Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders.

Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.

How brain 'cleaners' fail: Mechanisms and therapeutic value of microglial phagocytosis in Alzheimer's disease.

Microglia are the resident phagocytes of the brain, where they primarily function in the clearance of dead cells and the removal of un- or misfolded proteins. The impaired activity of receptors or proteins involved in phagocytosis can result in enhanced inflammation and neurodegeneration. RNA-seq and genome-wide association studies have linked multiple phagocytosis-related genes to neurodegenerative diseases, while the knockout of such genes has been demonstrated to exert protective effects against neurodegeneration in animal models. The failure of microglial phagocytosis influences AD-linked pathologies, including amyloid ß accumulation, tau propagation, neuroinflammation, and infection. However, a precise understanding of microglia-mediated phagocytosis in Alzheimer's disease (AD) is still lacking. In this review, we summarize current knowledge of the molecular mechanisms involved in microglial phagocytosis in AD across a wide range of pre-clinical, post-mortem, ex vivo, and clinical studies and review the current limitations regarding the detection of microglia phagocytosis in AD. Finally, we discuss the rationale of targeting microglial phagocytosis as a therapeutic strategy for preventing AD or slowing its progression.

Zinc finger protein 335 mediates lipopolysaccharide-induced neurodegeneration and memory loss as a transcriptional factor in microglia.

Zinc finger protein 335 (Zfp335) is a transcription factor that regulates mammalian neurogenesis and neuronal differentiation. It is a causative factor for severe microcephaly, small somatic size, and neonatal death. Here, we evaluated the effects of Zfp335 in the adult mouse brain after lipopolysaccharide (LPS) challenge. We used wild-type (WT) and Zfp335 knock-down (Zfp335+/- ) mice with LPS administered in the intracerebral ventricle in vivo and cultured microglia treated with LPS in vitro. The impact of Zfp335 was evaluated by RT-PCR, RNA-sequencing, western blotting, immunocytochemistry, ELISA, and the memory behavior tests. Knockdown of Zfp335 expression ameliorated microglia activation significantly, including reduced mRNA and protein expression of Iba1, reduced numbers of microglia, reduced cell diameter, and increased branch length, in the brains of 2-month-old mice after LPS treatment. Zfp335 was expressed in microglia and neurons, but increased in microglia, not neurons, in the brain of mice after LPS administration. LPS-induced microglia-mediated neurodegeneration was dependent upon microglial Zfp335 controlled by nuclear factor-kappa B. Microglial Zfp335 affected neuronal activity through transcriptional regulation of lymphocyte antigen-6M (Ly6M). Our data suggest that Zfp335 is a key transcription factor that exacerbates microglia-mediated neurodegeneration through upregulation of Ly6M expression. Inhibition of microglial Zfp335 may be a new strategy for preventing brain disease induced by microglia activation.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast-Schwann cell-signaling relay.

BACKGROUND: Although peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor. METHODS: We established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague-Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs). RESULTS: Transcriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery. CONCLUSIONS: These results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Research progress of cathepsin B in brain aging and Alzheimer's diseases.

Cathepsin B (CatB), a cysteine protease derived from lysosomes, was initially thought to non-selectively degrade proteins from phagocytosis and autophagy in lysosomes. However, CatB has been demonstrated to selectively degrade and specifically activate target proteins, thereby regulating the process of physiological and pathological responses. The expression, enzymatic activity, and cellular localization of CatB are significantly altered in brain aging and age-related neurodegenerative diseases. Therefore, the pathological function of CatB has attracted much attention in neuroscience research. In this review, we systematically summarize the molecular functions of CatB in brain aging and Alzheimer's disease and discuss the current problems in neuropathological studies of CatB, which lay a foundation for a comprehensive understanding of the pathogenesis of aging and Alzheimer's disease.

IL-33 induces orofacial neuropathic pain through Fyn-dependent phosphorylation of GluN2B in the trigeminal spinal subnucleus caudalis.

Orofacial neuropathic pain can cause considerable disruptions in patients' daily lives, especially because of a lack of effective medications as its underlying causative mechanisms are not fully understood. Here, we found neuron-specific expression of the interleukin (IL)-33 receptor in the trigeminal spinal subnucleus caudalis (Vc), distinct from the spinal dorsal horn. Reduction in head withdrawal threshold in response to von Frey filament stimulation of the whisker pad skin was inversely correlated with the upregulation of IL-33 in the Vc after infraorbital nerve injury (IONI). Neutralization of IL-33 in the Vc alleviated mechanical allodynia in the whisker pad skin after IONI; conversely, intracisternal administration of IL-33 elicited mechanical allodynia in the whisker pad skin, which was relieved by GluN2B antagonism. Moreover, IL-33 triggered the potentiation of GluN2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents and phosphorylation of synaptosomal GluN2B in the Vc, whereas IONI-induced GluN2B phosphorylation was inhibited by neutralization of IL-33 in the Vc. IL-33-induced GluN2B phosphorylation was mediated by phosphorylation of Fyn kinase, and inhibition of the Fyn kinase pathway prevented the development of IL-33-induced mechanical allodynia. Our findings provide insights into a new mechanism by which IL-33 directly regulates synaptic transmission and suggest that IL-33 signaling could be a candidate target for therapeutic interventions for orofacial neuropathic pain.

Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases.

Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood-brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer's disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood-brain barrier and neurodegenerative diseases.

Rab21 Protein Is Degraded by Both the Ubiquitin-Proteasome Pathway and the Autophagy-Lysosome Pathway.

Rab21 is a GTPase protein that is functional in intracellular trafficking and involved in the pathologies of many diseases, such as Alzheimer's disease (AD), glioma, cancer, etc. Our previous work has reported its interaction with the catalytic subunit of gamma-secretase, PS1, and it regulates the activity of PS1 via transferring it from the early endosome to the late endosome/lysosome. However, it is still unknown how Rab21 protein itself is regulated. This work revealed that Rab21 protein, either endogenously or exogenously, can be degraded by the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. It is further observed that the ubiquitinated Rab21 is increased, but the total protein is unchanged in AD model mice. We further observed that overexpression of Rab21 leads to increased expression of a series of genes involved in the autophagy-lysosome pathway. We speculated that even though the ubiquitinated Rab21 is increased due to the impaired proteasome function in the AD model, the autophagy-lysosome pathway functions in parallel to degrade Rab21 to keep its protein level in homeostasis. In conclusion, understanding the characters of Rab21 protein itself help explore its potential as a target for therapeutic strategy in diseases.

Receptor for advanced glycation end products up-regulation in cerebral endothelial cells mediates cerebrovascular-related amyloid ß accumulation after Porphyromonas gingivalis infection.

Cerebrovascular-related amyloidogenesis is found in over 80% of Alzheimer's disease (AD) cases, and amyloid ß (Aß) generation is increased in the peripheral macrophages during infection of Porphyromonas gingivalis (P. gingivalis), a causal bacterium for periodontitis. In this study, we focused on receptor for advanced glycation end products (RAGE), the key molecule involves in Aß influx after P. gingivalis infection to test our hypothesis that Aß transportation from periphery into the brain, known as "Aß influx," is enhanced by P. gingivalis infection. Using cultured hCMEC/D3 cell line, in comparison to uninfected cells, directly infection with P. gingivalis (multiplicity of infection, MOI = 5) significantly increased a time-dependent RAGE expression resulting in a dramatic increase in Aß influx in the hCMEC/D3 cells; the P. gingivalis-up-regulated RAGE expression was significantly decreased by NF-κB and Cathepsin B (CatB)-specific inhibitors, and the P.gingivalis-increased IκBα degradation was significantly decreased by CatB-specific inhibitor. Furthermore, the P. gingivalis-increased Aß influx was significantly reduced by RAGE-specific inhibitor. Using 15-month-old mice (C57BL/6JJmsSlc, female), in comparison to non-infection mice, systemic P. gingivalis infection for three consecutive weeks (1 × 108  CFU/mouse, every 3 days, intraperitoneally) significantly increased the RAGE expression in the CD31-positive endothelial cells and the Aß loads around the CD31-positive cells in the mice's brains. The RAGE expression in the CD31-positive cells was positively correlated with the Aß loads. These observations demonstrate that the up-regulated RAGE expression in cerebral endothelial cells mediates the Aß influx after P. gingivalis infection, and CatB plays a critical role in regulating the NF-κB/RAGE expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15073.

Cathepsin H deficiency decreases hypoxia-ischemia-induced hippocampal atrophy in neonatal mice through attenuated TLR3/IFN-ß signaling.

BACKGROUND: Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity. Its expression level is increased in activated immune cells including dendritic cells, macrophages, and microglia. We have previously reported that CatH deficiency impairs toll-like receptor 3 (TLR3)-mediated activation of interferon regulatory factor 3 (IRF3), and the subsequent secretion of interferon (IFN)-ß from dendritic cells. Furthermore, there is increasing evidence that IFN-ß secreted from microglia/macrophages has neuroprotective effects. These observations prompted further investigation into the effects of CatH deficiency on neuropathological changes. METHODS: In this study, neuropathological changes were examined using histochemical staining (both hematoxylin-eosin (H&E) and Nissl) of the hippocampus of wild-type (WT) and CatH-deficient (CatH-/-) mice after hypoxia-ischemia (HI). The density and the localization of CatH and TLR3 were examined by immunofluorescent staining. CatH processing in microglia was assayed by pulse-chase experiments, while immunoblotting was used to examine TLR3 expression and IRF3 activation in microglia/macrophages in the presence of poly(I:C). Microglial cell death was examined by fluorescence-activated cell sorting (FACS), and primary astrocyte proliferation in the presence of IFN-ß was examined using scratch wound assay. RESULTS: WT mice displayed severe atrophy in association with neuronal death and moderate astrogliosis in the hippocampus following neonatal HI. Somewhat surprisingly, CatH-/- mice showed marked neuronal death without severe atrophy in the hippocampus following HI. Furthermore, there was notable microglia/macrophages cell death and strong astrogliosis in the hippocampus. The TLR3 and phosphorylated IRF3 expression level in the hippocampus or splenocytes (mainly splenic macrophages); from CatH-/- mice was lower than in WT mice. In vitro experiments demonstrated that recombinant IFN-ß suppressed HI-induced microglial cell death and astrocyte proliferation. CONCLUSION: These observations suggest that CatH plays a critical role in the proteolytic maturation and stabilization of TLR3, which is necessary for IFN-ß production. Therefore, impaired TLR3/IFN-ß signaling resulting from CatH deficiency may induce microglial cell death after activation and astrogliosis/glial scar formation in the hippocampus following HI injury, leading to suppression of hippocampal atrophy.

GSK3ß is involved in promoting Alzheimer's disease pathologies following chronic systemic exposure to Porphyromonas gingivalis lipopolysaccharide in amyloid precursor proteinNL-F/NL-F knock-in mice.

In line with the strong association between periodontitis and Alzheimer's disease (AD) clinically, preclinical studies have shown that systemic exposure to Porphyromonas gingivalis (Pg) initiates AD pathologies. However, the involvement of periodontitis in promoting AD pathologies is unclear. In the present study, we provided evidence that chronic systemic exposure to lipopolysaccharide derived from Pg (PgLPS, 1 mg/kg, daily, intraperitoneally) prompted neuroinflammation and tau hyperphosphorylation in 10-month-old of amyloid precursor protein (APP) knock-in mice, a model of AD, carrying the Swedish and Beyreuther/Iberian mutation (APPNL-F/NL-F). The learning and memory function were assessed using the passive avoidance test. The production of APP, Amyloid (A)ß1-42, cytokines, synaptic proteins and the activation of glycogen synthase kinase (GSK)-3ß as well as phosphorylation of tau were analyzed by immunohistochemistry, Western blotting or an enzyme-linked immunosorbent assay (ELISA) in the cortex of APPNL-F/NL-F mice. We found that systemic exposure of PgLPS for three consecutive weeks induced learning and memory deficits with significantly reduced postsynaptic density protein (PSD95). Increased hyperphosphorylation of tau in multiple residues, including Ser202, Thr231 and Ser396, but not the accumulation of Aß1-42 was detected in the neurons of APPNL-F/NL-F mice. Furthermore, PgLPS increased the GSK3ß activity by reducing its phosphorylation of the serine residue at position 9 (Ser9) and promoted neuroinflammation by increasing the expression of interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α) while decreasing that of interleukin-10 (IL-10) and transforming growth factor (TGFß) in the cortex of APPNL-F/NL-F mice. Moreover, the PgLPS-increased GSK3ß activity was detected in both microglia and neurons, while the PgLPS-increased TNF-α expression was mainly detected in the microglia in the cortex of APPNL-F/NL-F mice. In in vitro studies, PgLPS (1 µg/ml) stimulation increased the mRNA and protein level of TNF-α in MG6 microglia, which were significantly inhibited by the GSK3ß-specific inhibitor TWS119. In contrast, the tau hyperphosphorylation and activation of GSK3ß in N2a neurons were enhanced after treatment with conditioned medium from PgLPS-stimulated microglia, which was attenuated after pre-treatment with TNF-α inhibitor. Taken together, these findings indicate that GSK3ß is involved in prompting microglia (TNF-α)-dependent tau hyperphosphorylation in neurons, resulting in learning and memory deficits in APPNL-F/NL-F mice without changes in the Aß expression during chronic systemic exposure to PgLPS. We propose that dampening GSK3ß activation may help delay the periodontitis-promoted pathological progression of AD.

Dual-Functionalized MSCs that Express CX3CR1 and IL-25 Exhibit Enhanced Therapeutic Effects on Inflammatory Bowel Disease.

Mesenchymal stem cells (MSCs) have shown great promise in inflammatory bowel disease (IBD) treatment, owing to their immunosuppressive capabilities, but their therapeutic effectiveness is sometimes thwarted by their low efficiency in entering the inflamed colon and variable immunomodulatory ability in vivo. Here, we demonstrated a new methodology to manipulate MSCs to express CX3C chemokine receptor 1 (CX3CR1) and interleukin-25 (IL-25) to promote their delivery to the inflamed colon and enhance their immunosuppressive capability. Compared to MSCs without treatment, MSCs infected with a lentivirus (LV) encoding CX3CR1 and IL-25 (CX3CR1&IL-25-LV-MSCs) exhibited enhanced targeting to the inflamed colon and could further move into extravascular space of the colon tissues via trans-endothelial migration in dextran sodium sulfate (DSS)-challenged mice after MSC intravenous injection. The administration of the CX3CR1&IL-25-LV-MSCs achieved a better therapeutic effect than that of the untreated MSCs, as indicated by pathological indices and inflammatory markers. Antibody-blocking studies indicated that the enhanced therapeutic effects of dual-functionalized MSCs were dependent on CX3CR1 and IL-25 function. Overall, this strategy, which is based on enhancing the homing and immunosuppressive abilities of MSCs, represents a promising therapeutic approach that may be valuable in IBD therapy.

Liquid chromatography-tandem mass spectrometry measurement of 26 steroid hormones in human serum and plasma samples.

The measurement of steroid hormones provided critical information in the clinical evaluation of endocrine disorders. In this study, we developed a high-throughput solid-phase extraction method for the analysis of 26 steroids in human serum and plasma samples by liquid chromatography-tandem mass spectrometry. Chromatographic conditions and sample preparation were optimized to achieve good separation and maximum sensitivity for these analytes. Under the optimum conditions, good linearities were achieved in the quantitative range for each steroid hormone with the correlation coefficients (r) larger than 0.99. The limits of quantitation of the method were in the range from 0.0005 to 0.7901 ng/mL. The recoveries were in the range of 87.2-114.2% with intra- and interday precision lower than 9.94%. This method has already been applied to series of samples from clinical trials, and there was no significant difference between serum and ethylenediaminetetraacetic acid plasma samples.

Rapid determination of 20 bile acids in human serum by ultra performance liquid chromatography-tandem mass spectrometry.

A method was established for the simultaneous determination of 20 kinds of bile acids in human serum employing ultraperformance liquid chromatography-tandem mass spectrometry. Chromatographic conditions and sample preparation were optimized to achieve good separation and maximum sensitivity for these analytes. The linearity, accuracy and repeatability of the development method were validated with a series of experiments. Under the optimum conditions, good linearities were achieved in the quantitative range for each bile acid with the correlation coefficients (r2 ) >0.9901. The limit of detections (signal-noise ratio 3) of the method were in a range from 0.02 to 0.57 nmol/L. The recoveries were in the range of 88.1-109.9%, RSD < 6.12%. This method was successfully applied for the determination of bile acids in a human serum sample with simple operation, high sensitivity and good accuracy, and provides a reference for the clinical determination of bile acid content.

Cathepsin B inhibition blocks neurite outgrowth in cultured neurons by regulating lysosomal trafficking and remodeling.

Lysosomes are known to mediate neurite outgrowth in neurons. However, the principal lysosomal molecule controlling that outgrowth is unclear. We studied primary mouse neurons in vitro and found that they naturally develop neurite outgrowths over time and as they did so the lysosomal cysteine protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we found that treating those neurons with CA-074Me, which inhibits CTSB, prevented neurites. As that compound also inhibits another protease, we evaluated a N2a neuronal cell line in which the CTSB gene was deleted (CTSB knockout, KO) using CRISPR technology and induced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me is a cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein 2-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB KO N2 a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN, and CREB. These findings show that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons. This adds valuable information regarding the physiological function of CTSB in neural development.

Modelling root growth and soil suction due to plant competition.

Previous experimental results show that planting spacing has significant effects on root distribution and soil suction (negative pore water pressure) due to inter-plant competition. However, there is a lack of theoretical study on this aspect. This study proposes a new physically based mathematical model to capture planting spacing effects on root growth and soil suction considering three key factors, namely hydrotropism, soil mechanical impedance and inter-plant competition. The model is mainly composed of four parts: (i) extension of root zone front; (ii) increase in root density; (iii) root water uptake and (iv) water flow in soil matrix. Root growth and root water uptake are fully coupled. In order to validate the model, laboratory and field tests were conducted on one tree (Schefflera heptaphylla) and one shrub species (Schefflera arboricola), respectively, with different planting spacings. Even though the investigated tree and shrub species had different values of leaf area index and root length density, consistent conclusions on planting spacing effects can be drawn. When planting spacing became smaller, the size of root system decreased while root density increased, hence causing higher soil suction. The model can capture the root distributions as well as induced soil suction during both evapotranspiration and rainfall events quite well for both tree and shrub species.

An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain.

BACKGROUND: Disturbances in clock genes affect almost all patients with Alzheimer's disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD. METHODS: In this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests. RESULTS: CLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice. CONCLUSION: Clock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.

[Trend of vitamin A and vitamin E among pregnancy of Beijing in 2013-2016].

OBJECTIVE: To describe the nutrition level of vitamin A and vitamin E among pregnancy of Beijing and the trends from 2013 to 2016. METHODS: A total of 5487 serum samples from pregnant women who simultaneous detection for the first time from January 2013 to December 2016 in Beijing were enrolled in the study, then high performance liquid chromatography(HPLC) method was used to determine the concentration of serum vitamin A and vitamin E, and it was compared with serum concentrations of vitamin A, vitamin E in normal people. Qualitative analysis of the trend of vitamin A and vitamin E nutrition in pregnant women from 2013 to 2016. RESULTS: From 2013 to 2016, the serum concentration of vitamin A(median(four guantile sapcing))was 1. 23(1. 05-1. 44), 1. 40(1. 19-1. 68), 1. 51(1. 30-1. 75) and 1. 54(1. 30-1. 79) µmol/L, respectively. The total abnormal rate for four years running was 22. 3%, 14. 6%, 8. 1% and 8. 6%, respectively, with main performance of lacking(21. 6%, 13. 4%, 6. 8% and 6. 4%, respectively). The serum concentration of vitamin E(median(four guantile sapcing)) was 36. 00(28. 80-45. 00), 30. 72(25. 44-37. 92), 30. 00(24. 96-35. 52) and 31. 68(26. 40-37. 92) µmol/L, respectively. The total abnormal rate from 2013 to 2016 was 17. 4%, 7. 5%, 3. 6% and 4. 3%, respectively, with excess(17. 4%, 7. 5%, 3. 6% and 4. 0%, respectively) as the main performance. CONCLUSION: For the pregnancy of Beijing between 2013 and 2016, the nutrition level of vitamin A showed an increasing trend and serum vitamin E level showed a tendency to decrease first and then rise. The overall nutritional status of vitamin A and vitamin E had improved.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

Cathepsin B plays a critical role in inducing Alzheimer's disease-like phenotypes following chronic systemic exposure to lipopolysaccharide from Porphyromonas gingivalis in mice.

A number of clinical and experimental studies have revealed a strong association between periodontitis and accelerated cognitive decline in Alzheimer's disease (AD); however, the mechanism of the association is unknown. In the present study, we tested the hypothesis that cathepsin (Cat) B plays a critical role in the initiation of neuroinflammation and neural dysfunction following chronic systemic exposure to lipopolysaccharide from Porphyromonas gingivalis (PgLPS) in mice (1mg/kg, daily, intraperitoneally). Young (2months old) and middle-aged (12months old) wild-type (WT; C57BL/6N) or CatB-deficient (CatB-/-) mice were exposed to PgLPS daily for 5 consecutive weeks. The learning and memory function were assessed using the passive avoidance test, and the expression of amyloid precursor protein (APP), CatB, TLR2 and IL-1ß was analyzed in brain tissues by immunohistochemistry and Western blotting. We found that chronic systemic exposure to PgLPS for five consecutive weeks induced learning and memory deficits with the intracellular accumulation of Aß in neurons in the middle-aged WT mice, but not in young WT or middle-aged CatB-/- mice. PgLPS significantly increased the expression of CatB in both microglia and neurons in middle-aged WT mice, while increased expression of mature IL-1ß and TLR2 was restricted to microglia in the hippocampus of middle-aged WT mice, but not in that of the middle-aged CatB-/- ones. In in vitro studies, PgLPS (1µg/ml) stimulation upregulated the mean mRNA expression of IL-1ß, TLR2 and downregulated the protein levels of IκBα in the cultured MG6 microglia as well as in the primary microglia from WT mice, which were significantly inhibited by the CatB-specific inhibitor CA-074Me as well as by the primary microglia from CatB-/- mice. Furthermore, the mean mRNA expression of APP and CatB were significantly increased in the primary cultured hippocampal neurons after treatment with conditioned medium from PgLPS-treated WT primary microglia, but not after treatment with conditioned medium neutralized with anti-IL-1beta, and not after treatment with conditioned medium from PgLPS-treated CatB-/- primary microglia or with PgLPS directly. Taken together, these findings indicate that chronic systemic exposure to PgLPS induces AD-like phenotypes, including microglia-mediated neuroinflammation, intracellular Aß accumulation in neurons and impairment of the learning and memory functions in the middle-aged mice in a CatB-dependent manner. We propose that CatB may be a therapeutic target for preventing periodontitis-associated cognitive decline in AD.