The pathogenetic mechanism of the mitochondrial tRNAuLuuR gene mutation responsible for the MELAS In the present work, the approach of mitochondria-mediated transformation utilizing human mtDNA-less (p0) cells as

Disorders of the central nervous system (CNS) are some of the most prevalent, devastating and yet poorly treated illnesses. The development of new therapies for CNS disorders such as Alzheimer's disease has the potential to provide patients with significant improvements in quality of life, as well as reduce the future economic burden on health-care systems. However, few truly innovative CNS drugs have been approved in recent years, suggesting that there is a considerable need for strategies to enhance the productivity of research and development in this field. In this article, using illustrative examples from neurological and psychiatric disorders, we describe various approaches that are being taken to discover CNS drugs, discuss their relative merits and consider how risk can be balanced and attrition reduced.

Disrupted-in-Schizophrenia-1 (DISC1), identified by positional cloning of a balanced translocation (1;11) with the breakpoint in intron 8 of a large Scottish pedigree, is associated with a range of neuropsychiatric disorders including schizophrenia. To model this mutation in mice, we have generated Disc1 tr transgenic mice expressing 2 copies of truncated Disc1 encoding the first 8 exons using a bacterial artificial chromosome (BAC). With this partial simulation of the human situation, we have discovered a range of phenotypes including a series of novel features not previously reported. Disc1 tr transgenic mice display enlarged lateral ventricles, reduced cerebral cortex, partial agenesis of the corpus callosum, and thinning of layers II/III with reduced neural proliferation at midneurogenesis. Parvalbumin GABAergic neurons are reduced in the hippocampus and medial prefrontal cortex, and displaced in the dorsolateral frontal cortex. In culture, transgenic neurons grow fewer and shorter neurites. Behaviorally, transgenic mice exhibit increased immobility and reduced vocalization in depression-related tests, and impairment in conditioning of latent inhibition. These abnormalities in Disc1 tr transgenic mice are consistent with findings in severe schizophrenia.

The The rules that govern selection, segregation, and complementation of different mtDNA molecules at the intracellular and cell population levels are poorly understood. In mammalian cells, until recently, the available evidence was limited to the behavior of drug-resistance-inducing mutations in cell hybrids and cybrids (cells fused to cytoplasts), and pointed to a random drift to the mutant or wild-type genotype in the absence of selection (1) and to a propagative advantage of the resistance-conferring mutant mtDNA in the presence of the drug (1, 2). The recent identification of human disorders associated with mtDNA mutations (3) has provided an opportunity for investigating the transmission of natural mutations in the patient's cells in vivo or in their in vitro derivatives. Thus, an accumulation of deleted mtDNA molecules relative to coexisting wild-type genomes has been reported in skeletal muscle fibers of patients with mitochondrial diseases (4, 5) and in cybrids derived from patient's cells (6).In the present work, cell lines containing in heteroplasmic form the mitochondrial tRNAI-u(uuR) gene mutation associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) (7-9) that were constructed by introducing patient mitochondria into human mtDNA-less (p0) cells (10) DNA Anaiysis. The presence and proportion of mutated mtDNA in total DNA samples (prepared by using a Applied Biosystems 340A extractor or by proteinase K: digestion of a 0.5% Tween 20 cell lysate) were determed y testing for the presence of the Apa I site created by the IMLAS mutation at position 3243 (7-9) in a fragment amplfied by a PCR using oligonucleotides corresponding to positions 3031-3050 and 3341-3360. The proportion of digested and undigested molecules was determined by laser densitometry after ethidium bromide staining of the agarose gel. To correct for possible resistance to digestion of heteroduplexes of wild-type and mutant mtDNA formed during the final stages of PCR amplification (17), a mixed-template standard curve was constructed by PCR amplification of a mtDNA fiagment containing the mutation from total DNA samples of 43B cells, containing 99% mutated mtDNA, and transformant 94C, containing exclusively wild-type mtDNA (12). Different ratio mixtures of the mutant and wild-type PCR products were then used for a second cycle of PCR amplification. Fig. 1 compares the mixed template standard curve (curve T) thus constructed with the mixed product standard curve (curve P), obtained by mixing the PCR products independently amplified from 43B-derived and 94C-derived PCR products in different ratios and then subjecting the mixtures to Apa I digestion.Abbreviation: MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. 11164The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. N...

Mitochondrial DNA from two genetically unrelated patients carrying the mutation at position 11778 that causes Leber's hereditary optic neuropathy has been transferred with mitochondria into human mtDNA-less 0 206 cells. As analyzed in several transmitochondrial cell lines thus obtained, the mutation, which is in the gene encoding subunit ND4 of the respiratory chain NADH dehydrogenase (ND), did not affect the synthesis, size, or stability of ND4, nor its incorporation into the enzyme complex. However, NADH dehydrogenasedependent respiration, as measured in digitonin-permeabilized cells, was specifically decreased by approximately 40% in cells carrying the mutation. This decrease, which was significant at the 99.99% confidence level, was correlated with a significantly reduced ability of the mutant cells to grow in a medium containing galactose instead of glucose, indicating a clear impairment in their oxidative phosphorylation capacity. On the contrary, no decrease in rotenone-sensitive NADH dehydrogenase activity, using a water-soluble ubiquinone analogue as electron acceptor, was detected in disrupted mitochondrial membranes. This is the first cellular model exhibiting in a foreign nuclear background mitochondrial DNA-linked biochemical defects underlying the optic neuropathy phenotype.

Our previous experience with abnormal fatty acid metabolism in several children with spinal muscular atrophy (SMA) prompted evaluation of fatty acid metabolism in a larger cohort. Thirty‐three infants with severe infantile SMA were shown to have a significantly increased ratio of dodecanoic to tetradecanoic acid in plasma compared with normal infants and 6 infants affected with equally debilitating, non‐SMA denervating disorders. Seventeen children with milder forms of SMA had normal fatty acid profiles. In addition, all 5 infants with severe SMA evaluated in a fasting state developed a distinctive and marked dicarboxylic aciduria, including saturated, unsaturated, and 3‐hydroxy forms, comparable in severity with the dicarboxylic aciduria of children with primary defects of mitochondrial fatty acid β‐oxidation. Nine children with chronic SMA and 23 control patients did not develop an abnormal dicarboxylic aciduria during fasting. No known disorder of fatty acid metabolism explains all of the abnormalities we find in SMA. Our data suggest, however, that the abnormalities are not a consequence of SMA‐related immobility, systemic illness, muscle denervation, or muscle atrophy. These abnormalities in fatty acid metabolism may be caused by changes in cellular physiology related to the molecular defects of the SMA‐pathogenic survival motor neuron gene or neighboring genes. Ann Neurol 1999;45:337–343

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Recent research has implicated the cerebellum in conjugate ocular motor control, including steady gaze-holding and accuracy of pursuit and saccades. Whether the cerebellum also has a role in the control of the alignment of the eyes during fixation and of the yoking of the eyes during movement i. less certain. We have studied binocular (disconjugate) ocular motor control in nine patients with cerebellar dysfunction and compared the results with those of normal subjects. Eye alignment during fixation and the yoking of the eyes during and immediately after saccades were quantified by recording the movements of both eyes using scleral search coils. Patients had disturbances of ocular alignment. All had an esophoria during monocular viewing and many an esotropia during binocular viewing, implying an increase in convergence tone. Most had a vertical misalignment that varied with horizontal eye position ('alternating skew deviation'). Patients showed conjugate dysmetria (saccade under- or overshoot and postsaccade drift) and disconjugate dysmetria (the eyes were poorly yoked during and immediately after saccades). Both the conjugate and disconjugate abnormalities were incommitant, i.e. they varied with orbital eye position. Correlations amongst the various abnormalities suggested that one part of the cerebellum, perhaps the dorsal vermis and the underlying posterior fastigial nucleus, controls the conjugate size of saccades and that another part of the cerebellum, perhaps the flocculus/paraflocculus, controls the yoking of the eyes during saccades and both the disconjugate and conjugate components of postsaccade drift.