Generation of a human induced pluripotent stem cell line (OGIi001) from peripheral blood mononuclear cells of a healthy male donor.

We have successfully derived a novel human induced pluripotent stem cell (hiPSC) line using non-integrative Sendai virus. This hiPSC line was generated from a healthy male adult donor, aged 55, and subjected to thorough characterization and extensive quality control. The analysis confirmed the expression of undifferentiated stem cell markers, demonstrated the ability to differentiate into the three germ layers, and revealed the absence of any chromosomal abnormalities.

Hexokinase inhibitor 2-deoxyglucose coordinates citrullination of vimentin and apoptosis of fibroblast-like synoviocytes by inhibiting HK2 /mTORC1-induced autophagy.

High hexokinase 2 (HK2) expression is associated with aberrant activation of fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA). However, the mechanism by which this occurs has not been fully elucidated. To investigate the role of HK2 and its underlying mechanism, adjuvant arthritis (AA) rats were treated with the HK2 inhibitor, 2-deoxyglucose (2-DG). In conjunction with HK2 knockdown experiments in FLSs, we evaluated the effect of HK2 on the citrullination of vimentin (cVIM), autophagy and apoptosis-associated protein expression, including that of cVIM, LC3, p62, Beclin1, Bax, Bcl2, and caspase 3. We further investigated the interaction of HK2 with downstream mTORC1 signaling effectors. Correlation analysis revealed that 2-DG treatment and HK2 knockdown upregulated the expression levels of caspase3, Bax, and p62 and downregulated the expression levels of LC3, Bcl2, and Beclin1, as well as decreasing vimentin citrullination. Furthermore, interactions between HK2 and mTOR decreased, coinciding with mTORC1 pathway activation. These findings suggest that the regulation of apoptosis and cVIM by HK2/mTORC1-dependent autophagy involves the inhibition of aberrant FLSs activation in the rat model of arthritis.

rAAV-mediated over-expression of acid ceramidase prevents retinopathy in a mouse model of Farber lipogranulomatosis.

Farber disease (FD) is a rare monogenic lysosomal storage disorder caused by mutations in ASAH1 that results in a deficiency of acid ceramidase (ACDase) activity and the abnormal systemic accumulation of ceramide species, leading to multi-system organ failure involving neurological decline and retinopathy. Here we describe the effects of rAAV-mediated ASAH1 over-expression on the progression of retinopathy in a mouse model of FD (Asah1P361R/P361R) and its littermate controls (Asah1+/+ and Asah1+/P361R). Using a combination of non-invasive multimodal imaging, electrophysiology, post-mortem histology and mass spectrometry we demonstrate that ASAH1 over-expression significantly reduces central retinal thickening, ceramide accumulation, macrophage activation and limits fundus hyper-reflectivity and auto-fluorescence in FD mice, indicating rAAV-mediated over-expression of biologically active ACDase protein is able to rescue the anatomical retinal phenotype of Farber disease. Unexpectedly, ACDase over-expression in Asah1+/+ and Asah1+/P361R control eyes was observed to induce abnormal fundus hyper-reflectivity, auto-fluorescence and retinal thickening that closely resembles a FD phenotype. This study represents the first evidence of a gene therapy for Farber disease-related retinopathy. Importantly, the described gene therapy approach could be used to preserve vision in FD patients synergistically with broader enzyme replacement strategies aimed at preserving life.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency.

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a "pre-retinal" injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation. Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy. Results: Under light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours. Conclusions: ERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

Uncovering a critical period of synaptic imbalance during postnatal development of the rat visual cortex: role of brain-derived neurotrophic factor.

KEY POINTS: With daily electrophysiological recordings and neurochemical analysis, we uncovered a transient period of synaptic imbalance between enhanced inhibition and suppressed excitation in rat visual cortical neurons from the end of the fourth toward the end of the fifth postnatal weeks. The expression of brain-derived neurotrophic factor (BDNF), which normally enhances excitation and suppresses inhibition, was down-regulated during that time, suggesting that this may contribute to the inhibition/excitation imbalance. An agonist of the BDNF receptor tropomyosin-related kinase B (TrkB) partially reversed the imbalance, whereas a TrkB antagonist accentuated the imbalance during the transient period. Monocular lid suture during the transient period is more detrimental to the function and neurochemical properties of visual cortical neurons than before or after this period. We regard the period of synaptic imbalance as the peak critical period of vulnerability, and its existence is necessary for neurons to transition from immaturity to a more mature state of functioning. ABSTRACT: The mammalian visual cortex is immature at birth and undergoes postnatal structural and functional adjustments. The exact timing of the vulnerable period in rodents remains unclear. The critical period is characterized by inhibitory GABAergic maturation reportedly dependent on brain-derived neurotrophic factor (BDNF). However, most of the studies were performed on experimental/transgenic animals, questioning the relationship in normal animals. The present study aimed to conduct in-depth analyses of the synaptic and neurochemical development of visual cortical neurons in normal and monocularly-deprived rats and to determine specific changes, if any, during the critical period. We found that (i) against a gradual increase in excitation and inhibition with age, a transient period of synaptic and neurochemical imbalance existed with suppressed excitation and enhanced inhibition at postnatal days 28 to 33/34; (ii) during this window, the expression of BDNF and tropomyosin-related kinase B (TrkB) receptors decreased, along with glutamatergic GluN1 and GluA1 receptors and the metabolic marker cytochrome oxidase, whereas that of GABAA Rα1 receptors continued to rise; (iii) monocular deprivation reduced both excitatory and inhibitory synaptic activity and neurochemicals mainly during this period; and (iv) in vivo TrkB agonist partially reversed the synaptic imbalance in normal and monocularly-deprived neurons during this time, whereas a TrkB antagonist accentuated the imbalance. Thus, our findings highlight a transitory period of synaptic imbalance with a negative relationship between BDNF and inhibitory GABA. This brief critical period may be necessary in transitioning from an immature to a more mature state of visual cortical functioning.

Role of brain-derived neurotrophic factor in the excitatory-inhibitory imbalance during the critical period of postnatal respiratory development in the rat.

The critical period of respiratory development in rats is a narrow window toward the end of the second postnatal week (P12-13), when abrupt neurochemical, electrophysiological, and ventilatory changes occur, when inhibition dominates over excitation, and when the animals' response to hypoxia is the weakest. The goal of this study was to further test our hypothesis that a major mechanism underlying the synaptic imbalance during the critical period is a reduced expression of brain-derived neurotrophic factor (BDNF) and its TrkB receptors. Our aims were to determine (1) that the inhibitory dominance observed in hypoglossal motoneurons during the critical period was also demonstrable in a key respiratory chemosensor, NTSVL; (2) if in vivo application of a TrkB agonist, 7,8-DHF, would prevent, but a TrkB antagonist, ANA-12, would accentuate the synaptic imbalance; and (3) if hypoxia would also heighten the imbalance. Our results indicate that (1) the synaptic imbalance was evident in the NTSVL during the critical period; (2) intraperitoneal injections of 7,8-DHF prevented the synaptic imbalance during the critical period, whereas ANA-12 in vivo accentuated such an imbalance; and (3) acute hypoxia induced the weakest response in both the amplitude and frequency of sEPSCs during the critical period, but it increased the frequency of sIPSCs during the critical period. Thus, our findings are consistent with and strengthen our hypothesis that BDNF and TrkB play a significant role in inducing a synaptic imbalance during the critical period of respiratory development in the rat.

Exercise intensity-dependent reverse and adverse remodeling of voltage-gated Ca(2+) channels in mesenteric arteries from spontaneously hypertensive rats.

Exercise can be regarded as a drug for treating hypertension, and the 'dosage' (intensity/volume) is therefore of great importance. L-type voltage-gated Ca(2+) (Cav1.2) channels on the plasma membrane of vascular smooth muscle cells have a pivotal role in modulating the vascular tone, and the upregulation of Cav1.2 channels is a hallmark feature of hypertension. The present study investigated the beneficial and adverse effects of exercise at different intensities on the remodeling of the Cav1.2 channel in mesenteric arteries (MAs) of spontaneously hypertensive rats (SHRs). Moderate- (SHR-M, 18-20 m min(-1)) and high-intensity (SHR-H, 26-28 m min(-1)) aerobic exercise training groups were created for SHRs and lasted for 8 weeks (1 h per day, 5 d per week). Age-matched sedentary SHRs and normotensive Wistar-Kyoto rats (WKY) were used as controls. The mesenteric arterial mechanical and functional properties were evaluated. Moderate-intensity exercise training induced a lower systolic blood pressure and heart rate in these rats compared with sedentary SHRs. BayK 8644 and nifedipine induced vasoconstriction and dose-dependent vasorelaxation, respectively, in the mesenteric arterial rings. Moderate-intensity exercise significantly suppressed the increase in BayK 8644-induced vasoconstriction, tissue sensitivity to nifedipine, Cav1.2 channel current density and Cav1.2 α1C-subunit protein expression in MAs from SHRs. However, high-intensity exercise training aggravated all of these hypertension-associated functional and molecular alterations of Cav1.2 channels. These results indicate that moderate-intensity aerobic training may act as a drug and effectively reverse the remodeling of Cav1.2 channels in hypertension to restore the vascular function in MAs, but that high-intensity exercise exaggerates the adverse remodeling of Cav1.2 channels and worsens the vascular function.

Chronic exercise normalizes changes in Cav 1.2 and KCa 1.1 channels in mesenteric arteries from spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Regular physical activity is an effective non-pharmacological therapy for prevention and control of hypertension. However, the underlying mechanisms are not fully understood. Accumulating evidence shows that the elevated vascular tone in hypertension is a consequence of the 'ion channel remodelling' that occurs during sustained high BP. The present study investigated the effects of aerobic exercise on the electrical remodelling of L-type Ca(2+) (Cav 1.2) and large-conductance Ca(2+) -activated K(+) (KCa 1.1) channels in mesenteric arteries (MAs) from spontaneously hypertensive rats (SHRs). EXPERIMENTAL APPROACH: SHRs and normotensive (Wistar-Kyoto) rats were subjected to aerobic training or kept sedentary, and vascular mechanical and functional properties were evaluated. KEY RESULTS: Exercise did not affect the heart weight, but reduced the heart rate and body weight in SHR. In mesenteric arterial myocytes, exercise normalized the increased Cav 1.2 and KCa 1.1 current density in SHRs. Exercise also ameliorated the increased open probability and mean open time of the single KCa 1.1 channel in hypertension. The isometric contraction study revealed that both nifedipine (Cav 1.2 channel blocker) and NS11021 (KCa 1.1 channel activator) induced concentration-dependent vasorelaxation in MAs precontracted with noradrenaline. Exercise normalized the increased sensitivity of tissues to nifedipine and NS11021 in SHR. Furthermore, protein expression of the Cav 1.2 α1C -subunit together with the KCa 1.1 α- and ß1-subunit was significantly increased in SHRs; and exercise ameliorated these molecular alterations in hypertension. CONCLUSIONS AND IMPLICATIONS: Chronic exercise reduces BP and restores vascular function in MAs from SHR, which might be related to the correction of the Cav 1.2 and KCa 1.1 channel remodelling during hypertension.