Stomata on the plant epidermis control gas and water exchange and are formed by MAPK-dependent processes. Although the contribution of MAP KINASE3 (MPK3) and MPK6 (MPK3/MPK6) to the control of stomatal patterning and differentiation in Arabidopsis () has been examined extensively, how they are inactivated and regulate distinct stages of stomatal development is unknown. Here, we identify a dual-specificity phosphatase, MAP KINASE PHOSPHATASE1 (MKP1), which promotes stomatal cell fate transition by controlling MAPK activation at the early stage of stomatal development. Loss of function of creates clusters of small cells that fail to differentiate into stomata, resulting in the formation of patches of pavement cells. We show that MKP1 acts downstream of YODA (a MAPK kinase kinase) but upstream of MPK3/MPK6 in the stomatal signaling pathway and that MKP1 deficiency causes stomatal signal-induced MAPK hyperactivation in vivo. By expressing MKP1 in the three discrete cell types of stomatal lineage, we further identified that MKP1-mediated deactivation of MAPKs in early stomatal precursor cells directs cell fate transition leading to stomatal differentiation. Together, our data reveal the important role of MKP1 in controlling MAPK signaling specificity and cell fate decision during stomatal development.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome of both acute and chronic liver disease. As a metabolic disorder, HE is considered to be reversible and therefore is expected to resolve following the replacement of the diseased liver with a healthy liver. However, persisting neurological complications are observed in up to 47% of transplanted patients. Several retrospective studies have shown that patients with a history of HE, particularly overt-HE, had persistent neurological complications even after liver transplantation (LT). These enduring neurological conditions significantly affect patient's quality of life and continue to add to the economic burden of chronic liver disease on health care systems. This review discusses the journey of the brain through the progression of liver disease, entering the invasive surgical procedure of LT and the conditions associated with the post-transplant period. In particular, it will discuss the vulnerability of the HE brain to peri-operative factors and post-LT conditions which may explain non-resolved neurological impairment following LT. In addition, the review will provide evidence; (i) supporting overt-HE impacts on neurological complications post-LT; (ii) that overt-HE leads to permanent neuronal injury and (iii) the pathophysiological role of ammonia toxicity on astrocyte and neuronal injury/damage. Together, these findings will provide new insights on the underlying mechanisms leading to neurological complications post-LT.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). The primary cause of hospital admissions for cirrhotic patients is an overt episode of HE. Precipitating factors of HE frequently lead to an increase in blood ammonia. Patients who have experienced multiple episodes of HE are associated with persisting neurological complications post-liver transplantation. Currently, the impact of HE episodes on neurological integrity is unknown. We hypothesize that multiple episodes of HE will accelerate and/or intensify neurological deterioration. To date, an animal model of episodic HE is lacking. Therefore, our goal was to characterize an animal model of episodic HE (precipitated with ammonia) and to evaluate the impact of cumulative episodes on neurological status in cirrhotic rats. METHODS: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. BDL and Sham rats were divided in two groups, episodic and non-episodic. Injection (i.p) of ammonium acetate was used to induce episodes of overt HE (pre-coma; loss of righting reflex) every 4 days starting 3-weeks post-BDL surgery (total 5 episodes). Saline was injected as vehicle for non-episodic groups. Two days following the last HE episode, we assessed motor-coordination (RotaRod), anxiety (elevated plus maze, EPMT), as well as short-term and long-term memory (novel object recognition) in all groups. Upon sacrifice, plasma ammonia was measured. RESULTS: The concentration of ammonia required to induce an episode of overt HE in BDL rats lessened with each subsequent episode, ranging from 7 to 4.5 mmol/kg. Short-term memory (P < 0.05) and motor-coordination (P < 0.05) were impaired in both non-episodic and episodic BDL groups compared to respective Sham-operated controls. Long-term memory impairment (P = 0.06) and increased anxiety (+60.0%, P < 0.05) were exclusively found in episodic BDL rats compared to non-episodic BDL rats. Moreover, there was an increase in blood ammonia (+30.4%, P = 0.06) in episodic compared to non-episodic BDL rats, suggesting that although episodic-BDL rats recover from each HE episode, baseline (pre-episode) ammonia remain higher than non-episodic BDL rats. CONCLUSIONS: Cumulative HE episodes escalate neurological impairments in cirrhotic-BDL rats. Thus, this new episodic HE model represents a good approach to explore the pathological mechanism arising from multiple episodes, as well as further investigate whether higher hyperammonemia and/or increased brain sensitivity to ammonia is responsible for more complex neurological manifestations in episodic-BDL rats. Moreover, this model is an excellent platform to investigate novel therapies to prevent/treat episodic HE.
BACKGROUND: Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering circulating ammonia. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert ammonia to arginine (SYNARG), and its administration to thioacetamide-treated mice resulted in a significant reduction of ammonia levels.1 SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory/anti-oxidant properties, and both strains were tested in an experimental model of cirrhosis and HE, the bile duct ligation (BDL). METHODS: One week post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3 × 10 11 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma ammonia and live markers were measured at 3 and 5 weeks. Recognition-memory, motor-coordination, muscle-strength, locomotion and anxiety were assessed in the 5-week BDL groups. RESULTS: BDL significantly increased ammonia over time, with levels of 109.1 ± 9.2 µM (Shams 56.7 ± 3.5 µM, P < 0.001) and 150.2 ± 25.6 µM (Shams 58.3 ± 3.0µM, P < 0.001) at 3- and 5-weeks, respectively. In addition, plasma liver markers alanine-transaminase, aspartate-transaminase, bilirubin, and gamma-glutamyl transferase were significantly increased in BDL rats at both timepoints while albumin was significantly lowered. As compared to BDL-Veh rats, hyperammonemia was attenuated by SYNARG (103.9 ± 12.3 µM) and SYNARG+BUT (110.8 ± 8.5 µM) at 5, but not 3 weeks post-surgery, while liver fibrosis (hydroxyproline content) was attenuated at 3, but not 5 weeks post-surgery. None of the circulating liver markers were changed by the treatments at any timepoint. Motor-coordination, muscle-strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (P < 0.001) and BDL-SYNARG (P < 0.05) versus Shams, while STM was resolved in BDL-SYNARG+BUT (P < 0.05 vs BDL-Veh). Long-term memory (LTM) was impaired in BDL-Veh vs Shams (P < 0.05), but BDL-SYNARG and BDL-SYNARG+BUT were protected. CONCLUSIONS: EcN, engineered to consume ammonia in the gut and synthesize butyrate, is an effective approach to lower plasma ammonia in a model of cirrhosis and HE. Moreover, the attenuation of hyperammonemia in cirrhotic rats is associated with a protective effect on memory in this model. The therapeutic potential of these engineered EcN strains should be further evaluated in patients with CLD and HE.
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