Patricia A. Trimmer

Neurotoxic Aβ 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 (Aβ) peptides accumulate in Alzheimers disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Aβ to increase oxygen radical production in vivo. The 1–40 Aβ 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 Aβ peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Aβ(25–35) and Aβ(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 Aβ aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Aβ(25–35) but not by Aβ(35–25). Neurotoxic Aβ peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Aβ levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimers disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed‐forward hypothesis of Aβ neurotoxicity.

Mitochondria in Sporadic Amyotrophic Lateral Sclerosis

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies

Many models of Parkinsons disease (PD) have succeeded in replicating dopaminergic neuron loss or α‐synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long‐term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to α‐synuclein, ubiquitin, parkin, synphilin‐1, neurofilament, β‐tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Creation and Characterization of Mitochondrial DNA-Depleted Cell Lines with “Neuronal-Like” Properties

Abstract: Mitochondrial dysfunction and attendant bioenergetic defects are increasingly recognized as playing an important role in neurodegenerative disorders. The increased attention on mitochondrial involvement points to the need for developing cell lines that have neuron‐like characteristics for the genetic analysis and modeling of these diseases. We describe the creation of respiratory‐deficient SH‐SY5Y neuroblastoma cell lines (ρ064/5) by selectively depleting mitochondrial DNA through prolonged exposure to ethidium bromide. Oxygen consumption in these cells and activities of the electron transport chain enzyme complexes I and IV that contain subunits encoded by the mitochondrial genome are eliminated. In contrast, the function of complex II, a nuclear‐encoded electron transport chain component, is largely intact in these cells. The ρ064/5 cells retain the ability to differentiate into cells with neuron‐like phenotypes following treatment with phorbol ester or retinoic acid. Normal respiratory function is recovered by repopulation of ρ064/5 cells with exogenous human platelet mitochondria. The ρ064/5 cell line serves as a valuable model for the study of neurologic diseases suspected of involving mitochondrial dysfunction.

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

Sporadic, non-familial Parkinsons 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 Parkinsons disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO‐mediated pathways. Fluorescently labeled mitochondria were studied using time‐lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re‐oxygenation or to diethyleneamine/nitric oxide adduct, DETA‐NO (100–500 µm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 ± 1.3% at 0 h and 7.4 ± 1.7% at 1 h of re‐oxygenation, versus 25.6 ± 1.4% in controls (p < 0.05). Mean mitochondrial velocity (µm s−1) decreased from 0.374 ± 0.01 in controls to 0.146 ± 0.01 at 0 h and 0.177 ± 0.02 at 1 h of re‐oxygenation (p < 0.001). Exposure to DETA‐NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG‐nitro‐l‐arginine methyl ester (l‐NAME) prevented the hypoxia‐induced decrease in mitochondrial movement at 0 h (30.1 ± 1.6%) and at 1 h (26.1 ± 9%) of re‐oxygenation. Exposure to either hypoxia/re‐oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO‐mediated pathway is at least partially involved.

Immunocytochemically defined astroglia from fetal, newborn and young adult rats express β-adrenergic receptors in vitro

Autoradiography of radioligand binding was used to assess the expression of beta-adrenergic receptors (beta-AR) by immunocytochemically identified astroglia cultured from the cerebral cortices of rats 16 days in gestation through 28 days postnatal (DPN). Polygonal astroglia isolated from animals at each age examined were found to exhibit large numbers of beta-AR. In contrast, only low levels of beta-AR could be detected on process-bearing astroglia and fibroblasts. Quantitative analysis showed that there was an increase in the density of beta-AR on polygonal astroglia between 16 days in gestation and 1 DPN. This increase in beta-AR receptor density was present whether the cells were grown for long periods of time in culture (8-22 days) or for short periods of time in culture (1-5 days). The results also suggest that differences in the level of receptor expression between cells grown in short-term and long-term culture may be due in part to culture methodology.

Genetic influences on cellular reactions to CNS injury: The reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration

This study compares the reactive changes in astrocytes in denervated neuropil regions in normal mice and in mice carrying the WldS mutation which leads to delayed Wallerian degeneration. In situ hybridization and immunocytochemical techniques were used to define the time course of changes in the levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA in the denervated neuropil of the hippocampus after unilateral aspiration lesions of the entorhinal cortex. In control mice, GFAP mRNA levels increased rapidly in the denervated neuropil to a peak that was about tenfold higher than control at 2–4 days, decreased between 6 and 8 days postlesion, and then increased again to a second peak at 10 days postlesion. Increases in immunostaining for GFAP were evident by 2 days, remained elevated until 12 days postlesion and then decreased slowly. In mice carrying the WldS mutation, the upregulation of GFAP mRNA levels in the denervated laminae was substantially delayed. Strikingly absent was the dramatic increase in labeling at 2–4 days postlesion which was such a prominent feature of the response in control animals. Peak labeling in the denervated laminae was not seen until 10–12 days postlesion. The development of a well‐defined band of intensely immunostained and hypertrophied astrocytes in the denervated zone was also delayed in the WldS animals, although there were modest increases in immunostaining as early as 2 days postlesion that were seen throughout the hippocampus ipsilateral to the lesion. These results suggest that degenerative changes in axons and synaptic terminals are the principal trigger for upregulating GFAP expression in the denervated neuropil, although other signals also play a role in the early postlesion response. J. Comp. Neurol. 380:70–81, 1997.

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture.

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.

Mitochondrial DNA-depleted neuroblastoma (Rho‡) cells exhibit altered calcium signaling

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.