Novel Synthetic Coumarin‐Chalcone Derivative (E)‐3‐(3‐(4‐(Dimethylamino)Phenyl)Acryloyl)‐4‐Hydroxy‐2H‐Chromen‐2‐One Activates CREB‐Mediated Neuroprotection in Aβ and Tau Cell Models of Alzheimer’s Disease

Abstract

Abnormal accumulations of misfolded Aβ and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer’s disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aβ and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.

Novel TRKB agonists activate TRKB and downstream ERK and AKT signaling to protect Aβ-GFP SH-SY5Y cells against Aβ toxicity

“…Thioflavin T is widely used to visualize and quantify the presence of misfolded amyloid protein aggregates in vitro [ 59 ]. The Aβ amyloid inhibiting potential of curcumin, LM-031 and analogs (5–20 μM) were assessed by using Aβ42 peptide (AnaSpec, Fremont, CA, USA) upon binding to thioflavin T as stated [ 60 ]. In addition, DPPH radical (Sigma-Aldrich) [ 61 ] was used to assay the free radical scavenging capacity of kaempferol, LM-031 and analogs (10–160 μM) as stated [ 60 ].…”

“…Neuronal differentiation of Aβ-GFP SH-SY5Y cells was induced by retinoic acid (10 μM; Sigma-Aldrich) [ 68 ]. Cells on 96-well plate (2.5 × 10 4 /well) were seeded on day 1, pre-treated with test compounds (1.2–5 μM) plus induced Aβ-GFP expression (5 μg/ml doxycycline) on day 2, and stained with Hoechst 33342 (0.1 μg/ml; Sigma-Aldrich) on day 8 as stated [ 60 ]. Cell images were then captured at 482 nm excitation/536 nm emission wavelengths (ImageXpress Micro Confocal high-content system) and analyzed (MetaXpress image acquisition and analysis software) (Molecular Devices, Sunnyvale, CA, USA).…”

“…As stated, Aβ-GFP SH-SY5Y cells were seeded (6 × 10 4 /24-well) with retinoic acid addition on day 1, treated with tested compounds (5 μM) plus induced Aβ-GFP expression (5 μg/ml doxycycline) on day 2, and stained with TUBB3 (tubulin beta 3 class III) primary antibody (1:1000; Covance #MMS-435P, Princeton, NJ, USA), goat anti-rabbit Alexa Fluor ® 555 secondary antibody (1:1000; Thermo Fisher Scientific #A27039), and DAPI (4′-6-diamidino-2-phenylindole, 0.1 μg/ml; Sigma-Aldrich) on day 8 [ 60 ]. Neuronal pictures were taken using the high-content system and analyzed for neurite length (μm), process (number of neurites protruding from neuronal cell body) and branch (number of neurites extending from process) (MetaXpress neurite outgrowth application module; Molecular Devices).…”

“…Cells on 6-well plate (5 × 10 5 /well) were subjected to the retinoic acid, test compound and doxycycline treatments as stated. Cells were collected on day 8 and cell lysates prepared for caspase 1 (BioVision, Milpitas, CA, USA) and AChE (Sigma-Aldrich) activity measurement [ 60 ].…”

A self-developed MyD88 inhibitor across the blood-brain barrier fully reverses acute cerebral ischemia-reperfusion injury

“…BBB is the interface that controls the exchange of substances between the central nervous system (CNS) and blood, which brings di culty in developing drugs that can cross such barrier [36]. Currently, not many drugs can enter the CNS e ciently through the BBB, which limits the development of therapies for brain diseases [37,38]. Microglia are resident macrophages in the brain.…”