regeneration of oxidized glutathione by glutathione reductase to its reduced form , glutathione ( gsh ) is vitally important in combating oxidative stress in neurons . to reduce oxidized glutathione , glutathione reductase uses nicotinamide adenine dinucleotide phosphate ( nadph ) as its cofactor . as levels of reactive oxygen species accumulate in neurons , the demand for nadph increases ( ben - yoseph et  al . , 1994 ) .
the levels of gsh in cells in culture are directly related to the production of nadph by the pentose phosphate pathway ( ppp ) ( salvemini et  al . , 1999 ) and it has been shown that the ppp is the primary source of nadph in cells ( pandolfi et  al . , 1995 ) .
importantly , in neurons the rate limiting glycolytic enzyme phosphofructokinase b3 is translated but subsequently ubiquitylated and degraded by the proteasome , with the result that neurons preferentially metabolize glucose via the ppp as opposed to glycolysis , in contrast to most other cell types ( herrero - mendez et  al . , 2009 ) . inhibiting glucose flux through the ppp in these experiments
was shown to cause increased levels of oxidative stress and cell death , emphasizing the extent to which neurons rely on the ppp for survival .
the effects of inhibiting the pentose phosphate and gluthathione pathways are similar to many of the pathologic changes associated with parkinson 's disease ( pd ) . depleting glutathione in rats using l - buthionine sulfoximine , has been shown to cause complex i and iv damage ( heales et  al . , 1995 ) , a phenomenon , which has been documented in postmortem pd brain tissue , with complex i damage in pd widely reported ( schapira et  al . , 1989 ) .
similarly , decreased levels of total gsh have been measured in pd brains ( perry et  al .
this loss of gsh is found in pd and lewy body disorders , but not multiple system atrophy , progressive supranuclear palsy , or huntington 's disease ( sian et  al . , 1994 ) .
one important , but less reported finding is that pharmacologic inhibition of the ppp in rats using the irreversible inhibitor 6-aminonicotinamide , has been shown to cause selective dopaminergic cell death in the striatum and muscle rigidity resembling bradykinesia ( herken , 1990 ) .
recent studies investigating metabolism in pd have continued to emphasize the importance of glucose usage in the disease process , with glucose hypometabolism shown in pd using magnetic resonance imaging and fludeoxyglucose ( 18f ) positron emission tomography studies ( borghammer , 2012 ) .
genetic microarray studies of the substantia nigra in pd show a strong association with the transcriptional regulator pgc1 , which is involved in the control of glucose usage ( zheng et  al . , 2010 ) and in animal models , knocking down -synuclein ( -syn )
a recent article - characterizing metabolism in cultured adipocytes suggests that glucose uptake may be regulated by -syn signaling via the lpar2/gab1/pi3k / akt pathway ( rodriguez - araujo et  al . ,
importantly , studies looking at the effect of glucagon - like - peptide 1 on animal models of pd , have shown that modulating glucose metabolism is able to reverse dopaminergic cell loss in rats ( harkavyi et  al . , 2008 ; li et  al . ,
2009 ) and clinical trials of the glucagon - like - peptide 1 agonist exendin-4 are currently at stage ii for use in pd ( tom foltyne , personal communication ) . through this study
comparison of these results with those from ad and control tissue ; will allow any differences in nadph production to  be identified .
we hypothesize that reduced glucose metabolism via the ppp in neurons , is an early event in sporadic pd pathogenesis .
we studied flash - frozen tissue from the frontal cortex ( brodmann area 4 ) , putamen , and cerebellar vermis .
cases with pathologically diagnosed pd were stratified according to the mckeith criteria ( mckeith et  al . ,
 mild  cases were grouped according to those with lewy body pathology in the brain stem only ( mckeith brainstem predominant ) . 
moderate and/or severe  cases were those with lewy body pathology visible in the limbic and neocortical areas ( mckeith limbic and diffuse neocortical lewy body pathology ) .
ad cases were similarly confirmed by postmortem analysis and cases with braak and braak stages iv - vi ( braak et  al . , 2003 ) were selected for these experiments . for each brain region 13
controls , 11 mild pd , 12 moderate and/or severe , and 13 ad cases were used . where tissue was available , cortex , putamen , and cerebellum samples were taken from the same individual .
controls were age matched and showed no lewy body pathology . cases with braak and braak stages ii and under were used for this study .
tissue was homogenized in an isolation buffer of 320 mm sucrose , 1 mm ethylenediaminetetraacetic acid and 10 mm tris - base , ph 7.4 using a mechanical homogenizer .
homogenates were centrifuged for 15 minutes at 16,000 g at 4 c and the supernatant removed for assaying .
we characterized ppp in pd by assessing the total nadph generated by glucose-6-phosphate dehydrogenase ( g6pd ) and 6-phosphogluconate dehydrogenase ( 6pgd ) .
this was done using a modified protocol , based on that described by ( ninfali et  al . , 1997 ) .
tissue homogenate was assayed under 2 separate conditions , 1 containing g6p and 6pg to calculate the total activity of the pentose phosphate pathway and 1 with 6pg alone .
g6pd activity was calculated by subtracting the nadph generated in the combined condition , from that in the 6pg condition .
reaction mixture contained a final concentration of 2 mm nadp , 1 mm glucose-6-phosphate , and 1 mm phosphogluconic acid reconstituted in 1 m tris - hcl , 5 mm ethylenediaminetetraacetic acid ph 7.6 , 15% ( vol / vol ) wst-8 dye supplemented with 0.2 mm 1-methoxy phenazinium methylsulfate ( all materials were purchased from sigma - aldrich , poole , uk ) .
samples were added to the reaction mixture and incubated at 37 c for 15 minutes .
the levels of the nadph - producing enzymes g6pd and 6pgd were calculated using elisa kits specific for human g6pd and 6pgd , according to the manufacturer 's instructions ( wuhan life sciences , houston , tx , usa ) . samples were run in duplicate .
protein concentration was estimated using the bio - rad dc protein assay kit according to the manufacturer 's instructions ( bio - rad , hemel hempstead , uk ) .
all results in this study were analyzed using a 1-way analysis of variance ( anova ) followed by a bonferroni posttest .
we set out to characterize utilization of the ppp in flash frozen postmortem pd brain tissue .
to look at glucose metabolism via this pathway over the course of disease progression , we identified 2 groups of pd samples , which were classified according to the extent of their lewy body pathology based on the mckeith lewy body pathology criteria ( mckeith et  al . , 2005 ) .
cases assigned to the  mild  group were those with lewy body pathology predominantly in the brain stem . 
moderate and/or severe  cases were those with lewy body pathology that had progressed to the limbic and neocortical areas .
all cases in the  mild  group showed a low level of lewy body deposition in the putamen .
ad cases were confirmed by postmortem neuropathological examination and cases with braak and braak stages iv - vi ( braak et  al . , 2003 ) were used for these experiments .
controls were age matched and confirmed to have braak and braak stages of ii and under .
total nadph produced by the ppp was calculated by addition of nadph production by g6pd and 6pgd . to assess the impact of enzyme expression on the production of nadph
data for 6pgd elisa and enzyme activity levels are shown as supplementary data . increased nadph production in the cortex
has been shown to occur in response to increased levels of oxidative stress in ad ( martins et  al . , 1986 ) .
calculation of nadph production by the ppp in our samples showed that generation of nadph is significantly increased in the ad and moderate and/or severe pd groups as compared with controls ( fig . 
1a gray bars , 1-way anova followed by bonferroni posttest vs. control ) . this increase was accompanied by higher levels of the rate - limiting enzyme g6pd ( fig . 
1b , gray bars , 1-way anova followed by bonferroni posttest p < 0.05 ) . in the mild pd group , that is pd cases that do not have pathology in the cortex , there was no increase in either the amount of nadph produced or levels of g6pd enzymes ( fig . 
1a and b gray bars ) . calculating the efficiency of g6pd by expressing nadph production per elisa unit of g6pd protein showed that there was no significant change in any of the experimental groups characterized ( fig .  1c , gray bars , 1-way anova , p  = 0.8017 ) .
total nadph produced by the ppp in the putamen was significantly increased in the brains of those with ad and in the moderate and/or severe pd group ( fig . 
1a , black bars , 1-way anova followed by bonferroni posttest ) . in ad , the levels of g6pd enzyme
similarly the moderate and/or severe pd group showed no significant increase in protein levels of the rate - limiting enzyme g6pd however , the mild pd group showed a significant decrease , suggesting a down - regulation of this pathway at the protein level ( fig . 
calculating the efficiency of g6pd for all samples , showed an activation of g6pd in the mild pd and ad groups . in these groups , each elisa unit of the enzyme produced approximately twice as much nadph per minute compared with the control group ( fig . 
importantly , ad cases with braak and braak stages iv - vi have amyloid beta and tau pathology in the putamen and the activation of g6pd in these samples suggests that an oxidative stress response is occurring . while both the mild and moderate and/or severe pd cases used in this study showed lewy body pathology in the putamen , it is only the moderate and/or severe group which shows increased nadph production in response to this ( fig .  1a , black bars )
this would suggest that although the mild cases are able to match control nadph production levels , the ability to mount an oxidative stress responsive is decreased and that an increase in nadph is not seen until pd pathology is more extensive . to assess the function of the ppp pathway in a region not affected in pd , we measured nadph production and the enzyme levels of g6pd and 6pgd in the cerebellum .
nadph production across all 3 experimental groups showed no significant change in the cerebellum when compared with controls ( fig . 
interestingly however , quantification of g6pd protein levels , again showed a down - regulation of the rate - limiting enzyme that was present in both pd groups , with enzyme levels showing a significant decrease compared with control ( fig . 
1b , white bars , 1-way anova followed by bonferroni posttest ) . calculating the efficiency of g6pd in these samples showed a 2-fold increase in nadph production of about 2-fold in these samples when compared with controls ( fig .  1c , 1-way anova followed by bonferroni posttest ) .
as shown in the putamen of samples in the mild pd group , despite a significant decrease in nadph producing enzymes , net nadph production in both pd groups remains level with that of the controls , because of each unit of g6pd producing more nadph .
samples in the ad group showed no difference in the nadph produced ( fig .  1a , white bars ) , or in the levels of ppp enzymes measured ( fig . 
neurons are subjected to high levels of oxidative stress because of a relatively high level of oxidative phosphorylation and nitric oxide signaling from astrocytes ( bolanos and heales , 2010 . ) , leaving them vulnerable to damage .
it has been shown that production of nadph from the ppp is responsible for reduction of oxidative species and therefore reducing levels of oxidative stress , leaving neurons able to function normally .
inhibition of the ppp is deleterious to neurons ( filosa et  al . , 2003 ; pandolfi et  al . , 1995 )
. increased levels of oxidative stress are important in the pathogenesis of pd ( alam et  al .
, 1997 ; dexter et  al . , 1989 ; floor et  al . , 1998 ) and it has been suggested that oxidative stress could play an important role in the formation of lewy body pathology ( jenner and olanow , 1996 ) . as the ppp is the main source of defense against oxidative stress in neurons , we thought it important to characterize the usage of this pathway in pd .
studies showing increased activity of the ppp in ad ( martins et  al . , 1986 ) , neuromuscular diseases ( meijer , 1991 ) , myocardial infarction ( gupte , 2008 ) , and studies using hepatocyte cell models ( ursini et  al . , 1997 )
however , characterization of the ppp in our experiments has shown that nadph production is not increased in the putamen of samples with early stage pd and furthermore , ppp enzyme levels are actually decreased .
this is despite widely documented evidence that dopaminergic cell death in the striatum of pd cases is accompanied by increased markers for oxidative stress and neuroinflammation in postmortem samples ( alam et  al . , 1997 ; imamura et  al . , 2003 ; marttila et  al . , 1988 ; ouchi et  al . , 2005 ; sanchez - ramos et  al . ,
1994 ) and in  vivo , by positron emission tomography imaging studies ( ikawa et  al . , 2011 ) . similarly , although there are a number of published studies showing increased levels of oxidative stress in the frontal cortex of cases with both clinical and preclinical pd ( dalfo et  al .
2008 ) , the early stage cases in our experiments again did not show an increase in nadph levels
. this could be because of faulty oxidative stress - sensing mechanisms in affected neurons , or because of an active suppression mechanism that is stopping increased nadph production from occurring .
an article looking at the ppp in als , showed that neuroblastoma - spinal cord hybrid cell lines ( nsc34 ) carrying g93a or g37r sod1 mutations display decreased levels of g6pd enzyme , which affects the production of nadph by this pathway ( kirby et  al . , 2005 ) .
similarly ataxia telangiectasia mutated knockout mice also show a reduction of g6pd in the cerebellum ( cosentino et  al . , 2011 ) , suggesting that downregulation of the ppp in disease could be a key mechanism in some forms of neurodegeneration . taking our results together with data from cell and animal models , suggests that dysregulation of the ppp could be causing oxidative stress because of less efficient gsh recycling ( heales et  al . , 1995 ; herrero - mendez et  al . , 2009 ) and that dysregulation of the ppp could be causative factor in the increased levels of oxidative stress seen in pd .
the results of our experiments suggest that there are 2 stages of ppp involvement with oxidative stress in pd . in the early stages of disease ,
lower levels of ppp enzymes in the putamen could be causing increased levels of oxidative stress . in response to this , g6pd is activated , potentially by posttranslational modification of the enzyme as shown in other studies ( cosentino et  al . , 2011 ; ursini et  al . , 1997 ) and the increased demand for nadph met by increased activity per elisa unit of the protein . in the later stages of disease , where pathology has spread to the limbic system and  neocortex ,
our results show that there is a switch from activation  of g6pd at its existing levels , to upregulating the nadph - producing enzymes at a protein level .
interestingly , the exact mechanisms for regulation seem to differ between brain regions and despite evidence of increased oxidative stress in the cerebellum in pd , the cerebellum does not show evidence of neurodegeneration .
elucidating the mechanisms underlying these differences could be important in understanding the vulnerability of certain cell types over others in different neurodegenerative diseases .
the results of this study come with the caveat that the tissue was from postmortem brains and therefore only provides a snapshot of the changes occurring at that time .
despite this , further investigation into mechanisms that are potentially underlying our findings may prove important in understanding the pathogenesis of pd
the results of this study shed light on a potentially new mechanism in the pathogenesis of sporadic pd and when taken with further studies from cell and animal models , may lead to better understanding of these mechanisms .
our data suggest that down - regulation of the ppp in pd could be a primary event in disease progression and further research down this avenue may provide potential targets for future therapeutic avenues .
we hypothesize that mitochondrial damage in pd occurs as a direct result of ppp dysregulation and that -syn plays an important role in the altered metabolism of glucose via the ppp in this disease .
 unlike most other cell types , neurons preferentially metabolize glucose via the pentose phosphate pathway ( ppp ) to maintain their antioxidant status . 
 inhibiting the ppp in neuronal cell models causes cell death . in rodents , 
 inhibition of this pathway causes selective dopaminergic cell death leading to motor deficits resembling parkinsonism . using postmortem human brain tissue , we characterized glucose metabolism via the ppp in sporadic parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and controls . 
 ad brains showed increased nicotinamide adenine dinucleotide phosphate ( nadph ) production in areas affected by disease . in pd however , increased nadph production was only seen in the affected areas of late - stage cases . quantifying ppp nadph - producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by enzyme - linked immunosorbent assay , showed a reduction in the putamen of early - stage pd and interestingly in the cerebellum of early and late - stage pd . 
 importantly , there was no decrease in enzyme levels in the cortex , putamen , or cerebellum of ad . 
 our results suggest that down - regulation of ppp enzymes and a failure to increase antioxidant reserve is an early event in the pathogenesis of sporadic pd . 