Biomedical subjects

David R Lynch

Publications and source records attributed to David R Lynch.

At least 19 recordsLinked to original sources

PubMed2006

Polymicrogyria and deletion 22q11.2 syndrome: window to the etiology of a common cortical malformation.

Several brain malformations have been described in rare patients with the deletion 22q11.2 syndrome (DEL22q11) including agenesis of the corpus callosum, pachygyria or polymicrogyria (PMG), cerebellar anomalies and meningomyelocele, with PMG reported most frequently. In view of our interest in the causes of PMG, we reviewed clinical data including brain-imaging studies on 21 patients with PMG associated with deletion 22q11.2 and another 11 from the literature. We found that the cortical malformation consists of perisylvian PMG of variable severity and frequent asymmetry with a striking predisposition for the right hemisphere (P = 0.008). This and other observations suggest that the PMG may be a sequela of abnormal embryonic vascular development rather than a primary brain malformation. We also noted mild cerebellar hypoplasia or mega-cisterna magna in 8 of 24 patients. Although this was not the focus of the present study, mild cerebellar anomalies are probably the most common brain malformation associated with DEL22q11.

Cerebellum↗

PubMed2006

Cytosolic catechols inhibit alpha-synuclein aggregation and facilitate the formation of intracellular soluble oligomeric intermediates.

Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2006

Developmental and cell-selective variations in N-methyl-D-aspartate receptor degradation by calpain.

NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties.

Aging↗

PubMed2006

Senataxin, the yeast Sen1p orthologue: characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease.

A severe recessive cerebellar ataxia, Ataxia-Oculomotor Apraxia 2 (AOA2) and a juvenile onset form of dominant amyotrophic lateral sclerosis (ALS4) result from mutations of the Senataxin (SETX) gene. To begin characterization this disease protein, we developed a specific antibody to the DNA/RNA helicase domain of SETX. In murine brain, SETX concentrates in several regions, including cerebellum, hippocampus and olfactory bulb with a general neuronal expression profile, colocalizing with NeuN. In cultured cells, we found that SETX was cytoplasmically diffuse, but in the nucleus, SETX was punctate, colocalizing with fibrillarin, a marker of the nucleolus. In differentiated non-cycling cells, nuclear SETX was not restricted to the nucleolus but was diffuse within the nucleoplasm, suggesting cell-cycle-dependent localization. SETX missense mutations cluster within the N-terminus and helicase domains. Flag tagging at the N-terminus caused protein mislocation to the nucleoplasm and failure to export to the cytoplasm, suggesting that the N-terminus may be essential for correct SETX localization. We report here the first characterization of SETX protein, which may provide future insights into a new mechanism leading to neuron death.

Adult↗

PubMed2006

Human immunodeficiency virus (HIV)-induced neurotoxicity: roles for the NMDA receptor subtypes.

Neuronal damage in human immunodeficiency virus type 1 (HIV-1) infection in the brain is thought to occur at least in part through NMDA receptor (NMDAR) excitation initiated by soluble neurotoxins from HIV-infected brain macrophages. Furthermore, brain regions enriched in NMDAR-2A (NR2A) and NMDAR-2B (NR2B) subunits, such as the hippocampus, are particularly vulnerable. Using cultured rat hippocampal cells and HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we examined the role of NR2A and NR2B in HIV/MDM-induced hippocampal neuronal death. We used the primary HIV-1 strain Jago derived from the CSF of an individual with HIV-associated dementia and that robustly replicates in MDM. We found the following: (1) hippocampal neuronal susceptibility to HIV/MDM excitotoxins varies according to the developmental expression patterns of NR2A and NR2B; (2) NMDAR activation by HIV/MDM results in neuronal calpain activation, which results in neuronal death; and (3) selective antagonists of homomeric NR2B/NR2B- and heteromeric NR2A/NR2B-containing NMDARs, as well as an inhibitor of calpain activity, afford neuroprotection against HIV/MDM. These studies establish a clear link between macrophage HIV infection, neuronal NR2A and NR2B activation, and calpain-mediated hippocampal neuronal death. They further suggest a dominant role for NR2A and NR2B in determining neuronal susceptibility in HIV-infected brain. Antagonists of NR2A and NR2B subunits as well as inhibitors of calpain activation offer attractive neuroprotective approaches against HIV in both developing and mature brain.

Animals↗

PubMed2006

Calpain and synaptic function.

Proteolysis by calpain is a unique posttranslational modification that can change integrity, localization, and activity of endogenous proteins. Two ubiquitous calpains, mu-calpain and m-calpain, are highly expressed in the central nervous system, and calpain substrates such as membrane receptors, postsynaptic density proteins, kinases, and phosphatases are localized to the synaptic compartments of neurons. By selective cleavage of synaptically localized molecules, calpains may play pivotal roles in the regulation of synaptic processes not only in physiological states but also during various pathological conditions. Activation of calpains during sustained synaptic activity is crucial for Ca2+-dependent neuronal functions, such as neurotransmitter release, synaptic plasticity, vesicular trafficking, and structural stabilization. Overactivation of calpain following dysregulation of Ca2+ homeostasis can lead to neuronal damage in response to events such as epilepsy, stroke, and brain trauma. Calpain may also provide a neuroprotective effect from axotomy and some forms of glutamate receptor overactivation. This article focuses on recent findings on the role of calpain-mediated proteolytic processes in potentially regulating synaptic substrates in physiological and pathophysiological events in the nervous system.

Animals↗

PubMed2005

N-methyl-D-aspartate receptor subtype mediated bidirectional control of p38 mitogen-activated protein kinase.

N-methyl-d-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentration-dependent increase in p38 phosphorylation (phospho-p38). However, in more mature neurons (>DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.

Animals↗

PubMed2005

Cleavage of alpha-synuclein by calpain: potential role in degradation of fibrillized and nitrated species of alpha-synuclein.

Alpha-synuclein (alpha-syn) is a major protein component of the neuropathological hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Neither the mechanism of alpha-syn fibrillization nor the degradative process for alpha-syn has been elucidated. Previously, we showed that wild-type, mutated, and fibrillar alpha-syn proteins are substrates of calpain I in vitro. In this study, we demonstrate that calpain-mediated cleavage near and within the middle region of soluble alpha-syn with/without tyrosine nitration and oxidation generates fragments that are unable to self-fibrillize. More importantly, these fragments prevent full-length alpha-syn from fibrillizing. Calpain-mediated cleavage of alpha-syn fibrils composed of wild-type or nitrated alpha-syn generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length alpha-syn to co-assemble. Therefore, calpain-cleaved soluble alpha-syn inhibits fibrillization, whereas calpain-cleaved fibrillar alpha-syn promotes further co-assembly. These results provide insight into possible disease mechanisms underlying synucleinopathies since the formation of alpha-syn fibrils could be causally linked to the onset/progression of these disorders.

Calpain↗

PubMed2005

The E46K mutation in alpha-synuclein increases amyloid fibril formation.

The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of alpha-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of alpha-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of alpha-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu(46) for an Ala also increased the assembly of alpha-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu(83) in the sixth repeat of alpha-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu(83) to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of alpha-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant alpha-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease.

Alanine↗

PubMed2005

Performance measures in Friedreich ataxia: potential utility as clinical outcome tools.

Although several neuroprotective agents have been proposed as potential therapies in Friedreich ataxia (FA), clinical trials of their efficacy are limited by a lack of sensitive outcome measures. We assessed whether performance measures (nine-hole peg test, the timed 25-foot walk, and low-contrast letter acuity) provide valid measures of disease status in FA. Scores for each measure correlated significantly with neurologic disability and disease duration. Rank correlations between scores for performance measures were moderate in magnitude, suggesting that the each test captures separate yet related dimensions of neurological function in FA. Linear regression models demonstrated that scores from the nine-hole peg test and the timed 25-foot walk (after reciprocal transformation) were predicted by age and triplet repeat length in patients with FA. In addition, comparison of the temporal courses of change for each performance measure demonstrated that scores from the timed 25-foot walk change early in the course of FA, nine-hole peg test scores change slowly over the full course of the disorder, and low-contrast letter acuity scores change in the later stages of the disease. Thus, a composite scale derived from these performance measures may provide the best overall measure for assessing disease progression throughout the illness.

Adult↗

PubMed2005

Effects of COMT genotype on behavioral symptomatology in the 22q11.2 Deletion Syndrome.

The 22q11.2 Deletion Syndrome (DiGeorge/velocardiofacial syndrome) is associated with elevated rates of psychosis, and is also characterized by severe attentional difficulties and executive dysfunction. Behavioral manifestations of this syndrome could result from haploinsufficiency of the catechol-O-methyltransferase (COMT) gene, located within the 22q11 region. The goal of the present study was to examine COMT genotype in relation to behavioral symptomatology in this syndrome. Val158/108Met was genotyped in 38 patients (16 Met/-, 22 Val/-) with confirmed 22q11.2 deletions who had received the Child Behavior Checklist (CBCL) as part of a comprehensive evaluation. Results indicated that the Val genotype was associated with significantly greater internalizing and externalizing behavioral symptomatology in children with 22q11.2 deletions. Val allele status was associated with a greater-than-four-fold increase in risk for clinically significant behavior problems in children with this syndrome. These data are consistent with previous findings of increased psychopathology associated with the Val genotype in normal individuals and suggest that a functional genetic polymorphism in the 22q11 region may influence behavior in individuals with COMT haploinsufficiency.

Catechol O-Methyltransferase↗

PubMed2005

N-methyl-D-aspartate receptor subtypes: multiple roles in excitotoxicity and neurological disease.

N-methyl-D-aspartate (NMDA) receptors are the major mediator of excitotoxicity. Although physiological activation of the NMDA receptor is necessary for cell survival, overactivation is a signal for cell death. Several pathways are activated through NMDA receptor stimulation, most of which can contribute to excitotoxicity. These include events leading to mitochondrial dysfunction, activation of calcium-dependent enzymes, and activation of mitogen-activated protein kinase pathways. Understanding the role of these mechanisms is important in developing agents that block excitotoxicity without inhibiting functions necessary for survival. NMDA receptor subtypes may be responsible for mediating separate pathways, and subtype-specific inhibition has shown promising results in some neurological models. This review examines the roles of NMDA receptor subtypes in excitotoxicity and neurological disorders.

Animals↗

PubMed2004

Interactions of postsynaptic density-95 and the NMDA receptor 2 subunit control calpain-mediated cleavage of the NMDA receptor.

The calcium-dependent protease calpain cleaves the NMDA receptor 2 (NR2) subunit of the NMDA receptor both in vitro and in vivo and thus potentially modulates NMDA receptor function and turnover. We examined the ability of postsynaptic density-95 (PSD-95) protein to alter the calpain-mediated cleavage of NR2A and NR2B. Coexpression of PSD-95 with NMDA receptors in human embryonic kidney 293 cells blocked cleavage of NR2A and NR2B by NMDA receptor-activated calpain. NR2A cleavage by calpain occurred in the cell surface and intracellular fractions and required the presence of NR1 subunits. The blocking effect of PSD-95 did not result from decreased calpain activity, lowered intracellular calcium responses, or the blockade of internalization. Instead, this effect was eliminated by deletion of the C-terminal ESDV motif of NR2A or by overexpression of a palmitoylation-deficient PSD-95 mutant lacking the ability to cluster and to interact with NMDA receptors in situ, suggesting a role for association between the C terminus of NR2A and clustered PSD-95. Synapse-associated protein 102, a membrane-associated guanylate kinase interacting with NR2A but lacking palmitoylation motifs and the ability to cluster, did not protect NR2A from cleavage by calpain. Pharmacological inhibition of palmitoylation disrupted the interaction of PSD-95 with NMDA receptors in cortical neurons and allowed NR2A to be cleaved by calpain, whereas NR2A could not be cleaved in untreated neurons. These results indicate that PSD-95 clustering and direct association of NR2A and PSD-95 mediate the blocking effect of PSD-95 on calpain cleavage. PSD-95 could regulate the susceptibility of NMDA receptors to calpain-mediated cleavage during synaptic transmission and excitotoxicity.

Amino Acid Motifs↗

PubMed2004

Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation.

Previous studies have shown the presence of nitrated alpha-synuclein (alpha-syn) in human Lewy bodies and other alpha-syn inclusions. Herein, the effects of tyrosine nitration on alpha-syn fibril formation, lipid binding, chaperone-like function, and proteolytic degradation were systematically examined by employing chromatographically isolated nitrated monomeric, dimeric, and oligomeric alpha-syn. Nitrated alpha-syn monomers and dimers but not oligomers accelerated the rate of fibril formation of unmodified alpha-syn when present at low concentrations. Immunoelectron microscopy revealed that nitrated monomers and dimers are incorporated into the fibrils. However, the purified nitrated alpha-syn monomer by itself was unable to form fibrils. Nitration of the tyrosine residue at position 39 was largely responsible for decreased binding of nitrated monomeric alpha-syn to synthetic vesicles, which correlated with an impairment of the nitrated protein to adopt alpha-helical conformation in the presence of liposomes. The chaperone-like activity of alpha-syn was not inhibited by nitration or oxidation. Furthermore, the 20 S proteasome and calpain I degraded nitrated monomeric alpha-syn, although at a slower rate compared with control alpha-syn. Collectively, these data suggest that post-translational modification of alpha-syn by nitration can promote the formation of intracytoplasmic inclusions that constitute the hallmark of Parkinson disease and other synucleinopathies.

Animals↗

PubMed2004

Increased prevalence of unprovoked seizures in patients with a 22q11.2 deletion.

Many neurologic abnormalities have been identified in patients with a deletion of chromosome region 22q11.2, including recurrent, apparently unprovoked seizures. We reviewed the database of patients with a 22q11.2 deletion at the Children's Hospital of Philadelphia to assess the prevalence of idiopathic epilepsy in this population. The records of 383 patients with a confirmed 22q11.2 deletion were reviewed for documentation of seizures; precipitating events such as hypocalcemia, fever, and recent surgery; MRI and EEG findings (to aid in seizure classification); and potential risk factors for epilepsy. Of 348 patients with adequately detailed histories, 27 patients had apparently unprovoked seizures (7% of the total population). A disproportionate number of these patients met criteria for generalized epilepsy. Cardiac disease and prematurity were not risk factors for the development of unprovoked seizures in this population. The prevalence of unprovoked seizures in individuals meeting criteria for epilepsy in patients with a 22q11.2 deletion evaluated at our institution is much greater than in the general population. The association with generalized epilepsy suggests that this increased risk is a primary manifestation of the genetic syndrome, not a secondary result of the other manifestations of 22q11.2 deletions. These results suggest that diagnostic screening for the 22q11.2 deletion syndrome should be considered in patients with epilepsy and other signs suggestive of this interstitial deletion syndrome, and have implications for the identification of potential genetic loci for idiopathic epilepsy.

Adolescent↗

PubMed2004

Effects of a functional COMT polymorphism on prefrontal cognitive function in patients with 22q11.2 deletion syndrome.

OBJECTIVE: The 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) is associated with attentional problems and executive dysfunction, and is one of the highest known risk factors for schizophrenia. These behavioral manifestations of 22q11.2 deletion syndrome could result from haploinsufficiency of the catechol O-methyltransferase (COMT) gene, located within the 22q11 region. The goal of the present study was to examine COMT genotype as a predictor of prefrontal cognitive function in patients with 22q11.2 deletion syndrome. METHOD: Patients with confirmed 22q11.2 deletions (N=44) underwent neurocognitive testing following Val(158)Met genotyping (Met hemizygous: N=16; Val hemizygous: N=28). RESULTS: Analyses of covariance revealed that Met-hemizygous patients performed significantly better on a composite measure of executive function (comprising set-shifting, verbal fluency, attention, and working memory) than did Val-hemizygous patients. CONCLUSIONS: These data are consistent with those of previous studies in normal individuals, suggesting that a functional genetic polymorphism in the 22q11 region may influence prefrontal cognition in individuals with COMT haploinsufficiency.

Alleles↗

PubMed2003

Selective activation induced cleavage of the NR2B subunit by calpain.

Although activation of calcium-activated neutral protease (calpain) by the NMDA receptor has been suggested to play critical roles in synaptic modulation and neurologic disease, the nature of its substrates has not been completely defined. In this study, we examined the ability of calpain to cleave the NMDA receptor in cultured hippocampal neurons. Activation of the NMDA receptor by agonist application led to rapid calpain-specific proteolysis of spectrin and decreased levels of NR2A/2B subunits. Cleavage of the NR2A/2B subunit created a 115 kDa product that retained the ability to bind 125I-MK-801 and is predicted to be active. Increases in levels of this product appeared within 5 min of NMDA receptor activation and were stable for periods of >30 min. Subtype-specific antibodies demonstrated that the NR2B subunit was cleaved in these primary cultures, but the NR2A subunit was not. An inhibitor of calpain blocked both the decrease of intact NR2B and the increase of the low molecular weight form, whereas neither caspase nor cathepsin inhibitors had an effect on these events. Cell surface biotinylation experiments demonstrated that the 115 kDa fragment remained on the cell surface. This NR2B fragment was also found in the rat hippocampus after transient forebrain ischemia, showing that this process also occurs in vivo. This suggests that calpain-mediated cleavage of the NR2B subunit occurs in neurons and gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation. Such forms could contribute to excitotoxicity and synaptic remodeling.

Animals↗

PubMed2003

Lack of effect of polymorphisms in dopamine metabolism related genes on imaging of TRODAT-1 in striatum of asymptomatic volunteers and patients with Parkinson's disease.

SPECT scanning using (99)Tc-TRODAT-1, a ligand that binds to dopamine transporters, may be useful for detection of early Parkinson's disease (PD), diagnosis of presymptomatic individuals, and monitoring disease progression. Understanding whether genetic factors contribute to inter-individual variability is crucial for interpreting imaging results in the context of disease pathophysiology. We tested whether polymorphisms in the genes for catechol-O-methyltransferase (COMT), monoamine-oxidase B (MAO-B), and the dopamine transporter (DAT) influence dopamine uptake parameters in the striatum in vivo in asymptomatic volunteers and patients with PD as measured with (99)Tc-TRODAT-1. (99)Tc-TRODAT-1 binding declined with age in both asymptomatic volunteers and PD patients, and depended on disease duration in PD patients. We found no significant association between COMT, MAO-B, and DAT polymorphisms and results of (99)Tc-TRODAT-1 testing in asymptomatic volunteers or patients with PD. In PD patients, the age of disease onset and speed of progression did not differ based on these polymorphisms. These results demonstrate that these specific genetic variations do not alter the fidelity of (99)Tc-TRODAT-1 as a measure of dopaminergic function in asymptomatic volunteer individuals or patients with PD.

Adolescent↗