8-[2-(2-Pentyl-Cyclopropylmethyl)-Cyclopropyl]-Octanoic Acid and Its Diastereomers Improve Age-Related Cognitive Deterioration

Racemic 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a linoleic acid derivative with cyclopropane rings instead of cis-double bonds, contains possible four diastereomers such as α,α-, α,β-, β,α-, and β,β-DCP-LA. The present study examined the effect of racemic and diastereomeric DCP-LA on age-related learning and memory disorders using accelerated-senescence-prone mice 8 (SAMP8) and accelerated-senescence-resistant mice 1 (SAMR1). In the water maze test, the acquisition and retention latencies for SAMP8 mice were significantly longer than the latency for SAMR1 mice, indicating spatial learning and memory impairment for SAMP8 mice. All the racemic (1?mg/kg, per os) and diastereomeric DCP-LA (0.25?mg/kg, per os) significantly shortened the acquisition latency for SAMP8 mice, and racemic, α,α- and α,β-DCP-LA significantly shortened the retention latency, with the advantage greater than the acetylcholine (ACh) esterase inhibitor galanthamine. The results of the present study show that all the racemic and diastereomeric DCP-LA, has the potential to improve age-related learning and memory deterioration, the potential varying among them.     

Improvement of Cognitive Function in Ovariectomized Rats by Human Neural Stem Cells Overexpressing Choline Acetyltransferase via Secretion of NGF and BDNF

Menopause is associated with memory deficits attributed to reduced serum estrogen levels. We evaluated whether an increase in brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF) levels, through transplantation of choline acetyltransferase (ChAT)-overexpressing neural stem cells (F3.ChAT), improved learning and memory in ovariectomized rats. PD13 mouse neuronal primary culture cells were treated with estradiol or co-cultured with F3.ChAT cells; choline transporter1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) expression was evaluated using real-time PCR. The relationship between estrogen receptors (ERs) and neurotrophin family members was analyzed using immunohistochemistry. After the transplantation of F3.ChAT cells into OVx rats, we evaluated the memory, ACh level, and the expression of ER, neurotrophin family proteins, and cholinergic system. Estradiol upregulated CHT1, ChAT, and VAChT expression in ER; they were co-localized with BDNF, NGF, and TrkB. Co-culture with F3.ChAT upregulated CHT1, ChAT, and VAChT by activating the neurotrophin signalling pathway. Transplantation of F3.ChAT cells in OVX animals increased the ACh level in the CSF and improved memory deficit. In addition, it increased the expression of ERs, neurotrophin signaling, and the cholinergic system in the brains of OVX animals. Therefore, the estradiol deficiency induced memory loss by the down-regulation of the neurotrophin family and F3.ChAT could ameliorate the cognitive impairment owing to the loss or reduction of estradiol.     

Aging-Related Metabolic Dysfunction in the Salivary Gland: A Review of the Literature

Aging-related salivary dysfunction commonly induces the poor oral health, including decreased saliva flow and dental caries. Although the clinical significance of the salivary glands is well-known, the complex metabolic pathways contributing to the aging-dysfunction process are only beginning to be uncovered. Here, we provide a comprehensive overview of the metabolic changes in aging-mediated salivary gland dysfunction as a key aspect of oral physiology. Several metabolic neuropeptides or hormones are involved in causing or contributing to salivary gland dysfunction, including hyposalivation and age-related diseases. Thus, aging-related metabolism holds promise for early diagnosis, increased choice of therapy and the identification of new metabolic pathways that could potentially be targeted in salivary gland dysfunction.     

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated β-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.     

Immune Dysfunction Associated with Abnormal Bone Marrow-Derived Mesenchymal Stroma Cells in Senescence Accelerated Mice

Senescence accelerated mice (SAM) are a group of mice that show aging-related diseases, and SAM prone 10 (SAMP10) show spontaneous brain atrophy and defects in learning and memory. Our previous report showed that the thymus and the percentage of T lymphocytes are abnormal in the SAMP10, but it was unclear whether the bone marrow-derived mesenchymal stroma cells (BMMSCs) were abnormal, and whether they played an important role in regenerative medicine. We thus compared BMMSCs from SAMP10 and their control, SAM-resistant (SAMR1), in terms of cell cycle, oxidative stress, and the expression of PI3K and mitogen-activated protein kinase (MAPK). Our cell cycle analysis showed that cell cycle arrest occurred in the G0/G1 phase in the SAMP10. We also found increased reactive oxygen stress and decreased PI3K and MAPK on the BMMSCs. These results suggested the BMMSCs were abnormal in SAMP10, and that this might be related to the immune system dysfunction in these mice.     

Microglial Activation Damages Dopaminergic Neurons through MMP-2/-9-Mediated Increase of Blood-Brain Barrier Permeability in a Parkinson’s Disease Mouse Model

Chronic neuroinflammation has been considered to be involved in the progressive dopaminergic neurodegeneration in Parkinson’s disease (PD). However, the mechanisms remain unknown. Accumulating evidence indicated a key role of the blood–brain barrier (BBB) dysfunction in neurological disorders. This study is designed to elucidate whether chronic neuroinflammation damages dopaminergic neurons through BBB dysfunction by using a rotenone-induced mouse PD model. Results showed that rotenone dose-dependently induced nigral dopaminergic neurodegeneration, which was associated with increased Evans blue content and fibrinogen accumulation as well as reduced expressions of zonula occludens-1 (ZO-1), claudin-5 and occludin, three tight junction proteins for maintaining BBB permeability, in mice, indicating BBB disruption. Rotenone also induced nigral microglial activation. Depletion of microglia or inhibition of microglial activation by PLX3397 or minocycline, respectively, greatly attenuated BBB dysfunction in rotenone-lesioned mice. Mechanistic inquiry revealed that microglia-mediated activation of matrix metalloproteinases-2 and 9 (MMP-2/-9) contributed to rotenone-induced BBB disruption and dopaminergic neurodegeneration. Rotenone-induced activation of MMP-2/-9 was significantly attenuated by microglial depletion and inactivation. Furthermore, inhibition of MMP-2/-9 by a wide-range inhibitor, SB-3CT, abrogated elevation of BBB permeability and simultaneously increased tight junctions expression. Finally, we found that microglial depletion and inactivation as well as inhibition of MMP-2/-9 significantly ameliorated rotenone-elicited nigrostriatal dopaminergic neurodegeneration and motor dysfunction in mice. Altogether, our findings suggested that microglial MMP-2/-9 activation-mediated BBB dysfunction contributed to dopaminergic neurodegeneration in rotenone-induced mouse PD model, providing a novel view for the mechanisms of Parkinsonism.     

Effect of cholineacetyltransferase activity and choline recycle ratio on diffusion-reaction modeling,bifurcation and chaotic behavior of acetylcholine neurocycle and their relation to Alzheimer's and Parkinson's diseases

This paper is an extension of the previous two papers (Mustafa et al., 2009a, Mustafa et al., 2009b) on the modeling, simulation and analysis of bifurcation, dynamics, and chaotic characteristics of the acetylcholine (ACh) neurocycle. The effects of cholineacetyltransferase (ChAT) activity and choline recycle ratio as bifurcation parameters, on the system performance are studied through modifying the two-compartment model. It is found that as ChAT activity increases, ACh concentration in the model compartments increases continuously. The effect of choline recycle ratio shows that choline uptake represents the key role for providing choline as a substrate for ACh synthesis. It is found that the choline recycle ratio has a great influence on all state variables in both compartments. A detailed bifurcation and sensitive analyses over a wide range of ChAT activity and choline recycle ratio parameters are carried out in order to highlight the characteristics of ACh cholinergic system like non-linear dynamics and different solutions such as multiplicity of: stationery states as well as periodic and chaotic states. The relations between the results and the functions of ACh cholinergic neurons are investigated. A comparison between the results and other experimental and physiological measurements is performed. It is found that there is a good relation between cholinergic diseases such as Alzheimer's and Parkinson's diseases, and level of ACh in brains from one side and current results extracted from the disturbances in ChAT activity and choline recycle from the other side. It is found that choline recycled is the most critical factor in ACh processes in comparison with any of the following parameters: ChAT activity, as well as feed acetate and ACh concentrations.     

Understanding the Potential Role of Sirtuin 2 on Aging: Consequences of SIRT2.3 Overexpression in Senescence

Sirtuin 2 (SIRT2) has been associated to aging and age-related pathologies. Specifically, an age-dependent accumulation of isoform 3 of SIRT2 in the CNS has been demonstrated; however, no study has addressed the behavioral or molecular consequences that this could have on aging. In the present study, we have designed an adeno-associated virus vector (AAV-CAG-Sirt2.3-eGFP) for the overexpression of SIRT2.3 in the hippocampus of 2 month-old SAMR1 and SAMP8 mice. Our results show that the specific overexpression of this isoform does not induce significant behavioral or molecular effects at short or long term in the control strain. Only a tendency towards a worsening in the performance in acquisition phase of the Morris Water Maze was found in SAMP8 mice, together with a significant increase in the pro-inflammatory cytokine Il-1β. These results suggest that the age-related increase of SIRT2.3 found in the brain is not responsible for induction or prevention of senescence. Nevertheless, in combination with other risk factors, it could contribute to the progression of age-related processes. Understanding the specific role of SIRT2 on aging and the underlying molecular mechanisms is essential to design new and more successful therapies for the treatment of age-related diseases.     

The Lack of Amyloidogenic Activity Is Persistent in Old WT and APPswe/PS1ΔE9 Mouse Retinae

We have previously reported that vision decline was not associated with amyloidogenesis processing in aging C57BL/6J wild-type (WT) mice and in a mouse model of Alzheimer’s disease, the APP_swe/PS1ΔE9 transgenic mouse model (APP/PS1). This conclusion was drawn using middle-aged (10–13 months old) mice. Here, we hypothesized that compared with hippocampal and cortical neurons, the weak amyloidogenic activity of retinal neurons may result in a detectable release of amyloid β (Aβ) only in aged mice, i.e., between 14 and 24 months of age. The aim of the present study was thus to follow potential activity changes in the amyloidogenic and nonamyloidogenic pathways of young (4 months) and old (20–24 months) WT and APP/PS1 mice. Our results showed that in spite of retinal activity loss reported by electroretinogram (ERG) recordings, the level of amyloid beta precursor protein (APP) and its derivatives did not significantly vary in the eyes of old vs. young mice. Strikingly, the ectopic expression of human APP_swe in APP/PS1 mice did not allow us to detect Aβ monomers at 23 months. In contrast, Aβ was observed in hippocampal and cortical tissues at this age but not at 4 months of life. In contrast, optic nerve transection-induced retinal ganglion cell injury significantly affected the level of retinal APP and the secretion of soluble APP alpha in the vitreous. Collectively, these results suggest that the amyloidogenic and nonamyloidogenic pathways are not involved in visual function decline in aging mice. In WT and APP/PS1 mice, it is proposed that retinal neurons do not have the capacity to secrete Aβ in contrast with other cortical and hippocampal neurons.     

Urine-Derived Stem Cell-Secreted Klotho Plays a Crucial Role in the HK-2 Fibrosis Model by Inhibiting the TGF-β Signaling Pathway

Renal fibrosis is an irreversible and progressive process that causes severe dysfunction in chronic kidney disease (CKD). The progression of CKD stages is highly associated with a gradual reduction in serum Klotho levels. We focused on Klotho protein as a key therapeutic factor against CKD. Urine-derived stem cells (UDSCs) have been identified as a novel stem cell source for kidney regeneration and CKD treatment because of their kidney tissue-specific origin. However, the relationship between UDSCs and Klotho in the kidneys is not yet known. In this study, we discovered that UDSCs were stem cells that expressed Klotho protein more strongly than other mesenchymal stem cells (MSCs). UDSCs also suppressed fibrosis by inhibiting transforming growth factor (TGF)-β in HK-2 human renal proximal tubule cells in an in vitro model. Klotho siRNA silencing reduced the TGF-inhibiting ability of UDSCs. Here, we suggest an alternative cell source that can overcome the limitations of MSCs through the synergetic effect of the origin specificity of UDSCs and the anti-fibrotic effect of Klotho.     

Increased Indoleamine 2,3-Dioxygenase Levels at the Onset of Sjögren’s Syndrome in SATB1-Conditional Knockout Mice

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferon γ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.     

Thrombin Inhibition Prevents Endothelial Dysfunction and Reverses 20-HETE Overproduction without Affecting Blood Pressure in Angiotensin II-Induced Hypertension in Mice

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO₂⁻ quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.     

Salivary and Lacrimal Gland Alterations of the Epidermal Fatty Acid-Binding Protein (E-FABP) in Non-Obese Diabetic Mice

The purpose of this study was to investigate the changes in E-FABP in the salivary and lacrimal glands of the Sjögren syndrome (SS) model non-obese diabetic mice (NOD). Cotton thread and ocular vital staining tests were performed on 10-week NOD male mice (n = 24) and age- and sex-matched wild-type (WT) mice (n = 25). Tear and saliva samples were collected at sacrifice for E-FABP ELISA assays. Salivary and lacrimal gland specimens underwent immunohistochemistry stainings for E-FABP. Real-time RT-PCR was also performed for the quantification of mRNA expression levels in the salivary and lacrimal glands. Corneal vital staining scores in the NOD mice were significantly higher compared with those for the wild-type mice (p = 0.0001). The mean tear E-FABP level showed a significantly lower concentration in the NOD mice (p = 0.001). The mean saliva E-FABP level also showed a significantly lower concentration in the NOD mice (p = 0.04). Immunohistochemistry revealed intense E-FABP staining in the LG acinar epithelium and less intense staining in the acinar epitheliae of the SGs in the NOD mice compared to the WT mice. Real-time RT-PCR for the mRNA expression of E-FABP showed a significantly decreased expression in the SG and a significant increase in the LG of the NOD mice compared to the WT mice. In conclusion, the E-FABP showed marked alterations in the tear film, saliva, lacrimal, and salivary glands of the NOD mouse, which may help explain the ocular surface changes in relation to the dry eye disease in this SS model mouse and keratoconjunctivitis sicca in SS patients.     

Allosteric Binding Sites of Aβ Peptides on the Acetylcholine Synthesizing Enzyme ChAT as Deduced by In Silico Molecular Modeling

The native function of amyloid-β (Aβ) peptides is still unexplored. However, several recent reports suggest a prominent role of Aβ peptides in acetylcholine homeostasis. To clarify this role of Aβ, we have reported that Aβ peptides at physiological concentrations can directly enhance the catalytic efficiency of the key cholinergic enzyme, choline acetyltransferase (ChAT), via an allosteric interaction. In the current study, we further aimed to elucidate the underlying ChAT-Aβ interaction mechanism using in silico molecular docking and dynamics analysis. Docking analysis suggested two most probable binding clusters on ChAT for Aβ₄₀ and three for Aβ₄₂. Most importantly, the docking results were challenged with molecular dynamic studies of 100 ns long simulation in triplicates (100 ns × 3 = 300 ns) and were analyzed for RMSD, RMSF, RoG, H-bond number and distance, SASA, and secondary structure assessment performed together with principal component analysis and the free-energy landscape diagram, which indicated that the ChAT-Aβ complex system was stable throughout the simulation time period with no abrupt motion during the evolution of the simulation across the triplicates, which also validated the robustness of the simulation study. Finally, the free-energy landscape analysis confirmed the docking results and demonstrated that the ChAT-Aβ complexes were energetically stable despite the unstructured nature of C- and N-terminals in Aβ peptides. Overall, this study supports the reported in vitro findings that Aβ peptides, particularly Aβ₄₂, act as endogenous ChAT-Potentiating-Ligand (CPL), and thereby supports the hypothesis that one of the native biological functions of Aβ peptides is the regulation of acetylcholine homeostasis.     

Toll-Like Receptor 7 Is Required for Lacrimal Gland Autoimmunity and Type 1 Diabetes Development in Male Nonobese Diabetic Mice

Sjögren syndrome (SS) is an immunologically complex, chronic autoimmune disease targeting lacrimal and salivary glands. Nonobese diabetic (NOD) mice spontaneously develop inflammation of lacrimal and salivary glands with histopathological features similar to SS in humans including focal lymphocytic infiltrates in the affected glands. The innate immune signals driving lymphocytic infiltration of these glands are not well-defined. Here we evaluate the role of Toll-like receptor (TLR) 7 in the development of SS-like manifestations in NOD mice. We created a Tlr7 knockout NOD mouse strain and performed histological and gene expression studies to characterize the effects of TLR7 on autoimmunity development. TLR7 was required for male-specific lacrimal gland inflammation but not for female-specific salivary gland inflammation. Moreover, TLR7 was required for type 1 diabetes development in male but not female NOD mice. RNA sequencing demonstrated that TLR7 was associated with a type I interferon (IFN) response and a type I IFN-independent B cell response in the lacrimal glands. Together these studies identify a previously unappreciated pathogenic role for TLR7 in lacrimal gland autoimmunity and T1D development in male NOD mice adding to the growing body of evidence supporting sex differences in mechanisms of autoimmune disease in NOD mice.     

Therapeutic Effects of Hydrogen Gas Inhalation on Trimethyltin-Induced Neurotoxicity and Cognitive Impairment in the C57BL/6 Mice Model

Oxidative stress (OS) is one of the causative factors in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease (AD) and cognitive dysfunction. In the present study, we investigated the effects of hydrogen (H₂) gas inhalation in trimethyltin (TMT)-induced neurotoxicity and cognitive dysfunction in the C57BL/6 mice. First, mice were divided into the following groups: mice without TMT injection (NC), TMT-only injection group (TMT only), TMT injection + lithium chloride-treated group as a positive control (PC), and TMT injection + 2% H₂ inhalation-treated group (H₂). The TMT injection groups were administered a single dosage of intraperitoneal TMT injection (2.6 mg/kg body weight) and the H₂ group was treated with 2% H₂ for 30 min once a day for four weeks. Additionally, a behavioral test was performed with Y-maze to test the cognitive abilities of the mice. Furthermore, multiple OS- and AD-related biomarkers such as reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca²⁺), malondialdehyde (MDA), glutathione peroxidase (GPx), catalase, inflammatory cytokines, apolipoprotein E (Apo-E), amyloid β (Aβ)-40, phospho-tau (p-tau), Bcl-2, and Bcl-2- associated X (Bax) were investigated in the blood and brain. Our results demonstrated that TMT exposure alters seizure and spatial recognition memory. However, after H₂ treatment, memory deficits were ameliorated. H₂ treatment also decreased AD-related biomarkers, such as Apo-E, Aβ-40, p-tau, and Bax and OS markers such as ROS, NO, Ca²⁺, and MDA in both serum and brain. In contrast, catalase and GPx activities were significantly increased in the TMT-only group and decreased after H₂ gas treatment in serum and brain. In addition, inflammatory cytokines such as granulocyte colony-stimulating factors (G-CSF), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) were found to be significantly decreased after H₂ treatment in both serum and brain lysates. In contrast, Bcl-2 and vascular endothelial growth factor (VEGF) expression levels were found to be enhanced after H₂ treatment. Taken together, our results demonstrated that 2% H₂ gas inhalation in TMT-treated mice exhibits memory enhancing activity and decreases the AD, OS, and inflammatory-related markers. Therefore, H₂ might be a candidate for repairing neurodegenerative diseases with cognitive dysfunction. However, further mechanistic studies are needed to fully clarify the effects of H₂ inhalation on TMT-induced neurotoxicity and cognitive dysfunction.