Fasting upregulates the monocarboxylate transporter MCT1 at the rat blood-brain barrier through PPAR δ activation.

BACKGROUND: The blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters. METHODS: We used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion. RESULTS: Fasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting. CONCLUSIONS: Altogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain.

Modifications of physical and functional integrity of the blood-brain barrier in an inducible mouse model of neurodegeneration.

The inducible p25 overexpression mouse model recapitulate many hallmark features of Alzheimer's disase including progressive neuronal loss, elevated Aß, tau pathology, cognitive dysfunction, and impaired synaptic plasticity. We chose p25 mice to evaluate the physical and functional integrity of the blood-brain barrier (BBB) in a context of Tau pathology (pTau) and severe neurodegeneration, at an early (3 weeks ON) and a late (6 weeks ON) stage of the pathology. Using in situ brain perfusion and confocal imaging, we found that the brain vascular surface area and the physical integrity of the BBB were unaltered in p25 mice. However, there was a significant 14% decrease in cerebrovascular volume in 6 weeks ON mice, possibly explained by a significant 27% increase of collagen IV in the basement membrane of brain capillaries. The function of the BBB transporters GLUT1 and LAT1 was evaluated by measuring brain uptake of d-glucose and phenylalanine, respectively. In 6 weeks ON p25 mice, d-glucose brain uptake was significantly reduced by about 17% compared with WT, without any change in the levels of GLUT1 protein or mRNA in brain capillaries. The brain uptake of phenylalanine was not significantly reduced in p25 mice compared with WT. Lack of BBB integrity, impaired BBB d-glucose transport have been observed in several mouse models of AD. In contrast, reduced cerebrovascular volume and an increased basement membrane thickness may be more specifically associated with pTau in mouse models of neurodegeneration.

Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood-Brain Barrier Reveals Interspecies Differences.

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 µM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 µM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 µM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

Protein interacting with Amyloid Precursor Protein tail-1 (PAT1) is involved in early endocytosis.

Protein interacting with Amyloid Precursor Protein (APP) tail 1 (PAT1) also called APPBP2 or Ara 67 has different targets such as APP or androgen receptor and is expressed in several tissues. PAT1 is known to be involved in the subcellular trafficking of its targets. We previously observed in primary neurons that PAT1 is poorly associated with APP at the cell surface. Here we show that PAT1 colocalizes with vesicles close to the cell surface labeled with Rab5, Rab4, EEA1 and Rabaptin-5 but not with Rab11 and Rab7. Moreover, PAT1 expression regulates the number of EEA1 and Rab5 vesicles, and endocytosis/recycling of the transferrin receptor. In addition, low levels of PAT1 decrease the size of transferrin-colocalized EEA1 vesicles with time following transferrin uptake. Finally, overexpression of the APP binding domain to PAT1 is sufficient to compromise endocytosis. Altogether, these data suggest that PAT1 is a new actor in transferrin early endocytosis. Whether this new function of PAT1 may have consequences in pathology remains to be determined.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

Acute and chronic neurobehavioral effects of the designer drug and bath salt constituent 3,4-methylenedioxypyrovalerone in the rat.

BACKGROUND: The substantial increase in use of 3,4-methylenedioxypyrovalerone (MDPV), a popular recreational synthetic cathinone, has raised legitimate questions about its behavioral consequences and abuse liability. AIMS: The aim of this study was to study MDPV-induced neurobehavioral effects in the rat, using different paradigms traditionally developed to study drug-attributed addictive properties. METHODS: Different patterns of intraperitoneal 3 mg/kg MDPV administration were investigated. Consequences on rat horizontal locomotion and behavior of acute, intermittent (once daily dosing over 10 days), and binge (three-time daily dosing for 3 days) MDPV administration as well as challenge after 10 day MDPV withdrawal were studied. The dopamine receptor-D1 antagonist, SCH23390, was bilaterally infused in the nucleus accumbens to determine the role of D1-receptors in MDPV-related effects on the associative memory recall using the conditioned place preference paradigm. In addition, in a separate experience using western blot, we investigated the effects of chronic MDPV administration (four injections during 24 h) on ΔFosB expression in the nucleus accumbens, caudate putamen, and prefrontal cortex. RESULTS: Acute MDPV administration increased stereotypies and open arm entries in the elevated plus maze while SCH23390 abolished MDPV-induced enhancing effects on memory consolidation. Intermittent MDPV administration resulted in sensitization of MDPV-induced locomotor effects and tolerance during the following challenge. With binge MDPV administration, locomotor activity was not altered despite tolerance onset after challenge. SCH23390 abolished MDPV-induced conditioned place preference. Chronic MDPV administration induced ΔFosB accumulation in the nucleus accumbens, caudate putamen, and prefrontal cortex. CONCLUSIONS: Our findings clearly show that MDPV produces profound behavioral alterations mediated by the activation of the dopaminergic system similarly to other amphetamines.

Isolation and differential transcriptome of vascular smooth muscle cells and mid-capillary pericytes from the rat brain.

Brain mural cells form a heterogeneous family which significantly contributes to the maintenance of the blood-brain barrier and regulation of the cerebral blood flow. Current procedures to isolate them cannot specifically separate their distinct subtypes, in particular vascular smooth muscle cells (VSMCs) and mid-capillary pericytes (mcPCs), which differ among others by their expression of smooth muscle actin (SMA). We herein describe an innovative method allowing SMA+ VSMCs and SMA- mcPCs to be freshly isolated from the rat cerebral cortex. Using differential RNA-Seq analysis, we then reveal the specific gene expression profile of each subtype. Our results refine the current description of the role of VSMCs in parenchymal cortical arterioles at the molecular level and provide a unique platform to identify the molecular mechanisms underlying the specific functions of mcPCs in the brain vasculature.

Soluble Amyloid Precursor Protein Alpha Interacts with alpha3-Na, K-ATPAse to Induce Axonal Outgrowth but Not Neuroprotection: Evidence for Distinct Mechanisms Underlying these Properties.

Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble N-terminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPα-induced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.

Expression and function of Abcg4 in the mouse blood-brain barrier: role in restricting the brain entry of amyloid-ß peptide.

ABCG4 is an ATP-binding cassette transmembrane protein which has been shown, in vitro, to participate in the cellular efflux of desmosterol and amyloid-ß peptide (Aß). ABCG4 is highly expressed in the brain, but its localization and function at the blood-brain barrier (BBB) level remain unknown. We demonstrate by qRT-PCR and confocal imaging that mouse Abcg4 is expressed in the brain capillary endothelial cells. Modelling studies of the Abcg4 dimer suggested that desmosterol showed thermodynamically favorable binding at the putative sterol-binding site, and this was greater than for cholesterol. Additionally, unbiased docking also showed Aß binding at this site. Using a novel Abcg4-deficient mouse model, we show that Abcg4 was able to export Aß and desmosterol at the BBB level and these processes could be inhibited by probucol and L-thyroxine. Our assay also showed that desmosterol antagonized the export of Aß, presumably as both bind at the sterol-binding site on Abcg4. We show for the first time that Abcg4 may function in vivo to export Aß at the BBB, in a process that can be antagonized by its putative natural ligand, desmosterol (and possibly cholesterol).

Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons.

BACKGROUND: The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate. RESULTS: We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling. CONCLUSIONS: These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.

Cytoplasmic SET induces tau hyperphosphorylation through a decrease of methylated phosphatase 2A.

BACKGROUND: The neuronal cytoplasmic localization of SET, an inhibitor of the phosphatase 2A (PP2A), results in tau hyperphosphorylation in the brains of Alzheimer patients through mechanisms that are still not well defined. RESULTS: We used primary neurons and mouse brain slices to show that SET is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. The localization of SET in the cytoplasm, either by the translocation of endogenous SET or by internalization of the recombinant full-length SET protein, induced tau hyperphosphorylation. Cytoplasmic recombinant full-length SET in mouse brain slices induced a decrease of PP2A activity through a decrease of methylated PP2A levels. The levels of methylated PP2A were negatively correlated with tau hyperphosphorylation at Ser-202 but not with the abnormal phosphorylation of tau at Ser-422. CONCLUSIONS: The presence of full-length SET in the neuronal cytoplasm is sufficient to impair PP2A methylation and activity, leading to tau hyperphosphorylation. In addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. The translocation of SET to the neuronal cytoplasm, the low activity of PP2A, and tau hyperphosphorylation are associated in the brains of Alzheimer patients. Our data show a link between the translocation of SET in the cytoplasm and the decrease of methylated PP2A levels leading to a decrease of PP2A activity and tau hyperphosphorylation. This chain of events may contribute to the pathogenesis of Alzheimer disease.

Oatp1a4 and an L-thyroxine-sensitive transporter mediate the mouse blood-brain barrier transport of amyloid-ß peptide.

The influx of amyloid-ß peptide (Aß) across the blood-brain barrier is partly mediated by the receptor for advanced glycation end products (RAGE). But other transporters, like Oatp (organic anion transporter polypeptide, SLC21) transporters, could also be involved. We used in situ brain perfusion to show that rosuvastatin and taurocholate, two established Oatp1a4 substrates, decreased (5-fold) the Clup of [3H]Aß while L-thyroxine increased it (5.5-fold). We demonstrated an interaction between Aß and Oatp1a4 by co-immunoprecipitation and western blotting experiments, supporting the hypothesis that the rosuvastatin- and taurocholate-sensitive transporter was Oatp1a4. In conclusion, our results suggest that, in mice, the brain uptake of Aß is partly mediated by Oatp1a4 and that L-thyroxine may play a crucial role in the inhibition of brain Aß clearance.

New highly sensitive rodent and human tests for soluble amyloid precursor protein alpha quantification: preclinical and clinical applications in Alzheimer's disease.

BACKGROUND: Amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD), is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in AD pathology or the soluble form of APP (sAPPα). The level of amyloidogenic peptides in human cerebrospinal fluid (CSF) is considered to be a biomarker of AD, whereas the level of sAPPα in CSF as a biomarker has not been clearly established. sAPPα has neurotrophic and neuroprotective properties. Stimulating its formation and secretion is a promising therapeutic target in AD research. To this end, very sensitive tests for preclinical and clinical research are required. METHODS: The tests are based on homogenous time-resolved fluorescence and require no washing steps. RESULTS: We describe two new rapid and sensitive tests for quantifying mouse and human sAPPα. These 20 µl-volume tests quantify the levels of: i) endogenous mouse sAPPα in the conditioned medium of mouse neuron primary cultures, as well as in the CSF of wild-type mice, ii) human sAPPα in the CSF of AD mouse models, and iii) human sAPPα in the CSF of AD and non-AD patients. These tests require only 5 µl of conditioned medium from 5 × 10(4) mouse primary neurons, 1 µl of CSF from wild-type and transgenic mice, and 0.5 µl of human CSF. CONCLUSIONS: The high sensitivity of the mouse sAPPα test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sAPPα in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sAPPα in the CSF of treated wild-type mice. Active molecules could then be tested in the AD mouse models by quantifying human sAPPα in the CSF through the progression of the disease. Finally, the human sAPPα test could strengthen the biological diagnosis of AD in large clinical investigations. Taken together, these new tests have a wide field of applications in preclinical and clinical studies.

Functions of Aß, sAPPα and sAPPß : similarities and differences.

Amyloid peptide (Aß) is derived from the cleavage of amyloid precursor protein (APP), which also generates the soluble peptide APPß (sAPPß). An antagonist and major APP metabolic pathway involves cleavage by alpha secretase, which releases sAPPα. Although soluble Aß oligomers are neurotoxic, Aß monomers share similar properties with sAPPα. These include neurotrophic and neuroprotective effects, as well as stimulation of neural-progenitor proliferation. The properties of Aß monomers and the neurotrophic capacity of sAPPß to stimulate axonal outgrowth suggest that Aß production is not deleterious per se. Consequently, therapeutic strategies for Alzheimer's disease that are targeted at Aß-cleaving enzymes should modulate rather than inhibit Aß generation. These strategies should focus on the factors that induce the conversion of Aß monomers into toxic soluble oligomers. Another interesting therapeutic approach is to focus on the mechanisms of the different properties of sAPPα. Indeed, increasing sAPPα levels could shift proliferating cells towards tumorigenesis. In contrast to its neuroprotective effects, sAPPα is also able to activate microglia, leading to neurotoxicity. Understanding the mechanisms that underlie the different properties of sAPPα could therefore lead to the development of therapeutic strategies against Alzheimer's disease, which could be curative as well as preventive.

CSF Aß1₋42 levels and glucose metabolism in Alzheimer's disease.

Glucose dysmetabolism has been consistently associated with an increased risk of cognitive disorders, and brain insulin resistance could play a role in Alzheimer's disease (AD) pathogenesis. Recent evidence suggests that cerebrospinal fluid (CSF) biomarkers may reflect the brain pathology in AD. We have investigated the relationship between CSF concentrations of amyloid-ß peptide 1-42 (Aß1₋42), total tau, and phosphorylated tau (ptau-181) and plasma and CSF glucose levels in a cohort of 94 newly diagnosed non-diabetics AD patients. We report that CSF Aß1₋42 level was inversely associated with CSF to plasma glucose ratio (Spearman's coefficient = -0.27, p = 0.008). This relationship remained after adjustment for age, gender, body mass index, hypertension, and MMSE score (ß [SE] of linear regression = -0.93 [0.37], p = 0.01). In stratified analysis, this relationship was observed only in patients who did not carry the apolipoprotein E4 allele. No significant relationship was found between glucose levels and total tau or phosphorylated tau 181. These results support the idea that a link between glucose dysmetabolism and the amyloid pathway may exist in the pathogenesis of AD.

Secreted amyloid precursor protein ß and secreted amyloid precursor protein α induce axon outgrowth in vitro through Egr1 signaling pathway.

BACKGROUND: sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPß released at the first step of amyloid peptide cleavage, raising the possibility that sAPPß could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPß on primary neurons and identified a key signaling event required for the changes observed. METHODOLOGY/PRINCIPAL FINDINGS: Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPß added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPß induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPß stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 -/- mice, sAPPs affected dendritic length but did not induce any increase of axon length. CONCLUSION/SIGNIFICANCE: sAPPα and sAPPß decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPß stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPß is not deleterious per se. Since sAPPß and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage.

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines.

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

Paget's disease of bone in the French population: novel SQSTM1 mutations, functional analysis, and genotype-phenotype correlations.

UNLABELLED: Mutation screening of the SQSTM1 gene in 94 French patients with PDB revealed two novel point-mutations (A381V and L413F) and two new compound heterozygous genotypes (P392L/A381V and P392L/A390X). Functional analysis showed an increased level of SQSTM1/p62 protein in PDB patients and truncated forms of the protein encoded by the A390X allele. Clinical data indicate that PDB patients with SQSTM1 mutation are younger at PDB diagnosis and have more extensive bone lesions. INTRODUCTION: Paget's disease of bone (PDB) is a common chronic disease of the skeleton, with a strong genetic component. A recurrent mutation (P392L) was first identified on chromosome 5, in the Sequestosome 1 (SQSTM1) gene. Several other mutations of the SQSTM1 gene have been described in PDB patients, affecting the ubiquitin-associated domain (UBA) of the SQSTM1/p62 protein. The objectives of this study were to evaluate the frequency of the SQSTM1 mutations in French PBD patients, to study the expression of the SQSTM1/p62 protein, and to search for genotype-phenotype correlations. MATERIALS AND METHODS: Blood was obtained from 94 unrelated French PDB patients and 100 controls for mutation screening of exons 7 and 8, encoding for the UBA domain of SQSTM1. Epstein-Barr virus (EBV)-immortalized B-cell lymphocytes were established from 13 patients, giving access to functional analysis of the gene and the SQSTM1/p62 expressions using real-time PCR and Western blot. RESULTS: Mutations of the SQSTM1 gene were identified in 12 of the 94 PDB patients (13%). Eight patients carried P392L. Two novel missense mutations were identified: L413F and A381V. This A381V mutation and A390X were found in distinct patients already carriers of P392L. The SQSTM1/p62 protein expression in PDB patients increased when zero, one, or two mutations were present, and SQSTM1 truncated forms were associated with the A390X mutation. The mean age of PDB diagnosis was younger in patients with the SQSTM1 mutation. PDB was more extensive in patients who carried a SQSTM1 mutation. CONCLUSIONS: Mutations of SQSTM1 are present in the French population. PDB patients with and without the SQSTM1 mutation have an increased level of SQSTM1/p62, caused by overproduction of the protein, probably involved in the pathophysiology of PDB. The presence of the SQSTM1 mutation may be a worsening factor for PDB.