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1.  New highly sensitive rodent and human tests for soluble amyloid precursor protein alpha quantification: preclinical and clinical applications in Alzheimer’s disease 
BMC Neuroscience  2012;13:84.
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 × 104 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.
doi:10.1186/1471-2202-13-84
PMCID: PMC3418197  PMID: 22824057
Alzheimer’s disease; Soluble amyloid precursor protein alpha; Homogeneous time-resolved fluorescence; Rodent; Human; Cerebrospinal fluid; Primary neurons; Sensitivity
3.  Glycoform-Selective Prion Formation in Sporadic and Familial Forms of Prion Disease 
PLoS ONE  2013;8(3):e58786.
The four glycoforms of the cellular prion protein (PrPC) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrPSc) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrPSc in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJDV180I) or from Thr to Ala at residue 183 (fCJDT183A). Here we report that fCJDV180I, but not fCJDT183A, exhibits a proteinase K (PK)-resistant PrP (PrPres) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrPres species in both fCJDV180I and VPSPr is likewise attributable to the absence of PrPres glycosylated at the first N-linked glycosylation site at residue 181, as in fCJDT183A. In contrast to fCJDT183A, both VPSPr and fCJDV180I exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrPV180I with a typical glycoform profile from cultured cells generates detectable PrPres that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJDV180I share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrPC to PrPSc is inhibited, probably by a dominant-negative effect, or by other co-factors.
doi:10.1371/journal.pone.0058786
PMCID: PMC3602448  PMID: 23527023
4.  Cerebrospinal Fluid PKR Level Predicts Cognitive Decline in Alzheimer’s Disease 
PLoS ONE  2013;8(1):e53587.
The cerebrospinal fluid (CSF) levels of the proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR) are increased in Alzheimer’s disease (AD), but whether CSF PKR concentrations are associated with cognitive decline in AD patients remain unknown. In this study, 41 consecutive patients with AD and 11 patients with amnestic mild cognitive impairment (aMCI) from our Memory Clinic were included. A lumbar puncture was performed during the following month of the clinical diagnosis and Mini-Mental State Examination (MMSE) evaluations were repeated every 6 months during a mean follow-up of 2 years. In AD patients, linear mixed models adjusted for age and sex were used to assess the cross-sectional and longitudinal associations between MMSE scores and baseline CSF levels of Aβ peptide (Aβ 1-42), Tau, phosphorylated Tau (p-Tau 181), PKR and pPKR. The mean (SD) MMSE at baseline was 20.5 (6.1) and MMSE scores declined over the follow-up (-0.12 point/month, standard error [SE] = 0.03). A lower MMSE at baseline was associated with lower levels of CSF Aβ 1–42 and p-Tau 181/Tau ratio. pPKR level was associated with longitudinal MMSE changes over the follow-up, higher pPKR levels being related with an exacerbated cognitive deterioration. Other CSF biomarkers were not associated with MMSE changes over time. In aMCI patients, mean CSF biomarker levels were not different in patients who converted to AD from those who did not convert.These results suggest that at the time of AD diagnosis, a higher level of CSF pPKR can predict a faster rate of cognitive decline.
doi:10.1371/journal.pone.0053587
PMCID: PMC3539966  PMID: 23320095
5.  Cerebrospinal fluid biomarker supported diagnosis of Creutzfeldt–Jakob disease and rapid dementias: a longitudinal multicentre study over 10 years 
Brain  2012;135(10):3051-3061.
To date, cerebrospinal fluid analysis, particularly protein 14-3-3 testing, presents an important approach in the identification of Creutzfeldt–Jakob disease cases. However, one special point of criticism of 14-3-3 testing is the specificity in the differential diagnosis of rapid dementia. The constant observation of increased cerebrospinal fluid referrals in the national surveillance centres over the last years raises the concern of declining specificity due to higher number of cerebrospinal fluid tests performed in various neurological conditions. Within the framework of a European Community supported longitudinal multicentre study (‘cerebrospinal fluid markers’) we analysed the spectrum of rapid progressive dementia diagnoses, their potential influence on 14-3-3 specificity as well as results of other dementia markers (tau, phosphorylated tau and amyloid-β1–42) and evaluated the specificity of 14-3-3 in Creutzfeldt–Jakob disease diagnosis for the years 1998–2008. A total of 29 022 cerebrospinal fluid samples were analysed for 14-3-3 protein and other cerebrospinal fluid dementia markers in patients with rapid dementia and suspected Creutzfeldt–Jakob disease in the participating centres. In 10 731 patients a definite diagnosis could be obtained. Protein 14-3-3 specificity was analysed for Creutzfeldt–Jakob disease with respect to increasing cerebrospinal fluid tests per year and spectrum of differential diagnosis. Ring trials were performed to ensure the comparability between centres during the reported time period. Protein 14-3-3 test specificity remained high and stable in the diagnosis of Creutzfeldt–Jakob disease during the observed time period across centres (total specificity 92%; when compared with patients with definite diagnoses only: specificity 90%). However, test specificity varied with respect to differential diagnosis. A high 14-3-3 specificity was obtained in differentiation to other neurodegenerative diseases (95–97%) and non-neurological conditions (91–97%). We observed lower specificity in the differential diagnoses of acute neurological diseases (82–87%). A marked and constant increase in cerebrospinal fluid test referrals per year in all centres did not influence 14-3-3 test specificity and no change in spectrum of differential diagnosis was observed. Cerebrospinal fluid protein 14-3-3 detection remains an important test in the diagnosis of Creutzfeldt–Jakob disease. Due to a loss in specificity in acute neurological events, the interpretation of positive 14-3-3 results needs to be performed in the clinical context. The spectrum of differential diagnosis of rapid progressive dementia varied from neurodegenerative dementias to dementia due to acute neurological conditions such as inflammatory diseases and non-neurological origin.
doi:10.1093/brain/aws238
PMCID: PMC3470713  PMID: 23012332
rapid dementia; Creutzfeldt–Jakob disease; cerebrospinal fluid; 14-3-3; specificity; neurodegeneration; differential diagnosis in dementia
6.  A case of Gerstmann-Sträussler-Scheinker disease with a novel six octapeptide repeat insertion 
doi:10.1111/j.1365-2990.2011.01174.x
PMCID: PMC3135713  PMID: 21426368
Amyloid; Gerstmann-Sträussler-Scheinker (GSS) disease; Neuropathology; Octapeptide repeat insertion (OPRI); Prion protein
7.  Serotonin transporter gene polymorphisms and hyperserotonemia in autistic disorder 
Molecular Psychiatry  2002;7(1):67-71.
Previous studies have provided conflicting evidence regarding the association of the serotonin transporter (5-HTT) gene with autism. Two polymorphisms have been identified in the human 5-HTT gene, a VNTR in intron 21 and a functional deletion/insertion in the promoter region (5-HTTLPR) with short and long variants.2 Positive associations of the 5-HTTLPR polymorphism with autism have been reported by two family-based studies, but one found preferential transmission of the short allele3 and the other of the long allele.4 Two subsequent studies failed to find evidence of transmission disequilibrium at the 5-HTTLPR locus.5,6 These conflicting results could be due to heterogeneity of clinical samples with regard to serotonin (5-HT) blood levels, which have been found to be elevated in some autistic subjects.7–9 Thus, we examined the association of the 5-HTTLPR and VNTR polymorphisms of the 5-HTT gene with autism, and we investigated the relationship between 5-HTT variants and whole-blood 5-HT. The transmission/disequilibrium test (TDT) revealed no linkage disequilibrium at either loci in a sample of 96 families comprising 43 trios and 53 sib pairs. Furthermore, no significant relationship between 5-HT blood levels and 5-HTT gene polymorphisms was found. Our results suggest that the 5-HTT gene is unlikely to play a major role as a susceptibility factor in autism.
doi:10.1038/sj.mp.4001923
PMCID: PMC1896269  PMID: 11803447
Adolescent; Adult; Alleles; Autistic Disorder; blood; genetics; Blood Platelets; metabolism; Carrier Proteins; genetics; physiology; Child; Child, Preschool; Female; Genetic Heterogeneity; Genetic Predisposition to Disease; Haplotypes; genetics; Humans; Introns; genetics; Linkage Disequilibrium; Male; Membrane Glycoproteins; genetics; physiology; Membrane Transport Proteins; Minisatellite Repeats; Mutagenesis, Insertional; Nerve Tissue Proteins; Polymorphism, Genetic; Risk Factors; Sequence Deletion; Serotonin; blood; Serotonin Plasma Membrane Transport Proteins; Autistic disorder; serotonin; serotonin transporter; endophenotype; association; linkage disequilibrium
8.  Platelet serotonergic markers as endophenotypes for obsessive-compulsive disorder 
Neuropsychopharmacology   2005;30(8):1539-1547.
Background
Although compelling evidence has shown that obsessive-compulsive disorder (OCD) has a strong genetic component, its genetic basis remains to be elucidated. Identifying biological abnormalities in non-affected relatives is one of the strategies advocated to isolate genetic vulnerability factors in complex disorders. Because peripheral serotonergic disturbances are frequently observed in OCD patients, the aim of this study was to investigate if they could represent endophenotypes, by searching for similar abnormalities in the unaffected parents of OCD patients.
Methods
We assessed whole blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor binding characteristics, and platelet inositol trisphosphate (IP3) content in a sample of OCD probands (n = 48) and their unaffected parents (n = 65), and compared them with sex- and age-matched controls (n = 113).
Results
Lower whole blood 5-HT concentration, fewer platelet 5-HTT binding sites, and higher platelet IP3 content were found in OCD probands and their unaffected parents compared to controls. Whole blood 5-HT concentration showed a strong correlation within families (p<0.001). The only parameter that appeared to discriminate affected and unaffected subjects was 5-HT2A receptor binding characteristics, with increased receptor number and affinity in parents and no change in OCD probands.
Conclusions
The presence of peripheral serotonergic abnormalities in OCD patients and their unaffected parents supports a familial origin of these disturbances. These alterations may serve as endophenotypic markers in OCD, and could contribute to the study of the biological mechanisms and genetic underpinnings of the disorder.
doi:10.1038/sj.npp.1300752
PMCID: PMC1885456  PMID: 15886722
Adolescent; Adrenergic Uptake Inhibitors; pharmacokinetics; Adult; Biological Markers; Blood Platelets; drug effects; metabolism; Case-Control Studies; Child; Female; Genotype; Humans; Imipramine; pharmacokinetics; Inositol 1,4,5-Trisphosphate; blood; Iodine Isotopes; pharmacokinetics; Lysergic Acid Diethylamide; pharmacokinetics; Male; Middle Aged; Minisatellite Repeats; genetics; Obsessive-Compulsive Disorder; blood; genetics; Paroxetine; pharmacokinetics; Radioimmunoassay; methods; Receptor, Serotonin, 5-HT2A; metabolism; Serotonin; blood; Serotonin Agents; pharmacokinetics; Serotonin Uptake Inhibitors; pharmacokinetics; Statistics; Statistics, Nonparametric; Tritium; pharmacokinetics; serotonin; serotonin transporter; 5-HT2A receptor; binding; inositol triphosphate; intrafamilial correlation

Results 1-8 (8)