SAK3 Administration Improves Spine Abnormalities and Cognitive Deficits in AppNL-G-F/NL-G-F Knock-in Mice by Increasing Proteasome Activity through CaMKII/Rpt6 Signaling

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by neuropathological hallmarks consisting of accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFT). Recently, we have identified a new AD therapeutic candidate, ethyl-8′-methyl-2′,4-dioxo-2-(piperidin-1-yl)-2′H-spiro[cyclopentane-1,3′-imidazo [1,2-a] pyridin]-2-ene-3-carboxylate (SAK3), which ameliorates the AD-like pathology in AppNL-F/NL-F knock-in mice. However, the detailed mechanism underlying the therapeutic effects of SAK3 remains unclear. In this study, we found that SAK3 administration improved the reduced proteasome activity through the activation of CaMKII/Rpt6 signaling in AppNL-F/NL-F knock-in (NL-G-F) mice. Moreover, spine abnormalities observed in NL-G-F mice were significantly reversed by SAK3 administration. Along with this, cognitive impairments found in NL-G-F mice were markedly ameliorated by SAK3. In summary, our data suggest that SAK3 administration increases the activity of the proteasome via activation of the CaMKII/Rpt6 signaling pathway, contributing to improvements in spine abnormalities and cognitive deficits in NL-G-F mice. Overall, our findings suggest that SAK3 might be a new attractive drug candidate, representing a new mechanism for the treatment of AD pathology

Development and scale-up of perfusion cell culture process for higer productivity

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An α-synuclein decoy peptide prevents cytotoxic α-synuclein aggregation caused by fatty acid binding protein 3

α-synuclein (αSyn) is a protein known to form intracellular aggregates during the manifestation of Parkinson’s disease. Previously, it was shown that αSyn aggregation was strongly suppressed in the midbrain region of mice that did not possess the gene encoding the lipid transport protein fatty acid binding protein 3 (FABP3). An interaction between these two proteins was detected in vitro, suggesting that FABP3 may play a role in the aggregation and deposition of αSyn in neurons. In order to characterize the molecular mechanisms that underlie the interactions between FABP3 and αSyn that modulate the cellular accumulation of the latter, in this report, we used in vitro fluorescence assays combined with fluorescence microscopy, transmission electron microscopy, and quartz crystal microbalance assays to characterize in detail the process and consequences of FABP3-αSyn interaction. We demonstrated that binding of FABP3 to αSyn results in changes in the aggregation mechanism of the latter; specifically, a suppression of fibrillar forms of αSyn, and also the production of aggregates with an enhanced cytotoxicity toward mice neuro2A cells. Since this interaction involved the C-terminal sequence region of αSyn, we tested a peptide derived from this region of αSyn (αSynP130-140) as a decoy to prevent the FABP3-αSyn interaction. We observed that the peptide competitively inhibited binding of αSyn to FABP3 in vitro and in cultured cells. We propose that administration of αSynP130-140 might be used to prevent the accumulation of toxic FABP3-αSyn oligomers in cells, thereby preventing the progression of Parkinson’s disease

Combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient brain ischemia.

Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient brain ischemia. Keywords: Citicoline, DHA, Bilateral common carotid artery occlusion, Neuroprotection, Memor

A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing 4 cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87N. Whereas lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. Sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of quantitative bioluminescence assay

Sigma-1 receptor is involved in modification of ER-mitochondria proximity and Ca2+ homeostasis in cardiomyocytes

The Sigma-1 receptor (Sigmar1) is downregulated in heart failure model mice with mitochondrial dysfunction. However, the mechanism in detail has not been investigated. In this study, we investigated the role of Sigmar1 in ER-mitochondria proximity using Sigmar1-knockdown or -overexpressed neonatal rat ventricular myocytes (NRVMs). The endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy was aggravated with the dysregulation of mitochondrial function and ER-mitochondrial junctional formation in Sigmar1-knockdown NRVMs, whereas improved in Sigmar1 overexpressed NRVMs. Our data suggests that the reduction of the cardiac Sigmar1 results in decrease mitochondrial Ca2+ influx and promotes mitochondrial fission, followed by reduced ER-mitochondria proximity, exacerbating ET-1-induced cardiomyocyte injury