Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs.

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.

A Novel Cell-based ß-secretase Enzymatic Assay for Alzheimer's Disease.

BACKGROUND: Deposition of the amyloid ß protein (Aß) into neuritic plaques is the neuropathological hallmark of Alzheimer's Disease (AD). Aß is generated through the cleavage of the Amyloid Precursor Protein (APP) by ß-secretase and γ-secretase. Currently, the evaluation of APP cleavage by ß-secretase in experimental settings has largely depended on models that do not replicate the physiological conditions of this process. OBJECTIVE: To establish a novel live cell-based ß-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely replicates its cleavage by ß-secretase under physiological conditions. METHODS: We have developed a chimeric protein construct, ASGß, incorporating the ß-site cleavage sequence of APP targeted by ß-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by ß-secretase, ASGß releases a phosphatase-containing portion that can be measured in the culture medium, and an intracellular fraction that can be detected through Western Blot. Subsequently, we have generated a cell line stably expressing ASGß that can be utilized to assay ß-secretase in real time. RESULTS: ASGß is specifically targeted by ß-secretase, being cleaved exclusively at the site responsible for the generation of Aß. Dosage response to ß-secretase inhibitors shows that ß-secretase activity can be positively correlated to phosphatase activity in culture media. CONCLUSION: Our findings suggest this system could be a high-throughput tool to screen compounds that aim to modulate ß-secretase activity and Aß production under physiological conditions, as well as evaluating factors that regulate this cleavage.

NMDA preconditioning protects against quinolinic acid-induced seizures via PKA, PI3K and MAPK/ERK signaling pathways.

Preconditioning by N-methyl-d-aspartate (NMDA) may be promoted in vivo by the administration of a sub-convulsing dose of NMDA, with a neuroprotective effect against seizures and neuronal death induced by the infusion of quinolinic acid (QA) in mice. This study aimed to evaluate the participation of protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), Ca(2+)/calmodulin dependent protein kinase II (CaMKII) and phosphatidilinositol-3 kinase (PI3K) signaling pathways in this neuroprotection model. Adult Swiss male mice were preconditioned with NMDA 24 h before the infusion of QA, and were treated with inhibitors of the aforementioned signaling pathways either 15 min before the preconditioning or infusion of QA. Inhibition of the PKA and PI3K pathways abolished the protection evoked by NMDA, and inhibition of the MEK pathway significantly diminished this protection. Treatment with PKC and CaMKII inhibitors did not alter the protection rate. Inhibition of the MEK and PKC pathways resulted in an increased mortality rate when followed by the infusion of QA, or NMDA preconditioning and QA infusion, respectively. These results suggest that the PKA, PI3K and MEK pathways have a crucial role in the achievement of a neuroprotective state following preconditioning.

Atorvastatin prevents hippocampal cell death due to quinolinic acid-induced seizures in mice by increasing Akt phosphorylation and glutamate uptake.

Statins are cholesterol-lowering agents due to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Recent studies have shown statins possess pleiotropic effects, which appear to be independent from its cholesterol-lowering action. In this study, we investigated whether atorvastatin would have protective effects against hippocampal cell death promoted by quinolinic acid (QA)-induced seizures in mice. Mice were pretreated with Atorvastatin (1 or 10 mg/kg) or vehicle (saline, 0.9%), orally, once a day for 7 days before the intracerebroventricular (i.c.v.) QA infusion (36.8 nmol/site). Atorvastatin treatment with 1 mg/kg/day did not significantly prevent QA-induced seizures (13.34%). However, administration of atorvastatin 10 mg/kg/day prevented the clonic and/or tonic seizures induced by QA in 29.41% of the mice. Additionally, administration of atorvastatin 10 mg/kg/day significantly prevented QA-induced cell death in the hippocampus. Atorvastatin treatment promoted an increased Akt phosphorylation, which was sustained after QA infusion in both convulsed and non-convulsed mice. Moreover, atorvastatin pretreatment prevented the reduction in glutamate uptake into hippocampal slices induced by QA i.c.v. infusion. These results show that atorvastatin attenuated QA-induced hippocampal cellular death involving the Akt pathway and glutamate transport modulation. Therefore, atorvastatin treatment might be a useful strategy in the prevention of brain injury caused by the exacerbation of glutamatergic toxicity in neurological diseases such as epilepsy.

Evaluation of glutathione metabolism in NMDA preconditioning against quinolinic acid-induced seizures in mice cerebral cortex and hippocampus.

Brain preconditioning refers to a wide range of treatments that induce a neuronal tolerance state where neuronal tissue become more resistant to a subsequent lethal insult. The mechanisms underlying the preconditioning-induced brain tolerance are not fully understood, but up-regulation of antioxidant enzymes activity has been suggested to play an important role. In order to test this hypothesis, evaluation of glutathione (GSH) scavenger system was carried out in mice showing the neuroprotective effect of NMDA preconditioning against quinolinic acid (QA)-induced seizures. NMDA is known to prevent seizures in 53% of the animals and completely prevent neural damage against QA. Mice were preconditioned by a non-convulsant NMDA dose (75 mg/kg, 10 ml/kg i.p.) 24 h before QA infusion (4 microl, 9.2 mM i.c.v.). GSH content and enzymatic activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) were evaluated in the cerebral cortex and hippocampus 24 h after QA infusion. NMDA preconditioning and QA infusion did not alter GSH content, GR and G6PDH activities, however, an increase in GST activity was observed in the cerebral cortex from mice. Moreover, NMDA pretreatment was able to prevent the QA-induced decrease in hippocampal GPx activity, but it was not effective against the decreased cortical GPx activity. These results indicate that, although NMDA preconditioning and QA toxicity modulate the activity of some GSH related enzymes, GSH metabolism is not directly linked to the neuroprotective effect induced by NMDA preconditioning.