genetic susceptibility and exposure to neurotoxins are contributors to brain tissue malfunction and neuronal cell loss .
environmental toxins influence pd onset ( rajput 2001 ) and are also suggested to be involved in atypical parkinsonian syndromes ( caparros - lefebvre and lees 2005 ) and progressive supranuclear palsy ( psp ) ( davis et al .
furthermore , the age - related neurodegenerative disorders are commonly characterised by the accumulation of insoluble neurotoxic ubiquinated proteins ( johnson 2000 ) .
the neurotoxin hypothesis states that detoxification enzymes can influence susceptibility to pd , but recently another mechanism has also been recognised as important for detoxification of the brain .
proteins that are involved in cellular efflux prevent penetration into the brain by various potentially toxic compounds or their accumulation .
they include p - glycoprotein ( p - gp ) ( cordon - cardo et al .
1989 ) and multidrug resistance associated protein ( mrp ) . at the blood  brain barrier ( bbb ) , p - gp is expressed in a high concentration at the luminal side of the brain vascular endothelium ( demeule et al .
2001 ) and functions as an efflux pump by translocating a substrate to the extracellular compartment .
p - gp in the endothelial cells of the choroid plexus has a transport direction towards the csf ( de lange 2004 ) .
the p - gp transporter is associated with the multidrug resistance gene ( mdr1 ) .
interestingly , a significant association was found between parkinsonian patients exposed to pesticides and c3435 t polymorphism of the mdr1 gene , related to lower p - gp function ( drozdzik et al .
thus the mutation of the mdr1 gene with diminished p - gp function increases the vulnerability to the damaging effects of pesticides .
it is assumed that neurotoxic substances such as certain pesticides are substrates of p - gp ( bain and leblanc 1996 ) and cns exposure to such agents may continue to cause neurodegenerative diseases . in a pilot study ( kortekaas et al .
2005 ) , impaired bbb p - gp transport was found in the midbrain of five pd patients compared to five healthy control subjects , measured with [ c]-verapamil pet , pointing to a role of p - gp in the brain to prevent accumulation of toxic substances causing pd neurodegeneration .
this finding , however , could not be replicated in a study that measured p - gp function with [ c]-(r)-verapamil pet in ten early stage pd patients compared to healthy controls ( bartels et al .
decreased expression of p - gp in bbb vasculature has been found in other neurodegenerative diseases , like alzheimer s disease ( ad ) ( vogelgesang et al . 2001 ) and creutzfeldt  jakob disease ( cjd ) ( vogelgesang et al
. 2006a ) . in ad , p - gp up - regulation in early pathogenesis
was suggested as a compensatory mechanism to increase the clearance of harmful proteins , while at later disease stages p - gp expression was lost ( vogelgesang et al .
we hypothesize that p - gp - mediated transport out of the brain is involved in clearance of harmful substances and dysfunction of this system also occurs in late stage parkinsonian neurodegeneration . in the present study
, we performed [ c]-verapamil - pet brain imaging of new groups of de novo pd patients , pd patients in more advanced disease stages , progressive supranuclear palsy ( psp ) , and multisystem atrophy ( msa ) patients and a healthy control group , to study regional bbb p - gp function in patients with different parkinsonian pathology .
pet with [ c]-verapamil has been shown to be an appropriate methodology for the in vivo assessment of the functional p - gp status in human bbb ( hendrikse et al .
the distribution volume ( dv ) of the p - gp substrate verapamil can be taken as a measure of p - gp efflux function , higher [ c]-verapamil uptake meaning less p - gp efflux function .
patients were selected from the movement disorders clinic of the university medical centre groningen ; healthy volunteers were asked to participate by advertisement .
the study was approved by the medical ethics committee of the university medical centre groningen and all subjects gave their informed consent .
ten de novo pd patients ( diagnosed in the past year and without pd medication ) , and ten pd patients in more advanced disease stage ( hoehn and yahr stages 23 ) participated in the study ( table  1 ) .
furthermore , five patients with psp and four patients with msa , compliant with the criteria for psp or msa according to the ninds - spsp international workshop ( litvan et al . 1996 ) and the consensus statement for the diagnosis of msa ( gilman et al , 1999 ) were included .
nine healthy age - matched subjects , who did not use medication , participated as controls .
patients did not use known p - gp modulating agents [ ( cardiovascular drugs , antimalarial drugs , cyclosporine a , phenothiazines , hormones ( e.g. , tamoxifen ) ] and certain antibiotics such as cefoperazone , ceftriaxone , and erythromycine [ see for a detailed list ( hendrikse et al . 2001 ) ] .
t1 weighted mri scans were performed on all subjects and did not show abnormalities or significant atrophy of cortical regions . 

table  1patient characteristicsdiagnosis ( n)mean agedisease durationupdrs iii medicationpd ( 10)63 sd 85  years sd 221 sd 7 (  onstate)levodopa  +  dopamine - agonist ( ropinirole or pramipexole or pergolide)pd de novo ( 10)66 sd 9<1  year19 sd 8xpsp ( 5)63 sd 83  years sd 220 sd 8amantadine ( 2 patients)msa ( 4)59 sd 113  years
sd 227 sd 3levodopa ( 2 patients)controls ( 10)59 sd 10none0nonemean values for the groups are given with standard deviations ( sd ) patient characteristics mean values for the groups are given with standard deviations ( sd ) [ c]-verapamil was synthesised as described before ( wegman et al .
pet scans were performed with an ecat exact hr  +  positron camera ( siemens / cti , knoxville , tn ) .
after cannulation of the radial artery , all subjects underwent 1  h dynamic pet scanning consisting of 21 frames after injection of [ c]-verapamil ( 200400  mbq ) .
serial arterial blood sampling to define the input function for [ c]-verapamil was done using a computerised sampling programme ; six remaining samples were drawn manually . these samples ( collected at intervals of 10  min )
were processed by high liquid chromatography to measure the fraction of unmetabolised parent compound . in this way
pet data were reconstructed to a 128    128    63 matrix with a plane separation of 2.425 and a bin size of 2.250  mm . for anatomical demarcation , the frames 1221 of the dynamic [ c]-verapamil scans were summated using clinical application programming package ( capp ; siemens , erlangen , germany ) . on this summated image ,
capp regions of interest ( rois ) were manually drawn over the midbrain and whole brain to calculate distribution volume ( dv ) in the rois .
using matlab ( the mathworks inc . , natick , usa ) and a linearization according to logan ( 2000 ) , starting at t  =  5  min , dv images were constructed from the [ c]-verapamil scans , which was done for the whole brain roi as well as pixelwise to obtain parametric dv images .
logan analysis is a graphical method of analysis , applicable to ligands that bind reversibly to receptors or enzymes , calculating the dv for dynamic pet data before steady state is actually reached . for comparison of regional differences between the groups
1991 ) ( spm99 , wellcome department of cognitive neurology , london , uk ) .
spm99 instead of the latest spm version was used to compare the results with those of the pilot study ( kortekaas et al .
individual dv images were spatially normalised to the [ c]-verapamil dv template that was constructed in the pilot study .
after spatial smoothing by convolution with a gaussian kernel of 20  mm , a two - sample t test was done . to visualize the statistical parametric maps , the threshold was set to p  <  0.001 uncorrected . because higher variation in dv between individuals may render smaller differences between the groups statistically insignificant in spm , t tests were repeated after global calculation of the dv images to 100 in spm .
we also used this spm analysis method to compare our results with the previous pilot study ( kortekaas et al .
2005 ) . from each subject 10  ml blood was collected in an edta coated vial for determination of mdr1 c3435 t polymorphism on exon 26 . a polymerase chain reaction ( pcr)-based restriction fragment length polymorphism assay was performed with the primers
mdr1f 5-tgctggtcctgaagttgatctgtaac-3 and mdr1r 5-acattaggcagtgactcgatgaaggca-3. pcr was carried out with an initial denaturation of 12  min at 95c followed by 30 cycles of denaturation at 94c for 1  min , primer annealing for 1  min at 60c , and 1  min extension at 70c .
when the mutation was present ( 3435 t ) , digesting of the 248  bp pcr - product produced the 172- , 60  bp and 16-bp fragments .
the digested pcr products were analysed on a 2% agarose gel with ethidium bromide staining .
whole brain verapamil dv of all subjects was plotted against the c3435 t polymorphism ( cc , ct and tt genotypes ) , and correlation coefficient of genotype with dv was calculated in all subjects and in the separate groups .
[ c]-verapamil uptake was high in the pituitary , ventricles and skin , moderate in grey matter and low in white matter and bone .
the logan analysis provides the dv of a tracer , and is defined by a ratio between the influx of the tracer k1 and the efflux out of the brain k2 , of which the latter reflects the efflux function of p - gp .
the influx k1 of [ c]-verapamil was of the same magnitude in all groups , which confirms that differences in dv reflect differences in p - gp efflux function . mean dv in the more advanced pd group was 0.56 ( sd 0,12 ) , de novo pd showed 0.50 ( sd 0,08 ) , psp 0.66 ( sd 0,05 ) , msa 0.66 ( sd 0,07 ) and controls 0.59 ( sd 0,13 ) .
k1 in the pd group gave 0.037 ( sd 0,013 ) , psp 0.038 ( sd 0,005 ) , msa 0.036 ( sd 0,006 ) and controls 0.036 ( sd 0,006 ) .
pixel by pixel t test ( p  <  0.001 uncorrected ) in spm did not show increased midbrain [ c]-verapamil uptake in either of the pd groups as compared to the control group .
the advanced pd group showed significantly increased [ c]-verapamil uptake in frontal white matter regions compared to controls ( spm coordinates 34 , 30 , 26 and 16 , 48 , 12 , near grey matter brodmann areas 9 and 10 , p corrected at cluster level 0.000 , fig . 
1 ) ; a cluster with decreased uptake was seen in the cerebellar tonsils ( maximum effect at coordinates 40 , 54 , 46 ) .
the de novo pd group had lower uptake in brainstem and cerebellar tonsils ( brainstem coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 , fig .  2 ) compared to the control group and did not show clusters with higher uptake .
plots of the local spm responses showed a considerable separation of the groups , although there are some overlapping cases , where the pd groups show larger variation in local response values .
comparison of de novo and advanced pd groups revealed a cluster of pixels reflecting higher uptake in the advanced pd group in the anterior cingulate cortex ( coordinates 4 , 28 , 6 ) and in the caudate head ( coordinates 14 , 14 , 2 with p corrected at cluster level 0.01 ) . 

increased [ c]-verapamil uptake in the frontal lobe in advanced pd patients compared to healthy controls ; spm coordinates 34 , 30 , 26 and 16 , 48 , 12 ( brodmann areas 9 and 10 ) with p corrected at cluster level 0.000 .
b plot of the frontal effect ( pd patients scan number 110 and controls 1120 ) shows little overlapfig . 
2a de novo pd patients showed lower brainstem [ c]-verapamil uptake in the brainstem compared to controls , p  <  0.001 ; spm coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 .
b plot of the brainstem effect shows more variation in pd de novo values ( scan number 110 ) with some overlap with the control group ( scan number 1120 ) a spm t test on mri overlay showing p  <  0.001 . increased [ c]-verapamil uptake in the frontal lobe in advanced pd patients
compared to healthy controls ; spm coordinates 34 , 30 , 26 and 16 , 48 , 12 ( brodmann areas 9 and 10 ) with p corrected at cluster level 0.000 .
b plot of the frontal effect ( pd patients scan number 110 and controls 1120 ) shows little overlap a de novo pd patients showed lower brainstem [ c]-verapamil uptake in the brainstem compared to controls , p  <  0.001 ; spm coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 .
b plot of the brainstem effect shows more variation in pd de novo values ( scan number 110 ) with some overlap with the control group ( scan number 1120 ) in the psp group , significantly increased uptake was seen in basal ganglia regions ( maximum effect in lentiform nucleus , coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 , fig . 
when plotting the effect , one psp patient did not show higher uptake compared to the healthy controls ( fig . 
notably , this was also the patient with shortest disease duration ( < 1  year ) and only mild parkinsonian symptoms ( updrs motor part score 12 ) .
the psp group had also increased uptake in frontal regions compared both to controls and the advanced pd group , but this did not reach significance and plotting the effect showed high variance between the psp patients .
msa patients did not show significant differences compared to controls , however after lowering the threshold to p  <  0.01 , also increased uptake in basal ganglia regions was seen with p corrected at cluster level 0.98 and uncorrected p at 0.056 in putamen ( 28 , 6 , 2 ) .
plot of the spm effect showed more variance between the msa patients with one patient in the control range .
this patient had lower disease severity compared to the other msa patients , with 2  years disease duration and updrs motor score 24 . 

3a psp patients showed higher [ 11c]-verapamil uptake in lentiform nucleus and putamen compared to controls , p  <  0.001 ; spm coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 .
b plot of the basal ganglia effect shows higher values in four of the five psp patients ( scan number 15 ) .
psp patient number 5 had shortest disease duration ( < 1  year ) a psp patients showed higher [ 11c]-verapamil uptake in lentiform nucleus and putamen compared to controls , p  < 
0.001 ; spm coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 .
b plot of the basal ganglia effect shows higher values in four of the five psp patients ( scan number 15 ) .
psp patient number 5 had shortest disease duration ( < 1  year ) all results did not remain statistically significant after correction for multiple comparisons in spm ( fdr correction with level of significance set at p  <  0 .
t - tests in spm were repeated after global calculation of the dv images to 100 , which showed comparable results and did not reveal additional foci with relative dv differences between the groups .
the frequencies of the determined mdr1 polymorphism in our study group did not differ from the frequencies seen in the general population ( tan et al .
furthermore , c3435 t genotypes did not significantly correlate with the differences in [ c]-verapamil dv in the brain in all groups .
we compared [ c]-verapamil pet data of the present study with data of the previously published pilot study that was performed by our group .
mean injected dose was lower in the pilot study with 111  mbq given as lowest dose ; for a better signal - to - noise ratio , the cut - off dose for the current study was set at 200  mbq .
the previous pilot control group showed relatively lower midbrain uptake compared to the nine control subjects of this study , which , however , only was shown in spm after global calculation of the mean dv to 100 .
neither group used medication nor was there a significant age difference between both the control groups , although the pilot group had one outlier of 86  years .
we could not demonstrate a definite methodological explanation for the midbrain effect in the pilot control subjects .
however in our opinion , global calculation of the mean may have affected the results in the midbrain area , which is a small region with a low uptake signal and is thereby prone to artefacts . finally , because of the lower injected tracer dose , data of the previous pilot study were not further used for the present study .
the results of this study do not support the idea of a primary dysfunctional bbb p - glycoprotein efflux system in the pathogenesis of parkinson s disease , but finds decreased p - gp function at later disease stages .
this is in line with a most recent study that measured bbb p - gp function in mostly early stage pd patients using [ c]-(r)-verapamil , where higher variance in tracer uptake was seen in several regions in the pd group , but no significant decreased p - gp function was found ( bartels et al .
more advanced pd patients showed frontal regions with diminished p - gp function relative to normal controls and de novo pd patients .
regional frontal decreases in both advanced pd and advanced psp possibly reflects the affected frontostriatal neuronal projections with frontal dysfunction in these disease stages ( green et al . 2002 ) .
the increased uptake lies in the white matter regions rather than the cortex . in general ,
involvement of white matter regions and vasculature in neurodegenerative disorders that can be seen as  proteinopathies  , involving accumulation of misfolded proteins , is a topic of important discussion .
it was shown in vitro that -amyloid is transported by p - gp ( kuhnke et al .
interestingly , the deposition of a140 in cerebral amyloid angiopathy ( caa ) , which is found in around 90% of ad patients , is predominantly found in the penetrating arterioles that supply the periventricular white matter ( lee and markus 2006 ) .
another study associated the failure of interstitial fluid drainage from the white matter in ad with the deposition of a in the perivascular fluid drainage pathways ( roher et al .
thus , decreased p - gp function in white matter regions may influence protein removal and accumulation at sites more distant from the white matter . furthermore
, it is suggested that lewy body - related pathology initially involves the neuronal perikarya , dendrites , and axons , with abundantly present 
these authors also find that impairment of axonal transport and synaptic transmission leads to the formation of lewy bodies , a hallmark of functional disturbance long before neuronal cell death .
white matter p - gp dysfunction might also influence removal of toxic substances such as pesticides , which could cause harm to distant sites of the white matter after alterations of axonal transport , leading to  dying - back  patterns of neurodegeneration ( morfini et al .
greater disease severity and a more rapid neurodegenerative process have been associated with white matter hyperintensities ( piccini et al .
however , although specific methodology or rating scales were not used to assess white matter pathology on the mri s in this study , we could not detect any visually apparent white matter hyperintensities in any subject .
both de novo and more advanced pd patients showed lower [ c]-verapamil uptake in the cerebellum . the cerebellum is closely linked to midbrain nuclei , but is by itself not affected with progression of pd pathology ( braak and braak 2000 ) .
possibly , p - gp function in these regions is increased in pd , but does not deteriorate in later stages , as these regions are not vulnerable to pd pathology . on the other hand , in msa cerebellar degeneration can be part of the disease spectrum .
we did not find changed p - gp function in the cerebellum in the msa patients .
however , the msa patients in this study did not show symptoms of cerebellar dysfunction and mri scans did not show cerebellar atrophy .
psp and msa patients showed basal ganglia regions with increased [ c]-verapamil uptake , pointing at decreased p - gp function in regions with neuropathological features in these disorders .
brain atrophic changes are not likely to play a role in these results , because mri scans did not show overt atrophy in these regions .
psp patients also showed decreased p - gp function in frontal regions . although this result was not significant , plotting this effect showed higher variation between the psp patients with one psp patient in early disease stage who showed values overlapping with the controls ( fig . 
these results may possibly point at regionally decreased p - gp function with advancing disease stage . regionally decreased p - gp function could thus occur not only in more advanced pd , but may also play a role in other neurodegenerative diseases with parkinsonism , and likely plays a role in advancing neurodegenerative processes in general .
it has been argued that decreased cerebrovascular p - gp impairs the ability of the brain to expel excess proteins , and that the resulting accumulation of protein in cells could overwhelm cellular ubiquitin - proteasomal degradation ( vogelgesang et al .
p - gp was even found to interact actively with the proteasome complex , and a role for p - gp in the transport of proteasome - derived peptides was suggested ( begley et al .
loss of p - gp function may possibly also lead to increased accumulation of insoluble -synuclein with ongoing neurodegeneration in pd .
p - gp dysfunction in late disease stage may also contribute to neuronal damage due to increased accumulation of toxins .
it is known that environmental factors causing damage , such as pesticides , are closely linked to mechanisms underlying the formation of -synuclein pathology and pd - like neurodegeneration ( norris et al . 2007 ) .
furthermore , it is recognised that the prevalence of dementia is higher in pd patients than in age - matched controls , which often is found to be defined by ad pathology in conjunction to the lewy bodies ( burn 2006 ) .
decreased p - gp function to expel proteins in advanced pd could also play a role in the co - existence of -synuclein and -amyloid in pd with dementia ( pdd ) , a patient group that was not investigated in the present study . in the study presented here , de novo pd patients showed lower [ c]-verapamil uptake compared to normal controls in the brainstem , which may indicate a regional up - regulation of p - gp function in early stage pd .
the brainstem regions lies posterior to the pons , comprising the locus coeruleus and lying slightly superior to the dorsal motor vagal nucleus , a region that is affected early in pd pathology ( del et al .
2002 ) , containing projection neurons of the intermediate reticular zone , coeruleus  subcoeruleus complex and caudal raphe nuclei .
a limitation of the methods , using [ c]-verapamil uptake as a measure of p - gp efflux function , is the low uptake signals of this tracer in the normal condition , as it is removed by p - gp .
intersubject variation in tracer uptake can be substantial , whereby small regional effects may be overlooked in the statistical comparison .
on the other hand , global calculation can be applied to investigate regional patterns within disease groups rather than absolute p - gp measures .
this should be applied with care , as small regions with low uptake signals may be prone to artefactual changes . in the case of the present study ,
the brainstem effect was also seen without proportional scaling in spm , and is therefore more likely a significant result . initial up - regulation of p - gp expression or increase of p - gp function in reaction to neuronal damage may be associated with a neuroinflammatory process in parkinson s disease . in pd as well as in other neurodegenerative diseases , extensive microglia activation , the key event in neuroinflammation ,
is seen ( gerhard et al . 2001 ; mcgeer and mcgeer 1998 , 2004 ) .
microglia themselves express p - gp , which increases upon microglia activation ( langford et al .
several factors that are involved in inflammatory processes , like tnf- , il-6 and nitric oxide ( no ) may increase p - gp function ( fernandez et al . 2004 ; mcrae et al .
2002 ) showed that exposure of bbb endothelial cells to activated t - lymphocytes induced endothelial cell death , whereas p - gp expression and function was increased in surviving cells .
the authors conclude that p - gp could play a regulatory role in promoting survival of cerebral endothelial cells and maintenance of bbb function . in conclusion , neuronal damage with a subsequent neuroinflammatory reaction
could , by activating the p - gp system promote the clearance of the brain of potentially toxic compounds . an initially compensatory mechanism with up - regulation of p - gp expression
has also been suggested in the early pathogenesis of alzheimer s disease ( ad ) . at later disease stages ,
p - gp in the brain could thus form a compensatory mechanism to increase clearance of neurotoxic substances that play a role in neurodegenerative diseases .
jakob disease ( cjd ) , where decreased p - gp expression was suggested to facilitate the accumulation of prp prions ( vogelgesang et al .
whether p - gp also transports -synuclein , a major constituent of lewy bodies that causes neurodegeneration in pd when aggregated , has so far not been investigated .
furthermore , it is known that p - gp contributes to the cellular efflux of some pesticides ( bain and leblanc 1996 ) and environmental toxins influence pd onset ( rajput , 2001 ) . however , decreased p - gp function was not seen in de novo pd patients , indicating that impaired bbb p - gp efflux function does not play a primary role in the initiation of pathology in idiopathic pd .
the findings of our study could have implications in the therapeutic options in pd and other proteopathies .
p - gp activity can be modulated by a variety of substances , but most studies focus on p - gp inhibition , e.g. , in the prevention of multidrug resistance in oncological treatments ( tan et al .
. however , p - gp activity can also be up regulated , for example by st johns worth or rifampicin ( fromm 2000 ; hennessy et al . 2002 ) .
selectively increasing p - gp mediated bbb efflux function would provide a possible novel therapeutic means to prevent further accumulation of insoluble toxic compounds or proteins leading to ongoing neurodegeneration .
we conclude that cerebrovascular p - gp function is regionally decreased with advanced stages in several parkinsonian neurodegenerative diseases , implying decreased regulatory and protective function of the bbb with ongoing neurodegeneration .
 decreased blood  brain barrier ( bbb ) efflux function of the p - glycoprotein ( p - gp ) transport system could facilitate the accumulation of toxic compounds in the brain , increasing the risk of neurodegenerative pathology such as parkinson s disease ( pd ) . 
 this study investigated in vivo bbb p - gp function in patients with parkinsonian neurodegenerative syndromes , using [ 11c]-verapamil pet in pd , psp and msa patients . 
 regional differences in distribution volume were studied using spm with higher uptake interpreted as reduced p - gp function . 
 advanced pd patients and psp patients had increased [ 11c]-verapamil uptake in frontal white matter regions compared to controls ; while de novo pd patients showed lower uptake in midbrain and frontal regions . 
 psp and msa patients had increased uptake in the basal ganglia . decreased bbb p - gp function seems a late event in neurodegenerative disorders , and could enhance continuous neurodegeneration . 
 lower [ 11c]-verapamil uptake in midbrain and frontal regions of de novo pd patients could indicate a regional up - regulation of p - gp function . 
Introduction
Materials and methods
Results
Discussion