Journal: Journal of Pharmaceutical Analysis

Article Title: Altered synaptic currents, mitophagy, mitochondrial dynamics in Alzheimer's disease models and therapeutic potential of Dengzhan Shengmai capsules intervention

doi: 10.1016/j.jpha.2023.10.006

Figure Lengend Snippet: Dengzhan Shengmai (DZSM) alleviates Aβ 42 -induced mitochondrial dysfunction in primary neural cells. (A) Representative flow cytograms and quantitative analysis of cellular reactive oxygen species (ROS). (B) Representative flow cytograms of cellular mitochondrial membrane potential (MMP) and the quantitative analysis of the ratio of red to green fluorescence. (C) Statistical histogram of cellular ATP content. (D) Representative images of MitoTracker Red CMXRos staining and the quantitative analysis of mitochondrial length (magnification = 400 ×; 80–100 mitochondria were selected for analysis, n = 3). (E) Representative transmission electron microscopy (TEM) images of cells (magnification = 7,000 ×; 20–30 mitochondria were selected for analysis, n = 3), and the quantitative analysis of the length and area of mitochondria. Experimental data are expressed as the mean ± standard error of mean (SEM), n = 3. ∗∗ P < 0.01 vs. Control group, and # P < 0.05 and ## P < 0.01 vs. Aβ 42 group. Mdivi1: mitochondrial division inhibitor 1.

Article Snippet: Trypsin (T1360), CCK8 kit (cat: CA1210), ATP detection kit (cat: BC0300), MMP Assay Kit with JC-1 (M8650), ROS assay kit (cat: CA1410), and BCA protein assay kit (cat: PC0020) were purchased from Solarbio Science & Technology (Beijing, China).

Techniques: Membrane, Fluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: American Journal of Translational Research

Article Title: Knockdown of miR-27a reduces TGFβ-induced EMT and H 2 O 2 -induced oxidative stress through regulating mitochondrial autophagy

doi:

Figure Lengend Snippet: Downregulating miR-27a reduced H2O2-induced oxidative stress. A. ESCs were treated with or without H2O2. The miR-27a RNA level was examined by qPCR. ESCs, transfected with or without inhibitor-NC and inhibitor-27, were then intervened by 100 μM H2O2. B. Intracellular ROS production was detected with DCFH-DA assay. C. Western blotting was conducted to examine Bcl-2 and Bax protein levels. D. Flow cytometry was carried out for cell apoptosis examination. E. Total p65 and phosphorylated p65 levels was accessed by western blotting. Data were presented as mean ± SD. **P<0.01, ***P<0.001.

Article Snippet: The ROS Assay Kit (Solarbio, China) was utilized to determine intracellular ROS levels.

Techniques: Transfection, DCFH-DA Assay, Western Blot, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Knockdown of miR-27a reduces TGFβ-induced EMT and H 2 O 2 -induced oxidative stress through regulating mitochondrial autophagy

doi:

Figure Lengend Snippet: miR-27a regulates mitophagy in H2O2-treated ESCs. A. Mimic-NC, mimic-27, inhibitor-NC, and inhibitor-27 were transfected into ESCs, respectively. PINK1, Parkin, and LC3 protein levels were examined by western blotting. ESCs, transfected with inhibitor-NC, inhibitor-27, and inhibitor-27+si-PINK1, were then intervened by 100 μM H2O2. B. PINK1, Parkin, and LC3 protein levels were examined by western blotting. C. Intracellular ROS production was detected by DCFH-DA assay. D. Western blotting was carried out to determine Bcl-2 and Bax protein levels. E. Cell apoptosis was examined by flow cytometry. Data were presented as mean ± SD. *, **, and *** represent P<0.05, P<0.01, and P<0.001, respectively.

Article Snippet: The ROS Assay Kit (Solarbio, China) was utilized to determine intracellular ROS levels.

Techniques: Transfection, Western Blot, DCFH-DA Assay, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 reduces Aβ/tau aggregate formation, Aβ levels, and AD pathology in vitro and in vivo. A Graph of fluorescence intensity versus concentration of CA140 in the presence of aggregated Aβ42 (average fluorescence measurements from three independent experiments). B Real-time monitoring of the inhibitory effects of CA140 on amyloid formation. C Quantification of the ThT intensity of Aβ42 at the final time point. D , E Aβ levels in primary cortical neurons and APP-overexpressing CHO cells. F–I Eight-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg) daily for 14 days, and immunofluorescence (IF) staining of brain slices was conducted with an anti-Aβ 17-24 (4G8) antibody (n = 16 brain slices from 4 mice/group). J , K Quantification of the ThT intensity of 2N4R full-length tau at the final time point. L , M 8-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and IF staining of brain slices was conducted with an anti-Tau Thr212/Ser214 (AT100) antibody (Veh, n = 18 brain slices from 4 mice; CA140, n = 20 brain slices from 4 mice). Scale bar = 200 μm. * p < 0.05, ** p < 0.01

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: In Vitro, In Vivo, Fluorescence, Concentration Assay, Injection, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: Identification of genes affected by CA140 in 3-month-old 5xFAD mice. A Heatmap of 183 upregulated and 639 downregulated genes in CA140-injected 5xFAD mice compared with vehicle-injected 5xFAD mice. B Volcano plot showing differentially expressed genes (DEGs) in CA140-treated 5xFAD mice. The X- and Y-axes present the log 2 -fold-change and –log 10 ( p value), respectively. Red and green dots represent upregulated and downregulated genes, respectively. Gray dots represent genes without significant differences in expression. C , D Gene ontology biological processes (GOBPs) represented by the downregulated ( C ) and upregulated ( D ) genes. The dotted line indicates the p value cutoff used. The number of genes in each biological process is indicated in parentheses. E DEGs involved in the inflammatory response, glial cell activation/proliferation, and Nlrp3 inflammasome signaling. The color bar shows the z-score gradient. F Relative mRNA levels of the indicated genes in CA140- or vehicle-treated 5xFAD mice were analyzed by real-time PCR (n = 2 mice/group). G Network model describing the interactions among inflammatory response-related signaling pathways. The node colors represent downregulation (green) and no change (yellow) of the corresponding genes in CA140-injected 5xFAD mice. Nodes are arranged and connected according to the activation (arrows) information in the KEGG pathway and WikiPathways databases. Solid and dotted lines denote direct and indirect interactions, respectively. “+p”, phosphorylation. “+u”, ubiquitination. * p < 0.05, ** p < 0.01

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Protein-Protein interactions, Phospho-proteomics, Ubiquitin Proteomics

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 injection regulates microglial activation and astrocytic morphology in 8-month-old 5xFAD mice. A–H Eight-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was conducted with anti-Iba-1 and anti-GFAP antibodies (Iba-1: Veh, n = 23–24 brain slices from 4 mice; CA140, n = 17 brain slices from 4 mice; GFAP: Veh, n = 24 brain slices from 4 mice; CA140, n = 17 brain slices from 4 mice). Scale bar = 200 μm. * p < 0.05, ** p < 0.01

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection, Activation Assay, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 administration reduces NLRP3, IL-1β levels and reactive gliosis in 8-month-old 5xFAD mice. A–H Eight-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was performed with anti-NLRP3 ( A , B ), anti-IL-1β ( C , D ), anti-CXCL10 ( E , F ) and anti-C1QA ( G , H ) antibodies (n = 16–23 brain slices from 4 mice). Scale bar = 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 treatment regulates reactive gliosis in primary astrocytes (PACs) and primary microglia (PMC) from 5xFAD mice. A–F PACs from 5xFAD mice were treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and the relative mRNA levels of the indicated genes were analyzed by real-time PCR (n = 12–15/group). G Summary illustration of the regulatory effect of CA140 on reactive astrogliosis in PACs from 5xFAD mice. H – M PMCs from 5xFAD mice were treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and the relative mRNA levels of the indicated genes were analyzed by real-time PCR (n = 5–6/group). N Summary illustration of the regulatory effect of CA140 on microglial reactive state in PMCs from 5xFAD mice. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 treatment reverses scopolamine (SCO)-induced impairments in long-term memory, dendritic spine number, and LTP in wild-type (WT) mice. A , B WT mice were injected daily with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) for 14 days. On days 3–14, the mice were also injected daily with PBS or SCO (1 mg/kg, i.p.). Y-maze and NOR tests were performed on days 12 and 14, respectively (n = 8 mice/group). C–E Representative AO and BS dendrites from the hippocampal CA1 region and cortical layer V region of mice treated with CA140 and/or SCO (hippocampus AO: Veh, n = 77 neurons from 8 mice; SCO, n = 76 neurons from 8 mice; SCO + CA140, n = 76 neurons from 8 mice; hippocampus BS: Veh, n = 76 neurons from 8 mice; SCO, n = 77 neurons from 8 mice; SCO + CA140, n = 76 neurons from 8 mice; cortex AO and BS: n = 24 neurons from 4 mice/group). Scale bar = 10 μm. F WT mice were injected daily with CA140 (30 mg/kg, i.p.) or vehicle (10% DMSO) for 14 days and were also injected with SCO (1 mg/kg, i.p.) or PBS on days 3–14. G Representative excitatory postsynaptic current (EPSC) traces from the vehicle, SCO, and SCO + CA140 treatment groups. H Input–output curves from the vehicle, SCO, and SCO + CA140 treatment groups (Veh, n = 18 cells from 5 mice; SCO, n = 17 cells from 5 mice; SCO + CA140, n = 14 cells from 5 mice). I Representative EPSC traces before and after LTP induction in the vehicle, SCO, and SCO + CA140 treatment groups (1: before LTP; 2: after LTP). J , K Effects of SCO on LTP induction in the presence/absence of CA140. L Overlay of the two graphs in J , K . M Summary statistics for LTP induction (last 5 min: Veh, n = 20 cells from 8 mice; SCO, n = 22 cells from 8 mice; SCO + CA140, n = 22 cells from 7 mice). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 improves long-term memory and dendritic spine number in 5xFAD mice. A , B Y-maze and NOR tests of 8-month-old 5xFAD mice injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days (n = 10 mice/group). C , D Representative hippocampal AO and BS dendrites of 8-month-old 5xFAD mice injected with vehicle (10% DMSO) or 30 mg/kg CA140 daily for 14 days (AO: Veh, n = 39 neurons from 5 mice; CA140, n = 35 neurons from 5 mice; BS: Veh, n = 35 neurons from 5 mice; CA140, n = 37 neurons from 5 mice). Scale bar = 10 μm. E Measurement of LTP in ex vivo hippocampal slices from 8-month-old 5xFAD mice injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days. Representative excitatory postsynaptic current (EPSC) traces before and after long-term potentiation (LTP) induction are shown (1: before LTP; 2: after LTP). F Effects of CA140 on LTP induction in 8-month-old 5xFAD mice. G Summary statistics for LTP induction (last 5 min: Veh, n = 26 cells from 7 mice; CA140, n = 23 cells from 7 mice). H Representative traces of mEPSCs of 6-month-old 5xFAD mice injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days. I , J Summary graphs of mEPSC amplitude and frequency in the vehicle and CA140-treated groups (Veh, n = 19 cells from 4 mice; CA140, n = 24 cells from 5 mice). K – N Eight-month-old 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining was conducted with anti-synaptophysin ( K , L ) or anti-PSD-95 antibodies ( M , N ) (n = 16 brain slices from 4 mice/group). Scale bar = 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection, Ex Vivo, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 alleviates cognitive function/Aβ pathology through DRD1 signaling in an aged AD mouse model. A HEK293 cells were transfected with DRD1 plasmid DNA, pretreated with a DRD1 antagonist (LE300, 10 μM) or vehicle (1% DMSO) for 30 min, and treated with A77636 (5 μM), dopamine (10 or 100 μM), or CA140 (10, 50, or 100 μM) for 1 h. cAMP levels were then measured by ELISA (n = 3/group). B–E Eight-month-old 5xFAD mice were injected with AAV-DRD1 shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region. Three weeks after AAV injection, the mice were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was performed with an anti-DRD1 antibody (n = 16 brain slices from 4 mice/group). Y-maze and NOR tests were performed on days 15–17 (n = 8–9 mice/group) F–H 8-month-old 5xFAD mice were injected with AAV-DRD1 shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region. Three weeks after AAV injection, the mice were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days, and western blotting of brain lysates was performed with anti-p-Elk1, anti-p-ERK, anti-p-CaMKIIα, or β-actin antibodies (n = 6–8 mice/group). I , J Eight-month-old 5xFAD mice were injected with AAV-DRD1shRNA or AAV-control shRNA in the bilateral hippocampal CA1 region. Three weeks after AAV injection, the mice were administered vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 14 days, and immunofluorescence staining of brain slices was performed with anti-Aβ 17-24 (4G8) antibody (n = 16 brain slices from 4 mice/group). Scale bar = 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection, shRNA, Control, Immunofluorescence, Staining, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 modulates DRD1 signaling to alter cognitive and synaptic function in wild-type (WT) mice. A , B WT mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10 days, and Y-maze and novel object recognition (NOR) tests were conducted (n = 10 mice/group). C–E Y-maze and NOR tests of WT mice injected with AAV-Control shRNA or AAV-DRD1 shRNA, followed by daily injections of CA140 (30 mg/kg, i.p.) or vehicle (1% DMSO) for 17 days (n = 4 mice/group). F–I WT mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 17 days, and western blotting of brain lysates was conducted with anti-p-ELK-1, anti-p-CaMKIIα, or anti-p-ERK antibodies (n = 8 mice/group). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Injection, Control, shRNA, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 enhances dendritic spine formation by regulating functional synapses in primary hippocampal neurons (PHNs). A , C Dendritic spine density was measured in GFP-transfected PHNs treated with vehicle (1% DMSO) or CA140 (1 or 5 μM) for 24 h on DIV14 and DIV21. B , D Quantification of data from A and C (DIV14: Veh, n = 30; 1 μM CA140, n = 47; 5 μM CA140, n = 48; DIV21: Veh, n = 161; 1 μM CA140, n = 60; 5 μM CA140, n = 50). E–H PHNs were transfected with GFP for 24 h, treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h, and immunostained with anti-synaptophysin ( E ) or anti-PSD-95 ( G ) antibodies (synaptophysin intensity: Veh, n = 11; CA140, n = 12; synaptophysin puncta number: Veh, n = 55; CA140, n = 42; PSD-95 intensity: Veh, n = 16; CA140, n = 16; PSD-95 puncta number: Veh, n = 15; CA140, n = 14). Scale bar = 20 μm. ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Functional Assay, Transfection

Journal: Journal of Neuroinflammation

Article Title: The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta

doi: 10.1186/s12974-024-03180-x

Figure Lengend Snippet: CA140 promotes dendritic spine formation through DRD1/CaMKII/ERK signaling in primary hippocampal neurons (PHNs). A , B DRD1 levels in GFP-transfected PHNs treated with vehicle (1% DMSO) or CA140 (5 μM) for 24 h were measured by immunostaining with anti-DRD1 antibodies (Veh, n = 152; CA140, n = 155). C , D Dendritic spine number in GFP-transfected PHNs pretreated with DRD1 inhibitor (LE300, 10 μM) or vehicle (1% DMSO) for 1 h and treated with CA140 (5 μM) or vehicle (1% DMSO) for 23 h (Veh, n = 79; CA140, n = 54; LE300, n = 72; LE300 + CA140, n = 62). E–H Immunostaining of p-CaMKIIα or p-ERK in GFP-transfected PHNs treated with CA140 (5 μM) or vehicle (1% DMSO) for 24 h (p-CaMKIIα: Veh, n = 93; CA140, n = 98; p-ERK: Veh, n = 29; CA140, n = 28). I – L Immunostaining of p-CaMKIIα or p-ERK in GFP-transfected PHNs treated with LE300 (10 μM) or vehicle (1% DMSO) for 24 h (p-CaMKIIα: Veh, n = 99; LE300, n = 85; p-ERK: Veh, n = 78; LE300, n = 93). Scale bar = 10 μm. M – P GFP-transfected PHNs were pretreated with vehicle (1% DMSO), CaMKIIα inhibitor (10 μM), or ERK inhibitor (PD98059) for 1 h and treated with CA140 (5 μM) or vehicle (1% DMSO) for 23 h. Then, dendritic spine number was measured (KN93: Veh, n = 37; CA140, n = 41; KN93 + Veh, n = 36; KN93 + CA140, n = 33; PD98059: Veh, n = 49; CA140, n = 45; PD98059 + Veh, n = 24; PD98059 + CA140, n = 34). Scale bar = 20 μm. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Collectively, these findings suggest that CA140 is a multitarget therapeutic drug for neurodegenerative diseases, including AD.

Techniques: Transfection, Immunostaining