Sookja K. Chung

Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is increasingly implicated in various neurodegenerative diseases. Deregulated Cdk5 activity has been associated with neuronal death, but the underlying mechanisms are not well understood. Here we report an unexpected role for Cdk5 in the regulation of induced autophagy in neurons. We have identified endophilin B1 (EndoB1) as a Cdk5 substrate, and show that Cdk5-mediated phosphorylation of EndoB1 is required for autophagy induction in starved neurons. Furthermore, phosphorylation of EndoB1 facilitates EndoB1 dimerization and recruitment of UVRAG (UV radiation resistance-associated gene). More importantly, Cdk5-mediated phosphorylation of EndoB1 is essential for autophagy induction and neuronal loss in models of Parkinson’s disease. Our findings not only establish Cdk5 as a critical regulator of autophagy induction, but also reveal a role for Cdk5 and EndoB1 in the pathophysiology of Parkinson’s disease through modulating autophagy.

Berberine ameliorates β-amyloid pathology, gliosis, and cognitive impairment in an Alzheimer's disease transgenic mouse model.

The accumulation of β-amyloid (Aβ) peptide derived from abnormal processing of amyloid precursor protein (APP) is a common pathological hallmark of Alzheimers disease (AD) brains. In this study, we evaluated the therapeutic effect of berberine (BBR) extracted from Coptis chinensis Franch, a Chinese medicinal herb, on the neuropathology and cognitive impairment in TgCRND8 mice, a well established transgenic mouse model of AD. Two-month-old TgCRND8 mice received a low (25 mg/kg per day) or a high dose of BBR (100 mg/kg per day) by oral gavage until 6 months old. BBR treatment significantly ameliorated learning deficits, long-term spatial memory retention, as well as plaque load compared with vehicle control treatment. In addition, enzyme-linked immunosorbent assay (ELISA) measurement showed that there was a profound reduction in levels of detergent-soluble and -insoluble β-amyloid in brain homogenates of BBR-treated mice. Glycogen synthase kinase (GSK)3, a major kinase involved in APP and tau phosphorylation, was significantly inhibited by BBR treatment. We also found that BBR significantly decreased the levels of C-terminal fragments of APP and the hyperphosphorylation of APP and tau via the Akt/glycogen synthase kinase 3 signaling pathway in N2a mouse neuroblastoma cells stably expressing human Swedish mutant APP695 (N2a-SwedAPP). Our results suggest that BBR provides neuroprotective effects in TgCRND8 mice through regulating APP processing and that further investigation of the BBR for therapeutic use in treating AD is warranted.

Protection of Retinal Ganglion Cells and Retinal Vasculature by Lycium Barbarum Polysaccharides in a Mouse Model of Acute Ocular Hypertension

Acute ocular hypertension (AOH) is a condition found in acute glaucoma. The purpose of this study is to investigate the protective effect of Lycium barbarum polysaccharides (LBP) and its protective mechanisms in the AOH insult. LBP has been shown to exhibit neuroprotective effect in the chronic ocular hypertension (COH) experiments. AOH mouse model was induced in unilateral eye for one hour by introducing 90 mmHg ocular pressure. The animal was fed with LBP solution (1 mg/kg) or vehicle daily from 7 days before the AOH insult till sacrifice at either day 4 or day 7 post insult. The neuroprotective effects of LBP on retinal ganglion cells (RGCs) and blood-retinal-barrier (BRB) were evaluated. In control AOH retina, loss of RGCs, thinning of IRL thickness, increased IgG leakage, broken tight junctions, and decreased density of retinal blood vessels were observed. However, in LBP-treated AOH retina, there was less loss of RGCs with thinning of IRL thickness, IgG leakage, more continued structure of tight junctions associated with higher level of occludin protein and the recovery of the blood vessel density when compared with vehicle-treated AOH retina. Moreover, we found that LBP provides neuroprotection by down-regulating RAGE, ET-1, Aβ and AGE in the retina, as well as their related signaling pathways, which was related to inhibiting vascular damages and the neuronal degeneration in AOH insults. The present study suggests that LBP could prevent damage to RGCs from AOH-induced ischemic injury; furthermore, through its effects on blood vessel protection, LBP would also be a potential treatment for vascular-related retinopathy.

Exchange protein activated by cAMP 1 (Epac1)-deficient mice develop β-cell dysfunction and metabolic syndrome

Previously, exchange protein directly activated by cAMP 2 (Epac2) and PKA were known to play a role in glucose‐stimulated insulin secretion (GSIS) by pancreatic β cells. The present study shows that Epac1 mRNA is also expressed by β cells. Therefore, we generated mice and embryonic stem (ES) cells with deletion of the Epac1 gene to define its role in β‐cell biology and metabolism. The homozygous Epac1‐ knockout (Epac1–/–) mice developed impaired glucose tolerance and GSIS with deranged islet cytoarchitecture, which was confirmed by isolated islets from adult Epac1–/– mice. Moreover, Epac1–/– mice developed more severe hyperglycemia with increased β‐cell apoptosis and insulitis after multiple low‐dose streptozotocin (MLDS; 40 mg/kg) treatment than Epac1+/+ mice. Interestingly, Epac1–/– mice also showed metabolic defects, including increased respiratory exchange ratio (RER) and plasma triglyceride (TG), and more severe diet‐induced obesity with insulin resistance, which may contributed to β‐cell dysfunction. However, islets differentiated from Epac1–/– ES cells showed insulin secretion defect, reduced Glut2 and PDX‐1 expression, and abolished GLP‐1‐stimulated PCNA induction, suggesting a role of Epac1 in β‐cell function. The current study provides in vitro and in vivo evidence that Epac1 has an important role in GSIS of β cells and phenotype resembling metabolic syndrome. Kai, A. K. L., Lam, A. K. M., Chen, Y., Tai, A. C. P., Zhang, X., Lai, A. K. W., Yeung, P. K. K., Tam, S., Wang, J., Lam, K. S., Vanhoutte, P. M., Bos, J. L., Chung, S. S. M., Xu, A., Chung, S. K. Exchange protein activated by cAMP 1 (Epac1)‐deficient mice develop β‐cell dysfunction and metabolic syndrome. FASEBJ. 27, 4122–4135 (2013). www.fasebj.org

Embryonic lethality in mice lacking the nuclear factor of activated T cells 5 protein due to impaired cardiac development and function.

Nuclear factor of activated T cells 5 protein (NFAT5) is thought to be important for cellular adaptation to osmotic stress by regulating the transcription of genes responsible for the synthesis or transport of organic osmolytes. It is also thought to play a role in immune function, myogenesis and cancer invasion. To better understand the function of NFAT5, we developed NFAT5 gene knockout mice. Homozygous NFAT5 null (NFAT5−/−) mouse embryos failed to develop normally and died after 14.5 days of embryonic development (E14.5). The embryos showed peripheral edema, and abnormal heart development as indicated by thinner ventricular wall and reduced cell density at the compact and trabecular areas of myocardium. This is associated with reduced level of proliferating cell nuclear antigen and increased caspase-3 in these tissues. Cardiomyocytes from E14.5 NFAT5−/− embryos showed a significant reduction of beating rate and abnormal Ca2+ signaling profile as a consequence of reduced sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and ryanodine receptor (RyR) expressions. Expression of NFAT5 target genes, such as HSP 70 and SMIT were reduced in NFAT5−/− cardiomyocytes. Our findings demonstrated an essential role of NFAT5 in cardiac development and Ca2+ signaling. Cardiac failure is most likely responsible for the peripheral edema and death of NFAT5−/− embryos at E14.5 days.

Aldose reductase deficiency reduced vascular changes in neonatal mouse retina in oxygen-induced retinopathy.

PURPOSE Retinal neovascularization is the major pathologic process in many ocular diseases and is associated with oxidative stress. Deficiency of aldose reductase (AR), the first enzyme in the polyol pathway for glucose metabolism, has been shown to reduce oxidative stress and blood vessel leakage. The present study aimed to investigate the effect of AR deficiency on retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model. METHODS Seven-day-old wild-type (WT) and AR-deficient (AR(-/-)) mice were exposed to 75% oxygen for 5 days and then returned to room air. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. Immunohistochemistry for AR, nitrotyrosine (NT), poly(ADP-ribose) (PAR), glial fibrillary acidic protein (GFAP), and Iba-1, as well as Western blots for vascular endothelial growth factor (VEGF), phospho-Erk (p-Erk), phospho-Akt (p-Akt), and phospho-IκB (p-IκB) were performed. RESULTS Compared with WT OIR retinae, AR(-/-) OIR retinae displayed significantly smaller central retinal vaso-obliterated area, less neovascularization, and reduced blood vessel leakage. Significantly reduced oxidative stress and glial responses were also observed in AR(-/-) OIR retinae. Moreover, reduced microglial response in the avascular area but increased microglial responses in the neovascular area were found with AR deficiency. Furthermore, expression levels of VEGF, p-Erk, p-Akt, and p-IκB were significantly reduced in AR(-/-) OIR retinae. CONCLUSIONS Our observations indicated that AR deficiency reduced retinal vascular changes in the mouse model of OIR, indicating that AR can be a potential therapeutic target in ischemia-induced retinopathy.

Effect of Lycium barbarum polysaccharides on the expression of endothelin-1 and its receptors in an ocular hypertension model of rat glaucoma

Lycium barbarum, a traditional Chinese anti-aging herb, has been shown to protect retinal ganglion cells (RGCs) in a rat chronic ocular hypertension (COH) model. Here, we investigated the expression of endothelin-1 (ET-1), a strong vasoconstrictor, and its receptors, ETA and ETB, in the COH model and assessed the effects of Lycium barbarum on the ET-1 axis. Elevated intraocular pressure (IOP) was induced in the right eye of SD rats using argon laser photocoagulation. (1) The expression of ET-1, ETA and ETB in normal and COH retinas was studied. (2) Some COH rats were fed daily with Lycium barbarum Polysaccharides (LBP) using 1 mg/kg or phosphate-buffered saline (PBS) for 3 weeks (started 1 week before photocoagulation). The effects of LBP on the expression of ET-1 and its receptors, ETA and ETB, in COH retina were evaluated. A semi-quantitative analysis of staining intensity was used to evaluate the expression levels of ET-1, ETA and ETB in retinal vasculature. We found that (1) Under COH condition, the immunoreactivity of ET-1 was increased in retina associated with an increase of ETB receptor immunoreactivity and a decrease of ETA receptor immunoreactivity. (2) After feeding COH rats with LBP, the expression of ET-1 was decreased with an increase of ETA expression and a decrease of ETB expression in the retina, especially in RGCs. (3) By comparing the staining intensity in the vasculature of COH retina in LBP-fed group with PBS-fed group, there was a decrease in the expression of ET-1 and ETA and an increase in ETB. In summary, ET-1 expression was up-regulated in the retina in COH model. LBP could decrease the expression of ET-1 and modulate the expression of its receptors, ETA and ETB, under the condition of COH. The neuroprotective effect of LBP on RGCs might be related to its ability to regulate the ET-1-mediated biological effects on RGCs and retinal vasculature.

Effects of Huanglian-Jie-Du-Tang and its modified formula on the modulation of amyloid-β precursor protein processing in Alzheimer's disease models.

Huanglian-Jie-Du-Tang (HLJDT) is a famous traditional Chinese herbal formula that has been widely used clinically to treat cerebral ischemia. Recently, we found that berberine, a major alkaloid compound in HLJDT, reduced amyloid-β (Aβ) accumulation in an Alzheimer’s disease (AD) mouse model. In this study, we compared the effects of HLJDT, four single component herbs of HLJDT (Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP) and Fructus gardenia (FG)) and the modified formula of HLJDT (HLJDT-M, which is free of RS) on the regulatory processing of amyloid-β precursor protein (APP) in an in vitro model of AD. Here we show that treatment with HLJDT-M and its components RC, CP, and the main compound berberine on N2a mouse neuroblastoma cells stably expressing human APP with the Swedish mutation (N2a-SwedAPP) significantly decreased the levels of full-length APP, phosphorylated APP at threonine 668, C-terminal fragments of APP, soluble APP (sAPP)-α and sAPPβ-Swedish and reduced the generation of Aβ peptide in the cell lysates of N2a-SwedAPP. HLJDT-M showed more significant APP- and Aβ- reducing effects than berberine, RC or CP treatment alone. In contrast, HLJDT, its component RS and the main active compound of RS, baicalein, strongly increased the levels of all the metabolic products of APP in the cell lysates. The extract from FG, however, did not influence APP modulation. Interestingly, regular treatment of TgCRND8 APP transgenic mice with baicalein exacerbated the amyloid plaque burden, APP metabolism and Aβ production. Taken together, these data provide convincing evidence that HLJDT and baicalein treatment can increase the amyloidogenic metabolism of APP which is at least partly responsible for the baicalein-mediated Aβ plaque increase in the brains of TgCRND8 mice. On the other hand, HLJDT-M significantly decreased all the APP metabolic products including Aβ. Further study of HLJDT-M for therapeutic use in treating AD is warranted.

A modified formulation of Huanglian-Jie-Du-Tang reduces memory impairments and β-amyloid plaques in a triple transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is a degenerative disorder typified by progressive deterioration of memory and the appearance of β-amyloid peptide (Aβ)-rich senile plaques. Recently we have identified a novel function of a patented formulation of modified Huanglian-Jie-Tu-Tang (HLJDT-M), a Chinese herbal medicine, in treating AD in in vitro studies (US patent No. 9,375,457). HLJDT-M is a formulation composed of Rhizoma Coptitis, Cortex Phellodendri and Fructus Gardeniae without Radix Scutellariae. Here, we assessed the efficacy of HLJDT-M on a triple transgenic mouse model of AD (3XTg-AD). Oral administration of HLJDT-M ameliorated the cognitive dysfunction of 3XTg-AD mice and lessened the plaque burden. In addition, biochemical assays revealed a significant decrease in levels of detergent-soluble and acid-soluble Aβ via decreasing the levels of full length amyloid-β precursor protein (FL-APP) and C-terminal fragments of APP (CTFs) in brain lysates of HLJDT-M-treated mice. HLJDT-M treatment also significantly reduced the levels of FL-APP and CTFs in N2a/SweAPP cells. In contrast, treatment using the classical formula HLJDT did not reduce the memory impairment of 3XTg-AD mice and, rather, increased the Aβ/Fl-APP/CTFs in both animal and cell culture studies. Altogether, our study indicates that HLJDT-M is a promising herbal formulation to prevent and/or cure AD.

Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells

Monosialoganglioside 1 (GM1) is the main ganglioside subtype and has neuroprotective properties in the central nervous system. In this study, we aimed to determine whether GM1 alleviates neurotoxicity induced by moderate and high concentrations of propofol combined with remifentanil in the immature central nervous system. Hippocampal neural stem cells were isolated from newborn Sprague-Dawley rats and treated with remifentanil (5, 10, 20 ng/mL) and propofol (1.0, 2.5, 5.0 μg/mL), and/or GM1 (12.5, 25, 50 μg/mL). GM1 reversed combined propofol and remifentanil-induced decreases in the percentage of 5-bromodeoxyuridine(+) cells and also reversed the increase in apoptotic cell percentage during neural stem cell proliferation and differentiation. However, GM1 with combined propofol and remifentanil did not affect β-tubulin(+) or glial fibrillary acidic protein(+) cell percentage during neural stem cell differentiation. In conclusion, we show that GM1 alleviates the damaging effects of propofol combined with remifentanil at moderate and high exposure concentrations in neural stem cells in vitro, and exerts protective effects on the immature central nervous system.