prolidase deficiency ( pd ) ( omim 170100 ) is a rare autosomal recessive disorder whose incidence of 1 - 2:1,000,000 people is probably underestimated .
pd patients have a broad and variable phenotypic spectrum , as well as a diverse clinical onset ( as early as shortly after birth or as late as 22 years of age ) .
the principal clinical feature is dermatological manifestation with chronic , recurrent , slowly healing ulcerations mainly located on the lower legs and feet .
typically , the phenotype includes moderate to severe degree of mental retardation , rarely epileptic seizures , facial dysmorphism , splenomegaly , susceptibility to recurrent respiratory infections , hypotonia , skeletal and vascular anomalies .
the severity of the disease varies widely , but no relationship between genotype and phenotype has been established yet , mainly due to the limited number of mutations described .
prolidase is linked to the metabolism of many biologically important molecules and it has a central role in extracellular matrix ( ecm ) remodelling , being essential for normal physiology including embryonic development , tissue resorption , wound healing , cell migration , and cell differentiation . in particular ,
the ecm structure participating to the last two processes in the brain is the pial basement membrane ( bm ) .
one of its major constituents is laminin , which has adhesive properties anchoring the plasma membrane of cells adjacent to bm .
meningeal cells actively participate in ecm formation and bm remodelling , in fact , impaired meningeal integrity could be the cause of migration defects .
the dark - like mutant mouse ( dal ) , obtained through random mutagenesis ( mouse mutant resource web site , the jackson laboratory , 2008 ) , could be used as animal model for pd , because of a 4bp deletion in the 14 exon of the murine pepd gene .
moreover , mice develop congenital cardiomyocyte hypertrophy due to a significant reduction in the levels of several integrin transducers and , being collagen the major ligand of integrins , it was suggested that prolidase , through its effects on the ecm , influences cardiomyocyte growth . at present , no information are available on the functional correlation between pd and the mental retardation described in these patients .
therefore , this is the first neuromorphological study on postnatal brain development in dal mutant mice , representing a suitable model to study the morphological / molecular causes of brain defects .
mainly two brain regions were analyzed : cerebral and cerebellar cortices , with the challenging goal to find a parallelism between the human phenotype and brain defects we found in pd mouse model . to investigate the general neuroarchitecture organization haematoxylin and eosin staining and map2 immunoreaction
were used ; to evaluate a possible involvement of meningeal bm , immunoreaction for laminin was carried out ( this marker labels the ecm of bm and also wall blood vessels ) .
simultaneously , gfap antibody was used to understand the possible consequences of disorganized bm , since it is an important anchor point for astrocyte end - feet , resulting fundamental during brain development , because gfap - containing fibers provide a migratory scaffold for neurons .
dark - like mice and their wild - type ( wt ) littermates ( c3h / j ) used for this study were provided by prof .
t. gunn ( great falls , mt , usa ) . five animals per genotype , per age were sacrificed at 10 postnatal day ( p10 ) , 21 postnatal day ( p21 ) , 30 postnatal day ( p30 ) , and 60 postnatal day ( p60 ) .
mice were deeply anesthetized with an intraperitoneal injection of 35% chloral hydrate ( 100 l/100 g b.w . ; sigma , st .
the use of mice was approved by the local pavia city hall authorities ( reference no .
the samples were fixed in carnoy s solution ( 6 absolute ethanol/3 chloroform/1 glacial acetic acid ) for 48 h with a change with fresh solution after 1 h , then placed in absolute ethanol , in acetone , and then embedded in paraplast x - tra ( sigma - aldrich , st .
brain sections ( 8 m thick ) were obtained serially in the sagittal plane and collected on silan - coated slides .
paraplast embedded sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series and rinsed in distilled water .
then , sections were immersed for 10 min in carazzi s haematoxylin at room temperature , washed in running tap water for 20 min and immersed in eosin for 2 s. sections were washed in distilled water , dehydrated in ethanol , cleared in xylene , and mounted in eukitt ( kindler , freiburg , germany ) .
slides were observed with an olympus bx51 microscope , and images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
corrections to brightness and contrast were made with paint shop pro 7 ( jasc software inc . ) .
paraplast embedded sections were deparaffinized in xylene , rehydrated in a decreasing ethanol series and rinsed in phosphate - buffered saline ( pbs ; sigma ) .
the endogenous peroxidases were suppressed by incubation of sections with 3% h2o2 in 10% methanol in pbs for 7 min .
localization of map2 , was achieved by applying on brain sections a rabbit polyclonal anti - map2 ( 1:250 santa cruz biotechnology , santa cruz , ca , usa ) in pbs overnight in a dark moist chamber .
thereafter , the sections were sequentially incubated with biotinylated secondary antibodies ( anti - rabbit , 1:200 ; vector laboratories , burlingame , ca , usa ) for 30 min and horseradish peroxidase conjugated avidin - biotin complex ( vector laboratories ) for 30 min at room temperature . then , 0.05% 3,3-diaminobenzidine tetrahydrochloride ( dab ; sigma ) with 0.01% h2o2 in tris - hcl buffer ( 0.05 m , ph 7.6 ) was used as a chromogen . after each reaction step
, sections were washed thoroughly in pbs , then dehydrated in ethanol , cleared in xylene , and mounted in eukitt ( kindler ) .
the slides were observed with an olympus bx51 microscope , and the images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
corrections to brightness and contrast were made with paint shop pro 7 ( jasc software inc ) . for control staining
localization of laminin and gfap was achieved by applying on paraplast - embedded sections a rabbit polyclonal anti - laminin ( 1:100 ; sigma ) and a goat polyclonal anti - gfap ( 1:100 ; santa cruz biotechnology ) in pbs overnight in dark moist chamber .
fluor 488-conjugated anti - rabbit ( 1:100 ; molecular probes , milan , italy ) and alexa fluor 594-conjugated anti - rabbit ( 1:100 ; molecular probes ) .
after washing with pbs , nuclei were counterstained with 0.1 g / ml hoechst 33258 for 6 min and coverslips were lastly mounted in a drop of mowiol ( calbiochem , san diego , ca , usa ) .
slides were observed by fluorescence microscopy with an olympus bx51 equipped with a 100w mercury lamp used under the following conditions : 450 - 480 nm excf , 500 nm dm , and 515 nm bf for alexa 488 and 540 nm excf , 480 nm dm , and 620 nm bf for alexa 594 .
images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
images were optimized for colour , brightness and contrast by using paint shop pro 7 software ( jasc software inc ) . for control staining
paraplast embedded sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series and rinsed in distilled water . then
, sections were immersed for 10 min in carazzi s haematoxylin at room temperature , washed in running tap water for 20 min and immersed in eosin for 2 s. sections were washed in distilled water , dehydrated in ethanol , cleared in xylene , and mounted in eukitt ( kindler , freiburg , germany ) .
slides were observed with an olympus bx51 microscope , and images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
corrections to brightness and contrast were made with paint shop pro 7 ( jasc software inc . ) .
paraplast embedded sections were deparaffinized in xylene , rehydrated in a decreasing ethanol series and rinsed in phosphate - buffered saline ( pbs ; sigma ) .
the endogenous peroxidases were suppressed by incubation of sections with 3% h2o2 in 10% methanol in pbs for 7 min .
localization of map2 , was achieved by applying on brain sections a rabbit polyclonal anti - map2 ( 1:250 santa cruz biotechnology , santa cruz , ca , usa ) in pbs overnight in a dark moist chamber .
thereafter , the sections were sequentially incubated with biotinylated secondary antibodies ( anti - rabbit , 1:200 ; vector laboratories , burlingame , ca , usa ) for 30 min and horseradish peroxidase conjugated avidin - biotin complex ( vector laboratories ) for 30 min at room temperature . then
, 0.05% 3,3-diaminobenzidine tetrahydrochloride ( dab ; sigma ) with 0.01% h2o2 in tris - hcl buffer ( 0.05 m , ph 7.6 ) was used as a chromogen . after each reaction step
, sections were washed thoroughly in pbs , then dehydrated in ethanol , cleared in xylene , and mounted in eukitt ( kindler ) .
the slides were observed with an olympus bx51 microscope , and the images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
corrections to brightness and contrast were made with paint shop pro 7 ( jasc software inc ) . for control staining
localization of laminin and gfap was achieved by applying on paraplast - embedded sections a rabbit polyclonal anti - laminin ( 1:100 ; sigma ) and a goat polyclonal anti - gfap ( 1:100 ; santa cruz biotechnology ) in pbs overnight in dark moist chamber .
fluor 488-conjugated anti - rabbit ( 1:100 ; molecular probes , milan , italy ) and alexa fluor 594-conjugated anti - rabbit ( 1:100 ; molecular probes ) . after washing with pbs
, nuclei were counterstained with 0.1 g / ml hoechst 33258 for 6 min and coverslips were lastly mounted in a drop of mowiol ( calbiochem , san diego , ca , usa ) .
slides were observed by fluorescence microscopy with an olympus bx51 equipped with a 100w mercury lamp used under the following conditions : 450 - 480 nm excf , 500 nm dm , and 515 nm bf for alexa 488 and 540 nm excf , 480 nm dm , and 620 nm bf for alexa 594 .
images were recorded with an olympus camedia c-5050 digital camera and stored on a pc .
images were optimized for colour , brightness and contrast by using paint shop pro 7 software ( jasc software inc ) . for control staining
compared to wt , alterations here described were not strictly age - specific or genotype - specific ( dal/+ and dal / dal ) .
as it is shown , wt mice presented a smooth cerebral cortex ( a , c ) , while the altered phenotype was characterized by undulations usually localized above the hippocampal formation , where the overall organization was compromised ( b , d ) . in correspondence to these anomalies the normal layering seems to be damaged , as well as the dendrite organization and positioning marked by the map2 immunoreaction . in wt , neuron dendrites ran linear and parallel each other ( insert in a ) , while in altered phenotype ( insert in b ) they followed cortex undulations .
the immunopositivity for laminin remarked a completely different profile : bm was continuous in wt ( c and insert ) , and interrupted and thinner in dal/+ and dal / dal ( d and insert ) .
defects in cerebellar cortex were evident regarding lobulation in dal/+ and dal / dal mice ( f , h ) compared to wt ( e , g ) ; protruding cortical portions with a lack of laminin immunopositive bm were also found ( h , arrow ) .
these detachments of molecular layer were accompanied by penetration of large blood vessels ( inserts in f and h , asterisk ) and altered gfap - positive fibers whose endfeet were not visible and did not reach the surface of the cerebellar cortex ( insert in h , arrowhead ) . on the other hand , in wt the meningeal bm appears continuous shearing uniform thickness ( insert in g ) with well structured gfap - positive fibers anchored , through their endfeet , to the laminin - positive bm layer ( insert in g , arrowhead ) .
the spectrum of alterations include also the absence of granule cells ( insert in f , arrow ) or their ectopic position surrounded by molecular layer ( insert in f , arrowhead ) , fusion of cerebellar fissures , and presence of degenerating cells ( not shown ) .
pd is a rare autosomal recessive disorder caused by a mutation in the pepd gene , which results in a broad spectrum of phenotype including mental retardation .
therefore , we focused our attention on brain morphology and possible factors involved in these anomalies .
the presence of vascularisation defects in the cerebral and cerebellar cortices probably is linked to two different causes : i ) embryonic cardiomyocyte hypertrophy in dal mutant mice ; ii ) prolidase role in angiogenic signalling processes .
the cardiomyocyte hypertrophy results , physiologically , in net induction of angiogenesis , and as it is reported by previous studies , this can obstruct the normal migration of cells in the central nervous system ( cns ) , as we also detected . moreover , prolidase is involved in ecm remodelling , which acts as scaffold for cellular support in all tissues and organs , and plays crucial roles during development of the cns in the spatial organization of molecules and cells .
ecm , with its constituents , is also known to contribute to other neuronal functions such as cell proliferation , migration , morphological differentiation , synaptogenesis , synaptic stability , and cell signalling cascades .
the absence of a fully functional prolidase enzyme might result in an altered ecm remodelling leading to meningeal defects , one of the possible causes of the brain development impairments , the loss of pial bm integrity and the consequent disorganized cytoarchitecture in dal mutant mice .
further studies will be directed to understand the peculiar role of prolidase in brain development , and to investigate other meningeal bm components .
we will focus also on the detection of important molecules and cells involved in the proliferation , migration and differentiation pathways during neurogenesis . our experimental findings in dal mutant mice with inherited pd underline the importance of prolidase , being relevant also for understanding pd physiology in humans .
 prolidase gene ( pepd ) encodes prolidase enzyme , which is responsible for hydrolysis of dipeptides containing proline or hydroxypro - line at their c - terminal end . 
 mutations in pepd gene cause , in human , prolidase deficiency ( pd ) , a rare autosomal recessive disorder . 
 pd patients show reduced or absent prolidase activity and a broad spectrum of phenotypic traits including various degrees of mental retardation . 
 this is the first report correlating pd and brain damages using as a model system prolidase deficient mice , the so called dark - like ( dal ) mutant mice . 
 we focused our attention on dal postnatal brain development , revealing a panel of different morphological defects in the cerebral and cerebellar cortices , such as undulations of the cerebral cortex , cell rarefaction , defects in cerebellar cortex lobulation , and blood vessels overgrowth . 
 these anomalies might be ascribed to altered angiogenic process and loss of pial basement membrane integrity . 
 further studies will be directed to find a correlation between neuroarchitecture alterations and functional consequences . 
Introduction
Materials and Methods
Haematoxylin and eosin staining
Immunoperoxidase staining
Immunofluorescence reactions
Results
Discussion