Bilateral Implantation of Shear Stress Modifier in ApoE Knockout Mouse Induces Cognitive Impairment and Tau Abnormalities

Vascular cognitive impairment (VCI) encompasses all causes of cerebrovascular disease that lead to cognitive decline, or overt dementia, atherosclerotic disease being the most common contributor. However, few rodent models that mimic the pathology of VCI replicated the clinical cerebrovascular atherosclerosis. Here we aimed to investigate the mechanism underlying VCI in an Apolipoprotein E knockout (ApoE-KO) mouse model fed with western style food with implantation of bilateral shear stress modifiers. We established a cognitive decline in spatial learning and memory developed in the bilateral modifier treated mice. Brain imaging and pathological examinations demonstrated reduced glucose intake and neuronal loss in hippocampus. Although no amyloid plaques or neurofibrillary tangles (NFTs) were observed, tau pathology including hyperphosphorylation, paired helical filament formation and pathologic truncation were found at considerable higher extent in the bilateral modifier group 8 weeks post the procedure. In addition, gliosis and microglia activation were confirmed in corpus callosum (CC) and ventral striatum. Thus, this ApoE-KO mouse model faithfully replicates the stenosis of common carotid artery (CCA) and cognitive impairment following atherosclerotic deposition and global cerebral hypoperfusion. The close correlation of cognitive decline and tau pathology indicates the toxic tau species could be at least partially responsible for the neurodegenerative changes induced by the chronic hypoxia/ischemia

Antidyskinetic Effects of MEK Inhibitor Are Associated with Multiple Neurochemical Alterations in the Striatum of Hemiparkinsonian Rats

L-DOPA-induced dyskinesia (LID) represents one of the major problems of the long-term therapy of patients with Parkinson's disease (PD). Although, the pathophysiologic mechanisms underlying LID are not completely understood, activation of the extracellular signal regulated kinase (ERK) is recognized to play a key role. ERK is phosphorylated by mitogen-activated protein kinase kinase (MEK), and thus MEK inhibitor can prevent ERK activation. Here the effect of the MEK inhibitor PD98059 on LID and the associated molecular changes were examined. Rats with unilateral 6-OHDA lesions of the nigrostriatal pathway received daily L-DOPA treatment for 3 weeks, and abnormal involuntary movements (AIMs) were assessed every other day. PD98059 was injected in the lateral ventricle daily for 12 days starting from day 10 of L-DOPA treatment. Striatal molecular markers of LID were analyzed together with gene regulation using microarray. The administration of PD98059 significantly reduced AIMs. In addition, ERK activation and other associated molecular changes including ΔFosB were reversed in rats treated with the MEK inhibitor. PD98059 induced significant up-regulation of 418 transcripts and down-regulation of 378 transcripts in the striatum. Tyrosine hydroxylase (Th) and aryl hydrocarbon receptor nuclear translocator (Arnt) genes were down-regulated in lesioned animals and up-regulated in L-DOPA-treated animals. Analysis of protein levels showed that PD98059 reduced the striatal TH. These results support the association of p-ERK1/2, ΔFosB, p-H3 to the regulation of TH and ARNT in the mechanisms of LID, and pinpoint other gene regulatory changes, thus providing clues for identifying new targets for LID therapy

TRH Analog, Taltirelin Protects Dopaminergic Neurons From Neurotoxicity of MPTP and Rotenone

Dopaminergic neurons loss is one of the main pathological characters of Parkinson’s disease (PD), while no suitable neuroprotective agents have been in clinical use. Thyrotropin-releasing hormone (TRH) and its analogs protect neurons from ischemia and various cytotoxins, but whether the effect also applies in PD models remain unclear. Here, we showed that Taltirelin, a long-acting TRH analog, exhibited the neuroprotective effect in both cellular and animal models of PD. The in vitro study demonstrated that Taltirelin (5 μM) reduced the generation of reactive oxygen species (ROS) induced by MPP+ or rotenone, alleviated apoptosis and rescued the viability of SH-SY5Y cells and rat primary midbrain neurons. Interestingly, SH-SY5Y cells treated with Taltirelin also displayed lower level of p-tau (S396) and asparagine endopeptidase (AEP) cleavage products, tau N368 and α-synuclein N103 fragments, accompanied by a lower intracellular monoamine oxidase-B (MAO-B) activity. In the subacute MPTP-induced and chronic rotenone-induced PD mice models, we found Taltirelin (1 mg/kg) significantly improved the locomotor function and preserved dopaminergic neurons in the substantia nigra (SN). In accordance with the in vitro study, Taltirelin down-regulated the levels of p-tau (S396), p-α-synuclein (S129) tau N368 and α-synuclein N103 fragments in SN and striatum. Together, this study demonstrates that Taltirelin may exert neuroprotective effect via inhibiting MAO-B and reducing the oxidative stress and apoptosis, preventing AEP activation and its subsequent pathological cleavage of tau and α-synuclein, thus provides evidence for Taltirelin in protective treatment of PD

TRH Analog, Taltirelin Improves Motor Function of Hemi-PD Rats Without Inducing Dyskinesia via Sustained Dopamine Stimulating Effect

Thyrotropin-releasing hormone (TRH) and its analogs are able to stimulate the release of the endogenic dopamine (DA) in the central nervous system. However, this effect has not been tested in the Parkinson’s disease (PD), which is characterized by the DA deficiency due to the dopaminergic neurons loss in the substantia nigra. Here, we investigated the therapeutic effect of Taltirelin, a long-acting TRH analog on 6-hydroxydopamine-lesioned hemi-Parkinsonian rat model. 1–10 mg/kg Taltirelin i.p. administration significantly improved the locomotor function and halted the electrophysiological abnormities of PD animals without inducing dyskinesia even with high-dose for 7 days treatment. Microdialysis showed that Taltirelin gently and persistently promoted DA release in the cortex and striatum, while L-DOPA induced a sharp rise of DA especially in the cortex. The DA-releasing effect of Taltirelin was alleviated by reserpine, vanoxerine (GBR12909) or AMPT, indicating a mechanism involving vesicular monoamine transporter-2 (VMAT-2), dopamine transporter (DAT) and tyrosine hydroxylase (TH). The in vivo and in vitro experiments further supported that Taltirelin affected the regulation of TH expression in striatal neurons, which was mediated by p-ERK1/2. Together, this study demonstrated that Taltirelin improved motor function of hemi-PD rats without inducing dyskinesia, thus supporting a further exploration of Taltirelin for PD treatment

7,8-Dihydroxyflavone Ameliorates Cognitive Impairment by Inhibiting Expression of Tau Pathology in ApoE-Knockout Mice

7,8-Dihydroxyflavone (7,8-DHF), a tyrosine kinase B (TrkB) agonist that mimics the neuroprotective properties of brain-derived neurotrophic factor, which can not efficiently deliver into the brain, has been reported to be useful in ameliorating cognitive impairment in many diseases. Researches have indicated that apolipoprotein E-knockout (ApoE-KO) mouse was associated with cognitive alteration via various mechanisms. Our present study investigated the possible mechanisms of cognitive impairment of ApoE-KO mouse fed with western type diet and the protective effects of 7,8-DHF in improving spatial learning and memory in ApoE-KO mouse. 5-weeks-old ApoE-KO mice and C57BL/6 mice were chronically treated with 7,8-DHF (with a dosage of 5mg/kg) or vehicles orally for 25 weeks, and then subjected to Morris water maze at the age of 30 weeks to evaluate the cognitive performances. Afterwards, histology analysis and western blotting were performed. Spatial learning and memory deficits were observed in ApoE-KO mice, which were consistent with higher expression of active-asparaginyl endopeptidase (active-AEP) as well as AEP-derived truncated tauN368 compared with normal group. In addition to that, long-term treatment of 7,8-DHF dramatically ameliorated cognitive decline in ApoE-KO mice, accompanied by the activation in phosphorylated protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway and down-regulated expression of tau S396 and PHF-tau (phosphorylated tau at ser396 and ser404 epitope). These findings suggested that cognitive impairment of ApoE-KO mouse might associate with tau pathology and 7,8-DHF could activate AKT and then phosphorylate its downstream molecule to inhibit expression of abnormal tau, meanwhile, 7,8-DHF could reduce the expression of active-AEP and then inhibit production of truncated tauN368

Bridging integrator 1 fragment accelerates tau aggregation and propagation by enhancing clathrin-mediated endocytosis in mice.

The bridging integrator 1 (BIN1) gene is an important risk locus for late-onset Alzheimer's disease (AD). BIN1 protein has been reported to mediate tau pathology, but the underlying molecular mechanisms remain elusive. Here, we show that neuronal BIN1 is cleaved by the cysteine protease legumain at residues N277 and N288. The legumain-generated BIN1 (1-277) fragment is detected in brain tissues from AD patients and tau P301S transgenic mice. This fragment interacts with tau and accelerates its aggregation. Furthermore, the BIN1 (1-277) fragment promotes the propagation of tau aggregates by enhancing clathrin-mediated endocytosis (CME). Overexpression of the BIN1 (1-277) fragment in tau P301S mice facilitates the propagation of tau pathology, inducing cognitive deficits, while overexpression of mutant BIN1 that blocks its cleavage by legumain halts tau propagation. Furthermore, blocking the cleavage of endogenous BIN1 using the CRISPR/Cas9 gene-editing tool ameliorates tau pathology and behavioral deficits. Our results demonstrate that the legumain-mediated cleavage of BIN1 plays a key role in the progression of tau pathology. Inhibition of legumain-mediated BIN1 cleavage may be a promising therapeutic strategy for treating AD

BIN1 N277A/N288A attenuates synaptic loss induced by K18 fibrils.

(a) Electron microscopy of synapses. Arrows indicate synapses. Scale bar, 2 μm. (b) Quantification of synaptic density (mean ± SEM; n = 5 mice per group; *P t test). (c) Golgi staining revealed the dendritic spines from the apical dendritic layer of the CA1 region. Scale bar, 2.5 μm. (d) Quantification of spine density (mean ± SEM; n = 5; *P t test). Source data can be found in S1 Data. BIN1, bridging integrator 1; EGFP, enhanced green fluorescent protein; FL, full-length. (TIF)</p

BIN1 (1–277) enhances CME.

(a) Transferrin uptake assay of neurons expressing EGFP, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (b) Quantification of transferrin uptake in (a). The uptake of transferrin was calculated as the mean fluorescence density of 30 cells under each condition. The fluorescence density was normalized to the GFP-Vector group (mean ± SEM n = 30 cells; *P P c, d) Transferrin uptake assay of neurons expressing BIN1 (1–277) in the presence or absence of dynamin inhibitor dynasore. Scale bar, 5 μm. (d) Quantification of transferrin uptake in (c). Mean ± SEM n = 30 cells; ***P e) FM 4–64 uptake assay in neurons expressing EGFP-vector, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (f) FM 4–64 dye after KCl-induced loss of fluorescence in neurons expressing EGFP-vector, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (g) Quantification of FM 4–64 uptake. FM 4–64 labeling was calculated as the integral fluorescence intensity of 30 boutons under each condition. The fluorescence density was normalized to the control group (mean ± SEM n = 30 cells; *P P P h) Immunofluorescence showing the distribution of dynamin in neurons expressing EGFP, EGFP-BIN1, EGFP-BIN1 (1–277), and EGFP-BIN1 (278–594). Scale bar, 5 μm. (i, j) Quantification of the number and size of the dynamin puncta (mean ± SEM n = 30 cells; *P k) Immunofluorescence showing the expression of Rab5 in neurons expressing EGFP, EGFP-BIN1, EGFP-BIN1 (1–277), and EGFP-BIN1 (278–594). Scale bar, 5 μm. (l, m) Quantification of the number and size of the Rab5 puncta (mean ± SEM n = 30; *P P S1 Data. BIN1, bridging integrator 1; CME, clathrin-mediated endocytosis; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; FL, full-length; FM, FM 4-64 dye; GFP, green fluorescent protein.</p

BIN1 (1–277) binds tau and facilitates its assembly.

(a) Colocalization of AT8 and BIN1 (1–277) in the cortex and hippocampus of tau P301S mice. Scale bar, 20 μm. (b) His pull-down assay showing the interaction between BIN1 (1–277) and K18 fibrils. (c) BIN1 (1–277) enhances the seeding activity of K18 fibrils in clone 1 cells (upper panel) and primary neurons from tau P301S mice (lower panel). Scale bar, 20 μm. (d) The percentage of cells with inclusions in clone 1 cells (left panel) and primary neurons from tau P301S mice (right panel) (mean ± SEM n = 5 independent experiments. ***P e, f) The Clone1 cells expressing HA-Vector, HA-BIN1 FL, HA-BIN1 (1–277), or HA-BIN1 (278–594) were transduced with K18 fibrils (1 μM) for 24 h. Scale bar, 20 μm. The bar graph shows the percentage of cells with inclusions (mean ± SEM n = 5 independent experiments. ***P g) ThS assay showing the assembly kinetics of K18 in the presence of the BIN1 N277 peptide (265–277), BIN1 N288 peptide (265–287), and BIN1 spanning peptide (282–294), respectively. (h) Electron micrographs of the fibrils formed by incubating K18 for 12 h in the presence or absence of BIN1 N277 peptide. Scale bar, 120 nm. Source data can be found in S1 Data and S1 Raw Images. a.u., arbitrary unit; BIN1, bridging integrator 1; F, fibrils; FL, full-length; GFP, green fluorescent protein; HA, human influenza hemagglutinin; M, monomers; O, oligomers; WB, western blot.</p

Relationship between the N277 fragment and tau pathology.

(a, b) Western blot analysis and quantification of BIN1, BIN1 (1–277), and BIN1 (1–288) in young control subjects, old control subjects, and AD patients (mean ± SEM). n = 4 patients per group, one-way ANOVA, ***P c) IF of BIN1 N277 fragments and AT8 in brain sections from AD patients with different Braak stages. Scale bar, 20 μm. (d, e) Quantification of AT8 and N277 immunostaining (mean ± SEM; one-way ANOVA, n = 6 sections from 3 patients). **P P f) Correlation between the concentrations of BIN1 (1–277) and p-Tau181 as determined by ELISA (R-squared = 0.8841, P S1 Data and S1 Raw Images. AD, Alzheimer’s disease; BIN1, bridging integrator 1; IF, immunofluorescence. (TIF)</p