Timp-3 deficiency impairs cognitive function in mice

Extracellular matrix (ECM) degradation is performed primarily by matrix metalloproteinases (MMPs). MMPs have recently been shown to regulate synaptic activity in the hippocampus and to affect memory and learning. The tissue inhibitor of metalloproteinase (Timp) is an endogenous factor that controls MMP activity by binding to the catalytic site of MMPs. At present, four Timp isotypes have been reported (Timp-1 through Timp-4) with 35–50% amino-acid sequence homology. Timp-3 is a unique member of Timp proteins in that it is bound to the ECM. In this study, we used the passive avoidance test, active avoidance test, and water maze test to examine the cognitive function in Timp-3 knockout (KO) mice. Habituation was evaluated using the open-field test. The water maze test showed that Timp-3 KO mice exhibit deterioration in cognitive function compared with wild-type (WT) mice. The open-field test showed decreased habituation of Timp-3 KO mice. Immunostaining of brain slices revealed the expression of Timp-3 in the hippocampus. In situ zymography of the hippocampus showed increased gelatinolytic activity in Timp-3 KO mice compared with WT mice. These results present the first evidence of Timp-3 involvement in cognitive function and hippocampal MMP activity in mice. Moreover, our findings suggest a novel therapeutic target to be explored for improvement of cognitive function in humans

A DKP cyclo(L-Phe-L-Phe) found in chicken essence is a dual inhibitor of the serotonin transporter and acetylcholinesterase.

Diketopiperazines (DKPs) are naturally-occurring cyclic dipeptides with a small structure and are found in many organisms and in large amounts in some foods and beverages. We found that a chicken essence beverage, which is popular among Southeast Asians as a traditional remedy and a rich source of DKPs, inhibited the serotonin transporter (SERT) and suppressed serotonin uptake from rat brain synaptosomes, which prompted us to isolate and identify the active substance(s). We purified a SERT inhibitor from the chicken essence beverage and identified it as the DKP cyclo(L-Phe-L-Phe). Interestingly, it was a naturally occurring dual inhibitor that inhibited both SERT and acetylcholinesterase (AChE) in vitro. The DKP increased extracellular levels of the cerebral monoamines serotonin, norepinephrine, and dopamine in the medial prefrontal cortex and acetylcholine in the ventral hippocampus of freely moving rats when administered orally. Moreover, cyclo(L-Phe-L-Phe) significantly shortened escape latency in the water maze test in depressed mice previously subjected to a repeated open-space swimming task, which induces a depression-like state. Cyclo(L-Phe-L-Phe) also significantly improved accuracy rates in a radial maze test in rats and increased step-through latencies in a passive avoidance test in mice with scopolamine-induced amnesia. These animal test results suggest that cyclo(L-Phe-L-Phe), which is present abundantly in some foods such as chicken essence, may abrogate the onset of depression and, thus, contribute to preventing the development of Alzheimer's disease and other dementia, because senile depression is a risk factor for dementia

Intravenous Administration of Human Amniotic Mesenchymal Stem Cells in the Subacute Phase of Cerebral Infarction in a Mouse Model Ameliorates Neurological Disturbance by Suppressing Blood Brain Barrier Disruption and Apoptosis via Immunomodulation

Neuro-inflammation plays a key role in the pathophysiology of brain infarction. Cell therapy offers a novel therapeutic option due to its effect on immunomodulatory effects. Amniotic stem cells, in particular, show promise owing to their low immunogenicity, tumorigenicity, and easy availability from amniotic membranes discarded following birth. We have successfully isolated and expanded human amniotic mesenchymal stem cells (hAMSCs). Herein, we evaluated the therapeutic effect of hAMSCs on neurological deficits after brain infarction as well as their immunomodulatory effects in a mouse model in order to understand their mechanisms of action. One day after permanent occlusion of the middle cerebral artery (MCAO), hAMSCs were intravenously administered. RT-qPCR for TNFα, iNOS, MMP2, and MMP9, immunofluorescence staining for iNOS and CD11b/c, and a TUNEL assay were performed 8 days following MCAO. An Evans Blue assay and behavioral tests were performed 2 days and several months following MCAO, respectively. The results suggest that the neurological deficits caused by cerebral infarction are improved in dose-dependent manner by the administration of hAMSCs. The mechanism appears to be through a reduction in disruption of the blood brain barrier and apoptosis in the peri-infarct region through the suppression of pro-inflammatory cytokines and the M2-to-M1 phenotype shift

Early Reperfusion Following Ischemic Stroke Provides Beneficial Effects, Even After Lethal Ischemia with Mature Neural Cell Death

Ischemic stroke is a critical disease caused by cerebral artery occlusion in the central nervous system (CNS). Recent therapeutic advances, such as neuroendovascular intervention and thrombolytic therapy, have allowed recanalization of occluded brain arteries in an increasing number of stroke patients. Although previous studies have focused on rescuing neural cells that still survive despite decreased blood flow, expanding the therapeutic time window may allow more patients to undergo reperfusion in the near future, even after lethal ischemia, which is characterized by death of mature neural cells, such as neurons and glia. However, it remains unclear whether early reperfusion following lethal ischemia results in positive outcomes. The present study used two ischemic mouse models—90-min transient middle cerebral artery occlusion (t-MCAO) paired with reperfusion to induce lethal ischemia and permanent middle cerebral artery occlusion (p-MCAO)—to investigate the effect of early reperfusion up to 8 w following MCAO. Although early reperfusion following 90-min t-MCAO did not rescue mature neural cells, it preserved the vascular cells within the ischemic areas at 1 d following 90-min t-MCAO compared to that following p-MCAO. In addition, early reperfusion facilitated the healing processes, including not only vascular but also neural repair, during acute and chronic periods and improved recovery. Furthermore, compared with p-MCAO, early reperfusion after t-MCAO prevented behavioral symptoms of neurological deficits without increasing negative complications, including hemorrhagic transformation and mortality. These results indicate that early reperfusion provides beneficial effects presumably via cytoprotective and regenerative mechanisms in the CNS, suggesting that it may be useful for stroke patients that experienced lethal ischemia

Effects of cyclo(L-Phe-L-Phe) on the number of different arms chosen within the first eight trials in the radiaL-arm maze test (A) and the step-through latencies in the passive avoidance test (B) with scopolamine-induced learning impairment.

<p>Male Sprague–Dawley rats (6-weeks-old) and male C57BL/6N mice were used in the radiaL-arm maze and the passive avoidance tests, respectively. Two doses (20 and 200 mg/kg) of cyclo(L-Phe-L-Phe) suspended in 0.5% CMC-Na were administered orally 10 min before scopolamine treatment. Cyclo(L-Phe-L-Phe) was orally administered at doses of 20 or 200 mg/kg 30 min after each scopolamine administration. Donepezil (1 mg/kg in the radial maze test or 2 mg/kg in the passive avoidance test) suspended in 0.5% CMC-Na was intraperitoneally administered as the positive control, and 0.5% CMC-Na was used as the vehicle control. All values are shown as mean ± standard error (Bonferroni’s post-test; **<i>P</i><0.01, n = 10 rats in the radial maze test and n = 10–16 mice in the passive avoidance test).</p

Purification of a serotonin transporter (SERT) inhibitor from a chicken essence beverage.

<p>(A) Sephadex LH-20 column chromatography of the diethyl ether extract. Every 80 ml of elution was collected from a Sephadex LH-20 column. Dried weight (dotted line) and SERT inhibitory activity (solid line) are shown. (B) Develosil ODS-HG-5 column chromatography of the fraction 3 from (A). The column was eluted with a linear gradient of 8–48% acetonitrile at a flow rate of 6 ml/min (blue line). The black line represents the relative absorbance at 215 nm. The red line represents peak fractionation. The active peak shown by the arrow was evaporated and dissolved in distilled water. (C) SOURCE 15 RPC column chromatography of the active fraction in (D). The column was eluted with a linear gradient of 0–80% acetonitrile at a flow rate 0.5 ml/min (green line). The blue, pink, and red lines represent the relative absorbance at 215, 240, and 280 nm, respectively. Dotted blue-green line represents 25 milliabsorbance (mAbs). The active peak is shown by the arrow.</p

Effects of a 14-day administration of cyclo(L-Phe-L-Phe) on extracellular levels of serotonin (5-HT) (A), norepinephrine (NE) (B), and dopamine (DA) (C) in the medial prefrontal cortex (mPFC) and extracellular levels of acetylcholine (ACh) (D) in the ventral hippocampus (VHIPP) of freely moving rats measured using brain microdialysis.

<p>Cyclo(L-Phe-L-Phe) was orally administered to male Sprague–Dawley rats at doses of 2, 20, and 200 mg/kg after sub-chronic treatment for 13 consecutive days once daily at the same doses. Values of the treated groups were compared to that of the vehicle group at respective time points (two-way analysis of variance followed by Bonferroni’s post-test; *<i>P</i><0.05, **<i>P</i><0.01. All values are mean ± standard error, n = 6 rats).</p

Apparent permeability coefficient (Papp) of cyclo(L-Phe-L-Phe) measured using a blood–brain barrier (BBB) <i>in vitro</i> model.

<p>Papps of caffeine, sucrose, Na-F, Evans blue albumin (EBA), and cyclo(L-Phe-L-Phe) were measured and calculated.</p