Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology

Abstract

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.

Modeling and Targeting Neuroglial Interactions with Human Pluripotent Stem Cell Models

“…These methods summarize the different steps of human cortical development, such as neurogenesis, gene expression profile, and regional and layer organization [172], and new protocols to produce myelinating oligodendrocytes, cortical neurons, and astrocytes have been recently released [173]. Organoids have successfully modeled CNS disorders, such as autosomal recessive primary microcephaly, brain infection by Zika virus, prenatal cocaine or nicotine exposure, or neonatal hypoxic stress [174], or recently traumatic brain injury [175] or ALS overlapping with frontotemporal dementia [176].…”

Functional Characterization of Human Pluripotent Stem Cell-Derived Models of the Brain with Microelectrode Arrays

“…While the approach is valid and can provide a view of the network activity of the culture, the full benefit of having a 3D model is not achieved as the MEA can only provide data from a single 2D plane. It is also possible to slice an organoid for the recordings ( Figure 2 ) [ 208 , 209 , 210 ] but this is hardly helpful if the goal is to study an intact 3D network from multiple 2D planes. Furthermore, slicing a small organoid and handling the delicate slices ( Figure 2 ) requires specific protocols that include embedding the organoid in an agarose block for slicing and applying sufficient recovery periods before recording.…”

“…Furthermore, slicing a small organoid and handling the delicate slices ( Figure 2 ) requires specific protocols that include embedding the organoid in an agarose block for slicing and applying sufficient recovery periods before recording. The electrodes of certain arrays do penetrate the sample [ 69 , 208 , 210 ] ( Figure 2 ), which in itself is a distinct advantage as the dead cell layer on the surface of the sliced sample is overcome, but measurements are still made only from a single 2D plane.…”

“…Additionally, the cortical structure is otherwise challenging, as while organoids can develop rudimentary architecture and markers of cortical layers, they still lack the fine architecture of all six layers of the cortex. However, slicing forebrain organoids to 500 μm sections every four weeks was shown to promote the organization of layer-specific markers to resemble the proper layer structure when the sliced organoids were cultivated for more than 100 days [ 209 , 210 ]. Furthermore, a four-shank multi-electrode laminar probe recorded spontaneous firing of EAPs (SUA) and synchronous bursts, showing network activity across long distances both vertically and horizontally in the layer-like structures [ 209 ].…”

“…The above-mentioned repetitive organoid slicing is one option for allowing oxygen and nutrients to reach the whole culture and preventing necrosis [ 209 ]. Similar to classic organotypic cultures, the slices can also be maintained on membranes in air–liquid interfaces, which removes the need for repeating the slicing [ 208 , 210 ]. The membrane-grown slices can be transferred to MEA where they display firing and synchronized bursts.…”