect. To reveal the effects of TMAS treatment on amyloid-beta (Aß) plaque and synaptic plasticity in Alzheimer's disease, we conducted a comparative analysis of TMAS and transcranial ultrasound stimulation (TUS) based on acoustic effects in 5xFAD mice and BV2 microglia cells. We found that the TMAS-TUS treatment effectively reduced amyloid plaque loads and plaque-associated neurotoxicity. Additionally, TMAS-TUS treatment ameliorated impairments in long-term memory formation and long-term potentiation. Moreover, TMAS-TUS treatment stimulated microglial proliferation and migration while enhancing the phagocytosis and clearance of Aß. In 5xFAD mice with induced microglial exhaustion, TMAS-TUS treatment-mediated Aß plaque reduction, synaptic rehabilitation improvement, and the increase in phospho-AKT levels were diminished. Overall, our study highlights that stimulation of hippocampal microglia by TMAS treatment can induce anti-cognitive impairment effects via PI3K-AKT signaling, providing hope for the development of new strategies for an adjuvant therapy for Alzheimer's disease.

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-ß plaques in human brain tissue.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-ß (Aß) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aß plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aß plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aß plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aß plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aß plaque pathology in post-mortem human brain tissue.

L-DOPA regulates neuroinflammation and Aß pathology through NEP and ADAM17 in a mouse model of AD.

Dopamine plays important roles in cognitive function and inflammation and therefore is involved in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Drugs that increase or maintain dopamine levels in the brain could be a therapeutic strategy for AD. However, the effects of dopamine and its precursor levodopa (L-DOPA) on Aß/tau pathology in vivo and the underlying molecular mechanisms have not been studied in detail. Here, we investigated whether L-DOPA treatment alters neuroinflammation, Aß pathology, and tau phosphorylation in 5xFAD mice, a model of AD. We found that L-DOPA administration significantly reduced microgliosis and astrogliosis in 5xFAD mice. In addition, L-DOPA treatment significantly decreased Aß plaque number by upregulating NEP and ADAM17 levels in 5xFAD mice. However, L-DOPA-treated 5xFAD mice did not exhibit changes in tau hyperphosphorylation or tau kinase levels. These data suggest that L-DOPA alleviates neuroinflammatory responses and Aß pathology but not tau pathology in this mouse model of AD.

BIN1K358R suppresses glial response to plaques in mouse model of Alzheimer's disease.

INTRODUCTION: The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. METHODS: To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. RESULTS: The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1K358R variant. DISCUSSION: The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities. HIGHLIGHTS: BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.

Chimeric antigen receptor macrophages target and resorb amyloid plaques.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of ß amyloid (Aß) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aß-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting "reinforced CAR-Ms" have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aß in AD.

Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer's disease mice.

BACKGROUND: Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer's disease (AD) if these macromolecules can cross the blood-brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD). METHODS: Eight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics. RESULTS: We observed regional differences in proteins relevant to Aß, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice. From 64 target proteins studied using spatial proteomics, a comparison of the Aß-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aß and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (⁓ 33%). TfRMAb-TNFR significantly reduced mature Aß plaques and increased Aß-associated microglia around larger Aß deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aß plaque-associated microglial TREM2 in 3xTg-AD mice. CONCLUSION: Overall, despite the low visual Aß load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.

Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

The fluorescent ligand bTVBT2 reveals increased p-tau uptake by retinal microglia in Alzheimer's disease patients and AppNL-F/NL-F mice.

BACKGROUND: Amyloid beta (Aß) deposits and hyperphosphorylated tau (p-tau) accumulation have been identified in the retina of Alzheimer's disease (AD) patients and transgenic AD mice. Previous studies have shown that retinal microglia engulf Aß, but this property decreases in AD patients. Whether retinal microglia also take up p-tau and if this event is affected in AD is yet not described. In the current study, we use the p-tau-specific thiophene-based ligand bTVBT2 to investigate the relationship between disease progression and p-tau uptake by microglia in the retina of AD patients and AppNL-F/NL-F knock-in mice, an AD mouse model known to demonstrate extracellular Aß plaques and dystrophic neurites in the brain from 6 months of age. METHODS: Evaluation of bTVBT2 specificity and its presence within microglia was assessed by immunofluorescent staining of hippocampal sections and flat-mount retina samples from non-demented controls, AD patients, 3-, 9-, and 12-month-old AppNL-F/NL-F knock-in mice and 12- and 18-month-old wild type (WT) mice. We used ImageJ to analyze the amount of bTVBT2 inside Iba1-positive microglia. Co-localization between the ligand and p-tau variant Ser396/Ser404 (PHF-1), Aß, phosphorylated TAR DNA binding protein 43 (pTDP-43), and islet amyloid polypeptide (IAPP) in the brain and retina was analyzed using confocal imaging. RESULTS: Confocal imaging analysis showed that bTVBT2 binds to PHF-1- and AT8-positive aggregates inside retinal microglia, and not to Aß, pTDP-43, or IAPP. The density of bTVBT2-positive microglia was higher in cases with a high Aß load compared to those with a low Aß load. This density correlated with the neurofibrillary tangle load in the brain, but not with retinal levels of high molecular weight (aggregated) Aß40 or Aß42. Analysis of AppNL-F/NL-F knock-in mouse retina further showed that 50% of microglia in 3-month-old AppNL-F/NL-F knock-in mice contained bTVBT2. The percentage significantly increased in 9- and 12-month-old mice. CONCLUSION: Our study suggests that the microglial capability to uptake p-tau in the retina persists and intensifies with AD progression. These results also highlight bTVBT2 as a ligand of interest in future monitoring of retinal AD pathology.

Transgenic amyloid precursor protein mouse models of amyloidosis. Incomplete models for Alzheimer's disease but effective predictors of anti-amyloid therapies.

INTRODUCTION: Amyloid precursor protein (APP) transgenic mice are models of Alzheimer's disease (AD) amyloidosis, not all of AD. Diffuse, compacted, and vascular deposits in APP mice mimic those found in AD cases. METHODS: Most interventional studies in APP mice start treatment early in the process of amyloid deposition, consistent with a prevention treatment regimen. Most clinical trials treat patients with established amyloid deposits in a therapeutic treatment regimen. RESULTS: The first treatment to reduce amyloid and cognitive impairment in mice was immunotherapy. The APP mouse models not only predicted efficacy, but presaged the vascular leakage called ARIA. The recent immunotherapy clinical trials that removed amyloid and slowed cognitive decline confirms the utility of these early APP models when used in therapeutic designs. DISCUSSION: New mouse models of AD pathologies will add to the research armamentarium, but the early models have accurately predicted responses to amyloid therapies in humans.

Hyperspectral Raman Imaging for Automated Recognition of Human Renal Amyloid.

In the clinical setting, routine identification of the main types of tissue amyloid deposits, light-chain amyloid (AL) and serum amyloid A (AA), is based on histochemical staining; rarer types of amyloid require mass spectrometry analysis. Raman spectroscopic imaging is an analytical tool, which can be used to chemically map, and thus characterize, the molecular composition of fluid and solid tissue. In this proof-of-concept study, we tested the feasibility of applying Raman spectroscopy combined with artificial intelligence to detect and characterize amyloid deposits in unstained frozen tissue sections from kidney biopsies with pathologic diagnosis of AL and AA amyloidosis and control biopsies with no amyloidosis (NA). Raman hyperspectral images, mapped in a 2D grid-like fashion over the tissue sections, were obtained. Three machine learning-assisted analysis models of the hyperspectral images could accurately distinguish AL (types λ and κ), AA, and NA 93-100% of the time. Although very preliminary, these findings illustrate the potential of Raman spectroscopy as a technique to identify, and possibly, subtype renal amyloidosis.

T Lymphocytes in the Pathogenesis and Progression of Alzheimer's Disease: Pursuing Direct Neuropathological Evidence.

Multiple studies have proposed important roles of T cells in the pathogenesis of Alzheimer's disease. Given the successful application of immune-based therapy for cancer and a variety of diseases, T cell-modifying therapy becomes an attractive way to develop new therapies for Alzheimer's disease and perhaps neurodegenerative diseases in general. However, most of these studies address peripheral T cell responses, while direct pathological evidence documenting T cell infiltration relative to Alzheimer's disease pathological markers (i.e., amyloid plaque and neurofibrillary tangle) is sparse and at best, very preliminary in both human subjects and relevant animal models. Here, we concisely summarize the available pathological data that directly corresponds to T cell infiltration, critically analyze the current knowledge gaps, and thoughtfully propose several key recommendations for future research.

Inflammation and the pathological progression of Alzheimer's disease are associated with low circulating choline levels.

Deficiency of dietary choline, an essential nutrient, is observed worldwide, with ~ 90% of Americans being deficient. Previous work highlights a relationship between decreased choline intake and an increased risk for cognitive decline and Alzheimer's disease (AD). The associations between blood circulating choline and the pathological progression in both mild cognitive impairment (MCI) and AD remain unknown. Here, we examined these associations in a cohort of patients with MCI with presence of either sparse or high neuritic plaque density and Braak stage and a second cohort with either moderate AD (moderate to frequent neuritic plaques, Braak stage = IV) or severe AD (frequent neuritic plaques, Braak stage = VI), compared to age-matched controls. Metabolomic analysis was performed on serum from the AD cohort. We then assessed the effects of dietary choline deficiency (Ch-) in 3xTg-AD mice and choline supplementation (Ch+) in APP/PS1 mice, two rodent models of AD. The levels of circulating choline were reduced while pro-inflammatory cytokine TNFα was elevated in serum of both MCI sparse and high pathology cases. Reduced choline and elevated TNFα correlated with higher neuritic plaque density and Braak stage. In AD patients, we found reductions in choline, its derivative acetylcholine (ACh), and elevated TNFα. Choline and ACh levels were negatively correlated with neuritic plaque load, Braak stage, and TNFα, but positively correlated with MMSE, and brain weight. Metabolites L-Valine, 4-Hydroxyphenylpyruvic, Methylmalonic, and Ferulic acids were significantly associated with circuiting choline levels. In 3xTg-AD mice, the Ch- diet increased amyloid-ß levels and tau phosphorylation in cortical tissue, and TNFα in both blood and cortical tissue, paralleling the severe human-AD profile. Conversely, the Ch+ diet increased choline and ACh while reducing amyloid-ß and TNFα levels in brains of APP/PS1 mice. Collectively, low circulating choline is associated with AD-neuropathological progression, illustrating the importance of adequate dietary choline intake to offset disease.

Pathological Roles of INPP5D in Alzheimer's Disease.

Current hypothesis of Alzheimer's disease (AD) postulates that amyloid ß (Aß) deposition in the brain causes tau inclusion in neurons and leads to cognitive decline. The discovery of the genetic association between triggering receptor expressed on myeloid cells 2 (TREM2) with increased AD risk points to a causal link between microglia and AD pathogenesis, and revealed a crucial role of TREM2-dependent clustering of microglia around amyloid plaques that prevents Aß toxicity to facilitate tau deposition near the plaques. Here we review the physiological and pathological roles of another AD risk gene expressed in microglia, inositol polyphosphate-5-polyphosphatase D (INPP5D), which encodes a phosphoinositide phosphatase. Evidence suggests that its risk polymorphisms alter the expression level and/or function of INPP5D, while concomitantly affecting tau levels in cerebrospinal fluids. In ß-amyloidosis mice, INPP5D was upregulated upon Aß deposition and negatively regulated the microglial clustering toward amyloid plaques. INPP5D seems to exert its function by acting antagonistically at downstream of the TREM2 signaling pathway, suggesting that it is a novel regulator of the protective barrier by microglia. Further studies to elucidate INPP5D's role in AD may help in developing new therapeutic targets for AD treatment.

The gut microbiome regulates astrocyte reaction to Aß amyloidosis through microglial dependent and independent mechanisms.

BACKGROUND: Previous studies show that antibiotic-mediated (abx) alteration of the gut microbiome (GMB) results in a reduction of amyloid beta (Aß) plaques and proinflammatory microglial phenotype in male APPPS1-21 mice. However, the effect of GMB perturbation on astrocyte phenotypes and microglial-astrocyte communication in the context of amyloidosis has not been examined. METHODS: To study whether the GMB modulates astrocyte phenotype in the context of amyloidosis, APPPS1-21 male and female mice were treated with broad-spectrum abx leading to GMB perturbation. GFAP + astrocytes, plaque-associated astrocytes (PAA), PAA morphological parameters, and astrocyte complement component C3 levels were quantified using a combination of immunohistochemistry, immunoblotting, widefield microscopy, and confocal microscopy. Furthermore, these same astrocyte phenotypes were assessed in abx-treated APPPS1-21 male mice that received either fecal matter transplant (FMT) from untreated APPPS1-21 male donors to restore their microbiome or vehicle control. To assess complete absence of the GMB on astrocyte phenotypes, the same astrocyte phenotypes were quantified in APPPS1-21 male mice raised in germ-free (GF) or specific-pathogen free conditions (SPF). Lastly, we assessed whether microglia are necessary for abx-induced astrocyte phenotypes by depleting microglia in APPPS1-21 male mice via treatment with a colony-stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) and vehicle control or PLX5622 and abx. RESULTS: Herein, we demonstrate that postnatal treatment of male APPPS1-21 mice with broad-spectrum abx leading to GMB perturbation reduces GFAP + reactive astrocytes and PAAs, suggesting that the GMB plays a role in regulating reactive astrocyte induction and recruitment to Aß plaques. Additionally, we show that compared to controls, PAAs in abx-treated male APPPS1-21 mice exhibit an altered morphology with increased number and length of processes and reduced astrocytic complement C3, consistent with a homeostatic phenotype. GFAP + astrocyte reduction, PAA reduction, astrocyte morphological changes, and C3 levels are restored when abx-treated mice are subject to FMT from untreated APPPS1-21 male donor mice. Next, we found that APPPS1-21 male mice raised in GF conditions have similar astrocyte phenotypes as abx-treated male APPPS1-21 male mice. Correlational analysis revealed that pathogenic bacteria depleted by abx correlate with GFAP + astrocytosis, PAAs, and astrocyte morphological changes. Finally, we determined that abx-mediated reduction in GFAP + astrocytosis, PAAs, and astrocytic C3 expression is independent of microglia. However, abx-induced astrocyte morphological alterations are dependent on the presence of microglia, suggesting that there is both microglial independent and dependent GMB control of reactive astrocyte phenotypes. CONCLUSIONS: We show for the first time, in the context of amyloidosis, that the GMB plays an important role in controlling reactive astrocyte induction, morphology, and astrocyte recruitment to Aß plaques. GMB regulation of these astrocytic phenotypes is both independent and dependent on microglia.

Effects of Striatal Amyloidosis on the Dopaminergic System and Behavior: A Comparative Study in Male and Female 5XFAD Mice.

BACKGROUND: Nearly two-thirds of patients diagnosed with Alzheimer's disease (AD) are female. In addition, female patients with AD have more significant cognitive impairment than males at the same disease stage. This disparity suggests there are sex differences in AD progression. While females appear to be more affected by AD, most published behavioral studies utilize male mice. In humans, there is an association between antecedent attention-deficit/hyperactivity disorder and increased risk of dementia. Functional connectivity studies indicate that dysfunctional cortico-striatal networks contribute to hyperactivity in attention deficit hyperactivity disorder. Higher plaque density in the striatum accurately predicts the presence of clinical AD pathology. In addition, there is a link between AD-related memory dysfunction and dysfunctional dopamine signaling. OBJECTIVE: With the need to consider sex as a biological variable, we investigated the influence of sex on striatal plaque burden, dopaminergic signaling, and behavior in prodromal 5XFAD mice. METHODS: Six-month-old male and female 5XFAD and C57BL/6J mice were evaluated for striatal amyloid plaque burden, locomotive behavior, and changes in dopaminergic machinery in the striatum. RESULTS: 5XFAD female mice had a higher striatal amyloid plaque burden than male 5XFAD mice. 5XFAD females, but not males, were hyperactive. Hyperactivity in female 5XFAD mice was associated with increased striatal plaque burden and changes in dopamine signaling in the dorsal striatum. CONCLUSION: Our results indicate that the progression of amyloidosis involves the striatum in females to a greater extent than in males. These studies have significant implications for using male-only cohorts in the study of AD progression.

Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aß) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aß represents a major constituent of the amyloid plaque. The data show that pE3Aß formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aß accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aß3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aß and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.

The Alzheimer's disease risk factor INPP5D restricts neuroprotective microglial responses in amyloid beta-mediated pathology.

INTRODUCTION: Mutations in INPP5D, which encodes for the SH2-domain-containing inositol phosphatase SHIP-1, have recently been linked to an increased risk of developing late-onset Alzheimer's disease. While INPP5D expression is almost exclusively restricted to microglia in the brain, little is known regarding how SHIP-1 affects neurobiology or neurodegenerative disease pathogenesis. METHODS: We generated and investigated 5xFAD Inpp5dfl/fl Cx3cr1Ert2Cre mice to ascertain the function of microglial SHIP-1 signaling in response to amyloid beta (Aß)-mediated pathology. RESULTS: SHIP-1 deletion in microglia led to substantially enhanced recruitment of microglia to Aß plaques, altered microglial gene expression, and marked improvements in neuronal health. Further, SHIP-1 loss enhanced microglial plaque containment and Aß engulfment when compared to microglia from Cre-negative 5xFAD Inpp5dfl/fl littermate controls. DISCUSSION: These results define SHIP-1 as a pivotal regulator of microglial responses during Aß-driven neurological disease and suggest that targeting SHIP-1 may offer a promising strategy to treat Alzheimer's disease. HIGHLIGHTS: Inpp5d deficiency in microglia increases plaque-associated microglia numbers. Loss of Inpp5d induces activation and phagocytosis transcriptional pathways. Plaque encapsulation and engulfment by microglia are enhanced with Inpp5d deletion. Genetic ablation of Inpp5d protects against plaque-induced neuronal dystrophy.