Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal, and differentiation into a variety of cell types. They thus represent an attractive source of material for cell therapy. However, little is known about the mechanisms underlying the proliferation of BMMSCs. The purpose of this study was to identify the factors and signaling pathways involved in the proliferation of stem cell antigen-1(+) (Sca-1(+)) BMMSCs. Among the cytokines and growth factors examined in this study, fibroblast growth factor-2 (FGF-2) and FGF-4 significantly stimulated the proliferation of Sca-1(+) BMMSCs, as determined by bromodeoxyuridine incorporation. PI3K-Akt, ERK1/2, and JAK/STAT3 pathways were investigated after stimulation with FGF-2 or FGF-4 via Western blot analysis. No changes were observed in the total ERK1/2 and Akt; however, the pERK1/2 and pAkt levels were upregulated early within 15 min in the FGF-2- or FGF-4-treated Sca-1(+) BMMSCs. Moreover, the pERK1/2 and pAkt upregulation induced by FGF-2 and -4 were completely abolished by treatment with the MEK1/2 inhibitor, U0126 and the PI3K inhibitor, LY294002. However, no change in pJAK2 or total JAK2 levels was observed in the Sca-1(+) BMMSCs induced by FGF-2 or FGF-4. As a consequence of PI3K-Akt and ERK1/2, the upregulation of c-Jun in the Sca-1(+) BMMSCs, after stimulation with FGF-2 or FGF-4, was observed after 12 and 24 h. Moreover, the activation of c-Jun in FGF-2- and FGF-4-treated Sca-1(+) BMMSCs was significantly reduced by U0126. Taken together, these data suggest that FGF-2 and -4 promote the proliferation of Sca-1(+) BMMSCs by activation of the ERK1/2 and PI3K-Akt signaling pathways.

To assess the glymphatic activity in patients with idiopathic normal pressure hydrocephalus (NPH) using the "Diffusion Tensor Image-Analysis aLong the Perivascular Space (DTI-ALPS)" method, and determine the feasibility of non-invasive MRI for the evaluation of the glymphatic function. Methods: Between April 2017 and March 2019, 16 patients diagnosed with NPH and 16 age-and sex-matched controls were included. On 3T DTI-MRI, the diffusivities along x-, y-, and z-axes were measured, and the ALPS-indexa ratio that accentuated water diffusion along the perivascular spacewas calculated by two independent readers. The inter-observer agreement was tested using the interclass correlation coefficient. The differences in the diffusivities and the ALPS-index between the NPH and control groups were compared using the Mann-Whitney test. The values were also compared according to the treatment response to the cerebrospinal fluid drainage and correlated with the callosal angle using a correlation coefficient. Results: The inter-observer agreements were excellent for the diffusivities and the ALPS-index. The diffusivity along the x-axis in the projection fibers area and the ALPS-index were significantly lower in patients with NPH (median, 0.556/1.181) than in the controls (0.610/1.540), respectively (P = 0.032/< 0.0001). The ALPS-index was significantly lower in the NPH group who did not show treatment response than those who showed symptomatic relief (0.987/1.329; P < 0.0001). The ALPS-index showed a significant positive correlation with the callosal angle (r = 0.82, P = 0.0001). Conclusions: The DTI-ALPS method can be a useful imaging tool for identifying glymphatic dysfunction and for individually quantifying glymphatic activity in patients with NPH.

Lactobacillus paracasei is a major probiotic and is well known for its anti-inflammatory properties. Thus, we investigated the effects of L. paracasei-derived extracellular vesicles (LpEVs) on LPS-induced inflammation in HT29 human colorectal cancer cells and dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. ER stress inhibitors (salubrinal or 4-PBA) or CHOP siRNA were utilized to investigate the relationship between LpEV-induced endoplasmic reticulum (ER) stress and the inhibitory effect of LpEVs against LPS-induced inflammation. DSS (2%) was administered to male C57BL/6 mice to induce inflammatory bowel disease, and disease activity was measured by determining colon length, disease activity index, and survival ratio. In in vitro experiments, LpEVs reduced the expression of the LPSinduced pro-inflammatory cytokines IL-1α, IL-1β, IL-2, and TNFα and increased the expression of the anti-inflammatory cytokines IL-10 and TGFβ. LpEVs reduced LPS-induced inflammation in HT29 cells and decreased the activation of inflammation-associated proteins, such as COX-2, iNOS and NFκB, as well as nitric oxide. In in vivo mouse experiments, the oral administration of LpEVs also protected against DSS-induced colitis by reducing weight loss, maintaining colon length, and decreasing the disease activity index (DAI). In addition, LpEVs induced the expression of endoplasmic reticulum (ER) stress-associated proteins, while the inhibition of these proteins blocked the anti-inflammatory effects of LpEVs in LPS-treated HT29 cells, restoring the pro-inflammatory effects of LPS. This study found that LpEVs attenuate LPS-induced inflammation in the intestine through ER stress activation. Our results suggest that LpEVs have a significant effect in maintaining colorectal homeostasis in inflammation-mediated pathogenesis.

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Although endoplasmic reticulum (ER) stress-induced apoptosis has been associated with pathogenesis of neurodegenerative diseases, the cellular components involved have not been well delineated. The present study shows that matrix metalloproteinase (MMP)-3 plays a role in the ER stress-induced apoptosis. ER stress induced by brefeldin A (BFA) or tunicamycin (TM) increases gene expression of MMP-3, selectively among various MMP subtypes, and the active form of MMP-3 (actMMP-3) in the brain-derived CATH.a cells. Pharmacological inhibition of enzyme activity, small interference RNA-mediated gene knockdown, and gene knock-out of MMP-3 all provide protection against ER stress. MMP-3 acts downstream of caspase-12, because both pharmacological inhibition and gene knockdown of caspase-12 attenuate the actMMP-3 increase, but inhibition and knock-out of MMP-3 do not alter caspase-12. Furthermore, independently of the increase in the protein level, the catalytic activity of MMP-3 enzyme can be increased via lowering of its endogenous inhibitor protein TIMP-1. Caspase-12 causes liberation of MMP-3 enzyme activity by degrading TIMP-1 that is already bound to actMMP-3. TIMP-1 is decreased in response to ER stress, and TIMP-1 overexpression leads to cell protection and a decrease in MMP-3 activity. Taken together, actMMP-3 protein level and catalytic activity are increased following caspase-12 activation during ER stress, and this in turn plays a role in the downstream apoptotic signaling in neuronal cells. MMP-3 and TIMP-1 may therefore serve as cellular targets for therapy against neurodegenerative diseases. The endoplasmic reticulum (ER)2 is the organelle responsible for proper synthesis and folding of proteins as well as maintenance of intracellular calcium homeostasis. Various cellular stresses cause disruption of normal ER functions, leading to ER stress, and excessive and prolonged ER stress leads to accumulation of misfolded and/or unfolded proteins, and ultimately apoptosis (1).Neurons are particularly vulnerable to ER stress, and ample evidence exists in the literature that links ER stress with neurodegeneration (2-4). Therefore, identification of cellular components and elucidation of the sequence of events following ER stress in neuronal cells would be of great importance in understanding the mechanism of neurodegeneration. The molecular connection between the ER stress response and apoptotic signaling, however, is not clearly understood. Although caspase-12, residing outside of ER membrane, has been shown to be specifically involved in the apoptotic signaling that results from ER stress (5, 6), the exact mechanism by which this occurs has not been elucidated and immediate downstream targets of caspase-12 remain to be identified.Matrix metalloproteinase (MMP)-3 belongs to a family of MMP enzymes known to participate in degradation of components of the extracellular matrix. MMP-3 has been associated with pathogenesis of a number of diseases such as Alzheimer disease, Parkinson disease, stroke, brain trauma, neuroinfl...

In Parkinson disease (PD), the dopaminergic (DAergic) neurons in the substantia nigra undergo degeneration. While the exact mechanism for the degeneration is still not completely understood, neuronal apoptosis and inflammation are thought to play roles. We have recently obtained evidence that matrix metalloproteinase (MMP)-3 plays a crucial role in the apoptotic signal in DAergic cells as well as activation of microglia. The present study tested whether doxycycline might modulate MMP-3 and provide neuroprotection of DAergic neurons. Doxycycline effectively suppressed the expression of MMP-3 induced in response to cellular stress in the DAergic CATH.a cells. This was accompanied by protection of CATH.a cells as well as primary cultured mesencephalic DAergic neurons via attenuation of apoptosis. The active form of MMP-3, released under the cell stress condition, was also decreased in the presence of doxycycline. In addition, doxycycline led to downregulation of MMP-3 in microglial BV-2 cells exposed to lipopolysaccharide (LPS). This was accompanied by suppression of production of nitric oxide and TNF-alpha, as well as gene expression of iNOS, TNF-alpha, IL-1beta, and COX-2. In vivo, doxycycline provided neuroprotection of the nigral DAergic neurons following MPTP treatment, as assessed by tyrosine hydroxylase immunocytochemistry and silver staining, and suppressed microglial activation and astrogliosis as assessed by Iba-1 and GFAP immunochemistry, respectively. Taken together, doxycycline showed neuroprotective effect on DAergic system both in vitro and in vivo and this appeared to derive from anti-apoptotic and anti-inflammatory mechanisms involving downregulation of MMP-3.

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