Rapid and Robust Multi-Phenotypic Assay System for ALS Using Human iPS Cells with Mutations in Causative Genes.

Amyotrophic lateral sclerosis (ALS) is a major life-threatening disease caused by motor neuron degeneration. More effective treatments through drug discovery are urgently needed. Here, we established an effective high-throughput screening system using induced pluripotent stem cells (iPSCs). Using a Tet-On-dependent transcription factor expression system carried on the PiggyBac vector, motor neurons were efficiently and rapidly generated from iPSCs by a single-step induction method. Induced iPSC transcripts displayed characteristics similar to those of spinal cord neurons. iPSC-generated motor neurons carried a mutation in fused in sarcoma (FUS) and superoxide dismutase 1 (SOD1) genes and had abnormal protein accumulation corresponding to each mutation. Calcium imaging and multiple electrode array (MEA) recordings demonstrated that ALS neurons were abnormally hyperexcitable. Noticeably, protein accumulation and hyperexcitability were ameliorated by treatment with rapamycin (mTOR inhibitor) and retigabine (Kv7 channel activator), respectively. Furthermore, rapamycin suppressed ALS neuronal death and hyperexcitability, suggesting that protein aggregate clearance through the activation of autophagy effectively normalized activity and improved neuronal survival. Our culture system reproduced several ALS phenotypes, including protein accumulation, hyperexcitability, and neuronal death. This rapid and robust phenotypic screening system will likely facilitate the discovery of novel ALS therapeutics and stratified and personalized medicine for sporadic motor neuron diseases.

Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid ß peptide using CRISPR/Cas9-based screening system.

The aberrant metabolism of amyloid ß peptide (Aß) has been implicated in the etiology of Alzheimer disease (AD). Aß is produced via the sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. However, the precise regulatory mechanism of Aß generation still remains unclear. To gain a better understanding of the molecular mechanism of Aß production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aß production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aß production. The disruption of Cib1 significantly upregulated Aß levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aß production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.

Retrograde transport of γ-secretase from endosomes to the trans-Golgi network regulates Aß42 production.

The aberrant metabolism of amyloid-ß protein (Aß) in the human brain has been implicated in the etiology of Alzheimer disease (AD). γ-Secretase is the enzyme that generates various forms of Aß, such as Aß40 and Aß42, the latter being an aggregation-prone toxic peptide that is involved in the pathogenesis of AD. Recently, we found that clathrin-mediated endocytosis of γ-secretase affects the production and deposition of Aß42 in vivo, suggesting that the membrane trafficking of γ-secretase affects its enzymatic activity. However, the detailed intracellular trafficking pathway of γ-secretase and its contribution to Aß42 generation remain unclear. Here, we show that Retro-2, which inhibits the retrograde transport, elevated the Aß42-generating activity both in cultured cells and mice brain. However, the result of in vitro γ-secretase assay using a recombinant substrate suggested that Retro-2 did not elevate the intrinsic Aß42-production activity of γ-secretase. Immunocytochemistry and cell-surface biotinylation experiments revealed that γ-secretase is recycled via the endosome-to-trans-Golgi network transport. In addition, γ-secretase is retrogradely transported by syntaxin 5/6, known as targets of Retro-2, independent pathway. Conversely, TPT-260, which enhances the trafficking function of retromers, lowered Aß42 levels and the Aß42/(Aß40 + Aß42) ratio in secreted Aß from cultured cells. Our results strongly suggest that the endosome-to-trans-Golgi network trafficking of γ-secretase regulates its Aß42 production activity. Modulation of this trafficking pathway might be a potential target for the development of Aß42-lowering AD therapeutics. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

BIN1 regulates BACE1 intracellular trafficking and amyloid-ß production.

BIN1 is a genetic risk factor of late-onset Alzheimer disease (AD), which was identified in multiple genome-wide association studies. BIN1 is a member of the amphiphysin family of proteins, and contains N-terminal Bin-Amphiphysin-Rvs and C-terminal Src homology 3 domains. BIN1 is widely expressed in the mouse and human brains, and has been reported to function in the endocytosis and the endosomal sorting of membrane proteins. BACE1 is a type 1 transmembrane aspartyl protease expressed predominantly in neurons of the brain and responsible for the production of amyloid-ß peptide (Aß). Here we report that the depletion of BIN1 increases cellular BACE1 levels through impaired endosomal trafficking and reduces BACE1 lysosomal degradation, resulting in increased Aß production. Our findings provide a mechanistic role of BIN1 in the pathogenesis of AD as a novel genetic regulator of BACE1 levels and Aß production.

FTY720/fingolimod, a sphingosine analogue, reduces amyloid-ß production in neurons.

Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-ß peptide (Aß), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aß decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aß40, but an increase in Aß42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aß metabolisms that are active in vitro and in vivo.