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5 DIFFERENTIAL P ROTEIN ABUN DANCE

IN THE HUMAN A LCOHOLIC

CEREBELLAR VERMIS

K. Alexander-Kaufman, C. Harper, P. Wilce and I. Matsumoto, (2007)

“The cerebellar vermis proteome of chronic alcoholics”, Alcoholism: Clinical & Experimental

Research 31(8): 1286-96 – For Complete Article refer to Appendix IV

CHAPTER 5 THE CEREBELLAR VERMIS PROTEOME OF CHRONIC ALCOHOLICS

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The following chapter is based on the following article:

K. Alexander-Kaufman, C. Harper, P. Wilce and I. Matsumoto, (2007)“

The cerebellar vermis proteome of chronic alcoholics”, Alcoholism: Clinical & Experimental

Research 31(8): 1286-96

Each author was responsible for the following tasks:

K. Alexander-Kaufman: Sample preparation and 2D-GE, image analysis, statistics, mass

spectrometry, database searching, manuscript compilation.

C. Harper: Academic input, manuscript compilation and director of brain bank.

P. Wilce: Academic input.

I. Matusmoto: Academic input, manuscript compilation and supervision.


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5.1 Introduction

Alcohol dependence and abuse are costly health and social problems. Patterns of drinking

are changing with dramatic increases in per capita consumption in China (Cochrane et al.,

2003) and Eastern Europe (Rehm et al., 2003). Excessive drinking can lead to functional

and structural brain changes and one region that appears to be particularly vulnerable is

the cerebellum. Pathological changes in the cerebellum are commonly reported in

alcoholism and acute and chronic alcohol consumption produce profound impairments in

cerebellar function. In autopsy studies, approximately 40 percent of alcoholics show signs

of cerebellar degeneration (Torvik and Torp, 1986). This can be recognized in vivo using

MRI (Pfefferbaum and Rosenbloom, 1993; Sullivan, 2003) and the changes are

characterized by a general shrinkage or atrophy of the cerebellar foliae, particularly in the

anterior superior cerebellar vermis (Pfefferbaum and Rosenbloom, 1993; Harper, 1998a;

Andersen, 2004). Microscopically there is significant loss of Purkinje nerve cells in the

vermis (Harper, 1998a; Andersen, 2004) and reduced dendritic arbor (Ferrer et al., 1984;

Pentney, 1982). White matter atrophy is also commonly reported in chronic alcoholics

(Harper and Kril, 1993b) and white matter loss in the vermis appears to contribute to

ataxia in chronic alcoholics (Baker et al., 1999). Other histological studies have reported

a reduction in the volume of the molecular and medullary layers of the vermis (Phillips et

al., 1987; Torvik and Trop, 1986). Using proton magnetic resonance spectroscopy, Parks

and colleagues showed a significant reduction of N-acetylaspartate, a putative marker of

neuronal integrity, and choline-containing compounds in the cerebellar vermis of chronic

alcoholics (Parks et al., 2002), suggesting that the vermis is uniquely sensitive to

alcohol’s effects.


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It has been proposed that many of the pathological changes described in the cerebellum in

alcoholics may be consequent to other common alcohol-related medical complications,

particularly thiamine deficiency (Baker et al., 1999; Phillips et al., 1990). Thiamine

deficiency is common in alcoholics (Majumdar, 1980; Majumdar et al., 1981) and can

lead to the Wernicke-Korsakoff’s Syndrome (WKS) if proper supplementation is not

given (Kril, 1996). Cerebellar degeneration is seen in approximately one third of

alcoholics with WKS (Harper, 1983; Victor et al., 1989) as well as in non-alcoholic WKS

(Kril, 1996). Hence, the relative contribution of alcohol toxicity and thiamine deficiency

to cerebellar degeneration is debatable (Martin et al., 2003), although it should be noted

that many alcoholics without overt WKS also have deficient or marginal thiamine status

(Damton-Hill and Truswell, 1990). The involvement of thiamine in cerebellar disease is

supported by a study which demonstrated a correlation between serum thiamine level and

cerebellar volume on MRI, which held true for thiamine levels within the normal range

(Maschke et al., 2005).

Although the cerebellum is primarily involved in motor control and co-ordination, it is

increasingly recognized for its role in various aspects of cognitive and sensory

functioning (Martin et al., 2003). Neuroanatomical studies demonstrate that the superior

cerebellar hemispheres are well connected to frontal and prefrontal areas (Schmahmann

and Pandya, 1997) and functional neuroimaging has shown that prefrontal cortical and

contralateral cerebellar activations occur in tandem (Diamond, 2000). The

corticopontocerebellar and cerebellothalamocortical circuits underlie a wide range of

neuropsychological processes that mediate not only traditional cerebellar functions, such


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as motor control, but also perceptual motor tasks, executive functions, and learning and

memory, all of which are compromised by alcoholism (Parks et al., 2002; Sullivan,

2003). Accordingly, alcohol–induced damage to the cerebellar vermis could indirectly

affect neurocognitive functions attributed to the frontal lobe (Martin et al., 2003).

Using a proteomics-based approach, we have compared the protein abundance profiles of

the cerebellar vermis from alcoholics (both uncomplicated and complicated with hepatic

cirrhosis) to healthy individuals. By identifying changes in protein abundance in the

cerebellar vermis, hypotheses may draw upon more mechanistic explanations as to how

chronic alcohol consumption causes the structural and functional changes associated with

alcohol-related brain damage. Comparing these results to other proteomics studies, we

may be able to isolate disturbances in molecular pathways specific to the brain damage

caused by alcohol, hepatic encephalopathy (HE) and thiamine deficiency.


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5.2 Materials & Methods

The NSW TRC, University of Sydney, provided tissue from the cerebellar vermis from

26 freshly frozen human brains. Cases were classified into 3 groups, (A) Normal control

cases (<20g of ethanol/day; n=13), (B) Uncomplicated alcoholics (>80g of ethanol/day;

no postmortem finding of cirrhosis or WKS; n=7), (C) Alcoholics complicated with

hepatic cirrhosis (>80g of ethanol/day; hepatic cirrhosis confirmed postmortem, no

indication of WKS; n=6). Patient demographics are listed in Chapter 2, Table 2.2.1.

Detailed protocols are outlined in Chapter 2, Part III. Briefly, 2D-GE was used to profile

proteins from human BA9 grey matter samples described above. Prepared samples were

separated by 2D-GE (immobilized pH 3-10 gradient 11cm strips; 2D Gel-Chip 6-15%,

10x15cm, run in duplicates; n=16 each group). Once sub-gels were fixed they were

visualized using colloidal Coomassie staining, scanned and analysed using Phoretix 2D

Expression Software. Protein spots with statistically significant changes in abundance

were excised from the sub-gels and identified using MALDI-TOF-MS.


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5.3 Results

The normal control averaged gel showed 744 spots (26 gels); the uncomplicated

alcoholics averaged gel had 788 spots (14 gels); the alcoholics complicated with hepatic

cirrhosis averaged gel had 751 spots (12 gels). Comparison between gels can be limited

by the ability to match spots between case groups or averaged gels. In this study, more

than 89% of all spots were matched to the reference gel allowing excellent comparison

across all case groups (736, 707 and 670 matches in control, uncomplicated alcoholics

and cirrhosis complicated alcoholics respectively).

The relative abundance of 43 and 75 protein ‘spots’ was identified to change in the

cerebellar vermis of uncomplicated alcoholics and alcoholics with cirrhosis respectively

(p<0.05). Forty-one of these protein spots were common to both alcohol groups (see

Figure 5.3.1). Of the protein spots significantly altered in the uncomplicated alcoholic

group, 14 were over-expressed and 29 were under-expressed. In the cirrhosis-complicated

alcoholics, 38 and 37 were over and under-expressed respectively. Interestingly, many of

the protein spots (34) were unique to the alcoholics complicated with hepatic cirrhosis. A

similar proportion of cirrhosis-specific protein spots were isolated in the CC study

(Kashem et al., 2007), however, this observation was not reflected in the prefrontal

(Brodmann area 9; BA9) region studies (Alexander-Kaufman et al., 2006; Alexander-

Kaufman et al., 2007; see Chapter 4). Using Pearson’s correlations, 5 spots showed

significant correlation to the patients’ PMI (0.209≤ r2 ≤0.332; p<0.02), 5 spots to brain

pH (0.211≤r2≤0.359; p<0.02) and 1 spot to the patient’s age (r2=0.334; p=0.002). The

remaining 66 ‘disease-related’ spots were identified by MALDI-TOF, of which 51 were


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identified as 40 different proteins (Table 5.3.1) These identified proteins are mapped to a

2D gel in Figure 5.3.2 and categorised in Figure 5.3.3. The FDR for the present data set

was calculated according to (Storey, 2002) and was determined as 0.129, thus any

inferences drawn from this data must be substantiated with further study

Figure 5.3.1: Summary of significant protein abundance level changes in the cerebellar vermis of

uncomplicated alcoholics and alcoholics complicated with hepatic cirrhosis. The relative abundance of 43

and 75 protein ‘spots’ was identified to change in the uncomplicated alcoholics and alcoholics with

cirrhosis respectively, of which 41 protein spots were common to both groups.


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Table 5.3.1: Proteins Identified in the Cerebellar Vermis of Alcoholics


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Spot no. and fold change were attained from Phoretix 2D Expression Software. Accession no. is the primary accession number obtained from SWISS/Prot and TrEMBL

databases. Sequence coverage (%) refers to the percentage of protein sequence coverage determined by number of matched peptides. Functional classes were determined by

searching the Human Protein Reference Database (www.HRPD.org). * Fold change did not reach significance (i.e. ANOVA, p>0.05)


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Figure 5.3.2: Map of identified proteins with differential abundance in the vermis from alcoholic patients. Protein spots are identified by primary accession numbers obtained

from SWISS/Prot and TrEMBL databases.

MW pH 3 10


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Uncomplicated Alcoholics Cirrhosis-Complicated Alcoholics

Figure 5.3.3: Protein categories identified in the cerebellar vermis of uncomplicated and alcoholics with

cirrhosis.

Energy Metabolism Protein Metabolism Signal Transduction Cytoskeletal

Oxidative Stress Ribonucleaoprotein Transport


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5.4 Comparison of Cerebellar Vermis Proteome to BA9 Region Studies

The following Venn diagrams (Figures 5.4.1-2) compare the cerebellar vermis proteome to

the BA9 white and grey matter studies.

Uncomplicated Alcoholics

Figure 5.4.1: Identified protein spots in 3 brain regions, the cerebellar vermis, BA9 grey matter and white

matter, which change significantly in uncomplicated alcoholics. Twenty-nine protein spots were identified by

MALDI-TOF in the uncomplicated alcoholic group in the vermis study, of which 8 were common to the BA9

grey matter study and 7 were common to the BA9 white matter study. Overall 6 protein spots were isolated in

all 3 studies.


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Cirrhosis Complicated Alcoholics

Figure 5.4.2: Comparison of identified, significant protein spot changes across the 3 brain regions in alcoholics

complicated with hepatic cirrhosis. Fifty protein spots were isolated in the alcoholics with liver cirrhosis group

in the vermis study, of which 11 and 9 protein spots were common to the BA9 grey and white matter studies

respectively. Overall 7 spots were common to all 3 studies.

Previously, 44 different proteins were identified in BA9 grey matter, 28 in BA9 white matter

(Alexander-Kaufman et al., 2006; Alexander-Kaufman et al., 2007) using the same methods

(see Chapter 4). Identified vermis proteins common to the dorsolateral prefrontal region study

are listed in Table 5.4.1.


Table 5.4.1: Identified Vermis Proteins Common to BA9 Studies

Protein Identified Vermis Vermis Change UA CA

BA9 GM BA9 GM Change UA CA

BA9 WM BA9 WM Change UA CA

Aconitase 42 √ √ 83 84

X √

√ √

171 √ √

Transketolase 79 √ √ 157 158 160

√ √ √

√ √ √

290 √ √

Dihydropyramidinase-related protein 2 92 111

√ X

X √

253 √ √ 319 322 389

1330

√ √ √ √

√ √ √ √

Phosphatidylethanolamine-binding protein 330 334 336 349 350

X X X √ X

√ √ √ √ √

704 √ √ 911 √ √

Transitional ER ATPase 28 691

√ √

√ √

930 √ √ 124 √ √

Fructose-bisphosphate Aldolase C 203 √ √ 1117 √ √ 579 590

√ √

√ √

Vacuolar ATPase subunit B (BA9 GM-subunit E) 123 X √ 559 √ √ 410 √ √ α-Enolase 144 X √ 309

322 √ √

√ √

Fumerate Hydratase 165 166

√ X

√ √

347 √ √

Pyruvate Dehydrogenase E1-β 236 √ √ 521 √ √ Pyruvate Kinase 942 X √ 199 X √ Creatine Kinase chain B 185

205 X √

√ √

576 √ √

Glial Fibrillary Acidic Protein (GFAP) 850 X √ 512 √ √

Spot numbers for all 3 studies were obtained from Phoretix 2D Expression Software. UA, Uncomplicated alcoholics; CA, Cirrhosis-complicated Alcoholics; BA9, Brodmann Area 9; GM, grey matter; WM, white matter. A tick (√) represents a significant abundance changes found in the corresponding study whereas a (X) represents no significant change. Some proteins showed more than one variant/isoform change, i.e. one form of tranketolase changed in the vermis and BA9 WM, whereas 3 forms were isolated in the BA9 GM study.


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5.5 Discussion

These results demonstrate that significant protein abundance level changes occur in

the cerebellar vermis in both uncomplicated alcoholics and alcoholics complicated

with hepatic cirrhosis. Of the protein spot changes common to both alcohol groups,

many of the differences were exacerbated in the cirrhotic alcoholic group. By way of

example, dihydropteridine reductase (Spot no. 787) had a 2.0 fold increase in the

uncomplicated alcoholic group and in the cirrhotic group levels of this protein were

even greater (3.1 fold) compared to controls (ANOVA, p=0.0003). Alcoholics

frequently develop severe hepatic dysfunction, i.e. hepatic cirrhosis, and this can

result in altered thiamine homeostasis and structural and functional changes to

astrocytes (Butterworth, 1995). Phillips et al., demonstrated that the presence of

cirrhosis influences the extent of vermal Purkinje cell loss, which may be associated

with alcohol-related cognitive impairments and motor dysfunction (Phillips et al.,

1990). However, unlike the BA9 grey and white matter studies (Alexander-Kaufman

et al., 2006; Alexander-Kaufman et al., 2007), almost half of protein abundance

changes isolated where unique to the cirrhosis-complicated group alone, perhaps

indicating the effects of liver dysfunction in the cerebellar vermis. These proteins may

be specifically vulnerable to changes in hepatic function or perhaps play a role in the

mechanisms underlying HE. The majority of identified proteins with differential

abundance in the CC splenium were also specific to cirrhosis–complicated alcoholics

(Kashem et al., 2007). Particular cirrhosis-specific proteins identified in the CC and

vermis are discussed below. Examining the levels of these proteins in the brains of

patients with non-alcoholic cirrhosis is necessary to substantiate these findings.


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Figures 5.4.1 and 5.4.2 show a comparison of all the proteins spots identified in 3

different brain areas; the vermis, BA9 grey and white matter. Clearly in both alcohol

groups, each brain region is reacting differently to chronic alcohol exposure (or other

related factors), as there is limited overlap between the studies. However, a few

proteins identified in the vermis were also documented to change in the white and

grey matter of BA9 (Table 5.4.1) perhaps indicating a disruption in common cellular

cascades underlying alcohol-related changes. Transketolase is one of these proteins,

and is an important co-enzyme in thiamine-related metabolism.

5.5.1 Thiamine Deficiency

As stated above, alcoholics frequently suffer thiamine deficiency (Majumdar, 1980;

Majumdar et al., 1981), which can lead to WKS (Kril, 1996). The abundance levels of

2 thiamine-dependent proteins, transketolase and pyruvate dehydrogenase E1β-

subunit, were found to change significantly in the BA9 region of uncomplicated and

cirrhotic alcoholics (Alexander-Kaufman et al., 2006; Alexander-Kaufman et al.,

2007). In the vermis, changes in these thiamine-dependent enzymes were also seen, as

well as changes in dihydrolipoamide dehydrogenase, which is a component of the

pyruvate dehydrogenase complex. In both the vermis and BA9 grey matter,

transketolase levels were much lower in alcoholics with hepatic cirrhosis than those in

uncomplicated alcoholics (-2.5 fold versus -1.7 fold in vermis; -5.2 to -8.5 fold versus

-1.7 to -2.4 fold respectively in BA9 grey matter; ANOVA p<0.05, no statistical tests

performed between alcohol groups alone). Hepatic cirrhosis has been shown to alter

thiamine homeostasis (Butterworth, 1995) and this co-morbidity may explain the

lower transketolase levels observed in these cases. Studies have found that the


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cerebellar vermis is particularly sensitive to the deleterious effects of thiamine

deficiency (Baker et al., 1999; Lavoie and Butterworth, 1995). From a structural point

of view, thiamine deficiency was shown to contribute to a reduction in the number

and size of Purkinje cells in the cerebellar vermis (Phillips et al., 1987). Reduced

transketolase and pyruvate dehydrogenase complex activities were demonstrated in

autopsied cerebellar vermis samples from alcoholic patients with WKS (Butterworth

et al., 1993) and in a later study, reduced transketolase activity was seen in various

brain areas, including the cerebellum and prefrontal cortex, of alcoholic patients with

hepatic cirrhosis but without WKS (Lavoie and Butterworth, 1995). Our data

indicated a marked decrease in thiamine-dependent enzyme levels not only in

cirrhosis-complicated alcoholics, but also in the brains of ‘neurologically

uncomplicated’ alcoholics. The changes in the levels of thiamine-dependent enzymes

reported in this study indicate that to some degree, all alcoholics may be thiamine

deficient and imply that the diagnostic criteria for WKS are not stringent enough to

pick up sub-clinical thiamine deficiencies or early stages of this syndrome. This

important issue should be a consideration for future postmortem studies as well as in

the clinical setting, as thiamine deficiency leading to WKS is readily and successfully

reversed by thiamine supplementation.

5.5.2 Energy Metabolism

Interestingly, almost half of the identified proteins are metabolic enzymes important

for energy transduction. Several of these are involved in glycolysis and the TCA

cycle. Disturbance of these enzymes/proteins may alter a cell’s capacity to fulfil its

metabolic functions, leading to energy deprivation and a loss of viability, resulting in


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cell death. Indeed, glucose hypometabolism was found in the superior cerebellar

vermis in patients with alcoholic cerebellar degeneration (Gilman et al., 1990). The

PPP is important for generating reducing equivalents for reductive biosynthesis and

ribose, an essential component of nucleic acids and ATP. Transketolase, an important

enzyme of the non-oxidative branch of this pathway, reversibly links the PPP to the

glycolysis pathway. The interconnection of these pathways may provide

compensatory shifts in energy metabolism whilst the cell is under stress. Although

precise evidence of the effect of ethanol on the major metabolic pathways and fluxes

in the brain is limited, a disturbance in transketolase levels together with changes in

glycolytic and TCA enzymes and proteins, suggests that a derangement in energy

metabolism may underlie vermal damage frequently observed in chronic alcoholics.

5.5.3 Oxidative Stress

There is strong evidence to suggest that chronic alcohol consumption may enhance

oxidative damage to neurons and result in cell death. Aconitase, a TCA enzyme found

in significantly reduced levels in alcoholic vermis, BA9 grey and white matter is

sensitive to inactivation by oxidation (Gardner et al., 1995). Inactivation of aconitase

may block normal electron flow to oxygen, leading to an accumulation of reduced

metabolites such as NADH (Yan et al., 1997), causing increased formation of ROS

(Renis et al,. 1996). Therefore oxidative inactivation of aconitase can initiate a

cascade with the potential to cause a dramatic increase in the cellular burden of

oxidative damage (Yan et al., 1997). Another protein found to change in all 3 regions

was V-ATPase. Changes in 2 different subunits of this protein were isolated (subunit

B, BA9 WM and vermis; subunit E, BA9 GM; Alexander-Kaufman et al., 2006;


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Alexander-Kaufman et al., 2007). V-ATPase is responsible for acidifying a variety of

intracellular compartments along the secretory pathway. Wang and Floor 1998,

showed that V-ATPase in bovine brain synaptic vesicles is highly sensitive to

inhibition by micromolar concentrations of H2O2 (Wang and Floor, 1998). NO has

also been proposed to inhibit V-ATPase (Wolosker et al., 1996). This suggests that V-

ATPase may function as a redox sensor that regulates transmitter storage in response

to oxidative stress (Wang and Floor, 1998).

Glucose-regulated proteins (GRPs) 75 (Stress-70 protein) and 94 (Endoplasmin),

calreticulin and protein disulfide isomerase (all identified to change in the alcoholic

vermis) are endoplasmic reticulum stress proteins, which play a cytoprotective role

against oxidative stress. For example, the over-expression of calreticulin, a

Ca2+binding protein and chaperone, increases the capacity of intracellular Ca2+ stores

and prevents Ca2+ toxicity (Liu et al., 1998). A proteomics study also using 2D-GE,

found changes in GRP-75 in the nucleus accumbens of alcohol-preferring rats

(Witzmann et al., 2003). Another study used northern blot hybridisation to verify

ethanol related increases in GRP-94 mRNA in a concentration dependent manner

(Miles et al., 1994). Other proteins identified in the cerebellar vermis of alcoholics,

including protein DJ-1 and peroxiredoxin-6, are also related to oxidative damage.

Protein DJ-1, reduced in the vermis of cirrhosis complicated alcoholics, has been

shown to play a role in anti-oxidative stress by eliminating ROS (Li et al., 2005) and

immunoblotting results revealed a marked increase in oxidised inactivated

peroxiredoxin (increased in both alcohol groups) in alcohol-exposed mouse livers and

ethanol-sensitive hepatoma cells (Kim et al., 2006). Ubiquinol cytochrome C

reductase Fe-S subunit is part of complex III, which is important for oxidative


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phosphorylation, was identified to change in the vermis of complicated alcoholics.

Complex III is thought to be involved in ethanol-related production of reactive

oxygen species (Bailey et al., 1999). Interestingly, it is suggested that ethanol

specifically interacts with the Fe-S cluster of complex III (Sharp et al., 1998). So

together changes in the all these proteins in the vermis and across different brain

regions support the notion that oxidative stress is an important mechanism

underpinning alcohol-related brain damage.

5.5.4 Liver Cirrhosis-Specific Changes

Several liver cirrhosis-specific proteins were identified in the vermis, perhaps

indicating the effects of liver dysfunction in this brain region. Similarly, many

cirrhosis-specific spots were identified to change in the CC of chronic alcoholics

(Kashem et al., 2007). Interestingly some of the cirrhosis-specific proteins identified

in this study were also found in the vermis; for example, β-actin, which was

significantly increased in both studies. A previous study by Wan et al., detected an

ethanol-induced expression of β-actin mRNA, which was proportional to blood

alcohol levels in chronic alcoholic liver disease in rats (Wan et al., 1995). HE is a

neuropsychiatric syndrome that results from severe liver dysfunction, i.e. cirrhosis,

and causes cognitive dysfunction as well as motor disturbances (Butterworth, 2003).

Alzheimer type II astrocytosis is a pathological hallmark of HE where astrocytes

undergo a characteristic morphological change and lose GFAP immunoreactivity

(Kril et al., 1997a). Interestingly, specific alterations in GFAP levels were identified

in both the vermis and the CC (Kashem et al., 2007) from alcoholics complicated with

hepatic cirrhosis. HE is precipitated by the liver’s inability to remove blood-borne


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neurotoxic agents, including ammonia and manganese (Butterworth, 2003). Ammonia

can be produced by at least 16 different enzymatic pathways, the most important of

which is glutamate dehydrogenase, which produces ammonia by reversible oxidative

deamination of glutamate (Felipo and Butterworth, 2002). Two protein spots, perhaps

isoforms, of glutamate dehydrogenase 1 (GDH1) were identified in the cerebellar

vermis from cirrhosis-complicated alcoholics. This may indicate a toxic load of

ammonia on the brain caused by liver compromise, however, one GDH1 spot change

also reached significance in the uncomplicated alcoholics in this brain area. Albrecht,

et al., demonstrated an increase in carbonic anhydrase (CA) activity in experimental

HE, supporting the idea that CA participates in ammonia detoxication in the

brain(Albrecht and Hilgier, 1984). Elevated CA-2 levels were also identified in the

complicated alcoholics in this study (non-significant elevation seen in uncomplicated

alcoholics), perhaps indicating an adaptive measure to clear excess ammonia.

Although none of our complicated cases demonstrated clear clinical or pathological

evidence of HE, all were diagnosed with hepatic cirrhosis and minor elevations of

blood ammonia may have precipitated the alterations seen in the protein profiles of

these cases.

5.6 Conclusions

Together, these results suggest that the alcohol-related pathology of the vermis is

more multi-factorial than other brain regions explored. Although we have isolated a

number of proteins changing significantly only in the cirrhosis complicated alcoholic

group, these results suggest that perhaps these uncomplicated alcoholic cases are not

in fact ‘uncomplicated’. Certainly clinically and pathologically so, but at the proteome


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level we seem to be isolating the confounding effects of nutritional deficiencies and

liver dysfunction and perhaps their role in alcohol-related brain damage in the

cerebellar vermis.

In summary, we have identified a number of proteins that appear to be altered in the

cerebellar vermis of uncomplicated alcoholics and alcoholics complicated with

hepatic cirrhosis. Some of the proteins identified were common to our previous

studies. A derangement in energy metabolism perhaps related to thiamine deficiency

seems to be important in the vermis as well as in prefrontal regions, even where there

are no clinical or pathological findings of WKS. These studies also suggest that

oxidative changes are important in all brain regions analysed. Interestingly, several

liver cirrhosis-specific proteins were identified in the vermis, perhaps indicating the

effects of liver dysfunction in this brain region. Together these results highlight the

complexity of this disease process in that a number of different proteins from different

cellular pathways appear to be affected.

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