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.

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.

Transdermal dopaminergic D(2) receptor agonist therapy in Parkinson's disease with N-0923 TDS: a double-blind, placebo-controlled study.

N-0923 is a non-ergot, dopaminergic D(2) agonist designed to be transdermally available. It has anti-parkinsonian effects when infused intravenously. An adhesive matrix patch was developed to deliver N-0923 transdermally (N-0923 TDS). In this phase II trial, we evaluated the effectiveness of various doses of N-0923 TDS at replacing levodopa. Eighty-five Parkinson's disease (PD) patients were randomized to placebo or one of four doses of N-0923 TDS for 21 days. Change in daily levodopa dose was the primary efficacy measure. Significantly greater reductions in levodopa dose were achieved as compared to placebo for the two highest doses of N-0923 TDS. Patients treated with 33.5 mg and 67 mg N-0923 TDS decreased levodopa use by 26% and 28%, vs. 7% for placebo. N-0923 TDS was safe and well tolerated.

Biochemical analysis of cybrids expressing mitochondrial DNA from Contursi kindred Parkinson's subjects.

Complex I activity is reduced in cytoplasmic hybrid (cybrid) cell lines that contain mitochondrial DNA (mtDNA) from sporadic Parkinson's disease (PD) patients. This implies that mtDNA aberration occurs in sporadic PD. To assess the integrity of mtDNA in autosomal dominant PD arising from mutation of the alpha-synuclein gene, we transferred mitochondrial genes from PD-affected members of the Italian-American Contursi kindred to cells previously depleted of their endogenous mtDNA. Unlike cybrid cell lines expressing mtDNA from persons with sporadic or maternally inherited PD, the resultant Contursi cybrid lines did not manifest complex I deficiency, indicating that in Contursi PD mtDNA integrity is relatively preserved. Compared to control cybrids, however, Contursi cybrid lines did show some evidence of oxidative stress. For reasons that are unclear, at least a limited amount of mtDNA damage may nevertheless develop in PD patients with alpha-synuclein mutation.

Gender ratio differences between Parkinson's disease patients and their affected relatives.

Mitochondrial dysfunction in Parkinson's disease (PD) is suspected to arise from either acquired or inherited mutation of mitochondrial DNA (mtDNA). If inherited, epidemiologic analysis may reveal maternal transmission. We looked for maternal inheritance bias in our PD clinical database. About 13% of 600 PD probands reported an affected parent. Although 60% of the PD probands were male, only 42% of the affected parents were. The gender ratios for the proband and affected parent generations were dissimilar (p<0.005), indicating an underrepresentation of affected fathers or an overrepresentation of affected mothers. To address these possibilities we analyzed a non-PD control cohort. Four percent of the controls had a PD affected parent, and 75% of these affected parents were male. Apparent maternal inheritance bias in our PD cohort is therefore more likely due to overrepresentation of affected mothers, and is consistent with mitochondrial inheritance in some of our ascertained cases.

Chronic reduction in complex I function alters calcium signaling in SH-SY5Y neuroblastoma cells.

Sporadic, non-familial Parkinson's disease is characterized by a 15-30% reduction in complex I activity of the electron transport chain. A pharmacological model of reduced complex I activity was created by prolonged treatment of SH-SY5Y cells with low doses (5-20 nM) of rotenone, a selective inhibitor of complex I. Short-term (less than 2 week) exposure to rotenone did not influence calcium signaling, production of reactive oxygen species, or mitochondrial morphology. However, following 2 weeks of rotenone exposure, SH-SY5Y cells showed unusual calcium dynamics, specifically multiple calcium responses to carbachol, a muscarinic agonist. These secondary calcium responses were not seen in control SH-SY5Y cells and were dependent upon calcium influx. Mitochondrial membrane potential was also reduced in low dose rotenone-treated cells. These results demonstrate that a chronic, partial reduction in complex I activity, such as that seen in Parkinson's disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Neurotoxic Abeta peptides increase oxidative stress in vivo through NMDA-receptor and nitric-oxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro.

Beta amyloid (Abeta) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Abeta to increase oxygen radical production in vivo. The 1-40 Abeta fragment increased 2,3-dehydroxybenzoic acid efflux more than the 1-28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Abeta peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Abeta(25-35) and Abeta(35-25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N-ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Abeta aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Abeta(25-35) but not by Abeta(35-25). Neurotoxic Abeta peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Abeta levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed-forward hypothesis of Abeta neurotoxicity.

Mitochondrial dysfunction in cybrid lines expressing mitochondrial genes from patients with progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative movement disorder of unknown etiology. We hypothesized that mitochondrial DNA (mtDNA) aberration could occur in this disease and contribute to its pathogenesis. To address this we created transmitochondrial cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from persons with PSP. The presence of cybrid mtDNA aberration was screened for by biochemical assay of mitochondrial gene products. Relative to a control cybrid set, complex I activity was reduced in PSP cybrid lines (p<0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.

Disrupted mitochondrial electron transport function increases expression of anti-apoptotic bcl-2 and bcl-X(L) proteins in SH-SY5Y neuroblastoma and in Parkinson disease cybrid cells through oxidative stress.

Death of dopamine neurons in Parkinson disease (PD) may arise from consequences of the complex I (C-I) defect in the mitochondrial electron transport chain (ETC). Whether cells activate programmed death (apoptosis) pathways derives, in part, from relative activities of proteins such as bcl-2 and bcl-X(L), that have anti-apoptotic actions. We studied the responses of bcl-2 and bcl-X(L) genes in pharmacologic (acute incubation with methylpyridinium (MPP+)) and mitochondrial transgenic ("cybrid") models of Parkinson disease C-I defects. MPP+ incubation increased levels of bcl-2 and bcl-X(L) proteins in native SH-SY5Y cells but not in rho(0) cells devoid of ETC activity. MPP+ increased bcl-2 mRNA levels by 40% at 8 hr. Confocal microscopic imaging showed that the intracellular distribution of immunoreactive bcl-2 was not significantly associated with mitochondrial membranes at baseline but was associated with mitochondria after 12 hr of MPP+. Immunoreactive bcl-X(L) protein was significantly and equally associated with mitochondrial membranes both at baseline and after MPP+. PD cybrids showed increased basal levels of bcl-2 and bcl-X(L) proteins, similar to the maximum levels found after MPP+ treatment of control SY5Y cells. After MPP+ exposure, bcl-2 protein levels increased in control cybrids but did not increase further in PD cybrids. Both pharmacologically generated and transgenically induced C-I inhibition increases levels of anti-apoptotic bcl proteins, possibly from increased gene transcription. Augmentation of bcl-2 and bcl-X(L) expression may delay neurodegeneration in PD.

Alzheimer's disease cybrids replicate beta-amyloid abnormalities through cell death pathways.

Alzheimer's disease (AD) is characterized by the deposition in brain of beta-amyloid (Abeta) peptides, elevated brain caspase-3, and systemic deficiency of cytochrome c oxidase. Although increased Abeta deposition can result from mutations in amyloid precursor protein or presenilin genes, the cause of increased Abeta deposition in sporadic AD is unknown. Cytoplasmic hybrid ("cybrid") cells made from mitochondrial DNA of nonfamilial AD subjects show antioxidant-reversible lowering of mitochondrial membrane potential (delta(gYm), secrete twice as much Abeta(1-40) and Abeta(1-42), have increased intracellular Abeta(1-40) (1.7-fold), and develop Congo red-positive Abeta deposits. Also elevated are cytoplasmic cytochrome c (threefold) and caspase-3 activity (twofold). Increased AD cybrid Abeta(1-40) secretion was normalized by inhibition of caspase-3 or secretase and reduced by treatment with the antioxidant S(-)pramipexole. Expression of AD mitochondrial genes in cybrid cells depresses cytochrome c oxidase activity and increases oxidative stress, which, in turn, lowers delta(psi)m. Under stress, cells with AD mitochondrial genes are more likely to activate cell death pathways, which drive caspase 3-mediated Abeta peptide secretion and may account for increased Abeta deposition in the AD brain. Therapeutic strategies for reducing neurodegeneration in sporadic AD can address restoration of delta(psi)m and reduction of elevated Abeta secretion.

Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease.

Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.

Abnormal mitochondrial morphology in sporadic Parkinson's and Alzheimer's disease cybrid cell lines.

Diseases linked to defective mitochondrial function are characterized by morphologically abnormal, swollen mitochondria with distorted cristae. Several lines of evidence now suggest that sporadic forms of Parkinson's disease (PD) and Alzheimer's disease (AD) are linked to mitochondrial dysfunction arising from defects in mitochondrial DNA (mtDNA). Human neuroblastoma (SH-SY5Y) cells that are deficient in mtDNA (Rho(0)) were repopulated with mitochondria from AD or PD patients or age-matched controls. These cytoplasmic hybrid (cybrid) cell lines differ only in the source of their mtDNA. Differences between cybrid cell lines therefore arise from differences in mtDNA and provide a model for the study of how impaired mitochondrial function alters the mitochondria themselves and how these changes adversely affect the neuronal cells they occupy. Cybrid cell mitochondria were labeled with the mitochondrial membrane potential-sensitive dye, JC-1. Analysis of these JC-1 labeled mitochondria by confocal microscopy revealed that mitochondrial membrane potential was significantly reduced in both PD and AD cybrid cells when compared with controls. Ultrastructural examination showed that control cybrid cells contained small, morphologically normal, round or oval mitochondria with a dark matrix and regular distribution of cristae. PD cybrid cells contained a significant and increased percentage of mitochondria that were enlarged or swollen and had a pale matrix with few remaining cristae (0.26-0.65 microm(2)). AD cybrid cells also contained a significantly increased percentage of enlarged or swollen mitochondria (0.25-5.0 microm(2)) that had a pale matrix and few remaining cristae. Other pathological features such as crystal-like intramitochondrial inclusions and cytoplasmic inclusion bodies were also found in PD and AD cybrids. These observations suggest that transfer of PD or AD mtDNA into Rho(0) cells was sufficient to produce pathological changes in mitochondrial ultrastructure that are similar to those seen in other mitochondrial disorders. These data were reported in abstract form (Trimmer et al., 1998, Soc. Neurosci. Abstr. 24: 476).

Heavy metals in wild rice from northern Wisconsin.

Wild rice grain samples from various parts of the world have been found to have elevated concentrations of heavy metals, raising concern for potential effects on human health. It was hypothesized that wild rice from north-central Wisconsin could potentially have elevated concentrations of some heavy metals because of possible exposure to these elements from the atmosphere or from water and sediments. In addition, no studies of heavy metals in wild rice from Wisconsin had been performed, and a baseline study was needed for future comparisons. Wild rice plants were collected from four areas in Bayfield, Forest, Langlade, Oneida, Sawyer and Wood Counties in September, 1997 and 1998 and divided into four plant parts for elemental analyses: roots, stems, leaves and seeds. A total of 194 samples from 51 plants were analyzed across the localities, with an average of 49 samples per part depending on the element. Samples were cleaned of soil, wet digested, and analyzed by ICP for Ag, As, Cd, Cr, Cu, Hg, Mg, Pb, Se and Zn. Roots contained the highest concentrations of Ag, As, Cd, Cr, Hg, Pb, and Se. Copper was highest in both roots and seeds, while Zn was highest just in seeds. Magnesium was highest in leaves. Seed baseline ranges for the 10 elements were established using the 95% confidence intervals of the medians. Wild rice plants from northern Wisconsin had normal levels of the nutritional elements Cu, Mg and Zn in the seeds. Silver, Cd, Hg, Cr, and Se were very low in concentration or within normal limits for food plants. Arsenic and Pb, however, were elevated and could pose a problem for human health. The pathway for As, Hg and Pb to the plants could be atmospheric.

16-year trends in elements of lichens at Theodore Roosevelt National Park, North Dakota.

An epiphytic lichen and a soil lichen in two very closely related genera (Parmelia sulcata and Xanthoparmelia chlorochroa, respectively) were sampled 16 years apart at Theodore Roosevelt National Park in North Dakota and measured for their elemental content. Mercury and cadmium decreased approximately 30% over the time period in both species. Sulfur decreased 8% in the epiphytic species, but increased 20% in the soil lichen. Factor analysis revealed that soil elements were higher in the soil lichen, indicating there was some soil contamination in that species. A relationship between iron and titanium was found only in the soil lichen. Sulfur and mercury were highly enriched in both species relative to the soil, which suggests that the atmosphere is a contributing source of these elements. New baseline values were calculated, 22 elements for both species, although it is not recommended that the soil lichen be sampled in the future.

Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va.

In mice a molecular motor of the myosin V class (designated myosin Va) is known to be the product of the dilute locus, where a mutation prevents melanosome transport in melanocytes. There is conflicting evidence about whether it has a role in dendrite outgrowth. We investigated its role by transiently transfecting antisense oligonucleotides to inhibit its expression in a melanocyte cell line. We demonstrated mRNA and protein expression of myosin Va in 3 mouse melanocyte lines and 1 human melanoma cell line, using RT-PCR and immunoblotting. Two splice variants were found in human cells whilst only the longer transcript, containing an additional exon, was present in mouse melanocyte lines. The shorter variant was detected in other mouse tissues. Myosin Va protein levels were similar in 3 melanocyte lines with differing amounts of pigmentation, indicating that expression of myosin Va is not tightly coupled to expression of melanin. Immunocytochemistry showed 2 types of myosin Va localisation. A punctate pattern of staining concentrated in the perinuclear region was indicative of organelle association, and the observation of occasional linear punctate staining aligned with F-actin bundles supported the idea that myosin Va has a role in transporting melanosomes along actin filaments. Staining was also intense at tips of dendrites and at sites of dendrite-cell contact, consistent with a possible role in dendrite growth. Transient transfection of antisense phosphorothioate oligodeoxynucleotides targeted against myosin Va mRNA reduced expression of myosin Va protein in cultured mouse melan-a melanocytes by over 70 % 20 h after transfection whereas a control (shuffled sequence) oligonucleotide did not. Upon trypsinisation and replating these cells the capacity of the transfected cells to extend new dendrites was reduced in the cells containing the specific antisense oligonucleotides but unaffected by the control oligonucleotide. Image analysis confirmed that the effect of transfection on morphology was statistically significant (P < 0.01). In contrast when cells were not trypsinised and replated following transfection so that previously existing dendrites could persist, the normal dendritic morphology continued to be observed. We conclude that in addition to its involvement in melanosome transport, myosin Va has a role in the extension of new dendrites by melanocytes but not in maintenance of pre-existing dendrites.

Characterization of cybrid cell lines containing mtDNA from Huntington's disease patients.

Electron transport chain (ETC) dysfunction may arise from mitochondrial genetic, nuclear genetic, or toxic etiologies. Cytoplasmic hybrid (cybrid) systems can help distinguish between these possibilities by facilitating expression of suspect mitochondrial DNA (mtDNA) within a nuclear and environmentally controlled context. Perpetuation of ETC dysfunction in cybrids is consistent with an mtDNA pathogenesis while defect correction is not. We previously used cybrids to screen sporadic Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis patients for mtDNA mutation with positive results. To further address the fidelity of these experiments, we created and characterized cybrids expressing mtDNA from persons with Huntington's disease (HD), an autosomal dominant, nuclear DNA-determined disorder in which mitochondrial ETC functioning is abnormal. On ETC, oxidative stress, and calcium homeostasis assays HD cybrid lines were indistinguishable from control cybrid lines. These data support the use of the cybrid technique for mtDNA mutation screening in candidate diseases.

Pramipexole--a new dopamine agonist for the treatment of Parkinson's disease.

Although L-DOPA is the current 'gold standard' for treatment of Parkinson's disease, its effectiveness fades rapidly and its use results in serious motor fluctuations (on-off, wearing off, freezing, involuntary movements) for most patients with Parkinson's disease. Pramipexole is an aminothiazole dopamine agonist with selective actions at dopamine receptors belonging to the D2 subfamily, where it possesses full activity similar to dopamine itself. Pramipexole's preferential affinity for the D3 receptor subtype could contribute to efficacy in the treatment of both the motor and psychiatric symptoms of Parkinson's disease. Both in vitro and in vivo studies in animals suggest that pramipexole possesses numerous neuroprotective properties, including dopamine autoreceptor agonist properties, antioxidant properties, ability to block the mitochondrial permeability transition pore and the ability to stimulate the release of trophic factors. Clinical studies have demonstrated that pramipexole has excellent pharmacokinetic properties and that it is an effective monotherapy in treating early Parkinson's disease and an effective adjunctive therapy with L-DOPA in treating late Parkinson's disease. In addition, pramipexole has demonstrated efficacy in a clinical trial for the treatment of major depression. In the early disease studies, pramipexole was able to retard the need for L-DOPA treatment for several years. Thus, a new 'L-DOPA-sparing' paradigm for treating Parkinson's disease may now be possible, whereby patients are initially treated with pramipexole and L-DOPA is added only as necessary.