Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3ß (GSK3ß). KLC2 phosphorylation by GSK3ß induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3ß on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-ß (TGFß) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFß-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

The potency of allospecific Tregs cells appears to correlate with T cell receptor functional avidity.

CD4(+) CD25(+) regulatory T cells (T(reg) cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T-cell receptor (TcR) activation is critical for T(reg) function, suggesting that the TcR avidity of T(reg) cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T(reg) lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T(reg) lines generated from CD4(+)CD25(+) T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T(reg) lines was confirmed in vitro. T(reg) lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T(reg) lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T(reg) cells, together with anti-CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T(reg) lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T(reg) function. It highlights the fact that strategies to select T(reg) with higher functional avidity might be beneficial for immunotherapy in transplantation.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Risk factors associated with low birth weight infants in the Malaysian population.

This study aimed to identify the risk factors which were significantly associated with low birth weight (LBW, <2500 g) infants among the Malaysian population. This was a case-control study carried out at the Tuanku Jaafar Hospital, Seremban, Malaysia over a five-month period. Cases were all infants born with birth weight less than 2500 g. Control infant were selected with the help a random sampling table from among infants with birth weight of > or =2500 g born on the same day in the hospital. Of 3341 livebirths delivered in the hospital, 422 (12.6%) were LBW infants. Logistic regression analysis showed that, after controlling for various potential confounders, the only significant risk factors associated with infants of LBW were gestational age (adjusted odds ratio (OR)=0.6, 95% C.I.: 0.5, 0.6; < 0.0001), maternal pre-pregnancy weight (adjusted OR = 0.97, 95% C.I.: 0.95, 0.99; p < 0.0001), nulliparity (adjusted OR = 3.4, 95% C.I.: 2.2, 5.1; p < 0.0001), previous history of LBW infants (adjusted OR = 2.3, 95% C.I.: 1.4, 3.8; p=0.001) and PIH during current pregnancy (adjusted OR=3.3, 95% C.I.: 1.6, 6.6; p = 0.001). A number of potentially preventable or treatable risk factors were identified to be associated with LBW infants in Malaysia.

Expression of the Fe65 adapter protein in adult and developing mouse brain.

Fe65 is a multimodular adaptor protein expressed mainly in the nervous system. Fe65 binds to the Alzheimer's disease amyloid precursor protein (APP) and the interaction is mediated via a phosphotyrosine binding domain in Fe65 and the carboxy-terminal cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP, including production of the beta-amyloid peptide that is believed to be central to the pathogenesis of Alzheimer's disease. Fe65 also facilitates translocation of a carboxy-terminal fragment of APP to the nucleus and is required for APP-mediated transcription events. In addition, Fe65 functions in regulation of the actin cytoskeleton and cell movement. Here we report the distribution profile of Fe65 immunoreactivity in adult mouse brain. Fe65 expression was found to be widespread in neurones in adult brain. The areas of highest expression included regions of the hippocampus in which the earliest abnormalities of Alzheimer's disease are detectable. Fe65 was also highly expressed in the cerebellum, thalamus and selected brain stem nuclei. Fe65 was evident in a sub-set of astrocytes within the stratum oriens and radiatum in the hippocampus. Expression of Fe65 was found to be developmentally regulated with levels reducing after embryonic day 15 and increasing again progressively from post-partum day 10 up to adulthood, a developmental pattern that partially parallels that of APP. These data indicate a widespread distribution of Fe65 in neurones throughout mouse brain and also suggest that Fe65 may have functions independent of APP and any potential role in the pathogenesis of Alzheimer's disease.

Intracranial venous thrombosis and pulmonary embolism with antiphospholipid syndrome in systemic lupus erythematosus.

The presence of antiphospholipid antibodies is associated with arterial and venous thrombosis. A 14-year-old girl, with systemic lupus erythematosus (SLE), developed headache and cough and was found to have intracranial venous sinus thrombosis with secondary pulmonary embolism associated with antiphospholipid syndrome. Clinical and radiological improvement occurred with anticoagulation therapy. Because SLE is commonly associated with antiphospholipid antibodies, thromboembolic events should be considered in the differential diagnosis of both cough and headache in children with SLE.

p35/cdk5 binds and phosphorylates beta-catenin and regulates beta-catenin/presenilin-1 interaction.

The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35). To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners. From one such screen, we identified beta-catenin as an interacting protein. Confirmation that p35 binds to beta-catenin was obtained by using glutathione S-transferase (GST)-beta-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that beta-catenin was present in p35 immunoprecipitates. p35 and beta-catenin also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that p35/cdk5 phosphorylates beta-catenin. beta-catenin also binds to presenilin-1 and altered beta-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD). Abnormal p35/cdk5 activity has also been suggested to contribute to AD. We therefore investigated how modulation of p35/cdk5 activity influenced beta-catenin/presenilin-1 interactions. Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either beta-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to beta-catenin. Thus, p35/cdk5 binds and phosphorylates beta-catenin and regulates its binding to presenilin-1. The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with beta-catenin and presenilin-1 in AD.

Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3).

Threonine(668) (thr(668)) within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) is believed to regulate APP function and metabolism. Thr(668) precedes a proline, which suggests that it is targeted for phosphorylation by proline-directed kinase(s). We have investigated the ability of four major neuronally active proline-directed kinases, cyclin dependent protein kinase-5, glycogen synthase kinase-3 beta, p42 mitogen-activated protein kinase and stress-activated protein kinase-1b, to phosphorylate APPthr(668) and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.

Molecular cloning and expression analysis of human glycogen synthase kinase-3 alpha promoter.

Human glycogen synthase kinase-3 alpha (GSK-3 alpha) is a serine/threonine kinase that phosphorylates a variety of cytoplasmic and nuclear proteins. It also phosphorylates components of the neuronal cytoskeleton including tau and neurofilament heavy chain. Hyperphosphorylated tau is found in neurofibrillary tangles, a hallmark of Alzheimer's disease and aberrant phosphorylation of neurofilament heavy chain is observed in motor neuron disease. Alterations in GSK-3 alpha activity may therefore contribute to the disease process in these disorders. As a first step to understand the transcriptional regulation of GSK-3 alpha, a 2-kb (p-1751/+243) DNA fragment upstream of the GSK-3 alpha initiation codon was obtained from a YAC clone and characterised. Using primer extension assays, a putative transcriptional start site was located to a G nucleotide 244 bp upstream of the ATG codon. Several transcription factor-binding sites were identified on the promoter region, but no TATA-like element was located close to the start site. Deletion mutants of the 2-kb DNA fragment were generated and fused to a promoterless chloramphenicol acetyltransferase (CAT) gene. Transfection study in a neuroblastoma cell line revealed the 1-kb (p-719/+243) fragment carried strong promoter activity, while the 2-kb construct that contains an Alu-like sequence was only 50% active.

Fe65 and X11beta co-localize with and compete for binding to the amyloid precursor protein.

The Fe65s and X11s are two families of adaptor proteins that bind to the Alzheimer's disease amyloid precursor protein (APP). Although both the X11s and Fe65s bind to similar regions of APP, they have opposing effects on Abeta production and hence may represent novel therapeutic targets. However, there is no evidence that the Fe65s and X11s are present within the same cell type or cell compartment and are thus capable of competing for binding to APP. Here we show that in neurones and transfected cells, APP, Fe65 and X11beta show overlapping subcellular distributions. Furthermore, we demonstrate that Fe65 and X11beta compete for binding to APP.

X11 alpha and x11 beta interact with presenilin-1 via their PDZ domains.

X11 alpha and X11 beta are two neuronal adaptor proteins that interact with the Alzheimer's disease amyloid precursor protein (APP). X11 alpha and X11 beta stabilise APP and inhibit production of proteolytic APP fragments including the A beta peptide that is deposited in the brains of Alzheimer's disease patients. The mechanisms by which X11 alpha and X11 beta modulate APP processing are not clear but one possibility is that they influence the activity of the secretases that cleave APP to give rise to A beta. Presenilin-1 is required for gamma-secretase activity and here we demonstrate that both X11 alpha and X11 beta interact with presenilin-1. X11/presenilin-1 binding is via two X11 PDZ domains and sequences within the carboxy-terminus of presenilin-1. We also demonstrate that both X11 alpha and X11 beta mediate the formation of complexes between APP and presenilin-1. These results suggest that the X11 regulation of APP processing is controlled, at least in part, via their interactions with APP and presenilin-1.

Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter.

Glycogen synthase kinase 3beta (GSK-3beta) is a proline-directed kinase that forms part of the wingless signaling pathway. Recent studies have shown that GSK-3beta phosphorylates the microtubule-associated protein tau in vitro and in cell culture. Tau is the principal component of the paired helical filaments (PHFs) found in the brains of patients with Alzheimer disease, and PHF-tau is hyperphosphorylated. GSK-3beta is therefore one of the candidate kinases for phosphorylating tau in Alzheimer disease. GSK-3beta activity is negatively regulated by phosphorylation on serine 9 and positively regulated by phosphorylation on tyrosine 216. However, since overexpression of GSK-3beta by transfection leads to increased activity in the absence of any stimuli, GSK-3beta activity may also be regulated at the transcriptional level. Indeed, increased GSK-3beta protein levels are found in Alzheimer disease brains, and GSK-3beta is found associated with PHFs in Alzheimer disease. To understand how GSK-3beta activity may be regulated at the transcriptional level, we have isolated the human GSK-3beta promoter. The GSK-3beta promoter does not contain a conventional TATA box although several other transcription factor binding sites were identified. A putative transcription start site was mapped by 5' RACE. Transfection of various GSK-3beta promoter CAT reporter genes into both COS-7 cells and SHSY5Y neuronal cells revealed that the GSK-3beta promoter is more active in neuronal cells. Such transfection studies involving promoter deletion mutants revealed that a negative transcriptional response element may be present at position -1421 to -1363 and an activator sequence at position -427 to -384. CP2 binding sites were also present within the promoter. CP2 has recently been shown to interact with the Alzheimer disease amyloid precursor protein binding protein Fe65. The significance of these results with respect to Alzheimer disease pathogenesis are discussed.

Expression analysis of glycogen synthase kinase-3 in human tissues.

Human glycogen synthase kinase-3 (GSK-3) is a multisubstrate, proline-directed kinase that phosphorylates tau, beta-amyloid and neurofilaments. In this study, the expression levels of the two GSK-3 isoforms, alpha and beta, RNA and proteins in different human tissues were examined. Northern analysis demonstrated that GSK-3alpha is encoded by a 2.6-kb mRNA and GSK-3beta by 8.3- and 2.8-kb mRNAs. The two GSK-3beta mRNA species were variably expressed in different tissues. Northern and quantitative polymerase chain reaction demonstrated that both GSK-3alpha and GSK-3beta mRNA were prominently expressed in testis, thymus, prostate and ovary but were low in adult lung and kidney. Western blot analysis showed that the 51-kDa GSK-3alpha protein was highly expressed in lung, ovary, kidney and testis, whereas the 46-kDa GSK-3beta protein was highly expressed in lung, kidney and brain. The differential expression of GSK-3alpha and GSK-3beta mRNA and proteins and the lack of relationship between transcription and translation in some tissues indicate that GSK-3alpha and GSK-3beta are subject to different means of regulation.

A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation, typically starting in early adulthood. IBD is subdivided into two subtypes, on the basis of clinical and histologic features: Crohn disease and ulcerative colitis (UC). Previous genomewide searches identified regions harboring susceptibility loci on chromosomes 1, 3, 4, 7, 12, and 16. To expand our understanding of the genetic risk profile, we performed a 9-cM genomewide search for susceptibility loci in 268 families containing 353 affected sibling pairs. Previous linkages on chromosomes 12 and 16 were replicated, and the chromosome 4 linkage was extended in this sample. New suggestive evidence for autosomal linkages was observed on chromosomes 1, 6, 10, and 22, with LOD scores of 2.08, 2.07, 2.30, and 1.52, respectively. A maximum LOD score of 1.76 was observed on the X chromosome, for UC, which is consistent with the clinical association of IBD with Ullrich-Turner syndrome. The linkage finding on chromosome 6p is of interest, given the possible contribution of human leukocyte antigen and tumor necrosis-factor genes in IBD. This genomewide linkage scan, done with a large family cohort, has confirmed three previous IBD linkages and has provided evidence for five additional regions that may harbor IBD predisposition genes.

Isolation and chromosomal mapping of human glycogen synthase kinase-3 alpha and -3 beta encoding genes.

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that exists as two isoforms, alpha and beta, encoded by separate genes. Phosphorylation targets include a variety of cytoplasmic and nuclear proteins. Recent studies found that neurofilaments, amyloid precursor protein, and tau proteins are substrates of GSK-3 and that aberrant phosphorylation of these proteins is implicated in pathologies of the nervous system. To analyse the organisation of these two genes, a YAC library was screened by polymerase chain reaction, using primers specific for human GSK-3 alpha and GSK-3 beta cDNA. Two clones, 220 and 285 kb in size, containing the complete GSK-3 alpha coding sequence, and two clones, 365 and 285 kb in size, containing the 5' coding sequence of GSK-3 beta, were isolated. By somatic cell hybrid panel DNA amplification and radiation hybrid mapping, GSK-3 alpha was found to be located at 19q13.2. On the other hand, by somatic cell hybrid panel DNA amplification and fluorescence in situ hybridisation using the 285-kb YAC clone, GSK-3 beta was mapped to 3q13.3.

Genetic analysis of inflammatory bowel disease in a large European cohort supports linkage to chromosomes 12 and 16.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a complex disorder of unknown etiology. Epidemiological investigations suggest a genetic basis for IBD. Recent genetic studies have identified several IBD linkages. The significance of these linkages will be determined by studies in large patient collections. The aim of this study was to replicate IBD linkages on chromosomes 12 and 16 in a large European cohort. METHODS: Three hundred fifty-nine affected sibling pairs from 274 kindreds were genotyped using microsatellite markers spanning chromosomes 12 and 16. Affection status of the sibling pairs was defined as Crohn's disease (CD) or ulcerative colitis (UC). RESULTS: Nonparametric statistical analyses showed linkage for both chromosomes. Two-point results for chromosome 12 peaked at D12S303 (logarithm of odds [LOD], 2.15; P = 0.003) for CD and at D12S75 (LOD, 0.92; P = 0.03) for UC. Multipoint analyses produced a peak LOD of 1.8 for CD. Chromosome 16 showed linkage for CD at marker D16S415 (LOD, 1.52; P = 0.007). Multipoint support peaked above markers D16S409 and D16S411 (LOD, 1.7). CONCLUSIONS: These data are consistent with linkage of IBD to chromosomes 12 and 16. The replication of genetic risk loci in a large independent family collection indicates important and common susceptibility genes in these regions and will facilitate identification of genes involved in IBD.