Neurotoxicity of cytarabine (Ara-C) in dorsal root ganglion neurons originates from impediment of mtDNA synthesis and compromise of mitochondrial function

Abstract

Peripheral Nervous System (PNS) neurotoxicity caused by cancer drugs hinders attainment of chemotherapy goals. Due to leakiness of the blood nerve barrier, circulating chemotherapeutic drugs reach PNS neurons and adversely affect their function. Chemotherapeutic drugs are designed to target dividing cancer cells and mechanisms underlying their toxicity in postmitotic neurons remain to be fully clarified. The objective of this work was to elucidate progression of events triggered by antimitotic drugs in postmitotic neurons. For proof of mechanism study, we chose cytarabine (ara-C), an antimetabolite used in treatment of hematological cancers. Ara-C is a cytosine analog that terminates DNA synthesis. To investigate how ara-C affects postmitotic neurons, which replicate mitochondrial but not genomic DNA, we adapted a model of Dorsal Root Ganglion (DRG) neurons. We showed that DNA polymerase γ, which is responsible for mtDNA synthesis, is inhibited by ara-C and that sublethal ara-C exposure of DRG neurons leads to reduction in mtDNA content, ROS generation, oxidative mtDNA damage formation, compromised mitochondrial respiration and diminution of NADPH and GSH stores, as well as, activation of the DNA damage response. Hence, it is plausible that in ara-C exposed DRG neurons, ROS amplified by the high mitochondrial content shifts from physiologic to pathologic levels signaling stress to the nucleus. Combined, the findings suggest that ara-C neurotoxicity in DRG neurons originates in mitochondria and that continuous mtDNA synthesis and reliance on oxidative phosphorylation for energy needs sensitize the highly metabolic neurons to injury by mtDNA synthesis terminating cancer drugs.

Keywords: Cytarabine (ara-C); DNA damage response; DNA polymerase γ; Dorsal root ganglion neurons; Mitochondria; Neurotoxicity; mtDNA.

Figure 1. mtDNA polymerase γ activity is inhibited by the cytosine analog ara-C

Reactions were assembled with end-labeled oligonucleotide substrates, dGTP and recombinant pol γ. (A) Substrate design is shown above the relevant lanes; * indicates 5’-end label; R - indicates ara-C residue. The one nucleotide extension [+1] product generated on non-modified substrate [lanes 2–4] is abolished on 3’-ara-C containing substrate [lanes 5–7]. Polymerase γ 3’-exonuclease activity [−1 product, lanes 3–4] is also abolished on 3’-ara-C containing substrate [lanes 5–7]. Likewise bypass extension reaction opposite ara-C residue incorporated in the template strand is inhibited [lanes 11–13]. 21-mer substrate (S) [lane #1] and the 20-mer and 22-mer products [P] are indicated. (B) Yields of [+1] extension products generated by pol γ on modified and non-modified substrates are presented as percent mean±SEM of 3 reactions sets. ***P<0.001 indicates different from either extension or bypass reaction assembled with non-modified substrates.

Figure 2. Ara-c exposure increases ROS in DRG neurons

In situ imaging of superoxide-mediated oxidation of dihydroethidium to 2-hydroxyethidium (2-HE) in ara-C exposed DRG neurons. Representative images: 2-HE is observed as red fluorescence and nuclei stain blue with DAPI; scale bar = 10 µm. Fluorescence intensity normalized to soma surface area and quantified by ImageJ, is presented as mean±SEM of three biological experiments; in each set #8 neurons were sequentially scored per each condition; *P<0.05.

Figure 3. Detection of oxidative DNA damage in mtDNA of ara-C exposed DRG neurons

Representative images show immunofluorescence of 8-oxoguanine (8-oxoG) in DRG neurons: (A) In DRG cultures exposed to ara-C for 24 h cytoplasmic punctate 8-oxoG immunoreactivity is observed (green); DRG neurons are identified by immunoreactivity of NF200 (red). 8-oxoG IF is observed also in small DRG neurons, which do not react with anti NF200 antibody (center panel, green; scale bar = 10 µm). (B) Bar graph shows percent of 8-oxoG positive DRG neurons as a function of exposure time. Values represent mean+SEM for four biological experiments. (C) Representative images of DRG neurons double stained with antibodies reacting with 8-oxoG (green) and mtDNA-binding mitochondrial transcription factor A (TFAM) (red). In ara-C exposed DRG neurons, TFAM largely co-localizes with the punctate green 8-oxoG immunofluorescence (merged image).

Figure 4. Depletion of mitochondrial content, reduction of mtDNA copy number and reduced expression of mtDNA encoded genes in ara-C exposed DRG neurons

(A) Following ara-C exposure IF of mitochondrial cytochrome oxidase 1 (COX1) showed reduced signal and aggregated distribution in soma of DRG neurons (green, arrowhead); DRGs are identified by NF200 (red), scale bar = 10 µm. (B) Reduced mtDNA copy number in DRG neurons following ara-C exposure. (C) Expression of mitochondria encoded genes was reduced in the course of ara-C exposure. Bars show changes relative to respective controls. Values represent mean±SEM for four biological experiments; *P<0.05 versus control.

Figure 5. Ara-C exposure compromises mitochondrial respiration and decreases NADPH and GSH levels in DRG neurons

(A) Respiratory profile for non-treated control DRG neurons (black) shows baseline OCR of ~180 pmoles O₂/min. Sequential, in port additions of mitochondrial effectors (vertical arrows), reveal that ~90% of total oxygen consumption feed mitochondrial respiration: of this OCR ~70% support ATP synthesis and ~30% is lost to proton leak. Spare respiratory capacity (SRC), i.e., FCCP-induced increase in OCR over the baseline is ~250% for control DRGs. Following ara-C exposure baseline OCR (red) drops by ~30% and maximal respiration by nearly 50% (FCCP-induced), with ~30% reduction in SRC and ~ 20% increase in OCR feeding the proton leak. (Bottom) Oxygen tension in control wells is reduced by the addition of FCCP and even further by 2-deoxyglucose (2DG). Oxygen tension is reduced to a lesser extent in ara-C exposed DRGs, reflecting diminished mitochondrial OCR. (B) Each parameter calculated for control cultures was assigned the value of 100%; effects of treatment were calculated as percent change relative to each respective control. Data are presented as mean±SEM of four independent biological experiments. (C) NADPH levels are decreased and NADP⁺/NADPH ratios increased by ara-C. Concomitant addition of 6AN increased these effects. Data are mean±SEM for 3 independent experiments; * different from control; **different from ara-C; P< 0.05. (D) GSH levels were reduced by ara-C and further depleted by 6AN. Data are given as mean±SEM calculated from 3 independent experiments; * different from control; **different from ara-C; P< 0.05

Figure 6. Temporal induction of nuclear γH2AX foci in ara-C exposed DRG neurons

Representative images of time dependent formation of nuclear γH2AX foci during ara-C exposure of DRG neurons are shown (A). DRG neurons are identified by NF200 (red) and γH2AX foci are observed in green; sparse foci emerge by 12 h with density increasing in the course of 72 h ara-C exposure (scale bar = 20 µm). B) Percent of γH2AX positive nuclei increased over time with positive DRGs reaching ~35%, 75% and 95% by 24 h, 48 h and 72 h, respectively. Data are obtained from three independent biological experiments and presented as mean±SEM percent of γH2AX positive nuclei versus time.

Figure 7. Nuclear γH2AX/γATM foci induced by ara-C exposure of DRG neurons are gradually cleared in the course of post ara-C recovery

Representative images of γH2AX/γATM foci formed following 48 h ara-C exposure and foci clearance in the course of 96 h recovery. (A) Merged images show largely colocalized γH2AX [red]/γATM [green] foci with gradual reduction in foci density in the course of post exposure recovery [scale bar = 10 µm]. (B) Bar graphs show the number of co-localized γH2AX/γATM foci per DRG nucleus as a function of recovery time. Data are mean±SEM values obtained from four independent biological experiments.