Amyloid-ß precursor protein processing and oxidative stress are altered in human iPSC-derived neuron and astrocyte co-cultures carrying presenillin-1 gene mutations following spontaneous differentiation.

INTRODUCTION: Presenilin-1 (PSEN1) gene mutations are the most common cause of familial Alzheimer's disease (fAD) and are known to interfere with activity of the membrane imbedded γ-secretase complex. PSEN1 mutations have been shown to shift Amyloid-ß precursor protein (AßPP) processing toward amyloid-ß (Aß) 1-42 production. However, less is known about whether PSEN1 mutations may alter the activity of enzymes such as ADAM10, involved with non-amyloidogenic AßPP processing, and markers of oxidative stress. MATERIALS AND METHODS: Control and PSEN1 mutation (L286V and R278I) Human Neural Stem Cells were spontaneously differentiated into neuron and astrocyte co-cultures. Cell lysates and culture media were collected and stored at -80 °C until further analysis. ADAM10 protein expression, the ratio of AßPP forms and Aß1-42/40 were assessed. In addition, cellular redox status was quantified. RESULTS: The ratio of AßPP isoforms (130:110kDa) was significantly reduced in neuron and astrocyte co-cultures carrying PSEN1 gene mutations compared to control, and mature ADAM10 expression was lower in these cells. sAßPP-α was also significantly reduced in L286V mutation, but not in the R278I mutation cells. Both Aß1-40 and Aß1-42 were increased in conditioned cell media from L286V cells, however, this was not matched in R278I cells. The Aß1-42:40 ratio was significantly elevated in R278I cells. Markers of protein carbonylation and lipid peroxidation were altered in both l286V and R278I mutations. Antioxidant status was significantly lower in R278I cells compared to control cells. CONCLUSIONS: This data provides evidence that the PSEN1 mutations L286V and R278I significantly alter protein expression associated with AßPP processing and cellular redox status. In addition, this study highlights the potential for iPSC-derived neuron and astrocyte co-cultures to be used as an early human model of fAD.

Astrocyte dysfunction and neuronal network hyperactivity in a CRISPR engineered pluripotent stem cell model of frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro FTD disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of FTD astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.