ALS spinal neurons show varied and reduced mtDNA gene copy numbers and increased mtDNA gene deletions

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.

Figure 1

Approximate location of the PCR products used for qPCR analysis of mtDNA genes ND2, CO3 and ND4.

Figure 2

Cumulative percentages of copy numbers for ND2, CO3 and ND4 genes in individual anterior spinal neurons from the ALS and CTL populations.

Figure 3

Cumulative percentages for ratios of ND4/ND2 and CO3/ND2, estimating relative abundances of deletions in ND4 and CO3, respectively, in individual anterior spinal neurons from the ALS and CTL populations.

Figure 4

Scatterplots relating the ND2 copy numbers to ratios of ND4/ND2 in the CTL (left) and ALS (right) individual anterior spinal neurons.

Figure 5

Scatterplots relating the ND2 copy numbers to ratios of CO3/ND2 in the CTL (left) and ALS (right) individual anterior spinal neurons.

Figure 6

Scatterplots showing how individual ALS cases display relatively greater deletion abundance for CO3 compared to ND4 (63835, 56550) or for ND4 compared to CO3 (63470, 63693).

Figure 7

Scatterplots relating the ND2 copy numbers to ratios of ND4/ND2 (top) and CO3/ND2 (bottom) in cerebellar Purkinje neurons from four CTL cases.