parkinson 's disease ( pd ) is a progressive neurodegenerative disorder affecting 1 - 2% of the elderly population .
in addition to the well - known motor problems of pd patients , that are related to nigrostriatal dopamine deficits , also nonmotor symptoms are common . these symptoms strongly affect quality of life of pd patients as they include autonomic dysfunction , sleep problems , cognitive and neuropsychiatric changes [ 25 ] , all changes unrelated to degeneration of the substantia nigra ( sn ) .
apart from dopaminergic cell loss , the deposition of -synuclein is a prominent neuropathological hallmark of pd . according to the staging concept described by braak
this coincides with the occurrence of nonmotor symptoms and can be observed already in early stages of pd , that is , in incidental lewy body disease ( ilbd ) .
moreover , -synuclein can activate microglial cells , and neuroinflammatory responses are indeed important pathological features of pd [ 810 ] .
accumulation of -synuclein has been reported in the hippocampus ( hc ) , which may contribute to the cognitive and depressive changes that represent prominent nonmotor symptoms in pd [ 11 , 12 ] ( figure 1 ) . of interest ,
the hc is also one of the few brain regions where stem cells reside . in the hippocampal subgranular zone ( sgz ) ,
stem cells undergo proliferation before they migrate through the granular cell layer ( gcl ) , where they eventually become newborn , functional neurons that contribute to network function .
in addition to this unique process of adult neurogenesis , which is largely confined to the dg , stem cells in the hc can proliferate and respond to neurodegenerative conditions .
for instance , overexpression of -synuclein in pd models induces aberrant differentiation of neural progenitors and alters cellular plasticity in the hippocampus [ 1315 ] . also striatal deafferentiation , or the loss of dopaminergic neurons , affects both neurogenesis   and proliferation in the hc .
in addition , several parkinson - related pathogens induce degeneration of human neural stem cells ( nscs )   or reduce neurogenesis .
stimulation of cellular plasticity on the other hand , for example through exercise , antidepressant treatment or high frequency stimulation , reverses impairments in neurogenesis in pd models   and may even benefit pd patients [ 21 , 22 ] .
the discovery that the human brain contains stem cells has instigated extensive research into their proliferative responses during neurodegeneration and into their potential for brain repair .
in related animal models for alzheimer 's disease ( ad ) , changes in cellular plasticity and proliferation appear to depend on the extent of pathology and the severity of the disorder .
regarding pd , stem cells and related cellular plasticity markers are also altered in relevant animal models [ 2426 ] , but only few studies exist on proliferative changes in human pd brain , most of which have focused on the sn [ 27 , 28 ] and subventricular zone ( svz ) [ 29 , 30 ] . in the human sn and svz , discrepant results have been obtained between different studies , and this topic remains subject of debate [ 26 , 29 , 3134 ] . thus far , little attention has been paid to cellular proliferative changes in the human hc , a brain region relevant for nonmotor symptoms in pd , like cognitive and depressive changes . moreover , besides proliferation of nscs , glial cells within the cns may also proliferate in response to neuropathological changes .
indeed , in the ad hippocampus or in experimental multiple sclerosis , for example , proliferation of cns resident microglia has been observed [ 35 , 36 ] .
here , we first studied proliferative changes in prodromal and established cases of pd and , given the differential -synuclein deposition , we include both the ca ( nonneurogenic ) and the dg ( neurogenic ) subregions .
as activated microglial cells are present in the hc in pd and implicated in pd pathogenesis [ 9 , 37 ] , we also questioned whether microglial cells proliferate in these conditions .
we selected hippocampal tissue of well - established and neuropathologically confirmed pd patients ( braak pd stages 46 ) , matched control subjects ( braak pd stage 0 ) , and incidental lewy body disease ( ilbd ) cases ( braak pd stages 13 ) ( figure 1 ) . as these latter cases lacked clinical symptoms of pd during their lives and did not receive dopaminergic medication , but displayed a neuropathological braak -synuclein deposition at autopsy , they can be considered a presymptomatic and prodromal state of pd . to assess cell proliferation
, we used the well validated marker minichromosome maintenance protein 2 ( mcm2 ) [ 38 , 39 ] and colabeled it with the microglial marker iba1 to investigate to what extent cell proliferation in the hc is accounted for by microglia .
human postmortem hippocampal tissue was obtained from the netherlands brain bank ( nbb , amsterdam , the netherlands ) . in compliance with all local ethical and legal guidelines , informed consent for brain autopsy and the use of brain tissue and clinical information for scientific research
fourteen clinically diagnosed and neuropathologically verified pd patients ( braak pd stages 46 ) were selected as well as six clinically healthy controls without neurological or psychiatric disease , that displayed some pd -synuclein pathology at autopsy ( braak pd stages 13 ) and were classified as ilbd cases . for the control group , nine healthy subjects without neurological or psychiatric disease and without any -synuclein pathology ( braak pd stage 0 ) were included .
the three groups were matched for gender and age ; age of the control subjects ranged from 62 to 92 years , in the ilbd cases from 56 to 91 , and in the pd patients from 59 to 96 .
also braak neurofibrillary tangles ( nft ) and amyloid - beta ( a ) plaques scores were matched between control subjects , ilbd cases , and pd patients , ruling out any possible difference in proliferation due to differences in ad pathology .
the clinicopathological data of the patients and the braak staging for pd and ad of all donors is summarized in table 1 .
braak scores for ad and pd neuropathology were provided by the nbb and had been obtained after careful neuropathological evaluation of disease - relevant brain areas by experienced neuropathologists .
the density and distribution of lbs / lns , nft , and a plaques were determined using classic bodian staining and immunohistochemistry for -synuclein ( clone km51 , novacastra , bioconnect bv ) and hyperphosphorylated tau ( clone at8 , pierce , rockford , il ) and a ( clone 6f/3d , dako , dakocytomation bv ) , respectively . at autopsy ,
brain regions were dissected and immersion - fixed in 4% formaldehyde for four weeks , after which they were embedded in paraffin . from the paraffin blocks that contained the hc ,
10-micrometer ( m ) sections were cut on a microtome , mounted , and dried in a stove overnight at 37c before immunohistochemical analysis .
sections were mounted on positively charged glass slides ( menzel - glaser superfrost plus , braunschweig , germany ) .
various markers are available to identify cell proliferation or specific phases of the cell cycle in human postmortem brain tissue [ 3942 ] . of these ,
the minichromosome maintenance protein 2 ( mcm2 ) is involved in the control of dna replication and commonly used in cancer research as a reliable marker for detecting dividing and slowly cycling putative stem cells in situ [ 38 , 43 , 44 ] .
mcm2 expression starts in early g1 and is maintained throughout the cell cycle , also in cells that proliferate without actually synthesizing dna , and is thus present in higher numbers than , for example , the short - lived proliferation marker ki-67 [ 38 , 45 , 46 ] . moreover , the majority of the cells that express mcm2 coexpress the immature neuronal marker doublecortin   and mcm2 was therefore also used to study cellular plasticity in comparable tissues .
various tests to validate and confirm specificity of mcm2 and related markers have been performed by us and others before [ 38 , 46 , 47 ] , for example , on samples of young rat brain and human colon , that were processed and embedded in the exact same way as the human brain tissue used in the current study [ 39 , 41 ] .
sections were heated in a stove for one hour at 56c , before they were deparaffinized in xylene and rehydrated through a graded series of ethanol ( 100% , 96% , 90% , and 70% , resp . ) and tbs . for subsequent antigen retrieval
, sections were rinsed in 10  mm tris buffer ( ph 9.0 ) containing 1  mm edta ( tris - edta ) and afterwards placed in preheated tris - edta buffer in a steamer at 9099c for 30 minutes .
after pretreatment , the sections were allowed to regain room temperature ( rt ) , rinsed in tris - buffered saline ( tbs , ph 7.6 ) , and incubated for 20  min in tbs containing 0.3% h2o2 and 0.1% sodium azide to block endogenous peroxidase activity .
nonspecific binding was blocked with 5% nonfat dried milk in tbs containing 0.5% triton ( tbs - t , ph 7.6 ; blocking solution ) for 30  min at rt .
subsequently , sections were incubated overnight at 4c with mouse anti - mcm2 ( bm28 , bd transduction lab 610700 , mouse 1:600 ) .
sections were then washed in tbs and incubated for 2  hr at rt in biotinylated goat anti - mouse iggs ( 1:400 ; jackson immunoresearch laboratories inc . , west grove , pa , usa ) followed by hrp - labeled avidin - biotin complex ( abc complex , 1:400 ; vector laboratories , burlingame , ca , usa ) in tbs - t for 1  hr at rt .
after dehydration in graded ethanol solutions , sections were cleared in xylene and coverslipped in entellan ( merck ) . for double - immunofluorescent labeling of microglial cells and mcm2 expression , iba1 ( wako chemicals , 019 - 19741 , rabbit , 1:300 ) and mcm2 ( 1:1000 ) were used .
sections were pretreated with tris - edta ( ph 9.0 ) and primary antibodies were diluted in the blocking solution , as indicated above .
after an overnight incubation at 4c , the sections were washed and subsequently incubated for 2  h at rt with donkey anti - rabbit alexa fluor 488 labeled donkey anti - rabbit iggs ( iba1 ) and biotinylated goat anti - mouse iggs ( mcm2 ) ( 1:400 , jackson immunoresearch , west grove , pa , usa ) , followed by abc complex ( 1:800 ) for 1  h and biotinylated tyramide enhancement in tbs for 20  min .
hereafter , sections were incubated with alexa fluor 594 labeled streptavidin ( 1:800 , jackson immunoresearch , west grove , pa , usa ) .
after washing , sections were coverslipped with vectashield and examined using a confocal microscope ( leica tsc - sp2-aobs ; leica microsystems , wetzlar , germany ) . for standardization purposes ,
hippocampal sections were collected only around the anterior to midlevel of the hc of every subject and only when large dentate gyrus ( dg ) and cornu ammonis ( ca ) subregions were both present .
furthermore , mcm2-positive cells were included in the subsequent analysis only when they displayed a clearly immature , mitotic , and/or migratory morphology , and were located in an isolated manner ( figure 2 ) .
semiquantification was performed by assessing the numbers of mcm2-positive cells present in the five main hc grey matter regions , that is dg , ca4 , ca3 , ca2 , and ca1 , at a 10x magnification and then expressed per surface area ( region of interest , roi , 1  mm ) using cell olympus soft imaging solutions gmbh software ( tokyo , japan ) .
statistical analyses were performed using the spps package version 20.0 ( statistical product and service solutions , chicago , il , usa ) .
the mean and standard deviation of the number of mcm2-positive cells were calculated for each group and within each subject for all the different subregions of the hc .
the data did not meet criteria for normal distribution ; thus statistical analyses were performed using nonparametric tests .
statistical analysis was executed with the nonparametric kruskal - wallis test to examine main group effects between controls , ilbd , and pd patients .
results were considered significant if they fell below an alpha of p  0.016 , after bonferroni correction . within the same cases , statistical analyses for the different subregions of the hc
were performed with the nonparametric related samples friedman 's analysis to examine main region effects , followed by paired wilcoxon tests as post hoc tests .
results were considered significant if they fell below an alpha of p  0.02 , family wise error- ( fwe- ) corrected .
this critical value was established with sisa ( http://www.quantitativeskills.com/sisa/calculations/bonhlp.htm ) , which uses the mean correlation between variables ( r = 0.65 ) that are mutually correlated ( i.e. , number of mcm2-positive cells ) for the alpha correction and allows one to perform a less stringent correction than the bonferroni method for multiple comparisons .
in the pyramidal layer and dg of the hc of all three patient groups , generally low numbers of mcm2-positive cells were found that displayed morphology typical for proliferating cells , such as a doublet shape and small size ( figure 2 and figures 5(b ) , 5(e ) , and 5(h ) ) . except for a significant main effect in the number of mcm2-positive cells between the hippocampal subregions within the control group ( figure 3(a ) ; p = 0.034 , related samples friedman 's analysis ) , no significant differences were found between the subregions within each of the 2 different patient groups ( figures 3(b ) and 3(c ) ) .
further analysis revealed a trend towards an increase in the dg compared to the ca3 , ca4 , and ca1 within the control group ( figure 3(a ) ; resp .
, p = 0.043 , p = 0.043 , and p = 0.08 ; nonparametric mann - whitney u test , significance reached at alpha of p  0.02 , fwe - corrected ; ctr dg mean = 2.45  1.03 ; ctr ca3 mean = 0.31  0.21 ; ctr ca4 mean = 0.35  0.25 ; ctr ca1 mean = 0.93  0.56 ) . taking all data together revealed a significant increase in the number of mcm2-positive cells in the total hippocampal grey matter ( ca1-ca4 , dg combined ) in the ilbd cases compared to control subjects ( figure 4(a ) ; p = 0.004 ; nonparametric mann - whitney u test , significance reached at alpha p  0.016 , bonferroni - corrected ; ctr mean = 5.21  2.23 ; ilbd mean = 45.24  25.29 ; pd mean = 26.51  9.25 ) .
when analyzing the subregions separately , a significant main group effect was found for ca3 and ca4 between control subjects , ilbd cases , and pd patients ( resp .
, p = 0.006 , p = 0.16 ; nonparametric independent samples kruskal - wallis analysis ) .
further analysis revealed there was a significant increase in the number of mcm2-positive cells in the ca3 and ca4 of the ilbd cases compared to the ca3 and ca4 of control subjects ( figures 4(d ) and 4(e ) ; resp .
, p = 0.001 , p = 0.003 ; nonparametric mann - whitney u test , significance reached at alpha p  0.016 , bonferroni - corrected ; ctr ca3 mean = 0.31  0.21 ; ctr ca4 mean = 0.35  0.25 ; ilbd ca3 mean = 13.05  8.77 ; ilbd ca4 mean = 4.95  1.67 ) and a trend towards an increase in the numbers of mcm2-positive cells in the other three hippocampal subregions of the ilbd cases compared to the control subjects ( figures 4(b ) , 4(c ) , and 4(f ) ; ca1 p = 0.097 ; ca2 p = 0.082 ; dg p = 0.191 ; ctr ca1 mean = 0.93  0.56 ; ctr ca2 mean = 1.17  0.62 ; ctr dg mean = 2.45  1.03 ; ilbd ca1 mean = 5.88  3.17 ; ilbd
ca2 mean = 14.82  9.58 ; ilbd dg mean = 6.45  4.04 ) .
no significant differences were found between pd patients and control or ilbd cases in total hc or within any of the subregions ( figure 4 ) . to determine
whether microglia are proliferating , double immunofluorescence and confocal microscopical analysis revealed that the majority of mcm2-positive cells colocalized with iba1-positive microglia ( representative examples are shown in figure 5 ) .
of each patient group , 3 cases were double stained for iba1 and mcm2 and the percentage coexpression was determined . in the control subjects ,
mcm2-positive cells were rare in the ca regions but always colocalized with iba1 ( 100% ) .
in ilbd cases , the highest number of mcm2-positive cells was present in the ca subregions .
an average of 14 positive mcm2 cells were counted , of which an average of 13 were iba1 positive , yielding 93% coexpression . in pd patients ,
an average of 6 positive mcm2 cells were counted , of which on average 5 were iba1 positive , that is , 83% . in the dg , overall less mcm2-positive cells were counted ; however , the percentages of colocalization with iba1 were similar to ca regions .
in general , quantification revealed > 90% of the mcm2-positive cells in the hc to be microglia in control subjects , ilbd cases , and pd patients ( representative examples are shown in figures 5 and 6 ) .
we studied proliferating cells in the hc of control subjects , ilbd cases , and established pd patients . using mcm2 as marker
, no difference was observed in the amount of proliferating cells between control subjects and pd patients .
however , in the presymptomatic ilbd cases , a clear increase in mcm2-positive cells was found .
interestingly , over 90% of the mcm2-positive cells were colabeled with iba1 , indicating that microglial cells are the main proliferating cells in the hc of ilbd cases and pd patients . in control subjects ,
the low number of mcm2-positive proliferating cells observed in the hc is consistent with the low rates of hippocampal proliferation and neurogenesis found before in the aged rodent , primate , and human hc and in comparable pd brain tissue [ 30 , 41 , 4851 ] . within the control group ,
mcm2-positive cells were increased in the dg compared to other hippocampal regions ( figure 3(a ) ) , which was of interest as most proliferation was beforehand expected to occur in this subregion .
interestingly , the differences between the dg and other subregions were no longer present in the ilbd and pd groups , with higher numbers of proliferating cells in all subregions ( figures 3(b ) and 3(c ) ) , suggesting that the disease process has triggered additional proliferative responses also outside the dg , and in fact throughout the hc . when total counts in the combined hc were compared between the three cohorts , significant increases in proliferation were found in the ilbd cases , but no difference was present between the pd group relative to the control group ( figure 4(a ) ) .
this was unexpected as , based on experimental studies , lowered dopamine levels were expected to reduce hippocampal proliferation .
also the increase in ilbd cases was unexpected as no clinical symptoms were present in this cohort ( yet ) and although not quantified , their dopamine levels are assumed to be unaltered . however , there is also some dat - spect data that reflects a decrease of striatal dopamine levels and nigral degeneration already in the premotor stage of pd [ 5254 ] .
whether these cases can be readily compared to our ilbd cases remains to be answered .
subdividing the different hc subregions revealed that the significant increase in the entire hc in ilbd was mainly due to the ca3 and ca4 and to a lesser extent to the ca2 subregion ( figures 4(c ) , 4(d ) , and 4(e ) ) .
since ilbd can be considered a presymptomatic state of pd , this result suggests that an early and disease - related induction of proliferation occurs in the hc .
 neurons that attempt to reenter the cell cycle , there are no indications that proliferation in the ca subregions will actually give rise to new neurons .
also the current morphology , consistent with a proliferative phenotype , and the localization of the mcm2 cells , that is , closely apposed to a pyramidal neuron and thus suggestive of a  satellite  cell ( figure 2 , arrowhead ) , hinted that proliferation in nonneurogenic subregions likely reflects that of a nonneuronal cell type , for example , microglia . to confirm this , we used immunofluorescent colabeling of mcm2 with iba1 and established that almost all proliferating cells in the pd hc represent microglia .
the increase in ilbd cases relative to control subjects and pd patients suggests that proliferation of microglia occurs early in pd , prior to actual deposition of -synuclein . in most pd cases , neuronal populations in the hc show
accumulation of -synuclein [ 56 , 57 ] which may activate the brain 's immune system through microglia activation .
it is still debatable whether neuronal -synuclein inclusions ( lewy bodies / lewy neurites ) cause microglial activation and/or neuronal death , or whether lbs / lns act as protective 
containers  and that it is the extracellular -synuclein oligomers and fibrils that are toxic and activate microglia . on
the other hand , neurons containing -synuclein inclusions could communicate with microglia and activate them by neuron to glia signaling . indeed ,
while different microglial phenotypes are present in the sn , hc , and ob in pd , their functional role and hence the implications of microglia proliferation for pd etiology remain elusive [ 37 , 5961 ] .
transgenic mice overexpressing wild - type -synuclein develop -synuclein inclusions shortly after birth but show unchanged cell proliferation in their svz and hc at later ages [ 14 , 62 , 63 ] .
in related prion disease models , proliferation of microglial cells is considered important in their turnover [ 64 , 65 ] and to a larger extent to account for the expansion of the resident microglia population during , for example , prion disease development . a related study on ad mouse models found changes in proliferation and microglia to coincide in time with increases in amyloid plaque load , suggesting that the accumulation of aberrant proteins like amyloid , and possibly also of -synuclein , may trigger microglial proliferation . while in vitro studies
had already demonstrated that the aggregated form of -synuclein can trigger microglial activation [ 10 , 68 ] , the hc of ilbd cases is largely devoid of -synuclein depositions , and a role for soluble forms or -synuclein oligomers can thus not be excluded .
-synuclein oligomers can , for example , activate microglia via toll - like receptor-2 ( tlr2 ) and thereby stimulate proinflammatory cytokine production [ 69 , 70 ] .
in agreement , in a separate study , we found tlr2 expression to be upregulated in microglia in the hc of ilbd cases .
in addition to increasing the cell population , an alternative , more speculative , interpretation of proliferation in microglia could be phagocytosis of -synuclein .
the release of -synuclein and the subsequent uptake by neighboring neurons or glia suggest possibilities for cell - to - cell transfer and propagation that was recently proposed as conceptual model for proteinopathies [ 71 , 72 ] and is consistent with the spatiotemporal progression of pd neuropathology over the brain .
interestingly , neuron - to - glia transmission is accompanied by microglia activation [ 26 , 74 ] and could thus underlie some of the proliferative changes observed in microglia in our current study .
for instance , -synuclein secreted by neurons is released into the extracellular space and can be taken up by microglia and phagocytosed .
several forms of -synuclein have been found to induce microglial activation [ 10 , 75 , 76 ] .
similar to amyloid - beta , such secreted proteins can be  sensed  by microglia through toll - like receptors that could lead to the activation of inflammatory response genes and their proliferation [ 61 , 69 , 71 , 7779 ] .
another possibility is that proliferative changes reflect altered calcium signaling that was , for example , found to be dysregulated in microglia in close vicinity to amyloid plaques in ad brain .
these options are still speculative , and ongoing phagocytosis of -synuclein inside microglia is technically difficult to visualize in postmortem human brain but may provide a possible explanation for the microglial proliferation we observed .
our current hippocampal data are in contrast with a previous study on proliferation in the svz in pd and pd models , indicating that cell proliferation in the svz , and possibly also in the dg , may be under dopaminergic control [ 16 , 29 , 81 , 82 ] .
these authors used nestin and beta tubulin as markers and found significant reductions in proliferation in the hc of 3 pd patients and 5 pd patients with dementia , which they compared to 3 controls .
although different markers were used , we did not find any indication for a reduction in proliferation in the hc our cohorts , together comprising 20 patients and 9 controls .
another study on the svz failed to find differences in the proliferative capacity between controls and pd patients too . while followed by an interesting debate [ 32 , 33 ] , this also suggested that proliferating progenitor cells are at least not reduced in pd .
the methodological limitations that exist for visualization of neuronal proliferation in human postmortem brain [ 8388 ] do not seem to hold explicitly for microglia proliferation .
mcm2 can thus be used as a marker to determine also nonneuronal cell proliferation in human postmortem material . in conclusion ,
the increase in numbers of proliferating cells in the hc of ilbd cases , a prodromal state of pd , but not in clinically established pd patients , indicates an early response to developing pathology in the pd hc . as almost all of the proliferating cells in the hc
are microglia , this implies that neuroinflammatory processes may play an important role in ongoing pd pathology .
 besides dopamine - deficiency related motor symptoms , nonmotor symptoms , including cognitive changes occur in parkinson 's disease ( pd ) patients , that may relate to accumulation of -synuclein in the hippocampus ( hc ) . 
 this brain region also contains stem cells that can proliferate . 
 this is a well - regulated process that can , for example , be altered by neurodegenerative conditions . 
 in contrast to proliferation in the substantia nigra and subventricular zone , little is known about the hc in pd . 
 in addition , glial cells contribute to neurodegenerative processes and may proliferate in response to pd pathology . in the present study , we questioned whether microglial cells proliferate in the hc of established pd patients versus control subjects or incidental lewy body disease ( ilbd ) cases as a prodromal state of pd . 
 to this end , proliferation was assessed using the immunocytochemical marker minichromosome maintenance protein 2 ( mcm2 ) . 
 colocalization with iba1 was performed to determine microglial proliferation . 
 mcm2-positive cells were present in the hc of controls and were significantly increased in the presymptomatic ilbd cases , but not in established pd patients . 
 microglia represented the majority of the proliferating cells in the hc . 
 this suggests an early microglial response to developing pd pathology in the hc and further indicates that neuroinflammatory processes play an important role in the development of pd pathology . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion