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Abstract Background Spinal cord neurons of ALS patients demonstrate reduced cytochrome oxidase histochemical activity, and ALS spinal cord tissues have increased mitochondrial DNA (mtDNA) point mutations and depleted mtDNA levels. It is presently unknown whether mtDNA abnormalities are present in single human ALS neurons. Results Using laser capture microdissection (LCM) we isolated several hundred individual anterior spinal neurons from unfixed, frozen sections of 10 ALS and 7 age-matched CTL cervical spinal cords. DNA from each individual neuron was analyzed with multiplex qPCR for ND2, CO3, and ND4, three mitochondrial DNA genes encoding respiratory proteins. Scatterplots of individual spinal neuron results showed extensive heterogeneity of mtDNA gene levels across 4-5 orders of magnitude that were much more clustered in single Purkinje neurons isolated from CTL cerebella. Plots of ratios of ND4/ND2 and CO3/ND2 showed that many but not all ALS neurons from individuals contained low ratios of these mtDNA genes, implying greater abundances of mtDNA deletions in the major arc. Single CTL cerebellar Purkinje neurons did not contain high levels of apparent mtDNA deletions observed in anterior spinal neurons. Conclusions At the time of ALS subjects' deaths, many but not all surviving anterior neurons in their cervical spinal cords have reduced mtDNA gene levels and increased mtDNA deletion abundances that arise for unclear reasons. If these anterior spinal neuron mtDNA gene deficiencies contribute to bioenergetic impairments, reduced synaptic function and increased risk of degeneration, then introduction into mitochondria and expression of intact mtDNA, now available through use of recently developed recombinant human TFAM, may reverse the course of ALS.

Abstract Background Sporadic Parkinson's disease (sPD) is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA), heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships. Results Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1) inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease® showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V). Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1α expression did not differ between sPD and CTL brains. Conclusion PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age-related increases in distribution of oxidative mtDNA damage in sPD but not CTL brains is not clear, tracks with but does not determine the sPD phenotype, and may indicate a unique consequence of aging present in sPD that could contribute to mtDNA deletion generation in addition to mtDNA replication, transcription and sequencing errors. sPD frontal cortex experiences a generalized bioenergetic deficiency above and beyond aging that could contribute to mood disorders and cognitive impairments.

Abstract 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.