ABSTRACT
Frontotemporal dementia and amyotrophic lateral sclerosis are closely related clinical syndromes with overlapping molecular pathogenesis. Several families have been reported with members affected by frontotemporal dementia, amyotrophic lateral sclerosis or both, which show genetic linkage to a region on chromosome 9p21. Recently, two studies identified the FTD/ALS gene defect on chromosome 9p as an expanded GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 gene (C9ORF72). In the present study, we provide detailed analysis of the clinical features and neuropathology for 16 unrelated families with frontotemporal dementia caused by the C9ORF72 mutation. All had an autosomal dominant pattern of inheritance. Eight families had a combination of frontotemporal dementia and amyotrophic lateral sclerosis while the other eight had a pure frontotemporal dementia phenotype. Clinical information was available for 30 affected members of the 16 families. There was wide variation in age of onset (mean = 54.3, range = 34-74 years) and disease duration (mean = 5.3, range = 1-16 years). Early diagnoses included behavioural variant frontotemporal dementia (n = 15), progressive non-fluent aphasia (n = 5), amyotrophic lateral sclerosis (n = 9) and progressive non-fluent aphasia-amyotrophic lateral sclerosis (n = 1). Heterogeneity in clinical presentation was also common within families. However, there was a tendency for the phenotypes to converge with disease progression; seven subjects had final clinical diagnoses of both frontotemporal dementia and amyotrophic lateral sclerosis and all of those with an initial progressive non-fluent aphasia diagnosis subsequently developed significant behavioural abnormalities. Twenty-one affected family members came to autopsy and all were found to have transactive response DNA binding protein with M(r) 43 kD (TDP-43) pathology in a wide neuroanatomical distribution. All had involvement of the extramotor neocortex and hippocampus (frontotemporal lobar degeneration-TDP) and all but one case (clinically pure frontotemporal dementia) had involvement of lower motor neurons, characteristic of amyotrophic lateral sclerosis. In addition, a consistent and relatively specific pathological finding was the presence of neuronal inclusions in the cerebellar cortex that were ubiquitin/p62-positive but TDP-43-negative. Our findings indicate that the C9ORF72 mutation is a major cause of familial frontotemporal dementia with TDP-43 pathology, that likely accounts for the majority of families with combined frontotemporal dementia/amyotrophic lateral sclerosis presentation, and further support the concept that frontotemporal dementia and amyotrophic lateral sclerosis represent a clinicopathological spectrum of disease with overlapping molecular pathogenesis.
Subject(s)
Chromosomes, Human, Pair 9/genetics , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Mutation/genetics , Proteins/genetics , Adult , Age of Onset , Aged , Aged, 80 and over , Alleles , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/psychology , Autopsy , C9orf72 Protein , DNA/genetics , Executive Function , Female , Frontotemporal Dementia/psychology , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Magnetic Resonance Imaging , Male , Memory Disorders/etiology , Memory Disorders/psychology , Middle Aged , Neuropsychological Tests , Polymerase Chain Reaction , Speech IntelligibilityABSTRACT
The nematode C. elegans uses mechanosensitive neurons to detect bacteria, which are food for worms. These neurons release dopamine to suppress foraging and promote dwelling. Through a screen of genes highly expressed in dopaminergic food-sensing neurons, we identify a K2P-family potassium channel-TWK-2-that damps their activity. Strikingly, loss of TWK-2 restores mechanosensation to neurons lacking the NOMPC-like channel transient receptor potential 4 (TRP-4), which was thought to be the primary mechanoreceptor for tactile food sensing. The alternate mechanoreceptor mechanism uncovered by TWK-2 mutation requires three Deg/ENaC channel subunits: ASIC-1, DEL-3, and UNC-8. Analysis of cell-physiological responses to mechanostimuli indicates that TRP and Deg/ENaC channels work together to set the range of analog encoding of stimulus intensity and to improve signal-to-noise characteristics and temporal fidelity of food-sensing neurons. We conclude that a specialized mechanosensory modality-tactile food sensing-emerges from coordination of distinct force-sensing mechanisms housed in one type of sensory neuron.
Subject(s)
Caenorhabditis elegans Proteins , Touch Perception , Animals , Caenorhabditis elegans/metabolism , Ion Channels/genetics , Touch/physiology , Sensory Receptor Cells/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Acid Sensing Ion Channels , Epithelial Sodium ChannelsABSTRACT
Two studies recently identified a GGGGCC hexanucleotide repeat expansion in a non-coding region of the chromosome 9 open-reading frame 72 gene (C9ORF72) as the cause of chromosome 9p-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In a cohort of 231 probands with ALS, we identified the C9ORF72 mutation in 17 familial (27.4%) and six sporadic (3.6%) cases. Patients with the mutation presented with typical motor features of ALS, although subjects with the C9ORF72 mutation had more frequent bulbar onset, compared to those without this mutation. Dementia was significantly more common in ALS patients and families with the C9ORF72 mutation and was usually early-onset FTD. There was striking clinical heterogeneity among the members of individual families with the mutation. The associated neuropathology was a combination of ALS with TDP-ir inclusions and FTLD-TDP. In addition to TDP-43-immunoreactive pathology, a consistent and specific feature of cases with the C9ORF72 mutation was the presence of ubiquitin-positive, TDP-43-negative inclusions in a variety of neuroanatomical regions, such as the cerebellar cortex. These findings support the C9ORF72 mutation as an important newly recognized cause of ALS, provide a more detailed characterization of the associated clinical and pathological features and further demonstrate the clinical and molecular overlap between ALS and FTD.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Brain/pathology , Frontotemporal Dementia/genetics , Mutation , Proteins/genetics , Spinal Cord/pathology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Brain/metabolism , C9orf72 Protein , Chromosomes, Human, Pair 9 , DNA-Binding Proteins/metabolism , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Humans , Proteins/metabolism , Spinal Cord/metabolismABSTRACT
We studied the self-assembly of inorganic nanocrystals (NCs) confined inside nanoliter droplets (plugs) into long-range ordered superlattices. We showed that a capillary microfluidic platform can be used for the optimization of growth conditions for NC superlattices and can provide insights into the kinetics of the NC assembly process. The utility of our approach was demonstrated by growing large (up to 200 µm) three-dimensional (3D) superlattices of various NCs, including Au, PbS, CdSe, and CoFe(2)O(4). We also showed that it is possible to grow 3D binary nanoparticle superlattices in the microfluidic plugs.
ABSTRACT
This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.
Subject(s)
Chromosome Mapping/instrumentation , DNA, Bacterial/genetics , Microfluidic Analytical Techniques/instrumentation , Microscopy, Fluorescence/instrumentation , Polymerase Chain Reaction/instrumentation , Signal Processing, Computer-Assisted/instrumentation , Staphylococcus aureus/genetics , Equipment Design , Equipment Failure AnalysisABSTRACT
INTRODUCTION: We developed an automated liquid chromatography-tandem mass spectrometry high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for multiplex quantification of wild-type (wt) amyloid ß (Aß) peptides 1-40 (Aß40) and 1-42 (Aß42) and detection of variant Aß peptides in cerebrospinal fluid. METHODS: The multiplex Aß HPLC-MS/MS assay was validated in a clinically accredited laboratory following regulatory guidelines, with Aß42 calibration assigned to the ERM/IFCC certified reference material; sequence variants were additionally multiplexed into the method. RESULTS: Sample preparation was fully automated on a liquid handler. The assay quantified wt-Aß42 and wt-Aß40 and detected sequence variants, when present, within the Aß42 sequence. DISCUSSION: Extension of the HPLC-MS/MS approach for quantification of wt-Aß42 and wt-Aß40 to include known sequence variants increases analytical accuracy of the mass spectrometric approach and enables identification of cases of autosomal dominant Alzheimer's disease. Development of an automated workflow and selection of appropriate instrumentation enabled deployment of this method in routine clinical testing.
ABSTRACT
Recent studies in our laboratory have demonstrated that angiotensin (ANG) II and endothelin (ET) 1 up-regulate the expression of arginine vasopressin V(2) receptor in the inner medullary collecting duct (IMCD) of the rat. The present studies were performed to explore the interaction between ANG II and ET-1 in up-regulating the expression of arginine vasopressin V(2) receptor in the IMCD of the rat. Two sets of studies were done. In the first set of studies, rat IMCD tissue was isolated and incubated with ANG II in combination with ET(A) or ET(B) antagonist. In the second set of experiments, rat IMCD tissue was incubated with ET-1 with ANG receptor antagonist saralasin. Tissue samples were then analyzed by means of quantitative reverse transcriptase polymerase chain reaction and Western blotting. The ANG II treatment resulted in increased V(2) messenger RNA (mRNA) from control level of 138 +/- 12 amol/microg of total RNA to 385 +/- 63 amol/microg of total RNA (P < .01). The ANG II/ET(A) treatment resulted in no significant decrease in V(2) mRNA expression (319 +/- 59 amol/microg of total RNA), whereas ET-1/ET(B) antagonist and ET-1/ET(A)/ET(B) antagonist treatments resulted in reducing V(2) mRNA to control levels of 214 +/- 25 and 176 +/- 22 amol/microg of total RNA, respectively. The ET-1 treatment increased V(2) mRNA expression from control level of 221 +/- 25 amol/microg of total RNA to 383 +/- 43 amol/microg of total RNA (P < .02). The ET-1-induced increase in V(2) mRNA expression was significantly reduced to control level (210 +/- 36 amol/microg of total RNA) after saralasin treatment. Western blotting revealed that changes in protein expression in the different treatment conditions were comparable with changes in V(2) mRNA expression. These data suggested that the up-regulation of V(2) receptor induced by ANG II and ET-1 is mediated by both vasoconstricting hormones. These 2 systems interact in up-regulating the expression of V(2) receptors in the kidney.
Subject(s)
Angiotensin II/pharmacology , Endothelins/pharmacology , Kidney Medulla/metabolism , Kidney Tubules, Collecting/metabolism , RNA, Messenger/biosynthesis , Vasopressins/biosynthesis , Vasopressins/genetics , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Blotting, Western , Endothelin A Receptor Antagonists , Endothelin B Receptor Antagonists , Kidney Medulla/drug effects , Kidney Tubules, Collecting/drug effects , RNA, Messenger/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction , Saralasin/pharmacology , Up-Regulation/drug effectsABSTRACT
The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.
Subject(s)
Actinin/metabolism , Actins/metabolism , Cytokinesis/physiology , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/metabolism , Actin Cytoskeleton/metabolism , Actomyosin/metabolism , HumansABSTRACT
We performed case-control studies using 2 Canadian cohorts to examine the role of 10 promising Alzheimer's disease (AD) loci identified in recent genomewide association studies. Patients age 65 years and older diagnosed with AD at baseline (prevalent cases) or who developed AD during follow-up assessments (incident cases) were compared with control subjects with no cognitive impairment. Our prevalent case study comparing prevalent AD cases (n = 428) with participants with no cognitive impairment (n = 524) revealed a significant association of rs6656401 and rs3818361 (CR1), rs2075650 (TOMM40), rs7561528 (BIN1), and rs3865444 (CD33) with late-onset AD that were robust to adjustment with age and apolipoprotein E ε4 genotype. The incident case study comparing patients who developed AD during longitudinal observation (n = 152) with participants with no cognitive impairment found that rs2075650 (TOMM40) and rs3865444 (CD33) influence the risk of developing AD in this population. In addition, pooled analysis of our AD patients confirmed that CR1, TOMM40, BIN1, and CD33 contribute to late-onset AD susceptibility, in addition to apolipoprotein E.
Subject(s)
Alzheimer Disease/epidemiology , Alzheimer Disease/genetics , Genetic Predisposition to Disease/epidemiology , Genetic Predisposition to Disease/genetics , Adaptor Proteins, Signal Transducing/genetics , Aged , Aged, 80 and over , Apolipoprotein E4/genetics , Canada/epidemiology , Case-Control Studies , Cohort Studies , Female , Genotype , Humans , Male , Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Nuclear Proteins/genetics , Polymorphism, Single Nucleotide , Prevalence , Receptors, Complement 3b/genetics , Risk , Sialic Acid Binding Ig-like Lectin 3/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Despite the growing number of genome-wide association studies, the involvement of polymorphisms in microRNA target sites (polymiRTS) in Alzheimer's disease (AD) remains poorly investigated. Recently, we have shown that AD-associated single-nucleotide polymorphisms (SNPs) present in the 3' untranslated region (3'UTR) of amyloid precursor protein (APP) could directly affect miRNA function. In theory, loss of microRNA (miRNA) function could lead to risk for AD by increasing APP expression and Aß peptide production. In this study, we tested the hypothesis that Nicastrin, a γ-secretase subunit involved in Aß generation, could be regulated by miRNAs, and consequently affected by 3'UTR polymorphisms. Bioinformatic analysis identified 22 putative miRNA binding sites located in or near Nicastrin 3'UTR polymorphisms. From these miRNA candidates, six were previously shown to be expressed in human brain. We identified miR-24, miR-186, and miR-455 as regulators of Nicastrin expression, both in vitro and under physiological conditions in human cells, which resulted in altered Aß secretion. Using luciferase-based assays, we further demonstrated that rs113810300 and rs141849450 SNPs affected miRNA-mediated repression of Nicastrin. Notably, rs141849450 completely abolished the miR-455-mediated repression of Nicastrin. Finally, the rs141849450 variant was identified in 1 out of 511 AD cases but not in 631 controls. These observations set the stage for future studies exploring the role of miRNAs and 3'UTR polymorphisms in AD.
ABSTRACT
OBJECTIVE: To assess the influence of rs5848 polymorphism in serum progranulin (PGRN) level in a cohort of subjects with Alzheimer and related dementias from a tertiary referral clinic. BACKGROUND: Mutations in the GRN gene cause autosomal dominant frontotemporal dementia (FTD) with TDP-43 pathology (FTLD-TDP) through haploinsufficiency. It has recently been shown that homozygous carriers of the T allele of rs5848 have an elevated risk of developing FTD, and this polymorphism may play a role in the pathogenesis of other dementia by modifying progranulin level. We hypothesize that genotype of rs5848 may influence serum PGRN level in AD, FTD, and other dementias. METHODS: Blood samples were obtained from patients with cognitive impairment and dementia referred to a tertiary dementia clinic, as well as samples from a cohort of healthy controls. Serum PGRN level was measured using an ELISA assay, and rs5848 genotype was determined by a TaqMan assay. RESULTS: We found that rs5848 SNP significantly influenced serum PGRN level, with TT genotype having the lowest levels, and CC as the highest. This relationship is observed in each of the subgroups. We also confirmed that GRN mutation carriers had significantly lower serum PGRN levels than all other groups. CONCLUSIONS: The rs5848 polymorphism significantly influences serum PGRN with TT carriers having a lower level of serum PGRN then CT and CC carriers. This is consistent with the finding that miR-659 binding to the high risk T allele of rs5848 may augment translational inhibition of GRN and alter risk of FTD and possibly other dementias.