Altered amyloid-ß structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion.

Deposition of amyloid beta (Aß) into plaques is a major hallmark of Alzheimer's disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aß. We recently identified the Uppsala APP mutation (APPUpp), which causes Aß pathology by a triple mechanism: increased ß-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aß conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aß pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aß pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased ß-secretase cleavage and suppressed α-secretase cleavage, resulting in AßUpp42 dominated diffuse plaque pathology appearing from the age of 5-6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [11C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aß pathology in all models, whereas the Aß protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aß pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AßUpp42 aggregates were found to affect their interaction with anti-Aß antibodies and profoundly modify the Aß-mediated glial response, which may be important aspects to consider for further development of AD therapies.

Astrocytic uptake of neuronal corpses promotes cell-to-cell spreading of tau pathology.

Tau deposits in astrocytes are frequently found in Alzheimer's disease (AD) and other tauopathies. Since astrocytes do not express tau, the inclusions have been suggested to be of neuronal origin. However, the mechanisms behind their appearance and their relevance for disease progression remain unknown. Here we demonstrate, using a battery of experimental techniques that human astrocytes serve as an intermediator, promoting cell-to-cell spreading of pathological tau. Human astrocytes engulf and process, but fail to fully degrade dead neurons with tau pathology, as well as synthetic tau fibrils and tau aggregates isolated from AD brain tissue. Instead, the pathogenic tau is spread to nearby cells via secretion and tunneling nanotube mediated transfer. By performing co-culture experiments we could show that tau-containing astrocytes induce tau pathology in healthy human neurons directly. Furthermore, our results from a FRET based seeding assay, demonstrated that the tau proteoforms secreted by astrocytes have an exceptional seeding capacity, compared to the original tau species engulfed by the cells. Taken together, our study establishes a central role for astrocytes in mediating tau pathology, which could be of relevance for identifying novel treatment targets for AD and other tauopathies.

Long-term effects of amyloid-beta deposits in human iPSC-derived astrocytes.

Growing evidence indicates that astrocytes are tightly connected to Alzheimer's disease (AD) pathogenesis. However, the way in which astrocytes participate in AD initiation and progression remains to be clarified. Our previous data show that astrocytes engulf large amounts of aggregated amyloid-beta (Aß) but are unable to successfully degrade the material. In this study, we aimed to evaluate how intracellular Aß-accumulation affects the astrocytes over time. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aß-fibrils and then cultured further for one week or ten weeks in Aß-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the media were screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy. Our data demonstrate that long-term astrocytes retained frequent Aß-inclusions that were enclosed within LAMP1-positive organelles and sustained markers associated with reactivity. Furthermore, Aß-accumulation resulted in endoplasmic reticulum and mitochondrial swelling, increased secretion of the cytokine CCL2/MCP-1 and formation of pathological lipid structures. Taken together, our results provide valuable information of how intracellular Aß-deposits affect astrocytes, and thereby contribute to the understanding of the role of astrocytes in AD progression.

Intracellular deposits of amyloid-beta influence the ability of human iPSC-derived astrocytes to support neuronal function.

BACKGROUND: Astrocytes are crucial for maintaining brain homeostasis and synaptic function, but are also tightly connected to the pathogenesis of Alzheimer's disease (AD). Our previous data demonstrate that astrocytes ingest large amounts of aggregated amyloid-beta (Aß), but then store, rather than degrade the ingested material, which leads to severe cellular stress. However, the involvement of pathological astrocytes in AD-related synaptic dysfunction remains to be elucidated. METHODS: In this study, we aimed to investigate how intracellular deposits of Aß in astrocytes affect their interplay with neurons, focusing on neuronal function and viability. For this purpose, human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Αß42 fibrils. The direct and indirect effects of the Αß-exposed astrocytes on hiPSC-derived neurons were analyzed by performing astrocyte-neuron co-cultures as well as additions of conditioned media or extracellular vesicles to pure neuronal cultures. RESULTS: Electrophysiological recordings revealed significantly decreased frequency of excitatory post-synaptic currents in neurons co-cultured with Aß-exposed astrocytes, while conditioned media from Aß-exposed astrocytes had the opposite effect and resulted in hyperactivation of the synapses. Clearly, factors secreted from control, but not from Aß-exposed astrocytes, benefited the wellbeing of neuronal cultures. Moreover, reactive astrocytes with Aß deposits led to an elevated clearance of dead cells in the co-cultures. CONCLUSIONS: Taken together, our results demonstrate that inclusions of aggregated Aß affect the reactive state of the astrocytes, as well as their ability to support neuronal function.

CRISPR-Cas9 treatment partially restores amyloid-ß 42/40 in human fibroblasts with the Alzheimer's disease PSEN 1 M146L mutation.

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-ß (Aß) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aß42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1 M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aß42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1 M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

Life cycle assessment during packaging of market-sized seabass and meagre: necessary adaptations toward GHG neutrality.

PURPOSE: Fish is a delicate and valuable source of protein, and aquaculture is expected to provide the required amount of fish needed at reasonable prices. Packaging is a vital stage to preserve hygiene, quality and freshness of aquaculture products. The purpose of this study was to identify environmental hotspots in farmed seabass and meagre during the packaging stage and to examine scenarios for reducing environmental impacts, aiming at greenhouse gas neutrality. METHODS: The life cycle assessment method was applied for the first time at three packaging plants of farmed seabass and meagre in western Greece, in order to assess their environmental impacts, taking into account the amount of electricity, expanded polystyrene, and other plastic and packaging materials needed. Moreover, different scenarios were designed involving the energy mix/alternative sources, recycling and reuse of packaging materials, and the replacement of standard polystyrene boxes and wooden pallets, in order to suggest environmentally friendly improvements in the packaging process. RESULTS AND DISCUSSION: Conventional operation of seabass and meagre packaging plants requires significant amounts of energy and packaging materials. In fact, electricity, boxes and pallets were documented as the main contributors to most of the environmental impact indicators. Seabass packaging had a slightly lower overall environmental impact compared to meagre, due to its smaller market size. Impact minimization scenarios resulted in various degrees of reduction of environmental burdens in both species; however, near-zero GHG emissions were achieved when energy from photovoltaic panels and recycling/reuse of packaging materials were combined with the use of non-fossil-based boxes and recyclable pallets. Such readily applicable adjustments to the conventional operation of packaging plants would contribute to overall environmental sustainability and to better consumer acceptability of the final product. CONCLUSIONS: In a nutshell, the life cycle assessment method proved to be a valuable tool for assessing the environmental performance of Mediterranean aquaculture packaging processes. Moreover, it served to identify critical factors directly related to the EU GHG neutrality target. Accordingly, environmentally friendly decision-making/strategic management in the farmed fish packaging sector will inevitably rely on alternative energy sources, recycling of packaging materials, and use of recyclable corrugated board boxes or similar non-fossil-based materials.

A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

Pattern discovery for microsatellite genome analysis.

Microsatellite loci comprise an important part of eukaryotic genomes. Their applications in biology as genetic markers are related to numerous fields ranging from paternity analyses to construction of genetic maps and linkage to human disease. Existing software solutions which offer pattern discovery algorithms for the correct identification and downstream analysis of microsatellites are scarce and are proving to be inefficient to analyze large, exponentially increasing, sequenced genomes. Moreover, such analyses can be very difficult for bioinformatically inexperienced biologists. In this paper we present Microsatellite Genome Analysis (MiGA) software for the detection of all microsatellite loci in genomic data through a user friendly interface. The algorithm searches exhaustively and rapidly for most microsatellites. Contrary to other applications, MiGA takes into consideration the following three most important aspects: the efficiency of the algorithm, the usability of the software and the plethora of offered summary statistics. All of the above, help biologists to obtain basic quantitative and qualitative information regarding the presence of microsatellites in genomic data as well as downstream processes, such as selection of specific microsatellite loci for primer design and comparative genome analysis.