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Brain cholinergic impairment in liver failure.

García-Ayllón MS, Cauli O, Silveyra MX, Rodrigo R, Candela A, Compañ A, Jover R, Pérez-Mateo M, Martínez S, Felipo V, Sáez-Valero J - Brain (2008)

Bottom Line: Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure.Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes.In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain.

ABSTRACT
The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

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Immunodetection of AChE subunits and detection of AChE and ChAT transcripts in brain cortex from NL and BDL rats. (A) Relative mRNA levels of the transcripts for AChE (T and R; labelled AChE-T and AChE-R in the figure) and ChAT (cholinergic and peripheral; cChAT and pChAT) were analysed by QRT-PCR. Values were calculated using relative standard curves and normalized to GAPDH control from the same cDNA preparations. Specifity of the PCR products was confirmed by dissociation curve analysis. (B) Three major AChE bands of ∼77, 70 and 60 kDa were identified with the antibody E-19 in NL and BDL brain cortical extracts (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing NL and BDL animals. (C) Representative immunoblot of individual G4 and G1 + G2 AChE peak-fractions separated by sucrose gradient centrifugation from NL and BDL brain cortical extracts (equivalent volume of G4 and G1 + G2 AChE peak-fractions were loaded in each lane).
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Figure 5: Immunodetection of AChE subunits and detection of AChE and ChAT transcripts in brain cortex from NL and BDL rats. (A) Relative mRNA levels of the transcripts for AChE (T and R; labelled AChE-T and AChE-R in the figure) and ChAT (cholinergic and peripheral; cChAT and pChAT) were analysed by QRT-PCR. Values were calculated using relative standard curves and normalized to GAPDH control from the same cDNA preparations. Specifity of the PCR products was confirmed by dissociation curve analysis. (B) Three major AChE bands of ∼77, 70 and 60 kDa were identified with the antibody E-19 in NL and BDL brain cortical extracts (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing NL and BDL animals. (C) Representative immunoblot of individual G4 and G1 + G2 AChE peak-fractions separated by sucrose gradient centrifugation from NL and BDL brain cortical extracts (equivalent volume of G4 and G1 + G2 AChE peak-fractions were loaded in each lane).

Mentions: Some of the molecular heterogeneity of AChE derives from alternative RNA splicing, generating different polypeptide encoding transcripts with the same catalytic domain, but distinct C-terminal peptides that determine the ability of the molecule to form oligomers (Massoulié et al., 1993; Taylor and Radic, 1994; Grisaru et al., 1999). To determine if AChE expression is altered in BDL rats, we performed QRT-PCR analysis of the AChE mRNA. The levels of the T-transcript, the major transcript in mammalian brain that encodes subunits which produce monomeric and tetrameric forms, was unaltered in cortices from BDL rats compared to controls (Fig. 5A). Similarly, levels of the R-transcript which encodes monomeric soluble subunits and is normally present at low levels in the mammalian brain (Kaufer et al., 1998) did not vary in BDL animals compared to controls (Fig. 5A). QRT-PCR analysis was used to determine the mRNA levels for both the conventional cholinergic ChAT transcript, the major variant found in both central and peripheral neurons [protein product is called ChAT of the common type (cChAT)], and also transcriptional levels for the minor splice variant [protein product designated ChAT of a peripheral type (pChAT)], which is predominantly localized in peripheral neurons (Tooyama and Kimura, 2000). As expected for the unmodified ChAT enzyme activity, the levels of both cChAT and pChAT were unaltered in cortices from BDL rats compared to NL controls (Fig. 5A).Fig. 5


Brain cholinergic impairment in liver failure.

García-Ayllón MS, Cauli O, Silveyra MX, Rodrigo R, Candela A, Compañ A, Jover R, Pérez-Mateo M, Martínez S, Felipo V, Sáez-Valero J - Brain (2008)

Immunodetection of AChE subunits and detection of AChE and ChAT transcripts in brain cortex from NL and BDL rats. (A) Relative mRNA levels of the transcripts for AChE (T and R; labelled AChE-T and AChE-R in the figure) and ChAT (cholinergic and peripheral; cChAT and pChAT) were analysed by QRT-PCR. Values were calculated using relative standard curves and normalized to GAPDH control from the same cDNA preparations. Specifity of the PCR products was confirmed by dissociation curve analysis. (B) Three major AChE bands of ∼77, 70 and 60 kDa were identified with the antibody E-19 in NL and BDL brain cortical extracts (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing NL and BDL animals. (C) Representative immunoblot of individual G4 and G1 + G2 AChE peak-fractions separated by sucrose gradient centrifugation from NL and BDL brain cortical extracts (equivalent volume of G4 and G1 + G2 AChE peak-fractions were loaded in each lane).
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Figure 5: Immunodetection of AChE subunits and detection of AChE and ChAT transcripts in brain cortex from NL and BDL rats. (A) Relative mRNA levels of the transcripts for AChE (T and R; labelled AChE-T and AChE-R in the figure) and ChAT (cholinergic and peripheral; cChAT and pChAT) were analysed by QRT-PCR. Values were calculated using relative standard curves and normalized to GAPDH control from the same cDNA preparations. Specifity of the PCR products was confirmed by dissociation curve analysis. (B) Three major AChE bands of ∼77, 70 and 60 kDa were identified with the antibody E-19 in NL and BDL brain cortical extracts (equivalent amounts of protein were loaded in each lane). None of the major AChE immunopositive bands were altered when comparing NL and BDL animals. (C) Representative immunoblot of individual G4 and G1 + G2 AChE peak-fractions separated by sucrose gradient centrifugation from NL and BDL brain cortical extracts (equivalent volume of G4 and G1 + G2 AChE peak-fractions were loaded in each lane).
Mentions: Some of the molecular heterogeneity of AChE derives from alternative RNA splicing, generating different polypeptide encoding transcripts with the same catalytic domain, but distinct C-terminal peptides that determine the ability of the molecule to form oligomers (Massoulié et al., 1993; Taylor and Radic, 1994; Grisaru et al., 1999). To determine if AChE expression is altered in BDL rats, we performed QRT-PCR analysis of the AChE mRNA. The levels of the T-transcript, the major transcript in mammalian brain that encodes subunits which produce monomeric and tetrameric forms, was unaltered in cortices from BDL rats compared to controls (Fig. 5A). Similarly, levels of the R-transcript which encodes monomeric soluble subunits and is normally present at low levels in the mammalian brain (Kaufer et al., 1998) did not vary in BDL animals compared to controls (Fig. 5A). QRT-PCR analysis was used to determine the mRNA levels for both the conventional cholinergic ChAT transcript, the major variant found in both central and peripheral neurons [protein product is called ChAT of the common type (cChAT)], and also transcriptional levels for the minor splice variant [protein product designated ChAT of a peripheral type (pChAT)], which is predominantly localized in peripheral neurons (Tooyama and Kimura, 2000). As expected for the unmodified ChAT enzyme activity, the levels of both cChAT and pChAT were unaltered in cortices from BDL rats compared to NL controls (Fig. 5A).Fig. 5

Bottom Line: Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure.Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes.In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain.

ABSTRACT
The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

Show MeSH
Related in: MedlinePlus