Loss of forebrain BIN1 attenuates hippocampal pathology and neuroinflammation in a tauopathy model.

Bridging integrator 1 (BIN1) is the second most prevalent genetic risk factor identified by genome-wide association studies (GWAS) for late-onset Alzheimer's disease. BIN1 encodes an adaptor protein that regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. In vitro evidence suggests that BIN1 can directly bind to tau in the cytosol. In addition, BIN1's function limits extracellular tau seed uptake by endocytosis and subsequent propagation as well as influences tau release through exosomes. However, the in vivo roles of BIN1 in tau pathogenesis and tauopathy-mediated neurodegeneration remain uncharacterized. We generated conditional knockout mice with a selective loss of Bin1 expression in the forebrain excitatory neurons and oligodendrocytes in P301S human tau transgenic background (line PS19). PS19 mice develop age-dependent tau neuropathology and motor deficits and are commonly used to study Alzheimer's disease tau pathophysiology. The severity of motor deficits and neuropathology was compared between experimental and control mice that differ with respect to forebrain BIN1 expression. BIN1's involvement in tau pathology and neuroinflammation was quantified by biochemical methods and immunostaining. Transcriptome changes were profiled by RNA-sequencing analysis to gain molecular insights. The loss of forebrain BIN1 expression in PS19 mice exacerbated tau pathology in the somatosensory cortex, thalamus, spinal cord and sciatic nerve, accelerated disease progression and caused early death. Intriguingly, the loss of BIN1 also mitigated tau neuropathology in select regions, including the hippocampus, entorhinal/piriform cortex, and amygdala, thus attenuating hippocampal synapse loss, neuronal death, neuroinflammation and brain atrophy. At the molecular level, the loss of forebrain BIN1 elicited complex neuronal and non-neuronal transcriptomic changes, including altered neuroinflammatory gene expression, concomitant with an impaired microglial transition towards the disease-associated microglial phenotype. These results provide crucial new information on in vivo BIN1 function in the context of tau pathogenesis. We conclude that forebrain neuronal BIN1 expression promotes hippocampal tau pathogenesis and neuroinflammation. Our findings highlight an exciting region specificity in neuronal BIN1 regulation of tau pathogenesis and reveal cell-autonomous and non-cell-autonomous mechanisms involved in BIN1 modulation of tau neuropathology.

Toluidine blue O attenuates tau phosphorylation in N2a-APPSwe cells.

Alzheimer's disease (AD) is characterized by extracellular amyloid plaques composed of amyloid-ß peptide (Aß), intracellular neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. Most of the FDA-approved AD drugs currently on the market are cholinesterase inhibitors, which are only effective in relieving the symptoms of AD. However, recent studies in AD drug discovery focus on multi-targeted strategies, including anti-amyloid and anti-tau therapy. In the current study, we have investigated the effects of toluidine blue O (TBO), a cholinesterase inhibitor, on amyloid precursor protein (APP) processing, tau phosphorylation, and tau kinases/phosphatase in N2a mouse neuroblastoma cells stably expressing the Swedish mutation of human APP695 (N2a-APPSwe). The results demonstrated that TBO reduces Aß40/42 levels by decreasing expression levels of ß-secretase 1 (BACE1), presenilin 1 (PS1) and total APP without causing cytotoxic effects in N2a-APPSwe cells. TBO also decreased the levels of both total tau and phosphorylated tau at residues Ser202/Thr205, Thr181, Ser396 and Ser 396/Ser404. Moreover, when the possible mechanisms underlying its effects on tau pathology were explored, TBO was found to decrease tau phosphorylation at those sites by reducing the expression levels of Akt, GSK-3ß, Cdk5, inactive p-PP2A and increasing the expression levels of p-Akt Ser473 and inactive p-GSK-3ß Ser9. Our new data support the idea that TBO may be a promising multi-target drug candidate for the treatment of AD.

Effects of toluidine blue O and methylene blue on growth and viability of pancreatic cancer cells.

Amyloid precursor-like protein-2 (APLP2) and its C-terminal fragments (CTFs) are expressed at high levels in pancreatic cancer cells and knockdown of APLP2 expression inhibits tumor growth. CTFs are released from APLP2 by beta-secretase (BACE). In this study, our goal was to determine whether methylene blue (MethB) and toluidine blue O (TBO) could be used to slow down the growth and viability of pancreatic cancer cells (Hs 766T). We found that TBO and MethB decreased the growth and viability of Hs 766T cells in a dose- and time-dependent manner compared to vehicle-treated control, as demonstrated by MTT and trypan blue exclusion assays. Although TBO led to decreased expression of APLP2, MethB did not show any significant effect on APLP2. However, both MethB and TBO reduced BACE activity and the levels of APLP2 CTFs in Hs 766T cells. In conclusion, MethB and TBO may be valuable candidates for the treatment of pancreatic cancer by targeting APLP2 processing.

Trafficking and proteolytic processing of amyloid precursor protein and secretases in Alzheimer's disease development: An up-to-date review.

Alzheimer's disease (AD), which is predicted to affect 1 in 85 persons worldwide by 2050, results in progressive loss of neuronal functions, leading to impairments in memory and cognitive abilities. As being one of the major neuropathological hallmarks of AD, senile plaques mainly consist of amyloid-ß (Aß) peptides, which are derived from amyloid precursor protein (APP) via the sequential cleavage by ß- and γ-secretases. Although the overproduction and accumulation of Aß peptides are at the center of AD research, the new discoveries point out to the complexity of the disease development. In this respect, it is crucial to understand the processing and the trafficking of APP, the enzymes involved in its processing, the cleavage products and their therapeutic potentials. This review summarizes the salient features of APP processing focusing on APP, the canonical secretases as well as the novel secretases and the cleavage products with an update of the recent developments. We also discussed the intracellular trafficking of APP and secretases in addition to their potential in AD therapy.

Azure B affects amyloid precursor protein metabolism in PS70 cells.

Alzheimer's disease (AD), the most common form of dementia, is characterized by abundant deposition of amyloid-ß (Aß) peptide that is the result of sequential cleavage of amyloid precursor protein (APP) by ß-secretase and γ-secretase. Several studies have documented that inhibition of Aß peptide synthesis or facilitating its degradation is one of the attractive therapeutic strategies in AD. Methylene blue (MethB), which has recently been investigated in Phase II clinical trials, is a prominent inhibitor in reducing Aß oligomers. Herein, we wonder whether the mitigating effects of MethB on amyloid metabolism are related to the activity of its major metabolite, azure B. The goal of this study was to investigate the effects of azure B, which is also a cholinesterase inhibitor, on APP processing by using Chinese hamster ovary cells stably expressing human wild-type APP and presenilin 1 (PS70). Azure B significantly decreased the levels of secreted APPα (sAPPα) and Aß40/42 in culture medium with a dose-dependent manner. A significant decrease was also observed in the levels of intracellular APP without affecting the cell viability. In parallel with the decrease of APP and APP metabolites, the activity of ß-secretase 1 (BACE1) was significantly attenuated compared to control. Overall, our results show that azure B has a large contribution for the pharmacological profile of MethB in APP metabolism.

Toluidine blue O modifies hippocampal amyloid pathology in a transgenic mouse model of Alzheimer's disease.

Recently, we have demonstrated that toluidine blue O (TBO), a phenothiazine dye, shows inhibitory effects on both cholinesterases and amyloid pathology in Alzheimer's disease (AD) cellular model. In the present study, we aimed to determine the effects of TBO (in a purity of 85%) on amyloid and tau pathologies in a triple transgenic mouse model of AD (3xTg-AD). Beginning at 7.5 (mild pathology) or 13 (severe pathology) months of age, 3xTg-AD mice were treated intraperitoneally with 4 mg/kg TBO or vehicle daily for 30 days. TBO treatment significantly reduced the levels of insoluble Aß40 and Aß42 in the hippocampi of mild and severe pathology groups compared to vehicle-treated counterparts. When the levels of full-length amyloid precursor protein (APP) and ß-site APP-cleaving enzyme 1 (BACE1) were assessed in 3xTg-AD mice at late pathological stage, no significant changes were observed after TBO treatment. Similarly, TBO did not recover hyperphosphorylation of tau at residues Thr181 and Ser202/Thr205 significantly in soluble and insoluble hippocampal fractions of 3xTg-AD mice. Taken together, the current study is the first in vivo report, to our knowledge, demonstrating that TBO mitigates amyloid pathology in 3xTg-AD mice with no apparent change on tau phosphorylation. Overall, the preliminary data presented here support the possible use of TBO as a disease-modifying drug for AD treatment.

Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model.

The excess accumulation of amyloid-ß (Aß) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aß, soluble APP-α (sAPPα), intracellular APP and ß-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aß40, Aß42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aß40, Aß42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aß pathology by lowering APP levels whereas reduced Aß caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro.