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Peter R Schofield

Publications and source records attributed to Peter R Schofield.

36 records · Page 2Linked to original sources
PubMed2004

Positional effects of presenilin-1 mutations on tau phosphorylation in cortical plaques.

Mutations in presenilin-1 (PS-1) account for the majority of familial Alzheimer's disease (AD). While increasing Abeta42 is one mechanism whereby PS-1 mutations are thought to exert their pathogenic effect, little is known about the role of tau in PS-1 AD. This study compares staining (AT8 and tau-2), morphology and quantity of tau-immunoreactive cortical plaques in six PS-1 and five sporadic AD cases. The densities of tau-positive plaques differentiated PS-1 from sporadic AD cases. All PS-1 cases demonstrated a greater than 6-fold increase in tau-2-positive plaques. In PS-1 cases with mutations in exons 5 and 6, there was an increase in classical AD plaques containing hyperphosphorylated tau (AT8- and tau 2-positive). However, cases with exon 8 and 9 mutations had numerous cotton wool plaques containing nonhyperphosphorylated tau (tau-2-positive, AT8-negative). These findings suggest that PS-1 mutations increase tau deposition while mutation-specific cellular responses determine phosphorylation events and may influence cell death mechanisms.

Adult↗
PubMed2004

Ligand-gated ion channels: mechanisms underlying ion selectivity.

Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABA(A) and GABA(C) receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it.

Animals↗
PubMed2004

In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system.

We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.

Animals↗
PubMed2003

The contribution of proline 250 (P-2') to pore diameter and ion selectivity in the human glycine receptor channel.

The glycine receptor-channel (GlyR) mediates neuronal inhibition by selectively allowing the passage of Cl(-) ions through its channel. The pore region for ion selectivity is localised to the constricted internal end of the M2 transmembrane domain. This paper investigates the contribution of the P-2' residue in determining pore diameter and ion charge selectivity of the GlyR. The deletion of this proline has been shown to decrease the anion/cation permeability ratio, with P(Cl)/P(Na) decreasing from approximately 27 to approximately 4. We show that the P-2' deletion by itself produces a GlyR with a larger pore diameter ( approximately 0.69 nm) than the wild type value ( approximately 0.54 nm). This confirms that the P-2' residue reduces pore size, which suggests that, in addition to electrostatic effects, pore size also contributes to ion-charge selectivity.

Amino Acid Sequence↗
PubMed2003

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor.

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.

Acetylcholine↗
PubMed2003

Genome scan meta-analysis of schizophrenia and bipolar disorder, part III: Bipolar disorder.

Genome scans of bipolar disorder (BPD) have not produced consistent evidence for linkage. The rank-based genome scan meta-analysis (GSMA) method was applied to 18 BPD genome scan data sets in an effort to identify regions with significant support for linkage in the combined data. The two primary analyses considered available linkage data for "very narrow" (i.e., BP-I and schizoaffective disorder-BP) and "narrow" (i.e., adding BP-II disorder) disease models, with the ranks weighted for sample size. A "broad" model (i.e., adding recurrent major depression) and unweighted analyses were also performed. No region achieved genomewide statistical significance by several simulation-based criteria. The most significant P values (<.01) were observed on chromosomes 9p22.3-21.1 (very narrow), 10q11.21-22.1 (very narrow), and 14q24.1-32.12 (narrow). Nominally significant P values were observed in adjacent bins on chromosomes 9p and 18p-q, across all three disease models on chromosomes 14q and 18p-q, and across two models on chromosome 8q. Relatively few BPD pedigrees have been studied under narrow disease models relative to the schizophrenia GSMA data set, which produced more significant results. There was no overlap of the highest-ranked regions for the two disorders. The present results for the very narrow model are promising but suggest that more and larger data sets are needed. Alternatively, linkage might be detected in certain populations or subsets of pedigrees. The narrow and broad data sets had considerable power, according to simulation studies, but did not produce more highly significant evidence for linkage. We note that meta-analysis can sometimes provide support for linkage but cannot disprove linkage in any candidate region.

Bipolar Disorder↗
PubMed2003

Kinetic determinants of agonist action at the recombinant human glycine receptor.

The amino acids glycine, beta-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, beta-alanine and taurine upon homomeric alpha1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10-90 % rise times determined from rapid application of 100 microM of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mM) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for beta-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for beta-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > beta-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.

Electrophysiology↗
PubMed2003

Genetic refinement and physical mapping of a 2.3 Mb probable disease region associated with a bipolar affective disorder susceptibility locus on chromosome 4q35.

A susceptibility locus for bipolar affective disorder has been mapped to chromosome 4q35 in a large multigenerational pedigree. We have expanded this analysis to include 55 pedigrees (674 individuals, 214 affecteds). The evidence for linkage to 4q35 was strengthened in this larger cohort, with a maximum two-point LOD score of 3.2 for marker D4S1652. Several other markers in the region gave LOD scores greater than 1.5. Non-parametric analysis provided additional support for linkage to the 4q35 region. To further refine this region, haplotype analysis was carried out in 16 of the 55 pedigrees that showed evidence of linkage. As there is no evidence for an ancestral haplotype, nor a one-to-one correspondence between the disease and putative disease haplotype, we undertook an analysis based on pedigree-specific, identical-by-descent allele-sharing in order to define a probable disease region. This analysis indicated that the percentage sharing of alleles, identical-by-descent, in affecteds of all linked pedigrees increases from 60% at the centromeric markers to 75% for markers at the telomere. Maximal allele sharing occurred between markers D4S3051 and 4qTEL13 with this 24 cM region defining a probable disease region. We have constructed a physical map of the 4q35 interval consisting of a YAC contig and BAC clones. Based on this map the probable disease region between D4S3051 and 4qTEL13 corresponds to only 2.3 Mb. This region is very gene poor with only three known genes indicated from the YAC/BAC map. The small number of genes will facilitate systematic screening for variations associated with bipolar disorder.

Bipolar Disorder↗
PubMed2003

Alzheimer's disease with spastic paraparesis and 'cotton wool' plaques: two pedigrees with PS-1 exon 9 deletions.

Several pedigrees have recently been reported in which dominantly inherited familial Alzheimer's disease is associated in some family members with spastic paraparesis and non-neuritic 'cotton wool' plaques. Here we report clinical, genetic and neuropathological findings in two further large pedigrees in which this combination of phenotypes is associated with a deletion of exon 9 of the presenilin-1 (PS-1) gene caused by mutations at the splice acceptor site. In both pedigrees, individuals with paraparesis at presentation had a later than average age at onset of symptoms. In addition, one subject with paraparesis had a much less prominent dementia syndrome than his dementia-affected siblings. As PS-1 mutations are almost always associated with a particularly aggressive form of presenile dementia, these findings suggest the existence of a protective or delaying factor in individuals with spastic paraparesis.

Adult↗
PubMed2003

Mutations in the tau gene that cause an increase in three repeat tau and frontotemporal dementia.

The majority of cases with frontotemporal dementia (FTD) have no tau deposition in the brain, yet mutations in the tau gene lead to a similar clinical phenotype with insoluble tau depositing in neuropathological lesions. We report two tau gene mutations at positions +19 and +29, in the intronic sequences immediately following the stem loop structure in exon 10, which segregate with FTD. Exon-trapping experiments showed that these gene mutations alter the splicing out of exon 10 and produce an increase in tau isoforms with three microtubule binding domains (three repeat tau). Mutagenesis experiments demonstrated that the +19 mutation was responsible for the increase in three repeat tau, possibly by altering an intron silencer modulator sequence element found at this region of the gene. Microtubule binding experiments revealed a significant decrease in microtubule assembly with increasing amounts of three and decreasing amounts of four repeat tau. Brain autopsy was available in one case. Analysis of the type of soluble tau isoforms revealed an increase in three repeat tau and an absence of tau isoforms with exon 3 inserts. No insoluble tau was isolated in the tissue fractions, consistent with the absence of tau-positive histopathology. There was also an increase in tau degradation products suggestive of increased proteolysis. This increase in tau breakdown products was associated with TUNEL- and activated caspase-3-positive neurons identified histologically. These studies show that increases in soluble three repeat tau can be responsible for FTD in cases with tau gene mutations in the intronic region immediately adjacent to the stem loop in exon 10. These cases of FTD have tau isoforms (without exon 3 inserts) that do not form abnormal aggregates and appear more prone to proteolysis. The increase in tau proteolysis was associated with increased evidence of apoptosis. This mechanism of neurodegeneration may be more applicable to the majority of FTD cases, which do not accumulate insoluble tau deposits.

Alternative Splicing↗
PubMed2002

Presenilin-1 mutation L271V results in altered exon 8 splicing and Alzheimer's disease with non-cored plaques and no neuritic dystrophy.

The mutation L271V in exon 8 of the presenilin-1 (PS-1) gene was detected in an Alzheimer's disease pedigree. Neuropathological examination of affected individuals identified variant, large, non-cored plaques without neuritic dystrophy, reminiscent of cotton wool plaques. Biochemical analysis of L271V mutation showed that it increased secretion of the 42-amino acid amyloid-beta peptide, suggesting a pathogenic mutation. Analysis of PS-1 transcripts from the brains of two mutation carriers revealed a 17-50% increase in PS-1 transcripts with deletion of exon 8 (PS-1deltaexon8) compared with unrelated Alzheimer's disease brains. Exon trapping analysis confirmed that L271V mutation enhanced the deletion of exon 8. Western blots of brain lysates indicated that PS-1deltaexon8 was overexpressed in an affected individual. Biochemical analysis of PS-1deltaexon8 in COS and BD8 cells indicate the splice isoform is not intrinsically active but interacts with wild-type PS-1 to generate amyloid-beta. Western blots of cell lysates immunoprecipitated with anti-Tau or anti-GSK-3beta antibodies indicated that PS-1deltaexon8, unlike wild-type PS-1, does not interact directly with Tau or GSK-3beta, potential modifiers of neuritic dystrophy. We postulate that variant plaques observed in this family are due in part to the effects of PS-1deltaexon8 and that interaction between PS-1 and various protein complexes are necessary for neuritic plaque formation.

Adult↗
PubMed2002

A missense mutation in kynurenine aminotransferase-1 in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR show elevated sympathetic nerve activity, and stimulation of the central blood pressure control centers with glutamate or nicotine results in exaggerated blood pressure responses, effects that appear to be genetically determined. Kynurenic acid, a competitive glutamate antagonist and a non-competitive nicotinic antagonist, can be synthesized in the brain by the enzyme kynurenine aminotransferase-1 (KAT-1). We have previously shown that KAT-1 activity is significantly reduced in SHR compared with normotensive Wistar Kyoto rats (WKY). Here we show that KAT-1 contains a missense mutation, E61G, in all the strains of SHR examined but not in any of the WKY or outbred strains. Previous studies on F2 rats from a cross of stroke-prone SHR and WKY have shown a suggestive level of linkage between elevated blood pressure and the KAT-1 locus on chromosome 3. In addition, the mutant enzyme expressed in Escherichia coli displays altered kinetics. This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in SHR that may be related to an underlying mechanism of hypertension and increased sensitivity to stroke.

Alleles↗
PubMed2002

Intraneuronal advanced glycation endproducts in presenilin-1 Alzheimer's disease.

The most frequently mutated gene resulting in dominantly inherited Alzheimer's disease is presenilin-1. We have used antibodies against advanced glycation endproducts (AGE) in brain tissue sections of four patients with three different presenilin I mutations. Accumulation of intracellular AGE was observed in 75-95% of pyramidal neurons in patients with presenilin-1 mutations, far exceeding the percentage of presenilin-1-, tau- or ubiquitin-positive neurons. This high level of AGE-modified proteins in vulnerable neurons is most likely explained by higher levels of their precursors (reactive (di)carbonyl products) or a slower turnover of the participating proteins. These conditions of carbonyl stress may contribute to increased neuronal dysfunction and vulnerability leading to the early disease onset.

Adult↗
PubMed2002

Hyperekplexia associated with compound heterozygote mutations in the beta-subunit of the human inhibitory glycine receptor (GLRB).

Hyperekplexia (MIM: 149400) is a neurological disorder characterized by an excessive startle response which can be caused by mutations in the alpha1-subunit (GLRA1) of the heteropentameric human inhibitory glycine receptor (hGlyR). These receptors facilitate fast-response, inhibitory glycinergic neurotransmission in the brainstem and spinal cord leading to a rapid modification and reduction of the excitatory startle response. Mutations in the beta-subunit of GlyR (glrb) occur in a murine model of hyperekplexia (spastic), but have not been detected in human hyperekplexia. Following mutation analysis of the human beta-subunit of hGlyR (GLRB) in a cohort of 22 hyperekplexia patients, we provide evidence to confirm that GLRB mutations can cause human hyperekplexia. A missense (G920A resulting in G229D) and a splice site mutation (IVS5+5G-->A) occurred together in a compound heterozygote with a transient hyperekplexia phenotype. Exon trap analysis revealed that IVS5+5G-->A results in the exclusion of exon 5 from GLRB transcripts. Electrophysiological studies showed reduced sensitivity to agonist mediated activation of the alpha1beta (G229D) GlyR suggesting that GlyR beta-subunits are not restricted to conferring modulatory influences and maintaining structural integrity, but may also play a functional role in hGlyR ligand binding.

Child, Preschool↗
PubMed2002

Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity.

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective alpha1 homomeric glycine receptor (alpha1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with P(Cl)/P(Na) = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'Delta mutant GlyR retained its anion selectivity (P(Cl)/P(Na) = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P(Cl)/P(Na) = 27.9). When the A-1'E and the P-2'Delta mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P(Cl)/P(Na) = 0.13). The SDM GlyR was also Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-permeable channel (P(Ca)/P(Na) = 0.73), without drastically altering monovalent charge selectivity (P(Cl)/P(Na) = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca(2+) permeability (P(Ca)/P(Na) = 0.92), with little effect on monovalent selectivity (P(Cl)/P(Na) = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the alpha1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.

Amino Acid Sequence↗
PubMed2002

Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels.

Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

Amino Acid Sequence↗
PubMed2001

Familial idiopathic basal ganglia calcification (Fahr's disease) without neurological, cognitive and psychiatric symptoms is not linked to the IBGC1 locus on chromosome 14q.

Idiopathic basal ganglia calcification (IBGC) is characterised by radiological, neurological, cognitive and psychiatric abnormalities. The associations between these abnormal phenotypes and abnormal genes remain unclear despite the recent mapping to chromosome 14q of a susceptibility locus for IBGC ( IBGC1). We identified two siblings, from a large multigenerational pedigree, who had both been diagnosed with radiological IBGC, dementia, bipolar affective disorder and Parkinsonism. We assessed (1) other family members to determine whether these four phenotypes were co-segregating as symptoms of IBGC, and (2) possible IBGC linkage to the IBGC1 locus on chromosome 14q or to any known or potential dementia genes. Nine second-generation and 21 third-generation members received radiological, neurological, neuropsychological and psychiatric assessments. We genotyped all family members for microsatellite markers at the IBGC1 locus and polymorphisms of the ApoE, VLDL, alpha1-ACT, BChE-K, APP, PS1, PS2 and tau genes and tested these for linkage to IBGC, dementia and bipolar disorder. Of the ten family members with radiological intracranial calcification, all except the two index cases were normal. There was no significant association between IBGC status and severe cognitive impairment or dementia ( P=0.335) or bipolar affective disorder or Parkinsonism ( P=1.0). Linkage to the IBGC1 locus was excluded. Of the eight dementia gene markers tested, the only positive LOD score was for the ApoE epsilon4 polymorphism and dementia/severe cognitive impairment. We have identified a form of IBGC in which calcification is inherited independently of neurological, cognitive and psychiatric symptoms. This may represent a second locus for this disorder.

Basal Ganglia Diseases↗