The ability to thoroughly investigate tau mRNA splicing and expression has been previously hindered by a lack of reliable tools for assessing tau mRNA through a histological lens. Since the majority of PSP cases are sporadic, there is an urgent need for a model system to study genetic risk factors, such as MAPT haplotypes. HiPSC models have emerged as a powerful tool to model and study human disease. The use of patient skin samples to derive hiPSCs is valuable because they can be reprogrammed into any disease-relevant cell type that retains the patient's genetic background, and autopsy-confirmed cases for hiPSC generation are crucial because any results can be validated. Many studies utilized 2D hiPSC-derived neuronal models, but these are limited in the capacity to recapitulate cell-to-cell interactions in the brain. A 3D culture system allows researchers to essentially derive brain tissue in vitro and has numerous applications for disease models and drug discovery. Organoids are unique because they contain many cell types and are capable of more complex patterning and layering than 2D cell cultures. 3D cell morphology is preserved within the organoids and differentiation occurs in a less directed, more self-organized manner than in 2D cell cultures. Organoids are grown in suspension, resulting in proliferation and growth rates that closely mimic human development, rather than 2D cell cultures grown in a monolayer attached to cell culture plates. Cerebral organoids can be generated to model multiple brain regions, including the forebrain and midbrain by distinct pharmacological differentiation protocols. An additional consideration for generating cell models is experimental proficiency and replicability. Suspension spinner flasks have the capacity to generate thousands of organoids per flask which are less variable than typical 2D cell culture methods. HiPSC-derived organoids have the potential to bridge the gap between 2D cell cultures and animal model studies in the future. In the context of neurodegenerative diseases, this gap poses a major barrier to understanding both the molecular mechanisms by which neurodegeneration occurs, as well as possible genetic drivers of protein upregulation that results in toxic accumulation. Organoid models have the potential to more closely recapitulate the cellular environment in which abnormal protein accumulation and degeneration occur in neurodegenerative diseases.
JADE1S protein interacts with tau containing four microtubule-binding domain repeats (4R) but not 3R in post-mortem human brain tissue. (A) Schematic of the two JADE1 isoforms, JADE1S and JADE1L. (B) Representative immunoblot using antisera targeting JADE1 in entorhinal cortex and cornu ammonis in individuals with primary age-related tauopathy (PART) and Alzheimer disease (AD) shows JADE1S but not JADE1L at the expected molecular weight. GAPDH was used as a loading standard. (C) Immunoprecipitation using JADE1 antisera co-immunoprecipitates tau the with a molecular weight near the 0N4R isoform (40 kDa). (D) Reverse immunoprecipitation using 0N tau antisera co-immunoprecipitates the JADE1S isoform. e Pulled down form of JADE1S molecular weight shifts downward after treatment with lambda protein phosphatase.
(F and G) Quantitative immunoblot analysis using PC12 cells transduced with the lentiviral miR-219 vector revealed reduced tau protein levels compared with those detected in the scrambled miRNA control when differentiated for 7 days. (H) qPCR showed reduced tau mRNA levels in PC12 cells transduced with the lentiviral miR-219 vector. (I) Immunofluorescence microscopy using tauC (red) showed neurite outgrowth in untransduced PC12 cells, but cells transduced with lentiviral miR-219 (green) failed to extend neurites, as judged by the merged image (lower panel; yellow). (J) Quantification of neurite extension revealed a significant decrease in the numbers of tau-positive neurites in cells transduced with miR-219 compared with that observed in untransduced cells. Data are representative of 3 experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by 2-tailed Student’s t test.
MOLECULAR & CELLULAR NEURODEGENERATION
miR-219 directly regulates tau expression and attenuates neurite outgrowth in mammalian cell cultures. (A) Cotransfection of human neuroblastoma cells (SH-SY5Y) with a miR-219 mimic and a dual-luciferase human tau 3′-UTR reporter demonstrated reduced expression compared with that seen in the scrambled miRNA control. Mutagenesis of the miR-219 recognition element abrogated silencing. (B) Immunofluorescence on rat pheochromocytoma (PC12) cells using antisera targeting total tau (tauC) showed extension of processes following treatment with 100 nM NGF. Scale bar: 25 μm. (C) Immunoblot using tauC demonstrated increased tau protein levels following NGF treatment. (D) An increase in tau mRNA levels was observed following NGF treatment. (E) A transient decrease in miR-219 levels (normalized to SNORD24) occurred 3 days after NGF treatment and returned to baseline levels by day 7.