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Diabetic corneal endothelial keratopathy is an intractable ocular complication characterized by corneal edema and endothelial decompensation, which seriously threaten vision. It has been suggested that diabetes is associated with pyroptosis, a type of programmed cell death via the activation of inflammation. Long noncoding RNA KCNQ1OT1 is commonly associated with various pathophysiological mechanisms of diabetic complications, including diabetic cardiomyopathy and diabetic retinopathy. However, whether KCNQ1OT1 is capable of regulating pyroptosis and participates in the pathogenesis of diabetic corneal endothelial keratopathy remains unknown. The aim of this study was to investigate the mechanisms of KCNQ1OT1 in diabetic corneal endothelial keratopathy. Here, we reveal that KCNQ1OT1 and pyroptosis can be triggered in diabetic human and rat corneal endothelium, along with the high glucose-treated corneal endothelial cells. However, miR-214 expression was substantially decreased in vivo and in experiments with cultured cells. LDH assay was also used to verify the existence of pyroptosis in high glucose-treated cells. Bioinformatics prediction and luciferase assays showed that KCNQ1OT1 may function as a competing endogenous RNA binding miR-214 to regulate the expression of caspase-1. To further analyze the KCNQ1OT1-mediated mechanism, miR-214 mimic and inhibitor were introduced into the high glucose-treated corneal endothelial cells. The results showed that upregulation of miR-214 attenuated pyroptosis; conversely, knockdown of miR-214 promoted it. In addition, KCNQ1OT1 knockdown by a small interfering RNA decreased pyroptosis factors expressions but enhanced miR-214 expression in corneal endothelial cells. To understand the signaling mechanisms underlying the prepyroptotic properties of KCNQ1OT1, si-KCNQ1OT1 was cotransfected with or without miR-214 inhibitor. The results showed that pyroptosis was repressed after silencing KCNQ1OT1 but was reversed by cotransfection with miR-214 inhibitor, suggesting that KCNQ1OT1 mediated pyroptosis induced by high glucose via targeting miR-214. Therefore, the KCNQ1OT1/miR-214/caspase-1 signaling pathway represents a new mechanism of diabetic corneal endothelial keratopathy progression, and KCNQ1OT1 could potentially be a novel therapeutic target.

The corneal endothelium (CE) is vital for the cornea to maintain its transparency. However, CE dysfunction occurs due to aging, intraocular surgery, trauma, dystrophy, etc. Corneal transplantation is the only method to clinically treat CE dysfunction; however, this treatment strategy faces the disadvantages of a global cornea shortage, graft failure, and severe side effects. There is a recognized need for a substitute for the CE. Stem cells are becoming increasingly common for the treatment of human diseases. In fact, several studies have documented the induction of corneal endothelial-like cells (CECs) from stem cells, but an ideal procedure has not yet been established. Thus, this study aimed at exploring a more efficient and robust differentiation method. We used a modified approach to differentiate induced pluripotent stem cells (iPSCs) into CECs. After the identification of differentiated CECs, the CECs were injected into the anterior chambers of the eyes of a rabbit model of bullous keratopathy. The rabbits were maintained in the eye-down position to ensure that the cells attached to the cornea. The results showed that corneal edema was alleviated in the rabbits injected with CECs compared with that in the rabbits belonging to the control group. This study extends the ability to differentiate iPSCs into CECs and provides a potential strategy for the treatment of reduced visual acuity caused by CE deficiency in the future.

Objective The therapeutic efficacy of apigenin in PC12 cells and rats remains uncertain. The aim of this study was to investigate the neuroprotective effects of apigenin against cerebral ischemia/reperfusion injury, both in vitro and in vivo. Methods We first treated PC12 cells with cobalt chloride (CoCl2) to create a model of oxidative stress injury. Cell viability was then determined using a multifunctional microplate reader. In addition, reactive oxygen species (ROS) levels, apoptosis, and mitochondrial membrane potentials (MMPs) were examined using high-content cytometer analysis. The efficacy of apigenin treatment was also analyzed in a rat middle cerebral artery occlusion (MCAO) model using TTC staining and neurological deficit scores. Results The half-inhibitory concentration of CoCl2 was 1.2 mM. Pretreatment with 10 µg ⋅ mL−1 apigenin significantly enhanced cell viability, reduced ROS levels, alleviated apoptosis, and improved MMP in PC12 cells with CoCl2-induced injury in vitro. In addition, apigenin treatment in vivo significantly improved neurological deficit scores and reduced infarct areas in MCAO rats. These results suggest that the neuroprotective mechanisms of apigenin may be related to mitochondrial activation. Conclusions Apigenin had excellent neuroprotective effects for the treatment of cerebral ischemia/reperfusion injury in vitro and in vivo.

Fibrillary α-synuclein (α-syn), which constitutes the major component of Lewy pathology characterized for Parkinson's disease (PD), is found also in the aged enteric nervous system (ENS) and central nervous system (CNS). However, what happens to oligomeric α-syn (o-α-syn) in the aged ENS and CNS remains poorly understood. Here, by using ELISA methods specific for o-α-syn and phosphorylated α-syn (p-α-syn), we measured the levels of o-α-syn and p-α-syn in the gastrointestinal (GI) tract and brain of aging cynomolgus monkeys. We detected a synchronously occurred age-dependent elevation of o-α-syn in the GI tract and brain, which was accompanied by an age-dependent increase of α-syn phosphorylated at serine 129, a modification that promotes α-syn aggregation. Correlation analysis revealed that alterations in p-α-syn and o-α-syn in the colon were positively correlated to those in some brain regions such as the striatum, hippocampus, thalamus and temporal lobe. The results obtained suggest that age-dependent elevations of p-α-syn and o-α-syn occur synchronously in various regions of the ENS and CNS.

Objective. To investigate prognostic values of serum biomarkers of soluble intercellular adhesion molecule 1 (sICAM-1), macrophage migration inhibitor factor (MIF), interleukin 1β (IL-1β), and soluble urokinase plasminogen activator receptor (su-PAR) in patients with acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Methods. From August 2017 to November 2019, 122 consecutive IPF patients treated in our center were classified as stable IPF and AE-IPF based on the newly published international guidelines. Serum levels of four biomarkers at admission were measured by the enzyme-linked immunosorbent assay (ELISA). The primary endpoint was 3-month mortality. The log-rank test and logistic regression analysis were used to evaluate the effects of these biomarkers for survival in patients with AE-IPF. Cox proportional hazards analysis was performed to evaluate the prognostic values of serological biomarkers and clinical data. Results. Eighty-one patients were diagnosed with stable IPF, and 41 AE-IPF patients were enrolled in the study. Serum levels of sICAM-1 (p<0.001), IL-1β (p<0.001), MIF (p<0.001), and su-PAR (p<0.001) in patients with IPF were significantly increased compared to those in healthy controls. All the four biomarkers were elevated in patients with AE-IPF compared to those with stable IPF. The 3-month mortality in AE-IPF was 56.1% (23/41). Increased levels of MIF (p=0.01) and IL-1β (>5 pg/mL, p=0.033) were independent risk factors for 3-month mortality in patients with AE-IPF. Conclusions. We showed the higher serum levels of IL-1β, and MIF had prognostic values for 3-month mortality in AE-IPF. This study provided a clue to promote our understanding in the pathogenesis of the disease.

The aging brain is associated with reduced cell surface expression of N-methyl-d-aspartate receptors (NMDARs), but the mechanism remains poorly understood. In the present study, we showed that in the striatum and hippocampus but not the cerebellum and parietal cortex, levels of α-synuclein monomers and oligomers increased with age, which correlated negatively with the expression of GluN1, and positively with the expression of total Rab5B. The oligomer-α-synuclein exhibited a stronger correlation with the expression of surface GluN1 and total Rab5B. In MES23.5 cells, the monomer- or oligomer-α-synuclein were shown to increase in a manner dependent on the concentrations of the added monomers and oligomers. Again, the oligomer-α-synuclein showed more potent effects than the monomer-α-synuclein on surface GluN1 and total Rab5B expression. Accordingly, the oligomer-treated cells showed a greater reduction in NMDA-evoked Ca2+ influx than the monomer-treated cells, which was largely inhibited by pistop2, a clathrin inhibitor. These results suggest that the age-dependent accumulation of α-synuclein monomers and oligomers differentially contributes to the reduction in surface NMDAR expression in selective brain regions.

Dopaminergic (DAergic) degeneration and abnormal α-synuclein (α-syn) expression, phosphorylation and aggregation are observed in both the nigrostriatal system (NSS) and enteric nervous system (ENS) of patients with Parkinson’s disease (PD). Whether these alterations in α-syn and DAergic neurons occur synchronously in the two nervous systems or follow a process that spreads from the gut to the brain remains a subject of debate. Here, in MPTP-intoxicated cynomolgus monkeys, we showed a parallel DAergic degeneration in the colon as well as in the substantia nigra and striatum (SN/STR), as indicated by reduced expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). In addition, we observed a simultaneous increase in the concentrations of total, phosphorylated, and oligomeric α-syn in the colon and SN/STR. Moreover, we identified that the above changes in α-syn were associated with an increase in the expression of polo-like kinase 2 (PLK2), an enzyme that promotes α-syn phosphorylation, and a decrease in the activity of protein phosphatase 2A (PP2A), an enzyme that facilitates α-syn dephosphorylation. Because the colonic ENS can be readily analyzed using routine biopsies, the shared pathological features between the colonic ENS and the brain NSS found in this study provide useful information for assessing and understanding the neuropathology in PD patients using colonic biopsies.

Exposure to early stressful adverse life events such as maternal separation severely impacts the development of the nervous system. Using immunohistochemistry, quantitative PCR and Western blot approaches, we found that alpha6 subunit-containing GABAA receptors (Gabra6-containing GABAA Rs) were expressed on hippocampal interneurons of adolescent rats. Maternal separation stress (MS) from postnatal day 2 to15 significantly reduced Gabra6 expression and provoked depressive behaviors such as anhedonia. Furosemide, the selective antagonist of Gabra6-containing GABAARs, strongly increased peak amplitude of evoked IPSCs at CA3-CA1 synapses and the frequency of miniature IPSPs recorded from CA1 pyramidal cells in naive control animals, and this effect was occluded in MS animals. Knockdown of Gabra6 expression in hippocampus mimicked furosemide's effect and was sufficient to produce similar depressive symptoms that were observed in MS animals. These results indicate that the Gabra6-containing GABAA R is a key modulator of hippocampal synaptic transmission and likely plays a crucial role in the pathophysiology of maternal separation-induced depression.

Hemoglobin (Hb) is the main component of red blood cells, and the majority of alpha-synuclein in the blood is present inside them. In Parkinson's disease and aging human brains, the levels of mitochondrial neuronal hemoglobin are significantly decreased in neurons where α-Syn has accumulated, suggesting a relationship between these two proteins. In the present study, we demonstrate that a complex comprising α-Syn and Hb exists in both peripheral RBCs and brain tissue in aging humans using Co-Immunoprecipitation and a newly established ELISA assay. The nHbα-Syn levels in the mitochondrial fraction of the human striatum reduced in an age-dependent manner which was negatively correlated with the nHbα-Syn complex levels in the cytoplasmic extraction. In contrast, no age-related alteration in the nHbα-Syn complex was found in the above subcellular fractions of the human cerebellum. Furthermore, the nHbα-Syn complex levels in RBCs increased with age, which agrees with the changes observed in the cytoplasm of the human striatum. Meanwhile, sequential window acquisition of all theoretical mass spectra-MS (SWATH-MS) was used to confirm the presence of the Hbα-Syn complex in RBCs and also found that nHbα-Syn levels increased notably in PD patients compared to healthy controls (HC). Receiver operating characteristic (ROC) curves indicated that Hbα-Syn in PD patients was distinct from HC, with areas under the curve (AUCs) of 0.875. The above results suggest that the alteration in nHbα-Syn levels in RBCs reflects those in select regions of the human brain, thereby providing a possibility of using Hbα-Syn as a biomarker to diagnose PD.

Aging is associated with an increased risk for Parkinson's disease and dementia with Lewy bodies, in which α-synuclein (α-syn) oligomerization plays key pathogenic roles. Here, we show that oligomeric α-syn levels increase with age in the human brain and are accompanied by a decrease in the activity of glucocerebrosidase (GCase), a lysosomal enzyme whose dysfunction is linked to the accumulation of oligomeric α-syn. The inverse relationship between oligomeric α-syn levels and GCase activity was more evident in brain regions susceptible to neurodegeneration (i.e., the striatum and hippocampus) than those that are less vulnerable (i.e., the cerebellum and occipital cortex). GCase could potentially regulate α-syn oligomerization, as demonstrated by the decrease in oligomeric α-syn levels caused by a GCase agonist. In vitro experiments showed that GCase activity was more potently inhibited by oligomeric than monomeric α-syn in the lysosome-enriched fractions isolated from brain tissues and cultured neuronal cells. Alterations in oligomeric α-syns and their association with GCase in aging brains may explain the vulnerability of certain brain regions to neurodegeneration in Parkinson's disease and dementia with Lewy bodies.

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Neuronal hemoglobin (nHb) plays a critical role in maintaining normal mitochondrial functioning in the brain. However, in aging and Parkinson's disease (PD) brains, mitochondrial nHb levels are greatly reduced in neurons that accumulate α-synuclein (α-syn), suggesting a link between the two proteins. In this study, we demonstrate that α-syn and Hb can form a complex in both brain tissue and peripheral red blood cells (RBCs) in aging cynomolgus monkeys. nHb-α-syn complex levels in the mitochondrial fraction of the striatum decreased with age; this was negatively correlated with levels in the cytoplasmic fraction and in RBCs and was accompanied by a reduction in mitochondrial free nHb. In contrast, no changes in nHb-α-syn complex formation or free nHb levels were detected in the cerebellum. In vitro studies using a cultured dopaminergic cell line showed that intracellular accumulation of α-syn caused an elevation in nHb-α-syn complex levels in both mitochondrial and cytoplasmic fractions as well as a reduction in mitochondrial free nHb. nHb overexpression increased free nHb levels in mitochondria, stabilized mitochondrial membrane potential, and reduced α-syn-induced apoptosis. The above results suggest that α-syn forms a complex with nHb in selected regions of the aging brain, thereby decreasing mitochondrial function and increasing the risk of PD.

Although numerous linkage maps have been constructed in the genus Populus, they are typically sparse and thus have limited applications due to low throughput of traditional molecular markers. Restriction-site associated DNA sequencing (RADSeq) technology allows us to identify a large number of single nucleotide polymorphisms (SNP) across genomes of many individuals in a fast and cost-effective way, and makes it possible to construct high-density genetic linkage maps. We performed RADSeq for 299 progeny and their two parents in an F1 hybrid population generated by crossing the female Populus deltoides 'I-69' and male Populus simonii 'L3'. A total of 2,545 high quality SNP markers were obtained and two parent-specific linkage maps were constructed. The female genetic map contained 1601 SNPs and 20 linkage groups, spanning 4,249.12 cM of the genome with an average distance of 2.69 cM between adjacent markers, while the male map consisted of 940 SNPs and also 20 linkage groups with a total length of 3,816.24 cM and an average marker interval distance of 4.15 cM. Finally, our analysis revealed that synteny and collinearity are highly conserved between the parental linkage maps and the reference genome of P. trichocarpa. We demonstrated that RAD sequencing is a powerful technique capable of rapidly generating a large number of SNPs for constructing genetic maps in outbred forest trees. The high-quality linkage maps constructed here provided reliable genetic resources to facilitate locating quantitative trait loci (QTLs) that control growth and wood quality traits in the hybrid population.

A pathological hallmark of Parkinson’s disease (PD) is formation of Lewy bodies in neurons of the brain. This has been attributed to the spread of α-synuclein (α-syn) aggregates, which involves release of α-syn from a neuron and its reuptake by a neighboring neuron. We found that treatment with plasma from PD patients induced more α-syn phosphorylation and oligomerization than plasma from normal subjects (NS). Compared with NS plasma, PD plasma added to primary neuron cultures caused more cell death in the presence of extracellular α-syn. This was supported by the observations that phosphorylated α-syn oligomers entered neurons, rapidly increased accumulated thioflavin S-positive inclusions, and induced a series of metabolic changes that included activation of polo-like kinase 2, inhibition of glucocerebrosidase and protein phosphatase 2A, and reduction of ceramide levels, all of which have been shown to promote α-syn phosphorylation and aggregation. We also analyzed neurotoxicity of α-syn oligomers relative to plasma from different patients. Neurotoxicity was not related to age or gender of the patients. However, neurotoxicity was positively correlated with H&Y staging score. The modification in the plasma may promote spreading of α-syn aggregates via an alternative pathway and accelerate progression of PD.