Establishment of DYT5 patient-specific induced pluripotent stem cells with a GCH1 mutation.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 20-year-old dystonia patient with a GCH1 mutation (DYT5). Episomal vectors were used to introduce reprogramming factors (OCT3/4, SOX2, KLF4, L-MYC, LIN28, and p53 carboxy-terminal dominant-negative fragment) to the PBMCs. The generated iPSCs expressed pluripotency markers, and were capable of differentiating into derivates of all three germ layers in vitro. The iPSC line also showed a normal karyotype and preserved the GCH1 mutation. This cellular model can provide opportunities to perform pathophysiological studies for aberrant dopamine metabolism-related disorders.

Genetic and pharmacological correction of aberrant dopamine synthesis using patient iPSCs with BH4 metabolism disorders.

Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders.

Cytoplasmic aggregates of dynactin in iPSC-derived tyrosine hydroxylase-positive neurons from a patient with Perry syndrome.

BACKGROUND: Perry syndrome is a rare autosomal dominant disorder clinically characterized by parkinsonism with depression/apathy, weight loss, and central hypoventilation. Eight mutations in DCTN1 gene have been reported. A novel disease model is required because the detailed pathogenesis remains unclear. METHODS: To develop a novel model, we generated induced pluripotent stem cells (iPSCs) from a Perry syndrome patient with F52L mutation in DCTN1, and describe clinical and neuroimaging investigations. We differentiated iPSCs into tyrosine hydroxylase (TH)-positive neurons. Immunocytochemistry analyses of control and mutant were performed. RESULTS: The patient displayed levodopa responsive parkinsonism. Dopamine transporter single photon emission tomography showed markedly decreased uptake in the striatum, and metaiodobenzylguanidine cardiac scintigraphy also showed decreased uptake. Perry syndrome TH-positive neurons showed dynactin aggregates in cytoplasm. CONCLUSIONS: TH-positive neurons from Perry syndrome iPSCs recapitulated an aspect of the disease phenotype of Perry syndrome.

Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aß production without affecting Notch cleavage.

γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid ß-peptide (Aß), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aß production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aß and these proteins were selected for further studies on Aß production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aß secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aß40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aß production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aß lowering therapy aimed at treating Alzheimer's disease.

Human brain proteins showing neuron-specific interactions with γ-secretase.

The transmembrane protease complex γ-secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid ß-peptide (Aß); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ-secretase inhibitors have been developed, but none have been released due to side-effects that appear to arise from reduced processing of Notch, one of many γ-secretase substrates. Hence, it is desirable to specifically inhibit Aß production. In our previous studies, we have identified several γ-secretase-associated proteins (GSAPs) from brain, which affect Aß production without having any major effects on Notch processing. In the present study using detergent-resistant membranes prepared from brain, we have identified four GSAPs that affect Aß production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ-secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ-secretase in neurons than in human embryonic kidney cells stably over-expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein-protein interactions in relevant cell types, and suggests that reducing Aß production by interfering with brain- or neuron-specific γ-secretase/GSAP interactions may reduce the risk of unwanted side-effects associated with treatment of Alzheimer disease.

PPARγ/RXRα-induced and CD36-mediated microglial amyloid-ß phagocytosis results in cognitive improvement in amyloid precursor protein/presenilin 1 mice.

Alzheimer's disease (AD) is characterized by the extracellular deposition of amyloid-ß (Aß), neurofibrillary tangle formation, and a microglial-driven inflammatory response. Chronic inflammatory activation compromises microglial clearance functions. Because peroxisome proliferator-activated receptor γ (PPARγ) agonists suppress inflammatory gene expression, we tested whether activation of PPARγ would also result in improved microglial Aß phagocytosis. The PPARγ agonist pioglitazone and a novel selective PPARα/γ modulator, DSP-8658, currently in clinical development for the treatment of type 2 diabetes, enhanced the microglial uptake of Aß in a PPARγ-dependent manner. This PPARγ-stimulated increase of Aß phagocytosis was mediated by the upregulation of scavenger receptor CD36 expression. In addition, combined treatment with agonists for the heterodimeric binding partners of PPARγ, the retinoid X receptors (RXRs), showed additive enhancement of the Aß uptake that was mediated by RXRα activation. Evaluation of DSP-8658 in the amyloid precursor protein/presenilin 1 mouse model confirmed an increased microglial Aß phagocytosis in vivo, which subsequently resulted in a reduction of cortical and hippocampal Aß levels. Furthermore, DSP-8658-treated mice showed improved spatial memory performance. Therefore, stimulation of microglial clearance by simultaneous activation of the PPARγ/RXRα heterodimer may prove beneficial in prevention of AD.

Erlin-2 is associated with active γ-secretase in brain and affects amyloid ß-peptide production.

The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid ß-peptide (Aß) from its precursor (APP). Aß has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aß production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aß production.