Aerobic Exercise Facilitates the Nuclear Translocation of SREBP2 by Activating AKT/SEC24D to Contribute Cholesterol Homeostasis for Improving Cognition in APP/PS1 Mice

Impaired cholesterol synthesizing ability is considered a risk factor for the development of Alzheimer’s disease (AD), as evidenced by reduced levels of key proteases in the brain that mediate cholesterol synthesis; however, cholesterol deposition has been found in neurons in tangles in the brains of AD patients. Although it has been shown that statins, which inhibit cholesterol synthesis, reduce the incidence of AD, this seems paradoxical for AD patients whose cholesterol synthesizing capacity is already impaired. In this study, we aimed to investigate the effects of aerobic exercise on cholesterol metabolism in the brains of APP/PS1 mice and to reveal the mechanisms by which aerobic exercise improves cognitive function in APP/PS1 mice. Our study demonstrates that the reduction of SEC24D protein, a component of coat protein complex II (COPII), is a key factor in the reduction of cholesterol synthesis in the brain of APP/PS1 mice. 12 weeks of aerobic exercise was able to promote the recovery of SEC24D protein levels in the brain through activation of protein kinase B (AKT), which in turn promoted the expression of mem-brane-bound sterol regulatory element-binding protein 2 (SREBP2) nuclear translocation and the expression of key proteases mediating cholesterol synthesis. Simultaneous aerobic exercise restored cholesterol transport capacity in the brain of APP/PS1 mice with the ability to efflux excess cholesterol from neurons and reduced neuronal lipid rafts, thereby reducing cleavage of the APP amyloid pathway. Our study emphasizes the potential of restoring intracerebral cholesterol homeostasis as a therapeutic strategy to alleviate cognitive impairment in AD patients

TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease

<div><p>Objective</p><p>To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.</p><p>Methods</p><p>Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF <i>in vitro</i>. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.</p><p>Results</p><p>Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.</p><p>Conclusions</p><p>Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.</p></div

Cell identification and detection of apoptosis in cultured chondrocytes.

<p><b>(a).</b> Chondrocytes identified by toluidine blue staining. <b>(b).</b> Aggrecan staining (Primary antibody: abcam, Ab36861. Secondary antibody: Zhongshan Co. Ltd, China, Cy3-conjugated goat anti-rabbit IgG). <b>(c).</b> Type II collagen dyeing (Primary antibody: Santa Cruz, sc7763. Secondary antibody: Zhongshan Co. Ltd, China, Cy3-conjugated donkey anti-goat IgG). <b>(d).</b> Type I collagen dyeing (Primary antibody: Abcam, ab90395. Secondary antibody: Zhongshan Co. Ltd, China, FITC-conjugated goat anti-mouse IgG). <b>(e).</b> Chondrocyte apoptosis detected by flow cytometry after stimulation with TNF at dosages of 10 ng/ml, 50 ng/ml or 100 ng/ml for 24 h.</p

The mRNA and protein levels of apoptosis <i>in vitro</i>.

<p>A comparison of the mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 <b>(b)</b> in the cultured chondrocytes between groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h. Comparison of the activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units between the groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h. Western blot measuring the release of cytochrome c from the mitochondria to the cytoplasm <b>(e)</b> and comparisons between the groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h <b>(f)</b>. The columns represent the mean values with 95% CIs. n = 3.</p

The mRNA and protein levels <i>in vitro</i> after treatment with TNF inhibitor.

<p>A comparison of the mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 <b>(b)</b> in the cultured chondrocytes stimulated for 24 h between the groups: control (Con), TNF 100 ng/ml (TNF) and TNF 100 ng/ml plus TNF inhibitor 10ug/ml (Inhib). Comparison of the activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units between the groups with stimulation for 24 h. Western blot measuring the release of cytochrome c from the mitochondria to the cytoplasm <b>(e)</b> and comparisons between the groups with stimulation for 24 h <b>(f)</b>. The columns represent the mean values with 95% CIs. n = 3.</p

Condylar cartilage Safranin O staining.

<p><b>(a).</b> Safranin O staining between groups: 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS). <b>(b).</b> Comparison of Safranin O staining positive areas in the central and posterior thirds. The data are expressed as the means and 95% CIs. n = 3.</p

Condylar cartilage histomorphology staining.

<p><b>(a).</b> A comparison of the TMJ condylar cartilage histomorphology between groups: 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS). Areas in 2w UAC and 4w UAC where cells have died are indicated by points. <b>(b).</b> Comparison of cartilage thickness in the central and posterior thirds. The data are expressed as the means and 95% CIs. n = 3.</p

Gene primers.

<p>Gene primers.</p

The mRNA and protein levels of apoptotic pathway components <i>in vivo</i>.

<p>A comparison of the apoptotic activity in the mandibular condylar cartilage between the 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS) groups. The mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 (<b>b</b>). The activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units. Western blot for the release of cytochrome c from the mitochondria into the cytoplasm <b>(e)</b> and its quantification <b>(f)</b>. The data are expressed as the means and 95% CIs. n = 3.</p