Characterization of monoamine oxidase-B (MAO-B) as a biomarker of reactive astrogliosis in Alzheimer's disease and related dementias.

Reactive astrogliosis accompanies the two neuropathological hallmarks of Alzheimer's disease (AD)-Aß plaques and neurofibrillary tangles-and parallels neurodegeneration in AD and AD-related dementias (ADRD). Thus, there is growing interest in developing imaging and fluid biomarkers of reactive astrogliosis for AD/ADRD diagnosis and prognostication. Monoamine oxidase-B (MAO-B) is emerging as a target for PET imaging radiotracers of reactive astrogliosis. However, a thorough characterization of MAO-B expression in postmortem control and AD/ADRD brains is lacking. We sought to: (1) identify the primary cell type(s) expressing MAO-B in control and AD brains; (2) quantify MAO-B immunoreactivity in multiple brain regions of control and AD donors as a proxy for PET radiotracer uptake; (3) correlate MAO-B level with local AD neuropathological changes, reactive glia, and cortical atrophy; (4) determine whether the MAOB rs1799836 SNP genotype impacts MAO-B expression level; (5) compare MAO-B immunoreactivity across AD/ADRD, including Lewy body diseases (LBD) and frontotemporal lobar degenerations with tau (FTLD-Tau) and TDP-43 (FTLD-TDP). We found that MAO-B is mainly expressed by subpial and perivascular cortical astrocytes as well as by fibrous white matter astrocytes in control brains, whereas in AD brains, MAO-B is significantly upregulated by both cortical reactive astrocytes and white matter astrocytes across temporal, frontal, and occipital lobes. By contrast, MAO-B expression level was unchanged and lowest in cerebellum. Cortical MAO-B expression was independently associated with cortical atrophy and local measures of reactive astrocytes and microglia, and significantly increased in reactive astrocytes surrounding Thioflavin-S+ dense-core Aß plaques. MAO-B expression was not affected by the MAOB rs1799836 SNP genotype. MAO-B expression was also significantly increased in the frontal cortex and white matter of donors with corticobasal degeneration, Pick's disease, and FTLD-TDP, but not in LBD or progressive supranuclear palsy. These findings support ongoing efforts to develop MAO-B-based PET radiotracers to image reactive astrogliosis in AD/ADRD.

An Insertion Within SIRPß1 Shows a Dual Effect Over Alzheimer's Disease Cognitive Decline Altering the Microglial Response.

Background: Microglial dysfunction plays a causative role in Alzheimer's disease (AD) pathogenesis. Here we focus on a germline insertion/deletion variant mapping SIRPß1, a surface receptor that triggers amyloid-ß(Aß) phagocytosis via TYROBP. Objective: To analyze the impact of this copy-number variant in SIRPß1 expression and how it affects AD molecular etiology. Methods: Copy-number variant proxy rs2209313 was evaluated in GERALD and GR@ACE longitudinal series. Hippocampal specimens of genotyped AD patients were also examined. SIRPß1 isoform-specific phagocytosis assays were performed in HEK393T cells. Results: The insertion alters the SIRPß1 protein isoform landscape compromising its ability to bind oligomeric Aß and its affinity for TYROBP. SIRPß1 Dup/Dup patients with mild cognitive impairment show an increased cerebrospinal fluid t-Tau/Aß ratio (p = 0.018) and a higher risk to develop AD (OR = 1.678, p = 0.018). MRIs showed that Dup/Dup patients exhibited a worse initial response to AD. At the moment of diagnosis, all patients showed equivalent Mini-Mental State Examination scores. However, AD patients with the duplication had less hippocampal degeneration (p < 0.001) and fewer white matter hyperintensities. In contrast, longitudinal studies indicate that patients bearing the duplication allele show a slower cognitive decline (p = 0.013). Transcriptional analysis also shows that the SIRPß1 duplication allele correlates with higher TREM2 expression and an increased microglial activation. Conclusions: The SIRPß1 internal duplication has opposite effects over MCI-to-Dementia conversion risk and AD progression, affecting microglial response to Aß. Given the pharmacological approaches focused on the TREM2-TYROBP axis, we believe that SIRPß1 structural variant might be considered as a potential modulator of this causative pathway.

Update on modifiable risk factors for Alzheimer's disease and related dementias.

PURPOSE OF REVIEW: All human beings undergo a lifelong cumulative exposure to potentially preventable adverse factors such as toxins, infections, traumatisms, and cardiovascular risk factors, collectively termed exposome. The interplay between the individual's genetics and exposome is thought to have a large impact in health outcomes such as cancer and cardiovascular disease. Likewise, a growing body of evidence is supporting the idea that preventable factors explain a sizable proportion of Alzheimer's disease and related dementia (ADRD) cases. RECENT FINDINGS: Here, we will review the most recent epidemiological, experimental preclinical, and interventional clinical studies examining some of these potentially modifiable risk factors for ADRD. We will focus on new evidence regarding cardiovascular risk factors, air pollution, viral and other infectious agents, traumatic brain injury, and hearing loss. SUMMARY: While greater and higher quality epidemiological and experimental evidence is needed to unequivocally confirm their causal link with ADRD and/or unravel the underlying mechanisms, these modifiable risk factors may represent a window of opportunity to reduce ADRD incidence and prevalence at the population level via health screenings, and education and health policies.

Monocyte-derived cells invade brain parenchyma and amyloid plaques in human Alzheimer's disease hippocampus.

Microglia are brain-resident myeloid cells and play a major role in the innate immune responses of the CNS and the pathogenesis of Alzheimer's disease (AD). However, the contribution of nonparenchymal or brain-infiltrated myeloid cells to disease progression remains to be demonstrated. Here, we show that monocyte-derived cells (MDC) invade brain parenchyma in advanced stages of AD continuum using transcriptional analysis and immunohistochemical characterization in post-mortem human hippocampus. Our findings demonstrated that a high proportion (60%) of demented Braak V-VI individuals was associated with up-regulation of genes rarely expressed by microglial cells and abundant in monocytes, among which stands the membrane-bound scavenger receptor for haptoglobin/hemoglobin complexes or Cd163. These Cd163-positive MDC invaded the hippocampal parenchyma, acquired a microglial-like morphology, and were located in close proximity to blood vessels. Moreover, and most interesting, these invading monocytes infiltrated the nearby amyloid plaques contributing to plaque-associated myeloid cell heterogeneity. However, in aged-matched control individuals with hippocampal amyloid pathology, no signs of MDC brain infiltration or plaque invasion were found. The previously reported microglial degeneration/dysfunction in AD hippocampus could be a key pathological factor inducing MDC recruitment. Our data suggest a clear association between MDC infiltration and endothelial activation which in turn may contribute to damage of the blood brain barrier integrity. The recruitment of monocytes could be a consequence rather than the cause of the severity of the disease. Whether monocyte infiltration is beneficial or detrimental to AD pathology remains to be fully elucidated. These findings open the opportunity to design targeted therapies, not only for microglia but also for the peripheral immune cell population to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying the progression of AD.

Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed Paraffin-Embedded Human Brain Sections.

There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aß plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.

Cytotoxicity and Effects on the Synapsis Induced by Pure Cylindrospermopsin in an E17 Embryonic Murine Primary Neuronal Culture in a Concentration- and Time-Dependent Manner.

Cylindrospermopsin (CYN) is a cyanotoxin whose incidence has been increasing in the last decades. Due to its capacity to exert damage at different levels of the organism, it is considered a cytotoxin. Although the main target organ is the liver, recent studies indicate that CYN has potential toxic effects on the nervous system, both in vitro and in vivo. Thus, the aim of the present work was to study the effects of this cyanotoxin on neuronal viability and synaptic integrity in murine primary cultures of neurons exposed to environmentally relevant concentrations (0-1 µg/mL CYN) for 12, 24, and 48 h. The results demonstrate a concentration- and time-dependent decrease in cell viability; no cytotoxicity was detected after exposure to the cyanotoxin for 12 h, while all of the concentrations assayed decreased this parameter after 48 h. Furthermore, CYN was also demonstrated to exert damage at the synaptic level in a murine primary neuronal culture in a concentration- and time-dependent manner. These data highlight the importance of studying the neurotoxic properties of this cyanotoxin in different experimental models.

Cyclic multiplex fluorescent immunohistochemistry and machine learning reveal distinct states of astrocytes and microglia in normal aging and Alzheimer's disease.

BACKGROUND: Astrocytes and microglia react to Aß plaques, neurofibrillary tangles, and neurodegeneration in the Alzheimer's disease (AD) brain. Single-nuclei and single-cell RNA-seq have revealed multiple states or subpopulations of these glial cells but lack spatial information. We have developed a methodology of cyclic multiplex fluorescent immunohistochemistry on human postmortem brains and image analysis that enables a comprehensive morphological quantitative characterization of astrocytes and microglia in the context of their spatial relationships with plaques and tangles. METHODS: Single FFPE sections from the temporal association cortex of control and AD subjects were subjected to 8 cycles of multiplex fluorescent immunohistochemistry, including 7 astroglial, 6 microglial, 1 neuronal, Aß, and phospho-tau markers. Our analysis pipeline consisted of: (1) image alignment across cycles; (2) background subtraction; (3) manual annotation of 5172 ALDH1L1+ astrocytic and 6226 IBA1+ microglial profiles; (4) local thresholding and segmentation of profiles; (5) machine learning on marker intensity data; and (6) deep learning on image features. RESULTS: Spectral clustering identified three phenotypes of astrocytes and microglia, which we termed "homeostatic," "intermediate," and "reactive." Reactive and, to a lesser extent, intermediate astrocytes and microglia were closely associated with AD pathology (≤ 50 µm). Compared to homeostatic, reactive astrocytes contained substantially higher GFAP and YKL-40, modestly elevated vimentin and TSPO as well as EAAT1, and reduced GS. Intermediate astrocytes had markedly increased EAAT2, moderately increased GS, and intermediate GFAP and YKL-40 levels. Relative to homeostatic, reactive microglia showed increased expression of all markers (CD68, ferritin, MHC2, TMEM119, TSPO), whereas intermediate microglia exhibited increased ferritin and TMEM119 as well as intermediate CD68 levels. Machine learning models applied on either high-plex signal intensity data (gradient boosting machines) or directly on image features (convolutional neural networks) accurately discriminated control vs. AD diagnoses at the single-cell level. CONCLUSIONS: Cyclic multiplex fluorescent immunohistochemistry combined with machine learning models holds promise to advance our understanding of the complexity and heterogeneity of glial responses as well as inform transcriptomics studies. Three distinct phenotypes emerged with our combination of markers, thus expanding the classic binary "homeostatic vs. reactive" classification to a third state, which could represent "transitional" or "resilient" glia.

Should We Open Fire on Microglia? Depletion Models as Tools to Elucidate Microglial Role in Health and Alzheimer's Disease.

Microglia play a critical role in both homeostasis and disease, displaying a wide variety in terms of density, functional markers and transcriptomic profiles along the different brain regions as well as under injury or pathological conditions, such as Alzheimer's disease (AD). The generation of reliable models to study into a dysfunctional microglia context could provide new knowledge towards the contribution of these cells in AD. In this work, we included an overview of different microglial depletion approaches. We also reported unpublished data from our genetic microglial depletion model, Cx3cr1CreER/Csf1rflx/flx, in which we temporally controlled microglia depletion by either intraperitoneal (acute model) or oral (chronic model) tamoxifen administration. Our results reported a clear microglial repopulation, then pointing out that our model would mimic a context of microglial replacement instead of microglial dysfunction. Next, we evaluated the origin and pattern of microglial repopulation. Additionally, we also reviewed previous works assessing the effects of microglial depletion in the progression of Aß and Tau pathologies, where controversial data are found, probably due to the heterogeneous and time-varying microglial phenotypes observed in AD. Despite that, microglial depletion represents a promising tool to assess microglial role in AD and design therapeutic strategies.

Amyloid-ß impairs the phagocytosis of dystrophic synapses by astrocytes in Alzheimer's disease.

Reactive astrocytes and dystrophic neurites, most aberrant presynaptic elements, are found surrounding amyloid-ß plaques in Alzheimer's disease (AD). We have previously shown that reactive astrocytes enwrap, phagocytose, and degrade dystrophic synapses in the hippocampus of APP mice and AD patients, but affecting less than 7% of dystrophic neurites, suggesting reduced phagocytic capacity of astrocytes in AD. Here, we aimed to gain insight into the underlying mechanisms by analyzing the capacity of primary astrocyte cultures to phagocytose and degrade isolated synapses (synaptoneurosomes, SNs) from APP (containing dystrophic synapses and amyloid-ß peptides), Tau (containing AT8- and AT100-positive phosphorylated Tau) and WT (controls) mice. We found highly reduced phagocytic and degradative capacity of SNs-APP, but not AT8/AT100-positive SNs-Tau, as compared with SNs-WT. The reduced astrocyte phagocytic capacity was verified in hippocampus from 12-month-old APP mice, since only 1.60 ± 3.81% of peri-plaque astrocytes presented phagocytic structures. This low phagocytic capacity did not depend on microglia-mediated astrocyte reactivity, because removal of microglia from the primary astrocyte cultures abrogated the expression of microglia-dependent genes in astrocytes, but did not affect the phagocytic impairment induced by oligomeric amyloid-ß alone. Taken together, our data suggest that amyloid-ß, but not hyperphosphorylated Tau, directly impairs the capacity of astrocytes to clear the pathological accumulation of oligomeric amyloid-ß, as well as of peri-plaque dystrophic synapses containing amyloid-ß, perhaps by reducing the expression of phagocytosis receptors such as Mertk and Megf10, thus increasing neuronal damage in AD. Therefore, the potentiation or recovery of astrocytic phagocytosis may be a novel therapeutic avenue in AD.

Enhancing microtubule stabilization rescues cognitive deficits and ameliorates pathological phenotype in an amyloidogenic Alzheimer's disease model.

In Alzheimer's disease (AD), and other tauopathies, microtubule destabilization compromises axonal and synaptic integrity contributing to neurodegeneration. These diseases are characterized by the intracellular accumulation of hyperphosphorylated tau leading to neurofibrillary pathology. AD brains also accumulate amyloid-beta (Aß) deposits. However, the effect of microtubule stabilizing agents on Aß pathology has not been assessed so far. Here we have evaluated the impact of the brain-penetrant microtubule-stabilizing agent Epothilone D (EpoD) in an amyloidogenic model of AD. Three-month-old APP/PS1 mice, before the pathology onset, were weekly injected with EpoD for 3 months. Treated mice showed significant decrease in the phospho-tau levels and, more interesting, in the intracellular and extracellular hippocampal Aß accumulation, including the soluble oligomeric forms. Moreover, a significant cognitive improvement and amelioration of the synaptic and neuritic pathology was found. Remarkably, EpoD exerted a neuroprotective effect on SOM-interneurons, a highly AD-vulnerable GABAergic subpopulation. Therefore, our results suggested that EpoD improved microtubule dynamics and axonal transport in an AD-like context, reducing tau and Aß levels and promoting neuronal and cognitive protection. These results underline the existence of a crosstalk between cytoskeleton pathology and the two major AD protein lesions. Therefore, microtubule stabilizers could be considered therapeutic agents to slow the progression of both tau and Aß pathology.

Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology.

Microglial cells are crucial players in the pathological process of neurodegenerative diseases, such as Alzheimer's disease (AD). Microglial response in AD has been principally studied in relation to amyloid-beta pathology but, comparatively, little is known about inflammatory processes associated to tau pathology. In the hippocampus of AD patients, where tau pathology is more prominent than amyloid-beta pathology, a microglial degenerative process has been reported. In this work, we have directly compared the microglial response in two different transgenic tau mouse models: ThyTau22 and P301S. Surprisingly, these two models showed important differences in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested mild microglial activation, P301S mice exhibited a strong microglial response in parallel with high phospho-tau accumulation. This differential phospho-tau expression could account for the different microglial response in these two tau strains. However, soluble (S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas the correspondent S1 fractions from P301S mice displayed low soluble phospho-tau levels and were not toxic for microglial cells. Therefore, not only the expression levels but the aggregation of phospho-tau should differ between both models. In fact, most of tau forms in the P301S mice were aggregated and, in consequence, forming insoluble tau species. We conclude that different factors as tau mutations, accumulation, phosphorylation, and/or aggregation could account for the distinct microglial responses observed in these two tau models. For this reason, deciphering the molecular nature of toxic tau species for microglial cells might be a promising therapeutic approach in order to restore the deficient immunological protection observed in AD hippocampus.

Microglia in Alzheimer's Disease: Activated, Dysfunctional or Degenerative.

Microglial activation has been considered a crucial player in the pathological process of multiple human neurodegenerative diseases. In some of these pathologies, such as Amyotrophic Lateral Sclerosis or Multiple Sclerosis, the immune system and microglial cells (as part of the cerebral immunity) play a central role. In other degenerative processes, such as Alzheimer's disease (AD), the role of microglia is far to be elucidated. In this "mini-review" article, we briefly highlight our recent data comparing the microglial response between amyloidogenic transgenic models, such as APP/PS1 and AD patients. Since the AD pathology could display regional heterogeneity, we focus our work at the hippocampal formation. In APP based models a prominent microglial response is triggered around amyloid-beta (Aß) plaques. These strongly activated microglial cells could drive the AD pathology and, in consequence, could be implicated in the neurodegenerative process observed in models. On the contrary, the microglial response in human samples is, at least, partial or attenuated. This patent difference could simply reflect the lower and probably slower Aß production observed in human hippocampal samples, in comparison with models, or could reflect the consequence of a chronic long-standing microglial activation. Beside this differential response, we also observed microglial degeneration in Braak V-VI individuals that, indeed, could compromise their normal role of surveying the brain environment and respond to the damage. This microglial degeneration, particularly relevant at the dentate gyrus, might be mediated by the accumulation of toxic soluble phospho-tau species. The consequences of this probably deficient immunological protection, observed in AD patients, are unknown.