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.

Plasma IAPP-Autoantibody Levels in Alzheimer's Disease Patients Are Affected by APOE4 Status.

Pancreas-derived islet amyloid polypeptide (IAPP) crosses the blood-brain barrier and co-deposits with amyloid beta (Aß) in brains of type 2 diabetes (T2D) and Alzheimer's disease (AD) patients. Depositions might be related to the circulating IAPP levels, but it warrants further investigation. Autoantibodies recognizing toxic IAPP oligomers (IAPPO) but not monomers (IAPPM) or fibrils have been found in T2D, but studies on AD are lacking. In this study, we have analyzed plasma from two cohorts and found that levels of neither immunoglobulin (Ig) M, nor IgG or IgA against IAPPM or IAPPO were altered in AD patients compared with controls. However, our results show significantly lower IAPPO-IgA levels in apolipoprotein E (APOE) 4 carriers compared with non-carriers in an allele dose-dependent manner, and the decrease is linked to the AD pathology. Furthermore, plasma IAPP-Ig levels, especially IAPP-IgA, correlated with cognitive decline, C-reactive protein, cerebrospinal fluid Aß and tau, neurofibrillary tangles, and brain IAPP exclusively in APOE4 non-carriers. We speculate that the reduction in IAPPO-IgA levels may be caused by increased plasma IAPPO levels or masked epitopes in APOE4 carriers and propose that IgA and APOE4 status play a specific role in clearance of circulatory IAPPO, which may influence the amount of IAPP deposition in the AD brain.

Contraction of human brain vascular pericytes in response to islet amyloid polypeptide is reversed by pramlintide.

The islet amyloid polypeptide (IAPP), a pancreas-produced peptide, has beneficial functions in its monomeric form. However, IAPP aggregates, related to type 2 diabetes mellitus (T2DM), are toxic not only for the pancreas, but also for the brain. In the latter, IAPP is often found in vessels, where it is highly toxic for pericytes, mural cells that have contractile properties and regulate capillary blood flow. In the current study, we use a microvasculature model, where human brain vascular pericytes (HBVP) are co-cultured together with human cerebral microvascular endothelial cells, to demonstrate that IAPP oligomers (oIAPP) alter the morphology and contractility of HBVP. Contraction and relaxation of HBVP was verified using the vasoconstrictor sphingosine-1-phosphate (S1P) and vasodilator Y27632, where the former increased, and the latter decreased, the number of HBVP with round morphology. Increased number of round HBVP was also seen after oIAPP stimulation, and the effect was reverted by the IAPP analogue pramlintide, Y27632, and the myosin inhibitor blebbistatin. Inhibition of the IAPP receptor with the antagonist AC187 only reverted IAPP effects partially. Finally, we demonstrate by immunostaining of human brain tissue against laminin that individuals with high amount of brain IAPP levels show significantly lower capillary diameter and altered mural cell morphology compared to individuals with low brain IAPP levels. These results indicate that HBVP, in an in vitro model of microvasculature, respond morphologically to vasoconstrictors, dilators, and myosin inhibitors. They also suggest that oIAPP induces contraction of these mural cells and that pramlintide can reverse such contraction.

Increased plasma and brain immunoglobulin A in Alzheimer's disease is lost in apolipoprotein E ε4 carriers.

BACKGROUND: Alzheimer's disease (AD) is foremost characterized by ß-amyloid (Aß)-extracellular plaques, tau-intraneuronal fibrillary tangles (NFT), and neuroinflammation, but over the last years it has become evident that peripheral inflammation might also contribute to the disease. AD patients often demonstrate increased levels of circulating proinflammatory mediators and altered antibody levels in the blood. In our study, we investigated the plasma Immunoglobulin A (IgA) levels in association with apolipoprotein E (APOE) ε4 status and Aß pathology. METHODS: IgA levels in antemortem-collected (cohort I) and postmortem-collected (cohort II) plasma samples from AD patients (n = 30 in cohort I and n = 16 in cohort II) and non-demented age-matched controls (NC) (n = 42 in cohort I and n = 7 in cohort II) were measured using ELISA. Hippocampal sections from cohort II were immunostained against IgA, and the IgA area fraction as well as the number of IgA positive (IgA+) cells in the cornu ammonis region were analysed using ImageJ. The relationship between plasma IgA levels and cognition, C-reactive protein (CRP), and cerebrospinal fluid (CSF) AD biomarkers in cohort I as well as neuropathology, IgA+ cell number, and IgA area fraction in cohort II was analysed before and after grouping the cohorts into APOEε4 carriers and APOEε4 non-carriers. RESULTS: Plasma IgA levels were higher in AD patients compared to NC in both cohorts. Also, AD patients demonstrated higher IgA area fraction and IgA+ cell number compared to NC. When APOEε4 status was considered, higher plasma IgA levels in AD patients were only seen in APOEε4 non-carriers. Finally, plasma IgA levels, exclusively in APOEε4 non-carriers, were associated with cognition, CRP, and CSF Aß levels in cohort I as well as with IgA area fraction, IgA+ cell number, and Aß, Lewy body, and NFT neuropathology in cohort II. CONCLUSIONS: Our study suggests that AD pathology and cognitive decline are associated with increased plasma IgA levels in an APOE allele-dependent manner, where the associations are lost in APOEε4 carriers.

Transgenic Mouse Models of Alzheimer's Disease: An Integrative Analysis.

Alzheimer's disease (AD) constitutes the most prominent form of dementia among elderly individuals worldwide. Disease modeling using murine transgenic mice was first initiated thanks to the discovery of heritable mutations in amyloid precursor protein (APP) and presenilins (PS) genes. However, due to the repeated failure of translational applications from animal models to human patients, along with the recent advances in genetic susceptibility and our current understanding on disease biology, these models have evolved over time in an attempt to better reproduce the complexity of this devastating disease and improve their applicability. In this review, we provide a comprehensive overview about the major pathological elements of human AD (plaques, tauopathy, synaptic damage, neuronal death, neuroinflammation and glial dysfunction), discussing the knowledge that available mouse models have provided about the mechanisms underlying human disease. Moreover, we highlight the pros and cons of current models, and the revolution offered by the concomitant use of transgenic mice and omics technologies that may lead to a more rapid improvement of the present modeling battery.

Animal and Cellular Models of Alzheimer's Disease: Progress, Promise, and Future Approaches.

Alzheimer's disease (AD) is an incurable neurodegenerative disease affecting over 45 million people worldwide. Transgenic mouse models have made remarkable contributions toward clarifying the pathophysiological mechanisms behind the clinical manifestations of AD. However, the limited ability of these in vivo models to accurately replicate the biology of the human disease have precluded the translation of promising preclinical therapies to the clinic. In this review, we highlight several major pathogenic mechanisms of AD that were discovered using transgenic mouse models. Moreover, we discuss the shortcomings of current animal models and the need to develop reliable models for the sporadic form of the disease, which accounts for the majority of AD cases, as well as human cellular models to improve success in translating results into human treatments.

Generation of a humanized Aß expressing mouse demonstrating aspects of Alzheimer's disease-like pathology.

The majority of Alzheimer's disease (AD) cases are late-onset and occur sporadically, however most mouse models of the disease harbor pathogenic mutations, rendering them better representations of familial autosomal-dominant forms of the disease. Here, we generated knock-in mice that express wildtype human Aß under control of the mouse App locus. Remarkably, changing 3 amino acids in the mouse Aß sequence to its wild-type human counterpart leads to age-dependent impairments in cognition and synaptic plasticity, brain volumetric changes, inflammatory alterations, the appearance of Periodic Acid-Schiff (PAS) granules and changes in gene expression. In addition, when exon 14 encoding the Aß sequence was flanked by loxP sites we show that Cre-mediated excision of exon 14 ablates hAß expression, rescues cognition and reduces the formation of PAS granules.

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.

Human Pluripotent Stem Cell-Derived Neural Cells as a Relevant Platform for Drug Screening in Alzheimer's Disease.

Extracellular amyloid-beta deposition and intraneuronal Tau-laden neurofibrillary tangles are prime features of Alzheimer's disease (AD). The pathology of AD is very complex and still not fully understood, since different neural cell types are involved in the disease. Although neuronal function is clearly deteriorated in AD patients, recently, an increasing number of evidences have pointed towards glial cell dysfunction as one of the main causative phenomena implicated in AD pathogenesis. The complex disease pathology together with the lack of reliable disease models have precluded the development of effective therapies able to counteract disease progression. The discovery and implementation of human pluripotent stem cell technology represents an important opportunity in this field, as this system allows the generation of patient-derived cells to be used for disease modeling and therapeutic target identification and as a platform to be employed in drug discovery programs. In this review, we discuss the current studies using human pluripotent stem cells focused on AD, providing convincing evidences that this system is an excellent opportunity to advance in the comprehension of AD pathology, which will be translated to the development of the still missing effective therapies.

Distinct disease-sensitive GABAergic neurons in the perirhinal cortex of Alzheimer's mice and patients.

Neuronal loss is the best neuropathological substrate that correlates with cortical atrophy and dementia in Alzheimer's disease (AD). Defective GABAergic neuronal functions may lead to cortical network hyperactivity and aberrant neuronal oscillations and in consequence, generate a detrimental alteration in memory processes. In this study, using immunohistochemical and stereological approaches, we report that the two major and non-overlapping groups of inhibitory interneurons (SOM-cells and PV-cells) displayed distinct vulnerability in the perirhinal cortex of APP/PS1 mice and AD patients. SOM-positive neurons were notably sensitive and exhibited a dramatic decrease in the perirhinal cortex of 6-month-old transgenic mice (57% and 61% in areas 36 and 35, respectively) and, most importantly, in AD patients (91% in Braak V-VI cases). In addition, this interneuron degenerative process seems to occur in parallel, and closely related, with the progression of the amyloid pathology. However, the population expressing PV was unaffected in APP/PS1 mice while in AD brains suffered a pronounced and significant loss (69%). As a key component of cortico-hippocampal networks, the perirhinal cortex plays an important role in memory processes, especially in familiarity-based memory recognition. Therefore, disrupted functional connectivity of this cortical region, as a result of the early SOM and PV neurodegeneration, might contribute to the altered brain rhythms and cognitive failures observed in the initial clinical phase of AD patients. Finally, these findings highlight the failure of amyloidogenic AD models to fully recapitulate the selective neuronal degeneration occurring in humans.

Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease.

Reactive astrogliosis, a complex process characterized by cell hypertrophy and upregulation of components of intermediate filaments, is a common feature in brains of Alzheimer's patients. Reactive astrocytes are found in close association with neuritic plaques; however, the precise role of these glial cells in disease pathogenesis is unknown. In this study, using immunohistochemical techniques and light and electron microscopy, we report that plaque-associated reactive astrocytes enwrap, engulf and may digest presynaptic dystrophies in the hippocampus of amyloid precursor protein/presenilin-1 (APP/PS1) mice. Microglia, the brain phagocytic population, was apparently not engaged in this clearance. Phagocytic reactive astrocytes were present in 35% and 67% of amyloid plaques at 6 and 12 months of age, respectively. The proportion of engulfed dystrophic neurites was low, around 7% of total dystrophies around plaques at both ages. This fact, along with the accumulation of dystrophic neurites during disease course, suggests that the efficiency of the astrocyte phagocytic process might be limited or impaired. Reactive astrocytes surrounding and engulfing dystrophic neurites were also detected in the hippocampus of Alzheimer's patients by confocal and ultrastructural analysis. We posit that the phagocytic activity of reactive astrocytes might contribute to clear dysfunctional synapses or synaptic debris, thereby restoring impaired neural circuits and reducing the inflammatory impact of damaged neuronal parts and/or limiting the amyloid pathology. Therefore, potentiation of the phagocytic properties of reactive astrocytes may represent a potential therapy in Alzheimer's disease.

Soluble phospho-tau from Alzheimer's disease hippocampus drives microglial degeneration.

The role of microglial cells in the development and progression of Alzheimer's disease (AD) has not been elucidated. Here, we demonstrated the existence of a weak microglial response in human AD hippocampus which is in contrast to the massive microglial activation observed in APP-based models. Most importantly, microglial cells displayed a prominent degenerative profile (dentate gyrus > CA3 > CA1 > parahippocampal gyrus), including fragmented and dystrophic processes with spheroids, a reduced numerical density, and a significant decrease in the area of surveillance ("microglial domain"). Consequently, there was a substantial decline in the area covered by microglia which may compromise immune protection and, therefore, neuronal survival. In vitro experiments demonstrated that soluble fractions (extracellular/cytosolic) from AD hippocampi were toxic for microglial cells. This toxicity was abolished by AT8 and/or AT100 immunodepletion, validating that soluble phospho-tau was the toxic agent. These results were reproduced using soluble fractions from phospho-tau-positive Thy-tau22 hippocampi. Cultured microglial cells were not viable following phagocytosis of SH-SY5Y cells expressing soluble intracellular phospho-tau. Because the phagocytic capacity of microglial cells is highly induced by apoptotic signals in the affected neurons, we postulate that accumulation of intraneuronal soluble phospho-tau might trigger microglial degeneration in the AD hippocampus. This microglial vulnerability in AD pathology provides new insights into the immunological mechanisms underlying the disease progression and highlights the need to improve or develop new animal models, as the current models do not mimic the microglial pathology observed in the hippocampus of AD patients.