INTRODUCTION: The Pathogen Infection Hypothesis proposes that β-Amyloid (Aβ) functions as an antimicrobial peptide, with pathogen-induced aggregation potentially contributing to Alzheimer's disease (AD) pathology. METHODS: We used human iPSC-derived 2D neurons and 3D cerebral organoids from wild-type and familial AD (PSEN1/2 mutant) lines to model acute infections with HSV-1 and TBEV and Aβ aggregation. Transcriptomic and proteomic analyses were conducted to assess molecular responses. RESULTS: HSV-1, but not TBEV, induced robust Aβ clustering, which was, however, dependent on extracellular amyloid peptides. Transcriptomic profiling revealed widespread HSV-1-induced changes, including activation of neurodegeneration-related pathways. Proteomic profiling confirmed enrichment of neurodegeneration- and senescence-associated secretome signatures. PSEN1/2 mutations did not alter the acute infection response. Reanalysis of independent datasets confirmed our findings and revealed a limited protective effect of acyclovir. DISCUSSION: Results directly support the Pathogen Infection Hypothesis and suggest that preventing viral infections via vaccinations may represent a feasible approach to reducing AD risk.

• Keywords
• Alzheimer’s disease, Cerebral organoids, Herpes virus, Senescence, Tick-borne Encephalitis,
• Publication type
• Journal Article MeSH
• Preprint MeSH

Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.

• Keywords
• 3Fn-domain, SORL1-associated Alzheimer’s disease, SORLA, dimerization, retromer,
• MeSH
• Alzheimer Disease * genetics   metabolism   pathology   MeSH
• Endosomes * metabolism   MeSH
• HEK293 Cells MeSH
• Middle Aged MeSH
• Humans MeSH
• Membrane Transport Proteins * genetics   metabolism   MeSH
• Mutation, Missense MeSH
• Protein Multimerization MeSH
• LDL-Receptor Related Proteins * genetics   metabolism   MeSH
• Pedigree * MeSH
• Aged MeSH
• Protein Transport MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Transport Proteins * MeSH
• LDL-Receptor Related Proteins * MeSH
• SORL1 protein, human MeSH Browser

Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.

• MeSH
• Centrioles metabolism   MeSH
• Cilia metabolism   MeSH
• Humans MeSH
• Pluripotent Stem Cells * metabolism   MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Hedgehog Proteins * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Serine-Threonine Kinases MeSH
• Hedgehog Proteins * MeSH
• tau-tubulin kinase MeSH Browser

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

• Keywords
• 3-sulfopropanoic acid, Aggregation, Alzheimer’s disease, Apolipoprotein E, Cerebral organoids, HDX-MS, Lipidomics, Molecular dynamics, Neurodegeneration, Protein crystallography, Proteomics, Tramiprosate,
• MeSH
• Alzheimer Disease * drug therapy   genetics   metabolism   MeSH
• Apolipoprotein E3 genetics   MeSH
• Apolipoprotein E4 * genetics   metabolism   MeSH
• Apolipoproteins E genetics   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Apolipoprotein E3 MeSH
• Apolipoprotein E4 * MeSH
• Apolipoproteins E MeSH
• tramiprosate MeSH Browser

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

• Keywords
• GLICO, cerebral organoids, glioblastoma, induced pluripotent stem cells,
• MeSH
• Cell Culture Techniques methods   MeSH
• Cell Line MeSH
• Glioblastoma * genetics   metabolism   MeSH
• Humans MeSH
• Brain MeSH
• Tumor Microenvironment MeSH
• Organoids metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Many dynamic interactions within the cell microenvironment modulate cell behavior and cell fate. However, the pathways and mechanisms behind cell-cell or cell-extracellular matrix interactions remain understudied, as they occur at a nanoscale level. Recent progress in nanotechnology allows for mimicking of the microenvironment at nanoscale in vitro; electron-beam lithography (EBL) is currently the most promising technique. Although this nanopatterning technique can generate nanostructures of good quality and resolution, it has resulted, thus far, in the production of only simple shapes (e.g., rectangles) over a relatively small area (100 × 100 μm), leaving its potential in biological applications unfulfilled. Here, we used EBL for cell-interaction studies by coating cell-culture-relevant material with electron-conductive indium tin oxide, which formed nanopatterns of complex nanohexagonal structures over a large area (500 × 500 μm). We confirmed the potential of EBL for use in cell-interaction studies by analyzing specific cell responses toward differentially distributed nanohexagons spaced at 1000, 500, and 250 nm. We found that our optimized technique of EBL with HaloTags enabled the investigation of broad changes to a cell-culture-relevant surface and can provide an understanding of cellular signaling mechanisms at a single-molecule level.

• Keywords
• biomimetic surface, cell adhesion and spreading, cell−cell interaction, electron-beam lithography, ligand clustering, nanopatterning, nanospacing,
• MeSH
• Cell Differentiation MeSH
• Cell Culture Techniques MeSH
• Extracellular Matrix MeSH
• Nanostructures * chemistry   MeSH
• Nanotechnology * methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer's disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery.

• Keywords
• Alzheimer’s disease, Astrocytes, Cerebral organoids, In vitro differentiation, Microglia, Neural differentiation, Neural progenitors, Neural stem cells, Neurons, iPSCs,
• MeSH
• Alzheimer Disease * genetics   metabolism   therapy   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Neural Stem Cells * metabolism   MeSH
• Neurons metabolism   MeSH
• Organoids pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The 'gold standard' treatment of severe neonatal jaundice is phototherapy with blue-green light, which produces more polar photo-oxidation products that are easily excreted via the bile or urine. The aim of this study was to compare the effects of bilirubin (BR) and its major photo-oxidation product lumirubin (LR) on the proliferation, differentiation, morphology, and specific gene and protein expressions of self-renewing human pluripotent stem cell-derived neural stem cells (NSC). Neither BR nor LR in biologically relevant concentrations (12.5 and 25 µmol/L) affected cell proliferation or the cell cycle phases of NSC. Although none of these pigments affected terminal differentiation to neurons and astrocytes, when compared to LR, BR exerted a dose-dependent cytotoxicity on self-renewing NSC. In contrast, LR had a substantial effect on the morphology of the NSC, inducing them to form highly polar rosette-like structures associated with the redistribution of specific cellular proteins (β-catenin/N-cadherin) responsible for membrane polarity. This observation was accompanied by lower expressions of NSC-specific proteins (such as SOX1, NR2F2, or PAX6) together with the upregulation of phospho-ERK. Collectively, the data indicated that both BR and LR affect early human neurodevelopment in vitro, which may have clinical relevance in phototherapy-treated hyperbilirubinemic neonates.

• Keywords
• bilirubin, neurodevelopment, phototherapy,
• Publication type
• Journal Article MeSH