Variants of CYP46A1 may interact with age and APOE to influence CSF Aβ42 levels in Alzheimer’s disease

Recent studies have suggested that variants of CYP46A1, encoding cholesterol 24-hydroxylase (CYP46), confer risk for Alzheimers disease (AD), a prospect substantiated by evidence of genetic association from several quantitative traits related to AD pathology, including cerebrospinal fluid (CSF) levels of the 42 amino-acid cleavage product of -amyloid (A42) and the tau protein. In the present study, these claims have been explored by the genotyping of previously associated markers in CYP46A1 in three independent northern European case-control series encompassing 1323 individuals and including approximately 400 patients with measurements of CSF A42 and phospho-tau protein levels. Tests of association in case-control models revealed limited evidence that CYP46A1 variants contributed to AD risk across these samples. However, models testing for potential effects upon CSF measures suggested a possible interaction of an intronic marker (rs754203) with age and APOE genotype. In stratified analyses, significant effects were evident that were restricted to elderly APOE 4 carriers for both CSF A42 (P=0.0009) and phospho-tau (P=0.046). Computational analyses indicate that the rs754203 marker probably does not impact the binding of regulatory factors, suggesting that other polymorphic sites underlie the observed associations. Our results provide an important independent replication of previous findings, supporting the existence of CYP46A1 sequence variants that contribute to variability in -amyloid metabolism.     

Common variants of ACE contribute to variable age-at-onset of Alzheimer’s disease

Studies on the role that genetic variation may play in a complex human disease can be empowered by an assessment of both disease risk in case-control or family models and of quantitative traits that reflect elements of disease etiology. An excellent example of this can be found for the 4 allele of APOE in relation to Alzheimers disease (AD) for which association with both risk and age-at-onset (AAO) is evident. Following a recent demonstration that variants of the gene encoding angiotensin I converting enzyme (ACE) contribute to AD risk, we have explored the potential influence of ACE upon AAO in AD. A total of 2861 individuals from three European populations, including six independent AD samples, have been examined in this study. Three single nucleotide polymorphisms (SNPs) previously demonstrated to have maximum effects upon ACE plasma levels and that span the ACE locus were genotyped in these materials. A strong effect upon AAO was observed for marker rs4343 in exon 17 (P<0.0001), but evidence was also obtained indicating a possible independent effect of marker rs4291 (P=0.0095) located in the ACE promoter. Effects were consistent with data from previous studies suggesting association with AD in case-control models, whereby alleles demonstrated to confer risk to disease also appear to reduce AAO. Equivalent effects were evident regardless of APOE 4 carrier status and in both males and females. These results provide an important complement to existing AD risk data, confirming that ACE harbors sequence variants that contribute to aspects of AD pathology.     

Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

Uracil phosphoribosyltransferase (UPRTase) catalyzes the conversion of 5-phosphate-alpha-1-diphosphate (PRPP) and uracil to uridine 5'-monophosphate (UMP) and diphosphate. The UPRTase from Sulfolobus solfataricus has a unique regulation by nucleoside triphosphates compared to UPRTases from other organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal differences in the subunits and their relative arrangement. In the UPRTase-UMP complex, the peptide bond between a conserved arginine residue (Arg80) and the preceding residue (Leu79) adopts a cis conformation in half of the subunits and a trans conformation in the other half and the tetramer comprises two cis-trans dimers. In contrast, four identical subunits compose the UPRTase-UMP-CTP tetramer. CTP binding affects the conformation of Arg80, and the Arg80 conformation in the UPRTase-UMP-CTP complex leaves no room for binding of the substrate PRPP. The different conformations of Arg80 coupled to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences in the interactions of the catalytically important Asp209. The active-site differences have been related to proposed kinetic models and provide an explanation for the regulatory significance of the C-terminal Gly216.     

Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons

The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.     

Decreased levels of soluble amyloid β-protein precursor and β-amyloid protein in cerebrospinal fluid of patients with systemic lupus erythematosus

Symptoms originating from the central nervous system (CNS) frequently occur in patients with systemic lupus erythematosus (SLE). These symptoms are extremely diverse, including a state of dementia. The aim of this study was to examine the cerebrospinal fluid (CSF) content of soluble molecules indicating axonal degeneration and amyloidogenesis. One hundred and fourteen patients with SLE and age-matched controls were evaluated clinically, with magnetic resonance imaging of the brain and CSF analyses. Levels of tau, amyloid precursor protein (APP), beta-amyloid protein (Abeta42), and transforming growth factor beta (TGF-beta) were all determined using sandwich ELISAs.APP and Abeta42 levels were significantly decreased in SLE patients irrespective of their CNS involvement, as compared with healthy controls. Patients with neuropsychiatric SLE who underwent a second lumbar puncture following successful cyclophosphamide treatment showed further decreases of Abeta42. CSF-tau levels were significantly increased in SLE patients showing magnetic resonance imaging-verified brain pathology as compared with SLE patients without such engagement. Importantly, tau levels displayed significant correlation to Abeta42 levels in the CSF. Finally, TGF-beta levels were significantly increased in patients with neuropsychiatric SLE as compared with those without. Low intrathecal levels of Abeta42 found in SLE patients seem to be a direct consequence of a diminished production of APP, probably mediated by heavy anti-inflammatory/immuno-suppressive therapy. Furthermore, our findings suggest that CSF tau can be used as a biochemical marker for neuronal degeneration in SLE. Finally, the increased TGF-beta levels observed may support a notion of an ongoing anti-inflammatory response counteracting tissue injury caused by CNS lupus.     

CSF total tau, Aβ42 and phosphorylated tau protein as biomarkers for Alzheimer’s disease

With the arrival of effective symptomatic treatments and the promise of drugs that may delay progression, we now need to identify Alzheimer’s disease (AD) at an early stage of the disease. To diagnose AD earlier and more accurately, attention has been directed toward peripheral biochemical markers. This article reviews promising potential cerebrospinal fluid (CSF) biomarkers for AD focussing on their role in clinical diagnosis. In particular, two biochemical markers, CSF total tau (t-tau) protein and the 42 amino acid form of β-amyloid (Aβ42), perform satisfactorily enough to achieve a role in the clinical diagnostic settings of patients with dementia together with the cumulative information from basic clinical work-up, genetic screening, and brain imaging. These CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. In order to discriminate AD from other primary dementia disorders, however, more accurate and specific markers are needed. Preliminary evidence strongly suggests that quantification of tau phosphorylated at specific sites in CSF improves early detection, differential diagnosis, and tracking of disease progression in AD.     

Eelgrass, Zostera marina, growth along depth gradients: upper boundaries of the variation as a powerful predictive tool

1200 measurements of eelgrass ( Zostera marina ) biomass, shoot density and cover along 19 depth gradients in Øresund, located between Denmark and Sweden, were analysed to characterise growth of eelgrass in relation to depth. The large data set allowed analyses of boundaries of distribution as well as of average trends. Natural variability is large in shallow water where populations are disturbed by wave action and other physical parameters. Models based on average values, therefore, did not adequately describe growth regulation by resources, and only explained a minor part (up to 30%) of the overall variation in data. In contrast, boundary functions, which describe the upper bounds of distributions, focus on the variation produced by the ultimately growth-regulating resource, and therefore provide models with high predictive power. An exponential model explained up to 90% of the variation in upper bounds of eelgrass shoot density as a function of depth and indicated that shoot density was ultimately regulated by light availability. The boundary functions demonstrated that eelgrass shoot density, biomass and cover followed markedly different patterns as functions of depth and were affected differently by the factors governing their distribution. In addition, boundary functions revealed informative spatial structures in data and illustrated whether a given general trend was caused by changes in maximum values, minimum values or both. For example, upper and lower boundaries of biomass-shoot density relations changed markedly with depth, demonstrating depth-related changes in intraspecific succession and competition patterns. Boundary functions are therefore suggested as a promising tool for analysing ultimate regulating factors of distribution and growth of organisms when large data sets are available.     

Mutations in the tryptophanyl-transfer ribonucleic acid ligase of E. coli causing temperature-sensitivity for growth.

Summary Strains of Escherichia coli K-12 with altered tryptophanyl-tRNA ligases, conferring temperature-sensitivity for growth, have been isolated as spontaneous one-step mutants. The mutated enzymes differ markedly in activity from the wildtype, even at the permissive temperature. When assayed at the non-permissive temperature, the mutant enzymes are completely inactive. In one of the mutant strains, growth can be completely inhibited by addition of L-phenylalanine or L-tyrosine to the medium.     

Cellular expression of neurotrophin mRNAs during tooth development

. Target-derived neurotrophins support and sustain peripheral sensory neurons during development. In addition, it has been suggested that these growth factors could have developmental functions in non-neuronal tissues. To further elucidate the possible roles of neurotrophins in tooth morphogenesis and innervation, we have used in-situ hybridization to determine the specific sites of neurotrophin gene activity in pre- and postnatal rat jaws from E16 to P7. All four neurotrophins were expressed during tooth development with specific temporospatial patterns. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal animals, and the pattern of expression correlated with the onset of dental innervation. In contrast, neurotrophin 3 (NT3) and neurotrophin 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Dental papilla NGF-mRNA expression was first seen in both epithelium and mesenchyme and later shifted to the odontoblast layer and the subodontoblast zone. BDNF-mRNA labeling was present in low levels in the early dental organ, but increased in the pulp and in the odontoblast cell layer of the developing teeth at later developmental stages. Both NT3 and NT4 mRNA were observed in the prenatal oral epithelium and the inner dental epithelium. NT3-mRNA labeling was seen mainly in the cervical loop region, fissure system depressions and cuspal tops, while NT4 mRNA was more evenly distributed in the dental epithelium. At P7, NT3-mRNA labeling was below detection level and NT4 mRNA expression was lower than at prior stages. Complementary to reports on the presence of low-affinity neurotrophin receptor (LANR), trkB and trkC mRNA in the developing teeth, our results suggest that neurotrophins may have multiple functions during tooth morphogenesis. Neurotrophins might participate in epithelial-mesenchymal interactions in early tooth morphogenetic events such as proliferation and differentiation of epithelial and mesenchymal cells. In addition, based on mRNA localization in postnatal animals, we also suggest that NGF and BDNF (beside glial cell line-derived neurotrophic factor) might participate in establishing and maintaining the innervation of the teeth, thus acting as classical neurotrophic factors.     

SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer’s disease

Background

Synaptic degeneration is an early pathogenic event in Alzheimer’s disease, associated with cognitive impairment and disease progression. Cerebrospinal fluid biomarkers reflecting synaptic integrity would be highly valuable tools to monitor synaptic degeneration directly in patients. We previously showed that synaptic proteins such as synaptotagmin and synaptosomal-associated protein 25 (SNAP-25) could be detected in pooled samples of cerebrospinal fluid, however these assays were not sensitive enough for individual samples.

Results

We report a new strategy to study synaptic pathology by using affinity purification and mass spectrometry to measure the levels of the presynaptic protein SNAP-25 in cerebrospinal fluid. By applying this novel affinity mass spectrometry strategy on three separate cohorts of patients, the value of SNAP-25 as a cerebrospinal fluid biomarker for synaptic integrity in Alzheimer’s disease was assessed for the first time. We found significantly higher levels of cerebrospinal fluid SNAP-25 fragments in Alzheimer’s disease, even in the very early stages, in three separate cohorts. Cerebrospinal fluid SNAP-25 differentiated Alzheimer’s disease from controls with area under the curve of 0.901 (P?<?0.0001).

Conclusions

We developed a sensitive method to analyze SNAP-25 levels in individual CSF samples that to our knowledge was not possible previously. Our results support the notion that synaptic biomarkers may be important tools for early diagnosis, assessment of disease progression, and to monitor drug effects in treatment trials.