Ex vivo analysis platforms for monitoring amyloid precursor protein cleavage

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder and the most common cause of dementia in the elderly. The presence of large numbers of senile plaques, neurofibrillary tangles, and cerebral atrophy is the characteristic feature of AD. Amyloid β peptide (Aβ), derived from the amyloid precursor protein (APP), is the main component of senile plaques. AD has been extensively studied using methods involving cell lines, primary cultures of neural cells, and animal models; however, discrepancies have been observed between these methods. Dissociated cultures lose the brain’s tissue architecture, including neural circuits, glial cells, and extracellular matrix. Experiments with animal models are lengthy and require laborious monitoring of multiple parameters. Therefore, it is necessary to combine these experimental models to understand the pathology of AD. An experimental platform amenable to continuous observation and experimental manipulation is required to analyze long-term neuronal development, plasticity, and progressive neurodegenerative diseases. In the current study, we provide a practical method to slice and cultivate rodent hippocampus to investigate the cleavage of APP and secretion of Aβ in an ex vivo model. Furthermore, we provide basic information on Aβ secretion using slice cultures. Using our optimized method, dozens to hundreds of long-term stable slice cultures can be coordinated simultaneously. Our findings are valuable for analyses of AD mouse models and senile plaque formation culture models

Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer's disease

Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'vertical bar disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported thatbCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between beta CTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and App(NLG-F/NLG-F) model mice. Furthermore, the T-RAP peptide derived from the beta CTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD

The Pursuit of the "Inside" of the Amyloid Hypothesis-Is C99 a Promising Therapeutic Target for Alzheimer's Disease?

Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer's disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-beta (A beta), which accumulates in the patient brain. The "amyloid hypothesis " based therapy that places the aggregation and toxicity of A beta at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. A beta is produced from a type-I transmembrane protein, A beta precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called beta CTF), is a direct precursor of A beta and accumulates in the AD patient's brain to demonstrate toxicity independent of A beta. Conventional drug discovery strategies have focused on A beta toxicity on the "outside " of the neuron, but C99 accumulation might explain the toxicity on the "inside " of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and A beta is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis

The Pursuit of the “Inside” of the Amyloid Hypothesis—Is C99 a Promising Therapeutic Target for Alzheimer’s Disease?

Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer’s disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-β (Aβ), which accumulates in the patient brain. The “amyloid hypothesis” based therapy that places the aggregation and toxicity of Aβ at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. Aβ is produced from a type-I transmembrane protein, Aβ precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called βCTF), is a direct precursor of Aβ and accumulates in the AD patient’s brain to demonstrate toxicity independent of Aβ. Conventional drug discovery strategies have focused on Aβ toxicity on the “outside” of the neuron, but C99 accumulation might explain the toxicity on the “inside” of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and Aβ is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis

Intravascular Free Tissue Factor Pathway Inhibitor Is Inversely Correlated With HDL Cholesterol and Postheparin Lipoprotein Lipase but Proportional to Apolipoprotein A-II

To elucidate the distribution and clinical implications of tissue factor pathway inhibitor (TFPI) concentrations, we measured TFPI levels consisting of preheparin free, lipoprotein-bound (Lp-bound), and endothelial cell-anchor pools in 156 patients with coronary artery disease (average age, 61.2+/-9.1 years; range, 32 to 78 years) by heparin infusion (50 IU/kg) and compared them with the preheparin TFPI levels of 229 healthy subjects (average age, 59. 6+/-9.4 years; range, 41 to 80 years). The patients had lower preheparin free TFPI and lower HDL cholesterol (HDL-C) levels than the healthy subjects with equivalent Lp-bound forms (free TFPI, 15. 9+/-6.5 versus 19.2+/-8.1 ng/mL). In a partial correlation analysis, the preheparin free TFPI levels were shown to be inversely correlated with the HDL-C concentrations in both the patients (r=-0. 454, P<0.001) and the healthy subjects (r=-0.136, P<0.05). As determined by comparison of preheparin and postheparin plasma, the patients generally showed preheparin free TFPI <10%, Lp-bound TFPI at 30%, and endothelial cell-anchor TFPI at 60%. When the patients were divided into 4 categories by their LDL cholesterol (LDL-C, 130 mg/dL) and HDL-C (40 mg/dL) levels to specify their coronary risks, the low-HDL-C groups had significantly increased preheparin and postheparin free TFPI levels and decreased postheparin LPL levels, whereas the high-LDL-C groups showed increased levels of Lp-bound TFPI. In a partial correlation analysis, we found a proportional relation between postheparin free TFPI and apolipoprotein A-II (r=0. 5327) and between HDL-C and LPL (r=0.4906), whereas postheparin free TFPI was inversely correlated with HDL-C (r=-0.4280) and postheparin LPL (r=-0.4791). The inverse relationship between TFPI and LPL suggests that increased free TFPI concentrations as a compensatory response of the endothelium to prevent atherothrombotic processes compete with and displace LPL on endothelial surface, resulting in reduced LPL and low HDL-C

Nuclear SphK2/S1P signaling is a key regulator of ApoE production and Aβ uptake in astrocytes

The link between changes in astrocyte function and the pathological progression of Alzheimer's disease (AD) has attracted considerable attention. Interestingly, activated astrocytes in AD show abnormalities in their lipid content and metabolism. In particular, the expression of apolipoprotein E (ApoE), a lipid transporter, is decreased. Because ApoE has anti-inflammatory and amyloid β (Aβ)-metabolizing effects, the nuclear receptors, retinoid X receptor (RXR) and LXR, which are involved in ApoE expression, are considered promising therapeutic targets for AD. However, the therapeutic effects of agents targeting these receptors are limited or vary considerably among groups, indicating the involvement of an unknown pathological factor that modifies astrocyte and ApoE function. Here, we focused on the signaling lipid, sphingosine-1-phosphate (S1P), which is mainly produced by sphingosine kinase 2 (SphK2) in the brain. Using astrocyte models, we found that upregulation of SphK2/S1P signaling suppressed ApoE induction by both RXR and LXR agonists. We also found that SphK2 activation reduced RXR binding to the APOE promoter region in the nucleus, suggesting the nuclear function of SphK2/S1P. Intriguingly, suppression of SphK2 activity by RNA knockdown or specific inhibitors upregulated lipidated ApoE induction. Furthermore, the induced ApoE facilitates Aβ uptake in astrocytes. Together with our previous findings that SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons, these results indicate that SphK2/S1P signaling is a promising multifunctional therapeutic target for AD that can modulate astrocyte function by stabilizing the effects of RXR and LXR agonists, and simultaneously regulate neuronal pathogenesis