Amyloid β-peptide-induced progressive neurodegeneration in an APP-transgenic mouse model for Alzheimer’s disease

The amyloid beta-protein (Abeta) is the main component of Alzheimer's disease (AD)-related senile plaques. In the human brain Abeta-deposition occurs in a hierarchical sequence in which different areas of the brain become involved. It is not clear whether this sequence shows the time course of Abeta-deposition or just different pathology in different individuals. Although Abeta is associated with the development of AD it has not been shown which forms of Abeta induce neurodegeneration in vivo, which types of neurons are vulnerable and whether Abeta-induced neurodegeneration increases with the progression of Abeta-pathology. To address these questions, DiI-crystals were implanted into the left frontocentral cortex of APP23 transgenic mice overexpressing mutant human APP and of wild-type littermates. In parallel, immunohistochemistry for Abeta-plaque detection was performed in 3-, 5-, 11-, 15- and 25-month-old APP23 mice and wild-type littermates. Traced commissural neurons in layer III of the right frontocentral cortex were quantified in 3-, 5-, 11-, and 15-month-old mice. Three different types of commissural neurons were traced. At 3 months of age no differences in the number of labeled commissural neurons were seen in APP23 mice compared to wild-type mice. A selective reduction of the heavily ramified type of neurons was observed in APP23 mice compared to wild-type animals at 5, 11, and 15 months of age, starting with the deposition of Abeta-plaques occurred in the frontocentral cortex at 5 months of age. The other two types of commissural neurons did not show alterations in 5- and 11-month animals. At 15 months of age, the number of traced sparsely ramified pyramidal neurons was reduced in addition to that of the heavily ramified neurons in APP23 mice compared with wild-type mice. At this point in time Abeta-deposits were seen in the neo- and allocortex as well as in the basal ganglia and the thalamus. At 25 months of age Abeta-deposits were also seen at the brainstem. In summary, the results show that 1) Abeta-deposition in APP23 mice follows a similar sequence as in human brain, in which the different areas become step-by-step involved in beta-amyloidosis, 2) this step-by-step regional involvement represents the time course of Abeta-deposition in the brain, and 3) Abeta thereby, induces progressive degeneration of distinct types of commissural neurons. Degeneration of the most vulnerable neurons starts in parallel with the occurrence of the first fibrillar Abeta deposits in the neocortex. The selective vulnerability of different types of neurons to Abeta is presumably related to the complexity of their dendritic morphology. In so doing, these results support Abeta to be the major therapeutic target for AD treatment in pre-clinical as well as in late stages of the disease

A Neuron, Microglia, and Astrocyte Triple Co-culture Model to Study Alzheimer’s Disease

Glial cells are essential to understand Alzheimer's disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.The authors acknowledge financial support by Basque Government (IT1203-19; ELKARTEK KK-2020/00034; PIBA_2016_1_0009; and PIBA_2020_1_0012), CIBERNED (CB06/0005/0076), MICINN (PID2019-109724RB-I00 and PID2019-108465RB-I00). CL and JZ-I were supported by Ph.D. Scholarships from the Tatiana Pérez de Guzmán el Bueno Foundation and Basque Government, respectively

Accumulation of Intraneuronal beta-Amyloid 42 Peptides Is Associated with Early Changes in Microtubule-Associated Protein 2 in Neurites and Synapses

Pathologic aggregation of beta-amyloid (A beta) peptide and the axonal microtubule-associated protein tau protein are hallmarks of Alzheimer's disease (AD). Evidence supports that A beta peptide accumulation precedes microtubule-related pathology, although the link between A beta and tau remains unclear. We previously provided evidence for early co-localization of A beta 42 peptides and hyperphosphorylated tau within postsynaptic terminals of CA1 dendrites in the hippocampus of AD transgenic mice. Here, we explore the relation between A beta peptide accumulation and the dendritic, microtubule-associated protein 2 (MAP2) in the well-characterized amyloid precursor protein Swedish mutant transgenic mouse (Tg2576). We provide evidence that localized intraneuronal accumulation of A beta 42 peptides is spatially associated with reductions of MAP2 in dendrites and postsynaptic compartments of Tg2576 mice at early ages. Our data support that reduction in MAP2 begins at sites of A beta 42 monomer and low molecular weight oligomer (M/LMW) peptide accumulation. Cumulative evidence suggests that accumulation of M/LMW A beta 42 peptides occurs early, before high molecular weight oligomerization and plaque formation. Since synaptic alteration is the best pathologic correlate of cognitive dysfunction in AD, the spatial association of M/LMW A beta peptide accumulation with pathology of MAP2 within neuronal processes and synaptic compartments early in the disease process reinforces the importance of intraneuronal A beta accumulation in AD pathogenesis

Linking Plasma Amyloid Beta and Neurofilament Light Chain to Intracortical Myelin Content in Cognitively Normal Older Adults

Evidence suggests that lightly myelinated cortical regions are vulnerable to aging and Alzheimer's disease (AD). However, it remains unknown whether plasma markers of amyloid and neurodegeneration are related to deficits in intracortical myelin content, and whether this relationship, in turn, is associated with altered patterns of resting-state functional connectivity (rs-FC). To shed light into these questions, plasma levels of amyloid-beta fragment 1-42 (A beta(1-42)) and neurofilament light chain (NfL) were measured using ultra-sensitive single-molecule array (Simoa) assays, and the intracortical myelin content was estimated with the ratio T1-weigthed/T2-weighted (T1w/T2w) in 133 cognitively normal older adults. We assessed: (i) whether plasma A beta(1-42) and/or NfL levels were associated with intracortical myelin content at different cortical depths and (ii) whether cortical regions showing myelin reductions also exhibited altered rs-FC patterns. Surface-based multiple regression analyses revealed that lower plasma A beta(1-42) and higher plasma NfL were associated with lower myelin content in temporo-parietal-occipital regions and the insular cortex, respectively. Whereas the association with A beta(1-42) decreased with depth, the NfL-myelin relationship was most evident in the innermost layer. Older individuals with higher plasma NfL levels also exhibited altered rs-FC between the insula and medial orbitofrontal cortex. Together, these findings establish a link between plasma markers of amyloid/neurodegeneration and intracortical myelin content in cognitively normal older adults, and support the role of plasma NfL in boosting aberrant FC patterns of the insular cortex, a central brain hub highly vulnerable to aging and neurodegeneration.This work was supported by the Spanish Ministry of Economy and Competitiveness (PID2020-119978RB-I00 to JLC and PID2020-118825GB-I00 to MA), CIBERNED (JLC, MA, CM, EC-Z, and FZ), Alzheimers Association (AARG-NFT-22-924702 to JLC), the Basque Government (IT1203-19; ELKARTEK KK-2020/00034 to EC-Z), the Research Program for a Long-Life Society of the Fundacion General CSIC (0551_PSL_6_E to JLC), the Junta de Andalucia (PY20_00858 to JLC), and the Andalucia-FEDER Program (UPO-1380913 to JLC)

Contribution of Neurons and Glial Cells to Complement-Mediated Synapse Removal during Development, Aging and in Alzheimer’s Disease

Synapse loss is an early manifestation of pathology in Alzheimer's disease (AD) and is currently the best correlate to cognitive decline. Microglial cells are involved in synapse pruning during development via the complement pathway. Moreover, recent evidence points towards a key role played by glial cells in synapse loss during AD. However, further contribution of glial cells and the role of neurons to synapse pathology in AD remain not well understood. This review is aimed at comprehensively reporting the source and/or cellular localization in the CNS-in microglia, astrocytes, or neurons-of the triggering components (C1q, C3) of the classical complement pathway involved in synapse pruning in development, adulthood, and AD.The authors thank Dr. Baleriola at Achucarro Basque Center for Neuroscience (Bilbao, Spain) and Dr. Sole-Domenech and Dr. Pipalia at Weill Cornell Medical College (Cornell University, New York, USA) for the helpful and critical revision of the manuscript. This study was supported by CIBERNED and by grants from Ministerio de Economia y Competitividad (SAF2016-75292-R), Gobierno Vasco (PIBA PI-2016-1-009-0016 and ELKARTEK 2016-00033), Ikerbasque, Basque Foundation for Science, and Universidad del Pais Vasco/Euskal Herriko Unibertsitatea UPV/EHU. Jone Zuazo held a fellowship from Gobierno Vasco and Celia Luchena from Fundacion Tatiana Perez de Guzman el Bueno

Selective vulnerability of different types of commissural neurons for amyloid β-protein-induced neurodegeneration in APP23 mice correlates with dendritic tree morphology

The amyloid β-protein (Aβ) is the main component of Alzheimer's disease-related senile plaques. Although Aβ is associated with the development of Alzheimer's disease, it has not been shown which forms of Aβ induce neurodegeneration in vivo and which types of neurons are vulnerable. To address these questions, we implanted DiI crystals into the left frontocentral cortex of APP23 transgenic mice overexpressing mutant human APP (amyloid precursor protein gene) and of littermate controls. Traced commissural neurons in layer III of the right frontocentral cortex were quantified in 3-, 5-, 11- and 15-month-old mice. Three different types of commissural neurons were traced. At 3 months of age no differences in the number of labelled commissural neurons were seen in APP23 mice compared with wild-type mice. A selective reduction of the heavily ramified type of neurons was observed in APP23 mice compared with wild-type animals at 5, 11 and 15 months of age, starting when the first Aβ-deposits occurred in the frontocentral cortex at 5 months. The other two types of commissural neurons did not show alterations at 5 and 11 months. At 15 months, the number of traced sparsely ramified pyramidal neurons was reduced in addition to that of the heavily ramified neurons in APP23 mice compared with wild-type mice. At this time Aβ-deposits were seen in the neo- and allocortex as well as in the basal ganglia and the thalamus. In summary, our results show that Aβ induces progressive degeneration of distinct types of commissural neurons. Degeneration of the most vulnerable neurons starts in parallel with the occurrence of the first fibrillar Aβ-deposits in the neocortex, that is, with the detection of aggregated Aβ. The involvement of additional neuronal subpopulations is associated with the expansion of Aβ-deposition into further brain regions. The vulnerability of different types of neurons to Aβ, thereby, is presumably related to the complexity of their dendritic morpholog

New, Fully Implantable Device for Selective Clearance of CSF-Target Molecules: Proof of Concept in a Murine Model of Alzheimer’s Disease

[EN] We have previously proposed a radical change in the current strategy to clear pathogenic proteins from the central nervous system (CNS) based on the cerebrospinal fluid (CSF)-sink therapeutic strategy, whereby pathogenic proteins can be removed directly from the CNS via CSF. To this aim, we designed and manufactured an implantable device for selective and continuous apheresis of CSF enabling, in combination with anti-amyloid-beta (Aβ) monoclonal antibodies (mAb), the clearance of Aβ from the CSF. Here, we provide the first proof of concept in the APP/PS1 mouse model of Alzheimer’s disease (AD). Devices were implanted in twenty-four mice (seventeen APP/PS1 and seven Wt) with low rates of complications. We confirmed that the apheresis module is permeable to the Aβ peptide and impermeable to mAb. Moreover, our results showed that continuous clearance of soluble Aβ from the CSF for a few weeks decreases cortical Aβ plaques. Thus, we conclude that this intervention is feasible and may provide important advantages in terms of safety and efficacy.This work was supported by the Instituto de Salud Carlos III, under Grant DTS19-00071 to M.M.-G. and by the Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT), under Grant AYUD/2021/57540, to C.T.-Z

Amyloid β / PKC-dependent alterations in NMDA receptor composition are detected in early stages of Alzheimer´s disease

[EN] Amyloid beta (Abeta)-mediated synapse dysfunction is an early event in Alzheimer's disease (AD) pathogenesis and previous studies suggest that NMDA receptor (NMDAR) dysregulation may contribute to these pathological effects. Although Abeta peptides impair NMDAR expression and activity, the mechanisms mediating these alterations in the early stages of AD are unclear. Here, we observed that NMDAR subunit NR2B and PSD-95 levels were aberrantly upregulated and correlated with Abeta42 load in human postsynaptic fractions of the prefrontal cortex in early stages of AD patients, as well as in the hippocampus of 3xTg-AD mice. Importantly, NR2B and PSD95 dysregulation was revealed by an increased expression of both proteins in Abeta-injected mouse hippocampi. In cultured neurons, Abeta oligomers increased the NR2B-containing NMDAR density in neuronal membranes and the NMDA-induced intracellular Ca2+ increase, in addition to colocalization in dendrites of NR2B subunit and PSD95. Mechanistically, Abeta oligomers required integrin beta1 to promote synaptic location and function of NR2B-containing NMDARs and PSD95 by phosphorylation through classic PKCs. These results provide evidence that Abeta oligomers modify the contribution of NR2B to NMDAR composition and function in the early stages of AD through an integrin beta1 and PKC-dependent pathway. These data reveal a novel role of Abeta oligomers in synaptic dysfunction that may be relevant to early-stage AD pathogenesis.We thank S. Marcos, L. Escobar, A Martínez and Z. Martínez for technical assistance. This study was supported by the Basque Government (IT1203-19; PIBA_2020_1_0012; ELKARTEK KK-2020/00034; fellowship to T.Q-L, U.B. and J.Z-I), University of the Basque Country (UPV/EHU; fellowship to C.O-S) CIBERNED, MICINN (PID2019-108465RB-I00) and Fundación Tatiana Pérez de Guzmán el Bueno (fellowship to C.L)

Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.E.A. was supported by MICINN (PID2019-108465RB-I00) and Basque Government (PIBA_2020_1_0012). C.M. was supported by MICINN (PID2019-109724RB-I00), Basque Government (IT1203-19) and CIBERNED (CB06/0005/0076). E.C.-Z. was supported by Basque Government (ELKARTEK KK-2020/00034; PIBA_2016_1_0009). J.L.Z. was supported by the Instituto de Salud Carlos III (PI18/00207), Basque Government (PIBA_2020_1_0048) and University of Basque Country Grant (US19/04)

Longitudinal evaluation of neuroinflammation and oxidative stress in a mouse model of Alzheimer disease using positron emission tomography

[EN] Background: Validation of new biomarkers of Alzheimer disease (AD) is crucial for the successful development and implementation of treatment strategies. Additional to traditional AT(N) biomarkers, neuroinflammation biomarkers, such as translocator protein (TSPO) and cystine/glutamine antiporter system (x(c)(-)), could be considered when assessing AD progression. Herein, we report the longitudinal investigation of [F-18]DPA-714 and [F-18]FSPG for their ability to detect TSPO and x(c)(-) biomarkers, respectively, in the 5xFAD mouse model for AD. Methods: Expression of TSPO and x(c)(-) system was assessed longitudinally (2-12 months of age) on 5xFAD mice and their respective controls by positron emission tomography (PET) imaging using radioligands [F-18]DPA-714 and [F-18]FSPG. In parallel, in the same mice, amyloid-beta plaque deposition was assessed with the amyloid PET radiotracer [F-18]florbetaben. In vivo findings were correlated to ex vivo immunofluorescence staining of TSPO and x(c)(-) in microglia/macrophages and astrocytes on brain slices. Physiological changes of the brain tissue were assessed by magnetic resonance imaging (MRI) in 12-month-old mice. Results: PET studies showed a significant increase in the uptake of [F-18]DPA-714 and [F-18]FSPG in the cortex, hippocampus, and thalamus in 5xFAD but not in WT mice over time. The results correlate with A beta plaque deposition. Ex vivo staining confirmed higher TSPO overexpression in both, microglia/macrophages and astrocytes, and overexpression of x(c)(-) in non-glial cells of 5xFAD mice. Additionally, the results show that A beta plaques were surrounded by microglia/macrophages overexpressing TSPO. MRI studies showed significant tissue shrinkage and microstructural alterations in 5xFAD mice compared to controls. Conclusions: TSPO and x(c)(-) overexpression can be assessed by [F-18]DPA-714 and [F-18]FSPG, respectively, and correlate with the level of A beta plaque deposition obtained with a PET amyloid tracer. These results position the two tracers as promising imaging tools for the evaluation of disease progression.J.L. and P.R. thank the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 (PID2020-117656RB-100 and PID2020-118546RBI00, respectively) and the Interreg Atlantic Area Programme (EAPA_791/2018). Abraham Martin acknowledges funding from the Spanish Ministry of Education and Science (RYC-2017-22412, PID2019-107989RB-I00), the Basque Government (BIO18/IC/006), and Fundacio La Marato de TV3 (17/C/2017). Estibaliz Capetillo-Zarate acknowledges funding from the Basque Government (IT120319; ELKARTEK KK-2020/00034) and CIBERNED (CB06/0005/0076). The work was performed under the Maria de Maeztu Units of Excellence Programme -Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/50110001103