Biomedical subjects
Publications and source records attributed to E Byrne.
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Age-associated changes in mitochondrial respiratory chain activity were investigated in human brain tissue collected at autopsy. Four brain regions, the frontal cortex, superior temporal cortex, cerebellum and putamen, were studied to map any regional variation. A significant decrease in cytochrome c oxidase activity was seen in all regions studied with increasing age (P<0.05). Although a small decrease in succinate dehydrogenase-cytochrome c oxidoreductase and NADH: ubiquinone oxidoreductase activities was observed, this was not statistically significant. This study has shown that the age-related fall in cytochrome c oxidase activity affects the frontal cortex, superior temporal cortex, cerebellum and putamen. The variation in the extent of age-related oxidative phosphorylation decline was striking. We hypothesize that individuals with more severe age-related decline may be predisposed to neuronal dysfunction, whereas individuals with well preserved oxidative phosphorylation may enjoy some degree of neuronal protection.
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This study aims to investigate the cellular distribution of human cytochrome c oxidase (COX) subunit II (CII) and COX subunit IV (CIV) in Alzheimer's disease relative to control brains. The levels of CIV and CII proteins in the cerebellar Purkinje cells were reduced in age-matched controls relative to young controls and in the Alzheimer's disease group relative to both age-matched and young controls. Results suggest that these age-associated changes are more marked in Alzheimer's disease.
Polymerase chain reaction (PCR) based methods for the diagnosis and screening of the mitochondrial disorders have been well established. A number of tissues are routinely used. In this study, we compared the detection rate for MELAS A3243G point mutation in muscle, blood and hair follicles. Ten subjects were studied; mean age was 47 years, (SD 16, range 23-73). All ten subjects had the MELAS A3243G point mutation detected in muscle and hair follicles, but only five had the abnormality in blood samples. The rate of detection of the point mutation in blood samples was age dependent. MtDNA analysis on hair follicles is as sensitive as muscle in detecting this mutation. Analysis using blood samples is not as sensitive, particularly in older subjects. The absence of the mutation in blood samples suggests that there is a preferential selection process for normal (wild type) mtDNA over time. This may be related to the rate of cell division and energy requirements of each tissue.
Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.
Understanding the pathobiology of mitochondrial (mt) DNA diseases involves both characterization of the effects of individual mutations on respiratory function and elucidation of the changes in mutation load and distribution (energy mosaicism) over serial cell generations. Whether a given mutation is stably maintained, or increases or decreases with cell growth, is one of the determinants as to whether a particular tissue will be affected by oxidative phosphorylation failure. In this study, we correlated mt genotype with biochemical phenotype in myoblasts from patients with pathogenic mtDNA mutations. The dominant process detected was a progressive elimination of mutant mtDNA genomes concomitant with an improvement in respiratory chain activity, suggesting that energetically normal cells have a growth advantage over those with a high mutation load. We propose that this elimination is by biased distribution of wild-type mtDNA to daughter cells, and that a similar mechanism could operate in vivo and contribute to both the clinical expression of mt disease and the maintenance of a predominantly wild-type mt genome pool across generations.
There have been few studies investigating the mechanism and nature of the hearing loss that occurs in the mitochondrial disorders. We studied 18 patients with the MELAS A3243G point mutation from four different kindreds. Pure tone audiometry, speech discrimination testing, acoustic reflexes, tympanometry, and brain stem auditory evoked responses were performed to localize the site of pathology in the auditory pathways. In 12 patients, we performed electrocochleography and otoacoustic emissions to assess cochlear involvement. Neuroimaging and promontory nerve stimulation were performed to exclude retrocochlear pathology. Audiological testing confirmed sensorineural hearing loss in 14 of the 18 patients studied; hearing loss was usually gradual in onset, was symmetrical, and initially affected the higher frequencies. In some patients, there were features that distinguished the hearing loss from presbyacusis, including a young age at onset, asymmetrical involvement, stepwise progression, and partial recovery. We treated one patient who had profound bilateral hearing loss with cochlear implantation; this restored good functional hearing. Hearing loss in MELAS syndrome appears to be due to dysfunction of the cochlea, probably resulting from metabolic failure of the stria vascularis and outer hair cells. Cochlear implantation is a therapeutic option worth considering in those patients who become deaf.
Growth factors are theoretically promising agents for ALS therapy, but have been disappointing in subcutaneous delivery due to either toxicity or lack of major efficacy. Leukaemia inhibitory factor (LIF), was named after its effect on haemopoietic cells, and belongs to a group of cytokines which includes CNTF, IL-6, CT-1, OM and IL-11. All group members use the gp130 signal transducing subunit for intracellular signalling, but show differences in biological effect. In vitro and in vivo studies on axotomy and nerve crush models demonstrate a powerful effect of LIF in the survival of both motor and sensory neurones, while reducing denervation induced muscle atrophy. Its effects in muscle also include stimulating myoblast proliferation in vitro, and up-regulation after muscle injury. LIF will also stimulate muscle regeneration in vivo when applied exogenously after injury. In published studies of both axotomy induced neuronal death and in the Wobbler mouse models LIF is active at doses of 10 microg/kg delivered systemically, well below the expected maximum tolerated dose suggested by primate safety studies. LIF is expressed in low levels by spinal cord neurones with significant up-regulation when the neurones are damaged by BOAA toxin, an excitatory amino acid associated with a form of ALS. This augments other evidence suggesting LIF is a trauma factor playing a role in the injury response of adult neuronal tissue, and may be more effective than related growth factors. Taken together, the data suggests LIF is a physiologically relevant trophic factor with implications in clinical medicine as a therapy for ALS, and a human recombinant form (AM424), entered human clinical trials during 1998.
We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.
OBJECTIVE: To determine the neuroradiological abnormalities associated with subjects carrying the mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) tRNA(Leu)(UUR) A3243G point mutation METHODS: Mitochondrial genetic analysis was performed on 24 subjects from six kindreds with the MELAS tRNA(Leu)(UUR) A3243G point mutation. Cerebral CT and MRI were performed on 24 patients and 15 patients respectively. Previous neuroradiological investigations including cerebral CT from four deceased members of the families were also reviewed. Histological examination of postmortem specimens of two patients within the kindreds was performed. RESULTS: The commonest radiological finding was basal ganglia calcification. Other abnormalities included focal lesions and cerebellar and cerebral atrophy. Basal ganglia calcification was progressive, symmetric, and asymptomatic. Histologically, basal ganglia calcification in one patient was found to be in the pericapillary regions of the globus pallidus, with no neuronal involvement. Focal lesions most commonly involved the grey matter of the parietal and occipital lobes and cerebellum. Histopathological examination suggested that these were due to cellular rather than vascular dysfunction. Enlargement of the fourth ventricle was the first sign of cerebellar atrophy. Cerebral and cerebellar atrophy were only present with severe disease. CONCLUSIONS: These radiological findings, when considered in the context of the clinical and pathological findings, seem to reflect two major disease processes: an intermittent abrupt loss of function associated with cell injury from which there is at least partial recovery and a slowly progressive degenerative process causing basal ganglia calcification, and cerebral and cerebellar atrophy. The clinical and radiological features resulting from these processes are distinctive and provide insight into the consequences of mitochondrial dysfunction on the brain.
Since its development in the late 1980's, the polymerase chain reaction (PCR) has revolutionised molecular genetic studies. It has provided direct access to genetic material in quantities sufficient for meaningful analyses to be performed. Adaptations to the basic technique have resulted in a wide range of applications from basic gene amplification to the estimation of DNA species quantities within cells. The study of human mitochondrial genetics is but one of the many disciplines to benefit from the rapid ascension of PCR based technology. In this communication we outline several uses of the PCR technique in the detection, quantification and characterisation of human mitochondrial genetic defects. The data presented in this communication highlight the versatility and applicability of PCR not only to mitochondrial research but to other disciplines of medical research.
High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to < or = 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (Neo(R)) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.
Heteroplasmic populations of mtDNA, consisting of normal mtDNA and mtDNA with large deletions, are found in the skeletal muscle and other tissues of certain patients with mitochondrial respiratory chain deficiencies, particularly in those with the CPEO (chronic progressive external ophthalmoplegia) phenotype. To study the developmental genetics of this mitochondrial disorder, the distribution of the deleted mtDNA in a wide range of tissues of different embryonic origins (total 34 samples from 27 tissues obtained at autopsy) was investigated in a patient with the CPEO syndrome. Three species of partially deleted mtDNA were observed, with deletions of 2.3 kb, 5.0 kb and 6.4 kb. Their tissue distribution suggests that the mtDNA deletions have occurred very early during embryonic development, prior to the differentiation events that lead to the formation of the three primary embryonic germ layers, and that the partially deleted mtDNA species were segregated during development mainly to the skeletal muscle and to tissues of the central nervous system.
Fourteen patients from four unrelated families were studied to determine the prevalence of retinal pigmentary abnormalities associated with the MELAS A to G 3243 point mutation. Neurologic and ophthalmic examinations, retinal photography, pattern shift visual evoked potentials, and electroretinography were performed in all patients. Eight of the 14 patients had retinal pigmentary abnormalities characterized by symmetric areas of depigmentation involving predominantly the posterior pole and midperipheral retina. None of the patients had optic atrophy and only one patient with pigmentary retinal abnormalities had impaired visual acuity. None of the diabetic subjects (n = 6) had signs of diabetic retinopathy. Fluorescein angiography demonstrated mottled hyper- and hypofluorescent areas indicating multiple window defects in the retinal pigmentary epithelium. Visual evoked potentials showed delayed P100 responses in four of the eight patients with retinal pigmentary abnormalities. We conclude that there is a high prevalence of retinal pigmentary abnormalities in patients with MELAS A to G 3243 point mutation. These abnormalities are usually asymptomatic and best detected by retinal photography.
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