To study the cellular and molecular events

To study the cellular and molecular events of FTD3, we derived a disease model using human iPSCs from patients carrying the 31449G > C mutation in CHMP2B and isogenic controls generated via the CRISPR/Cas9 system with subsequent in vitro neuronal differentiation. Our model not only confirmed partly prior-observed pathologies in animal models such as endosome dysregulation (Ghazi-Noori et al., 2012), but also revealed additional disease-relevant phenotypes in FTD3 by way of abnormal mitochondrial ultrastructure and function. Moreover, RNA sequencing (RNA-seq) uncovered that the CHMP2B mutation manifests in dysregulated peptide yy of key genes associated with Parkinson’s disease (PD) and Alzheimer’s disease (AD) and indicated that perurbed iron homeostasis is one of the underlying disease mechanisms.
Results
Discussion We identified enlargements of particularly the early endosome population in FTD3 neurons, indicating that perturbed endo-lysosomal fusion and degradation of substrates is key to the underlying FTD3 pathophysiology. Cellular components, including mitochondria, are degraded in the endosome; in amyotrophic lateral sclerosis, a progressive lysosomal deficit is causative for impaired degradation of damaged mitochondria in motor neurons (Xie et al., 2015). Defective recycling of large organelles, such as mitochondria, is the earliest detectable impairment in a chain of events initiated by defective recycling (Cannizzo et al., 2012), which clearly links endosome and mitochondrial dysfunction. Strikingly, in FTD3 neurons we identified a robust, significant upregulation of LRRK2 encoding the dardarin protein. One function of dardarin is the modulation of intracellular trafficking via recruitment of RAB7 (Greggio et al., 2006). Mutant CHMP2B impairs the recruitment of RAB7, crucial for endosome-lysosome fusion (Urwin et al., 2010). This relationship between CHMP2B, RAB7, and dardarin is supported by studies showing that dardarin negatively regulates RAB7-dependent localization of the lysosome (Dodson et al., 2012). Therefore, upregulation of LRRK2 as a consequence of CHMP2B mutation might contribute to deficient endo-lysosomal trafficking and fusion. Dardarin also plays a role in mitochondrial functionality and positioning, since induced expression of wild-type LRRK2 induces mitochondrial fragmentation via increased fission and upregulation of DLP1, which is critical for correct mitochondrial distribution (Wang et al., 2012). Furthermore, our analysis revealed APOE to be significantly downregulated in FTD3 neural cells. The cells analyzed via RNA-seq are a mixed population of neurons and glial cells, with an up to 90% enrichment of VGlut1+ neurons. Due to this mixed population of cells we were still able to detect APOE via RNA-seq, even though it is mainly expressed in astrocytes. APOE is a major cholesterol carrier in neurons that supports lipid transport and brain injury repair, and there is a key requirement for APOE in facilitating the clearance of soluble amyloid beta (Abeta) (Jiang et al., 2008). Furthermore, APOE suppresses microglia activation (Laskowitz et al., 2001), and reduced APOE levels might potentially trigger inflammatory events. Together, this suggests that downregulation of APOE in FTD3 neurons might be a contributing factor in the microglia activation seen in postmortem FTD3 brains. Interestingly, we observed another phenotype restricted to glial cells. It has previously been reported for FTD3 mouse models and patient brains that these display p62 inclusions, which are indicative for impaired autophagy, oxidative stress, and neurotoxicity (Ghazi-Noori et al., 2012; Holm et al., 2007). We identified these p62 inclusions only in the S100β+ glial cell population and not in our TUJ1+ neurons (Figure S4B). Both the APOE and the p62 phenotypes clearly show that, even though several disease features can be replicated in our FTD3 neurons, other neural cell types need to be investigated in the future to understand the full complexity and pathophysiology of the disease.