Mitochondrial quality, dynamics and functional capacity in Parkinson's disease cybrid cell lines selected for Lewy body expression.

BACKGROUND: Lewy bodies (LB) are a neuropathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates ("cybrid LB" or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB. RESULTS: Cloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63(CLB)), mitochondrial function declined compared to the original PD cybrid line (PD63(Orig)) due to low levels of mtDNA in nucleoids. In another cell line (PD61(Orig)), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61(CLB)). In the third cell line (PD67(Orig)), there was no change in function after selection for CLB expression (PD67(CLB)). CONCLUSIONS: Expression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial dysfunction in PD. Additional studies will be needed to assess the direct effect of CLB expression on cellular and mitochondrial function.

Reduced axonal transport in Parkinson's disease cybrid neurites is restored by light therapy.

BACKGROUND: It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined. RESULTS: The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT. CONCLUSION: The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.

Relationships among molecular genetic and respiratory properties of Parkinson's disease cybrid cells show similarities to Parkinson's brain tissues.

We have studied sporadic Parkinson's disease (sPD) from expression of patient mitochondrial DNA (mtDNA) in neural cells devoid of their own mtDNA, the "cybrid" model. In spite of reproducing several properties of sPD brain, it remains unclear whether sPD cybrid cells reflect more complex sPD brain bioenergetic pathophysiology. We characterized and correlated respiration of intact sPD cybrid cells with electron transport chain (ETC) protein assembly, complex I ETC gene expression and ETC protein levels in sPD brain. We also assayed expression for multiple ETC genes coded by mtDNA and nuclear DNA (nDNA) in sPD cybrid cells and brain. sPD cybrid cells have reduced levels of mtDNA genes, variable compensatory normalization of mitochondrial gene expression and show robust correlations with mitochondrial ETC gene expression in sPD brains. Relationships among ETC protein levels predict impaired complex I-mediated respiration in sPD brain. That sPD cybrid cells and sPD brain samples show very correlated regulation of nDNA and mtDNA ETC transcriptomes suggests similar bioenergetic physiologies. We propose that further insights into sPD pathogenesis will follow elucidation of mechanisms leading to reduced mtDNA gene levels in sPD cybrids. This will require characterization of the abnormalities and dynamics of mtDNA changes propagated through sPD cybrids over time.

Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells.

BACKGROUND: Parkinson's disease, the most common adult neurodegenerative movement disorder, demonstrates a brain-wide pathology that begins pre-clinically with alpha-synuclein aggregates ("Lewy neurites") in processes of gut enteric and vagal motor neurons. Rostral progression into substantia nigra with death of dopamine neurons produces the motor impairment phenotype that yields a clinical diagnosis. The vast majority of Parkinson's disease occurs sporadically, and current models of sporadic Parkinson's disease (sPD) can utilize directly infused or systemic neurotoxins. RESULTS: We developed a differentiation protocol for human SH-SY5Y neuroblastoma that yielded non-dividing dopaminergic neural cells with long processes that we then exposed to 50 nM rotenone, a complex I inhibitor used in Parkinson's disease models. After 21 days of rotenone, ~60% of cells died. Their processes retracted and accumulated ASYN-(+) and UB-(+) aggregates that blocked organelle transport. Mitochondrial movement velocities were reduced by 8 days of rotenone and continued to decline over time. No cytoplasmic inclusions resembling Lewy bodies were observed. Gene microarray analyses showed that the majority of genes were under-expressed. qPCR analyses of 11 mtDNA-encoded and 10 nDNA-encoded mitochondrial electron transport chain RNAs' relative expressions revealed small increases in mtDNA-encoded genes and lesser regulation of nDNA-encoded ETC genes. CONCLUSION: Subacute rotenone treatment of differentiated SH-SY5Y neuroblastoma cells causes process retraction and partial death over several weeks, slowed mitochondrial movement in processes and appears to reproduce the Lewy neuritic changes of early Parkinson's disease pathology but does not cause Lewy body inclusions. The overall pattern of transcriptional regulation is gene under-expression with minimal regulation of ETC genes in spite of rotenone's being a complex I toxin. This rotenone-SH-SY5Y model in a differentiated human neural cell mimics changes of early Parkinson's disease and may be useful for screening therapeutics for neuroprotection in that disease stage.

Differentiated Alzheimer's disease transmitochondrial cybrid cell lines exhibit reduced organelle movement.

The axonal transport and function of organelles like mitochondria and lysosomes may be impaired and play an important role in the pathogenesis of Alzheimer's disease (AD). Unique cybrid cell lines that model AD pathology were created by fusing platelets containing mitochondria from age-matched AD and control volunteers with mitochondrial DNA-free SH-SY5Y human neuroblastoma cells. These cybrid lines were differentiated to form process-bearing neuronal cells. Mitochondria and lysosomes in the neurites of each cybrid line were fluorescently labeled to determine the kinetics of organelle movement. The mitochondria in AD cybrid neurites were elongate, whereas the mitochondria in control cybrid neurites were short and more punctate. The mean velocity of mitochondrial movement, as well as the percentage of moving mitochondria, was significantly reduced in AD cybrids. The velocity of lysosomal movement was also reduced in the processes of AD cybrid cells, suggesting that the axonal transport machinery may be compromised in cybrid cell lines that contain mitochondrial DNA derived from AD patients. Reduced mitochondrial and lysosomal movement in susceptible neurons may compromise function in metabolically demanding structures like synaptic terminals and participate in the terminal degeneration that is characteristic of AD.

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies.

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-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 alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-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.