Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones.

We have purified a polyclonal antibody by affinity chromatography which binds specifically to the phosphorylated form of the regulatory light chain (Mr = 20,000) of smooth muscle myosin. This antibody does not stain relaxed, permeabilized smooth muscle cells isolated from guinea pig taenia coli. However, when these cells were stimulated to contract with CaCl2 (100 microM) and ATP (1 mM), the immunofluorescence staining was localized in a series of transverse bands. This distribution of activated myosin appears to reflect an underlying structural organization of the smooth muscle cell cytoskeleton into mechanically coupled contractile zones.

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event.

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.

Amino acid neurotransmitter candidates: sodium-dependent high-affinity uptake by unique synaptosomal fractions.

Glutamic and aspartic acids and glycine are accumulated by high-affinity uptake systems into synaptosomal preparations in central nervous tissue. Sodium is required by these high-affinity transports, but not by the low-affinity transports for these and other amino acids. The sodium-requiring amino acid uptake systems label unique synaptosomal fractions. Observations suggest that these amino acids serve specific synaptic functions, presumably as neurotransmitters.

Field responses of Prunus serotina and Asclepias syriaca to ozone around southern Lake Michigan.

Higher ozone concentrations east of southern Lake Michigan compared to west of the lake were used to test hypotheses about injury and growth effects on two plant species. We measured approximately 1000 black cherry trees and over 3000 milkweed stems from 1999 to 2001 for this purpose. Black cherry branch elongation and milkweed growth and pod formation were significantly higher west of Lake Michigan while ozone injury was greater east of Lake Michigan. Using classification and regression tree (CART) analyses we determined that departures from normal precipitation, soil nitrogen and ozone exposure/peak hourly concentrations were the most important variables affecting cherry branch elongation, and milkweed stem height and pod formation. The effects of ozone were not consistently comparable with the effects of soil nutrients, weather, insect or disease injury, and depended on species. Ozone SUM06 exposures greater than 13 ppm-h decreased cherry branch elongation 18%; peak 1-h exposures greater than 93 ppb reduced milkweed stem height 13%; and peak 1-h concentrations greater than 98 ppb reduced pod formation 11% in milkweed.

Visualization of cyclosporin A and Ca2+-sensitive cyclical mitochondrial depolarizations in cell culture.

Mitochondria not only facilitate chemiosmotic energy transduction, but also are excitable organelles that are important participants in intracellular Ca2+ signaling and are obligate participants in the active cell death cascade known as apoptosis. Underlying these functions is the cyclosporin A (CSA)-sensitive mitochondrial permeability transition pore (MTP), which can open transiently in a low conductance mode (MTPL) to relieve excess Ca2+, and irreversibly during the initiation of apoptosis. Here we image for the first time CSA- and Ca2+-sensitive cyclical mitochondrial depolarizations in cultures of the SH-SY5Y human neuroblastoma cell. In addition, we show that mitochondrial transmembrane potential (DeltaPsi) increases in response to CSA, indicating a baseline channel activity. Moreover, networks of mitochondria are shown to behave as an excitable system that may use Ca2+ as a diffusible messenger to recruit neighboring mitochondria to depolarize. We propose that these depolarizations represent MTPL activity. Our data further reinforce the notion that mitochondria are excitable organelles and suggest coordinated activation of MTPL.

Use of cytochalasin B to distinguish between early and late events in neutrophil activation.

Cytochalasin B greatly enhances secretion of beta-glucuronidase and generation of superoxide on stimulation of rabbit peritoneal neutrophils with the soluble chemotactic factor N-formylmethionylleucylphenylalanine (f-Met-Leu-Phe). There are smaller changes due to cytochalasin B on binding of f-Met-Leu-[(3)H]-Phe, stimulation of phosphatidylinositol turnover and the stimulated increase in the permeability of the cell membrane to Ca(2+). These latter changes are probably artefactual and arise as secondary consequences of cell stimulation. Our observations support the notion that changes in Ca(2+) permeability of membranes and stimulation of phosphatidylinositol turnover reflect early stages in the sequence of events initiated by f-Met-Leu-Phe binding to its receptor and which lead to cell activation phenomena such as secretion and superoxide production.

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism.

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson's disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.

Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease.

The dopaminergic neurotoxin N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) causes a syndrome in primates and humans which mimics Parkinson's disease (PD) in clinical, pathological, and biochemical findings, including diminished activity of complex I in the mitochondrial electron transport chain. Reduced complex I activity is found in sporadic PD and can be transferred through mitochondrial DNA, suggesting a mitochondrial genetic etiology. We now show that MPTP treatment of mice and N-methylpyridinium (MPP+) exposure of human SH-SY5Y neuroblastoma cells increases oxygen free radical production and antioxidant enzyme activities. Cybrid cells created by transfer of PD mitochondria exhibit similar characteristics; however, PD cybrids' antioxidant enzyme activities are not further increased by MPP+ exposure, as are the activities in control cybrids. PD mitochondrial cybrids are subject to metabolic and oxidative stresses similar to MPTP parkinsonism and provide a model to determine mechanisms of oxidative damage and cell death in PD.

The phospholipid headgroup specificity of an ATP-dependent calcium pump.

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37 degrees C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids. We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.

Neurotransmitter receptors in frontal cortex of schizophrenics.

Frontal cerebral cortex brain samples from schizophrenics and controls have been assayed for binding associated with muscarinic cholinergic, serotonin (5HT), gamma-aminobutyric acid (GABA), and beta-adrenergic receptors as well as for the activity of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Binding levels of tritium-LSD, presumably associated with postsynaptic 5HT receptors, were reduced 40% to 50% in samples from schizophrenics in three independent studies, whereas no other consistent alteration was observed in levels of binding associated with other receptors or in the activity of GAD. This change in receptor binding levels does not seem to be attributable to postmortem changes, to influence of drugs received by the patients, or to demographic features of the patient populations.

Circular ruffle formation in rat basophilic leukemia cells in response to antigen stimulation.

Rat basophilic leukemia cells have previously been described to undergo striking cell surface changes after IgE-mediated stimulation of histamine secretion, whereby the dorsal surface loses its microvilli and acquires characteristic wavy ruffles. We have found using scanning electron microscopy, phase contrast and immunofluorescence, that a proportion of these cells also exhibit the formation of circular membrane ruffles on their dorsal surface after exposure to an IgE-directed secretagogue; some cells also develop circular membrane ruffles following stimulation by phorbol myristate acetate or by calcium ionophore A23187. A flattened morphology appears to be linked to circular membrane ruffle formation in that these ruffles were found in areas of presumed cell spreading which are largely devoid of intermediate filaments and displaced to one side of the cell's nucleus, and they were not observed on rounded cells. This is in contrast to the wavy ruffles which are found on the entire cell surface including the region overlying the nucleus, and which are observed in rounded cells as well as spread cells. Circular ruffle formation and secretion are triggered by similar concentrations of antigen, but the circular ruffles are formed more slowly and only become abundant at times after most of the histamine has been released. The circular membrane ruffles showed no obvious association with endocytosis, as detected using fluorescein isothiocyanate-dextran as a fluid phase marker. The position of accumulation of endocytotic vesicles occurring subsequent to secretion was not found to be related to the circular membrane ruffles, but was observed around the nucleus. Circular membrane ruffles contain F-actin, and their formation is prevented by cytochalasin D. At least three types of myosin, types I, II and V are present and presumably play a role in circular ruffle formation.

Suppression of dyskinesias in advanced Parkinson's disease. I. Continuous intravenous levodopa shifts dose response for production of dyskinesias but not for relief of parkinsonism in patients with advanced Parkinson's disease.

We characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and 3-O-methyldopa levels in six patients with advanced Parkinson's disease (PD) and fluctuating responses to oral levodopa/carbidopa. Dose response to ramped intravenous levodopa infusion was measured after overnight drug withdrawal on two occasions: first after chronic, intermittent oral levodopa/carbidopa, and second after 3 to 5 days of continuous intravenous levodopa. Continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. Improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/3-O-methyldopa remained constant. Our results support the hypothesis that relief of parkinsonism and production of dyskinesia by levodopa occur by separate mechanisms.

Efficacy of pramipexole, a novel dopamine agonist, as monotherapy in mild to moderate Parkinson's disease. The Pramipexole Study Group.

A total of 335 patients with early Parkinson's disease (PD) were enrolled in a multicenter, randomized, double-blind trial designed to assess the efficacy and safety of pramipexole. Entry was restricted to patients with idiopathic PD who were not receiving levodopa. Pramipexole was administered according to an ascending dose schedule up to 4.5 mg/d. During the 7-week dose-escalation phase, each subject was titrated to his or her maximally tolerated dose of study medication. This was followed by a 24-week period of maintenance therapy. The mean daily dose during the maintenance period was 3.8 mg. Pramipexole significantly reduced the severity of PD symptoms and signs compared with placebo, as measured by decreases in parts II (Activities of Daily Living) and III (Motor Examination) of the Unified Parkinson's Disease Rating Scale at week 24 compared with baseline (p < or = 0.0001). Differences between the active drug and placebo groups emerged at week 3 (1.5 mg/d) in the ascending-dose interval and persisted throughout the maintenance phase (p < or = 0.0001). The majority of patients completed the study (pramipexole 83%, placebo 80%). In the assessment of adverse events, nausea, insomnia, constipation, somnolence, and visual hallucinations occurred more frequently in the pramipexole treatment group compared with placebo patients. No clinically significant changes were noted in blood pressure or pulse rate. Overall, these results indicate that pramipexole is safe and effective in the treatment of early PD.

Suppression of dyskinesias in advanced Parkinson's disease. II. Increasing daily clozapine doses suppress dyskinesias and improve parkinsonism symptoms.

We gave increasing daily doses of clozapine to six patients with advanced Parkinson's disease (PD) and levodopa-induced dyskinesias. Clozapine reduced the daily dyskinesia time five-fold, increased "on" time eight-fold, and doubled the serum [DOPA] producing half-maximal dyskinesia. Parkinsonism scores after overnight DOPA withdrawal improved with increasing daily clozapine intake, and there was no clozapine dose-related shift in levodopa dose response for relief of parkinsonism. Patients experienced sedation, sialorrhea, and orthostatic hypotension. Clozapine appears to be an effective agent for suppression of levodopa-induced dyskinesias in PD.

Clinical correlates of sleep benefit in Parkinson's disease.

The phenomenon of sleep benefit, a period of lessened disability or feeling "on" upon awakening from sleep in the morning, has received scant attention in the literature on Parkinson's disease. We interviewed 162 consecutive patients regarding disease onset, medication history, and symptoms, evaluated them using the Unified Parkinson's Disease Rating Scale, and assessed them as to the presence or absence of sleep benefit. Thirty-three percent reported experiencing sleep benefit. Compared with patients not having sleep benefit, patients with sleep benefit tended to be younger at disease onset, have longer disease duration, take higher total daily doses of levodopa, have longer duration of levodopa treatment, and exhibit less cognitive and physical disability. The findings of this study suggest that sleep benefit is a common phenomenon that may be anticipated in a subgroup of patients with Parkinson's disease. The mechanisms underlying sleep benefit do not appear to be simple and may be multifactorial. Clinicians need to be aware of the authenticity of patients' reports of sleep benefit and consider the existence of this phenomenon when prescribing or adjusting patients' medication schedules.

Early morning dystonia in Parkinson's disease.

We interviewed 383 patients with PD regarding disease onset and medication history and evaluated them using the Unified Parkinson's Disease Rating Scale. Sixteen percent of the sample reported the occurrence of early morning dystonia (EMD). Patients with EMD had been taking levodopa for a longer time, were taking higher daily levodopa doses, demonstrated more disability in carrying out their activities of daily living, exhibited dystonia more often before initiation of levodopa treatment, and experienced more peak-dose and diphasic dyskinesias with levodopa therapy.

Cybrids in Alzheimer's disease: a cellular model of the disease?

The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimer's disease (AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.

The metabolism of systemically-administered L-dihydroxyphenylalanine, by intact and dopamine-denervated striata, as revealed by brain microdialysis.

Idiopathic Parkinson's Disease arises from the progressive loss of dopamine (DA)-utilizing neurons of the nigrostriatum and responds to the replacement of DA with L-dihydroxyphenylalanine (L-DOPA). In awake rats, with unilateral lesions induced with 6-hydroxydopamine (6-OHDA) of the DA-utilizing nigrostriatal pathway, treatment with L-DOPA causes the rapid onset of brisk contralateral turning behaviour. In urethane-anesthetized rats with identical unilateral lesions of the nigrostriatum, dialysis of the striatum, performed before and after the systemic administration of L-DOPA (25 mg/kg i.p.), did not demonstrate any alteration in extracellular DA in the striatum which was DA-deprived compared to intact striata. After treatment with L-DOPA extracellular levels of the metabolites of DA. DOPAC and HVA increased several fold. These results suggest: (a) DA neurons surviving after extensive lesions with 6-OHDA can compensate for loss of DA in the striatum and maintain extracellular fluid (and presumably synaptic) concentrations of DA; (b) in striata with extensive depletion of DA L-DOPA undergoes rapid decarboxylation to DA, followed by catabolism to DOPAC and HVA; and (c) in urethane-anesthetized animals, DA formed from DOPA does not appear to enter a releasable pool.

Choline acetyltransferase- and substance P-like immunoreactive elements in fetal striatal grafts in the rat: a correlated light and electron microscopic study.

Fetal striatal neurons were transplanted into the ibotenic acid-lesioned rat striatum. Three months after transplantation, the graft tissue was processed for choline acetyltransferase- and substance P-like immunoreactivity and was subsequently examined at the light and electron microscopic levels. The study demonstrated that choline acetyltransferase- and substance P-like-immunoreactive neurons were homogenously present throughout fetal striatal grafts, although in decreased numbers compared with those in the normal rat striatum. The majority of the choline acetyltransferase-immunoreactive neurons had fusiform, oval, or polygonal somata with somatic diameters greater than 20 microns and contained deeply invaginated nuclei surrounded by copious cytoplasm. In addition, choline acetyltransferase-immunoreactive neurons with somatic diameters between 10 and 20 microns were also demonstrated. The grafts' substance P-like-immunoreactive neurons, which had somatic diameters between 10 and 25 microns and had oval or polygonal perikarya, could be classified into two types based on their ultrastructural characteristics. Type I neurons contained an unindented nucleus which was surrounded by a thin rim or moderate amount of cytoplasm, whereas Type II immunoreactive neurons contained an indented nucleus which was surrounded by copious cytoplasm. Choline acetyltransferase- and substance P-like-immunoreactive dendrites in the grafts' neuropil were contacted by multiple unlabeled axon terminals. In addition, choline acetyltransferase- and substance P-like-immunoreactive axon terminals forming symmetric contacts with unlabeled dendrites were present within the graft. The study demonstrated that many of the neuroanatomical features of choline acetyltransferase- and substance P-like-immunoreactive elements found in the normal rat striatum are present in mature fetal striatal grafts.