Correction to: Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

The original version of the article unfortunately contained an error in the unit of the protein concentrations under 'Stereotactic Intraparenchymal Injections' subsection in 'Methods' section.

Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

Pigment epithelium-derived factor (PEDF) is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. In the brain, blood-brain barrier (BBB) function is essential for homeostasis. Its impairment plays a crucial role in the pathophysiology of many neurological diseases, including ischemic stroke. We investigated (a) whether PEDF counteracted vascular endothelial growth factor (VEGF)-induced BBB disruption in the mouse brain, (b) the time course and route of BBB permeability and the dynamics of PEDF expression after cerebral ischemia, and (c) whether intraventricular infusion of PEDF ameliorated brain ischemia by reducing BBB impairment. C57Bl6/N mice received intraparenchymal injections of CSF, VEGF, or a combination of VEGF and PEDF. PEDF increased paracellular but not transcellular BBB integrity as indicated by an increase in the tight junction protein claudin-5. In another group of mice undergoing 60-min middle cerebral artery occlusion (MCAO), transcellular BBB permeability (fibrinogen staining in the absence of a loss of claudin-5) increased as early as 6 h after reperfusion. PEDF immunofluorescence increased at 24 h, which paralleled with a decreased paracellular BBB permeability (claudin-5). PEDF after MCAO originated from the blood stream and endogenous pericytes. In the third experiment, the intraventricular infusion of PEDF decreased edema and cell death after MCAO, potentially mediated by the improvement of the paracellular route of BBB permeability (claudin-5) in the absence of an amelioration of Evans Blue extravasation. Together, our data suggest that PEDF improves BBB function after cerebral ischemia by affecting the paracellular but not the transcellular route. However, further quantitative data of the different routes of BBB permeability will be required to validate our findings.

Re-thinking the Etiological Framework of Neurodegeneration.

Neurodegenerative diseases are among the leading causes of disability and death worldwide. The disease-related socioeconomic burden is expected to increase with the steadily increasing life expectancy. In spite of decades of clinical and basic research, most strategies designed to manage degenerative brain diseases are palliative. This is not surprising as neurodegeneration progresses "silently" for decades before symptoms are noticed. Importantly, conceptual models with heuristic value used to study neurodegeneration have been constructed retrospectively, based on signs and symptoms already present in affected patients; a circumstance that may confound causes and consequences. Hence, innovative, paradigm-shifting views of the etiology of these diseases are necessary to enable their timely prevention and treatment. Here, we outline four alternative views, not mutually exclusive, on different etiological paths toward neurodegeneration. First, we propose neurodegeneration as being a secondary outcome of a primary cardiovascular cause with vascular pathology disrupting the vital homeostatic interactions between the vasculature and the brain, resulting in cognitive impairment, dementia, and cerebrovascular events such as stroke. Second, we suggest that the persistence of senescent cells in neuronal circuits may favor, together with systemic metabolic diseases, neurodegeneration to occur. Third, we argue that neurodegeneration may start in response to altered body and brain trophic interactions established via the hardwire that connects peripheral targets with central neuronal structures or by means of extracellular vesicle (EV)-mediated communication. Lastly, we elaborate on how lifespan body dysbiosis may be linked to the origin of neurodegeneration. We highlight the existence of bacterial products that modulate the gut-brain axis causing neuroinflammation and neuronal dysfunction. As a concluding section, we end by recommending research avenues to investigate these etiological paths in the future. We think that this requires an integrated, interdisciplinary conceptual research approach based on the investigation of the multimodal aspects of physiology and pathophysiology. It involves utilizing proper conceptual models, experimental animal units, and identifying currently unused opportunities derived from human data. Overall, the proposed etiological paths and experimental recommendations will be important guidelines for future cross-discipline research to overcome the translational roadblock and to develop causative treatments for neurodegenerative diseases.

Alzheimer's disease: Elevated pigment epithelium-derived factor in the cerebrospinal fluid is mostly of systemic origin.

Pigment-epithelium derived factor (PEDF) is a neurotrophic factor with neuroprotective, anti-tumorigenic, and anti-angiogenic effects. Elevated levels of PEDF have previously been proposed as a cerebrospinal fluid (CSF) biomarker for Alzheimer's disease. However, the origin of PEDF in CSF, i.e. whether it is derived from the brain or from the systemic circulation, and the specificity of this finding hitherto remained unclear. Here, we analyzed levels of PEDF in paired CSF and serum samples by ELISA in patients with Alzheimer's disease (AD, n=12), frontotemporal dementia (FTD, n=6), vascular dementia (n=4), bacterial meningitis (n=8), multiple sclerosis (n=32), pseudotumor cerebri (n=36), and diverse non-inflammatory neurological diseases (n=19). We established CSF/serum quotient diagrams to determine the fraction of intrathecally synthesized PEDF in CSF. We found that PEDF is significantly increased in CSF of patients with AD, FTD, and bacterial meningitis. Remarkably, PEDF concentrations were also significantly elevated in serum of patients with AD. CSF/serum quotient diagrams demonstrated that elevated PEDF concentrations in CSF of patients with AD are mostly due to elevated PEDF concentrations in serum. These findings underscore the importance of relating concentrations of proteins in CSF to their respective concentrations in serum to avoid erroneous interpretations of increased protein concentrations in lumbar CSF.

Effects of pigment epithelium-derived factor on traumatic brain injury.

PURPOSE: Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, anti-inflammatory, neurotrophic and neurogenic properties. The effect of PEDF on traumatic brain injury (TBI) has not been explored. In this study, we aimed to show the in vivo effects of PEDF on lesion volume, cell death and cell proliferation after TBI. METHODS: Rats were subjected to controlled cortical impact injury (CCII). PEDF mRNA brain levels were measured by RT-PCR. The lesion volume, cell proliferation, cell death and microglia activation were assessed in the brains of lesioned animals with intraventricular alzet infusion of PEDF or aCSF, and intraperitoneal injections of BrdU. RESULTS: We detected a significant increase of PEDF mRNA levels after TBI. PEDF intraventricular infusion showed no significant effect on the contusion volume, whereas the number of dead cells, activated microglia, BrdU-positive cells around the lesion were significantly decreased. In contrast, PEDF application increased cell proliferation in the ipsilateral subventricular zone. No effect was found on cell proliferation in the dentate gyrus. CONCLUSION: The present work indicates that PEDF acts as a multifunctional agent after TBI influencing cell death, inflammation and cell proliferation.

Influence of pigment epithelium-derived factor on outcome after striatal cerebral ischemia in the mouse.

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.

Effects of pigment epithelium derived factor (PEDF) on malignant peripheral nerve sheath tumours (MPNSTs).

Neurofibromatosis type 1 (NF1) is an inherited genetic disease affecting 1 in 3,500 individuals. A prominent feature of NF1 is the formation of benign tumours of the peripheral nerve sheath (neurofibromas). However, these can become malignant and form highly metastatic malignant peripheral nerve sheath tumours (MPNST), which are usually fatal despite aggressive surgery, chemotherapy, and radiotherapy. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis in several kinds of tumours. The present study was designed to determine the in vitro and in vivo effects of PEDF on MPNST angiogenesis and tumour growth. PEDF inhibited proliferation and augmented apoptosis in S462 MPNST cells after 48 h of treatment in culture. In xenografts of S462 MPNST cells in athymic nude mice, PEDF suppressed MPNST tumour burden, due mainly to inhibition of angiogenesis. These results demonstrate for the first time inhibitory effects of PEDF on the growth of human MPNST via induction of anti-angiogenesis and apoptosis. Our results suggest that PEDF could be a novel approach for future therapeutic purposes against MPNST.

Protein expression of pigment-epithelium-derived factor in rat cochlea.

Pigment-epithelium-derived factor (PEDF) is a 50-kDa glycoprotein with well-recognised expression in various mammalian organs showing diverse (e.g. anti-angiogenic and neuroprotective) activities. However, at present, no information is available regarding the potential function of this cytokine in the inner ear. As a first approach to investigating whether PEDF is involved in cochlear function, we have explored its protein expression in the rat cochlea by immunocytochemistry. Our results show that PEDF expression in the cochlea is most prominent in the basilar membrane below the organ of Corti, in the lateral wall (especially in the stria vascularis), in ganglion neurons, and in the endothelia of blood vessels. Our findings on its distribution in the cochlea suggest that PEDF in the basilar membrane prevents blood vessel formation that would disturb cochlear micromechanics and would interfere with the mechano-electrical transduction in the organ of Corti. In cochlear ganglion neurons, PEDF might serve a neuroprotective function possibly protecting these neurons from excessive glutamate released by the inner hair cells. Our data constitute the first report on the morphological protein distribution of this multifunctional molecule in the rat cochlea and suggest its role in important functions of the internal ear.

The constitutively active orphan G-protein-coupled receptor GPR39 protects from cell death by increasing secretion of pigment epithelium-derived growth factor.

GPR39 is a constitutively active orphan G-protein-coupled receptor capable of increasing serum response element-mediated transcription. We found GPR39 to be up-regulated in a hippocampal cell line resistant against diverse stimulators of cell death and show that its overexpression protects against oxidative and endoplasmic reticulum stress, as well as against direct activation of the caspase cascade by Bax overexpression. In contrast, silencing GPR39 rendered cells more susceptible to cell death. An array analysis of transcripts induced by GPR39 revealed up-regulation of RGS16 (inhibitor of G-protein signaling 16), which suggested coupling to Galpha(13) and induction of serum response element-mediated transcription by the small GTPase RhoA. In line with this, co-expression of GPR39 with RGS16, dominant-negative RhoA, or serum response factor abolished cell protection, whereas overexpression of the serum response factor protected from cell death. Further downstream the signaling cascade, GPR39 overexpression leads to increased secretion of the cytoprotective pigment epithelium-derived growth factor (PEDF). Medium conditioned by cells overexpressing GPR39 contained 4-fold more PEDF, and when stripped off it lost most but not all of its protective properties. We conclude that GPR39 is a novel inhibitor of cell death, which might represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress like cancer, ischemia/reperfusion injury, and neurodegenerative disease.

Expression of pigment-epithelium-derived factor during kidney development and aging.

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development.

ICAM-1 and VCAM-1 expression following aneurysmal subarachnoid hemorrhage and their possible role in the pathophysiology of subsequent ischemic deficits.

BACKGROUND: The pathophysiology of ischemic cerebral lesions following aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. There is growing evidence that inflammatory reactions could be involved in the pathogenesis of such delayed occurring ischemic lesions. The aim of this study was to evaluate adhesion molecules with regard to these lesions following SAH. METHODS: Serum and cerebrospinal fluid (CSF) samples were taken daily from 15 patients up to day 9 after SAH and evaluated for intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). RESULTS: CSF and serum samples correlated well during nearly the whole time course (p < 0.0001). A secondary increase in ICAM-1 and VCAM-1 in the serum and CSF correlated with an increase in flow velocity in the transcranial Doppler (p > 0.0001 and p < 0.007) but not to a delayed lesion in the CT scan. CONCLUSION: We believe that inflammatory processes are involved in the pathogenesis of cerebral vasospasm but they might only be a part of a multifactorial pathogenesis.

Possible role of the C-reactive protein and white blood cell count in the pathogenesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage.

The delayed ischemic neurologic deficit (DIND) is a common and potentially devastating complication in patients who have sustained subarachnoid hemorrhage (SAH). Recent evidence suggests that various constituents of the inflammatory response may be critical in the pathogenesis of this ischemic complication. The aim of this study was to evaluate the possible relationship between the C-reactive protein (CRP)/white blood cell (WBC) count and DIND. A total of 88 patients with acute SAH were included. CRP and WBC count were estimated on a daily basis. Outcome was evaluated 1 year after the initial ictus according to the Glasgow Outcome Scale. CRP levels on days 5, 6, 7, and 8 were statistically significantly higher in the group of patients developing a DIND (P < 0.025, P < 0.016, P < 0.011, P < 0.0002). WBC counts were higher in this patient group on days 1, 4, 5, 6, and 7 (P < 0.0253, P < 0.0087, P < 0.00167, P < 0.0026, P < 0.0045). Overall CRP values were higher with increasing severity of the initial ictus according to the Hunt and Hess Scale and to the outcome according to the Glasgow Outcome Scale from day 3 on. A statistically significant relationship between WBCs and outcome could not be observed. The presented data do not prove that WBCs and CRP values have a direct contribution to the pathogenesis of ischemic complications following SAH, but it supports the assertion that inflammation may present a common pathogenic pathway in the development of such complications.

Multilayered retinal microglial response to optic nerve transection in rats.

PURPOSE: Microglia normally exist in several layers across the retinal thickness. When retinal ganglion cells undergo apoptosis after lesion to their axons, microglial cells proliferate and promptly clear the debris. We have previously reported on the phagocytic response following optic nerve axotomy. Here, we present how microglial cells of deeper retinal layers are affected by transection of the optic nerve. METHODS: Normal and reactive microglia in the retina of adult rats whose optic nerves had been lesioned were labeled by using antibodies OX42 and ED1. Analysis of the time course (between 1 and 180 days post-axotomy) of appearance and distribution of microglial cells in the retinal layers was performed. RESULTS: In normal retinas, microglia were found in the ganglion cell layer (GCL), the superficial inner nuclear layer (INL) and the outer plexiform layer (OPL). Increases in numbers of microglia occurred maximally in the GCL at day 12 post-axotomy. Increases were also detected in the superficial INL. The proliferation of these phagocytic cells led to their highest numbers in the more central eccentricities in the two most superficial layers. Microglia in the OPL remained undisturbed. Microglial normal histology is restored over a period of 6 months as dying ganglion cells disappear. CONCLUSIONS: Histological characteristics of normal rat retinal microglia are uniform across different retinal eccentricities for each of the three laminae where they exist. Retinal microglia of various layers respond to optic nerve damage. Their increase in numbers and morphological transformation follow retinal ganglion cell death. Their morphology, density, and layered distribution slowly return to normal, confirming that retinal ganglion cells, or their densities, do not play any role in dictating microglial distribution within the different retinal layers.

A missense mutation in GUCY2D acts as a genetic modifier in RPE65-related Leber Congenital Amaurosis.

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous severe retinal dystrophy presenting in infancy. To explain the phenotypical variability observed in two affected siblings of a consanguineous pedigree diagnosed with LCA and establish a genotype-phenotype correlation, we screened GUCY2D, RPE65, CRX, AIPL1, and RPGRIP1 for mutations. The more severely affected sibling carried a heterozygous missense mutation in the GUCY2D gene (Ile539Val), which did not segregate with the disease phenotype. Subsequently, a homozygous nonsense mutation (Glu102STOP) in the RPE65 gene was identified in both affected siblings, thus identifying the causative gene. This data provides evidence for the presence of genetic modulation in LCA. It appears that the heterozygous GUCY2D mutation further disrupts the already compromised photoreceptor function resulting in more severe retinal dysfunction in the older sibling. We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65 mutation.

Functional analyses of mutant recessive GUCY2D alleles identified in Leber congenital amaurosis patients: protein domain comparisons and dominant negative effects.

PURPOSE: Recessive mutations in GUCY2D, the gene encoding the retinal guanylyl cyclase protein, RetGC-1, have been shown to cause Leber Congenital Amaurosis (LCA), a severe retinal dystrophy. The purpose of this study was to determine the functional consequences of selected mutations in GUCY2Dlinked to LCA. The mutations investigated in this study map to the catalytic domain (P858S, L954P) and the extracellular domain (C105Y, L325P) of RetGC-1. METHODS: All four mutations were introduced into the in vitro expression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293 cells. We assayed the abilities of the mutant cyclases to generate cGMP (basal activity), and to be activated by guanylyl cyclase activating proteins (GCAP-1 and GCAP-2). Additionally, we co-expressed the catalytic domain mutations (P858S and L954P) with a wild-type allele to test for dominant negative effects on wild-type RetGC-1. RESULTS: The P858S and L954P mutations, both in highly conserved residues of the catalytic domain of RetGC-1, severely impair basal, GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. In addition, when co-expressed with the wild-type allele, both catalytic domain mutations act as dominant negative proteins and reduce the activity of wild-type RetGC-1. The basal activities of the C105Y and L325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclase activities are reduced approximately 50%. CONCLUSIONS: GUCY2D mutations from LCA patients have distinct functional consequences on RetGC-1 catalytic activity in vitro. Our analyses showed that the catalytic domain mutations cause a marked reduction in cyclase activity, while the extracellular domain mutations moderately reduce activity. The catalytic domain mutant alleles cause dominant negative effects, indicating that the functionality of RetGC-1 is compromised even in heterozygotes. This is consistent with abnormalities in cone electroretinograms (ERGs) detected in obligate heterozygous GUCY2D parents that carry the L954P mutation.

A three base pair deletion encoding the amino acid (lysine-270) in the alpha-cone transducin gene.

PURPOSE: Cone transducin plays an important role in interacting with the cone photoreceptor visual pigments and activating the cGMP-dependent phosphodiesterase. The human gene for the alpha-subunit of cone transducin (GNAT2) has been cloned and characterized. Recently achromatopsia has been associated with mutations in this gene. Cone and cone-rod dystrophies are a genetically heterogeneous group of photoreceptor diseases, in which mutations of a single gene may cause a variety of phenotypes. In this study we tested the hypothesis that mutations in GNAT2 cause cone-rod degeneration (CRD). METHODS: PCR-SSCP and heteroduplex analysis combined with automated sequencing was used for mutation detection in GNAT2 in 13 independent pedigrees with CRD. We used co-segregation analysis to establish or reject causation, when possible. Molecular computer modeling was utilized to examine the possible consequences of mutations onto GNAT2 protein structure. RESULTS: We found a novel 3 base-pair deletion, predicted to cause the loss of a highly conserved lysine at position 270 (K270del) in a French-Canadian CRD pedigree. We detected this deletion in a CRD proband, but also in his unaffected son, the proband's unaffected father and the proband's unaffected brother. However, we did not find this defect in 12 other CRD pedigrees, nor in 100 normal, culturally matched chromosomes. According to literature and our molecular computer modeling, the K270 plays an important role in securing the guanine ring in the nucleotide binding cleft of the molecule and in creating a salt bridge between the helical and GTPase domains of GNAT2. However, the K270del in GNAT2 does not appear to have extensive consequences to the structure and the function of the GNAT2. Apparently, there is a compensatory effect of lysine (K-271), which forms a hydrogen bond with the N1 ring nitrogen substituting for the loss of the lysine at position 270. CONCLUSIONS: We detected a deletion of a highly conserved lysine at codon 270 in a critical functional area of the alpha-cone transducin molecule. The co-segregation analysis showed that the deletion is not co-inherited with the disease phenotype and therefore is not the disease causing mutation. Apparently the function of this molecule is not altered by this mutation, due to a compensatory effect of aminoacid 271. Taken together, the presence of this deletion in healthy individuals, and our protein modeling results, predict that alpha-cone transducin molecule is able to tolerate structurally and functionally the K270del.