Inhibiting mtDNA transcript translation alters Alzheimer's disease-associated biology

Abstract

Introduction: Alzheimer's disease (AD) features changes in mitochondrial structure and function. Investigators debate where to position mitochondrial pathology within the chronology and context of other AD features.

Methods: To address whether mitochondrial dysfunction alters AD-implicated genes and proteins, we treated SH-SY5Y cells and induced pluripotent stem cell (iPSC)-derived neurons with chloramphenicol, an antibiotic that inhibits mtDNA-generated transcript translation. We characterized adaptive, AD-associated gene, and AD-associated protein responses.

Results: SH-SY5Y cells and iPSC neurons responded to mtDNA transcript translation inhibition by increasing mtDNA copy number and transcription. Nuclear-expressed respiratory chain mRNA and protein levels also changed. There were AD-consistent concordant and model-specific changes in amyloid precursor protein, beta amyloid, apolipoprotein E, tau, and α-synuclein biology.

Discussion: Primary mitochondrial dysfunction induces compensatory organelle responses, changes nuclear gene expression, and alters the biology of AD-associated genes and proteins in ways that may recapitulate brain aging and AD molecular phenomena.

Highlights: In AD, mitochondrial dysfunction could represent a disease cause or consequence. We inhibited mitochondrial translation in human neuronal cells and neurons. Mitochondrial and nuclear gene expression shifted in adaptive-consistent patterns. APP, Aβ, APOE, tau, and α-synuclein biology changed in AD-consistent patterns. Mitochondrial stress creates an environment that promotes AD pathology.

Keywords: Alzheimer's disease; amyloid; mitochondria; neurons; translation.

FIGURE 1

Chloramphenicol inhibits SH‐SY5Y ρ+ cell mtDNA transcript translation, induces compensatory mtDNA copy number and transcription changes, and alters bioenergetic flux and enzyme function. (A) Chloramphenicol untreated and treated cells appeared equally viable. (B) Treated cells contained less MT‐CO2 and MT‐ND3 protein. (C) Treated cells had a higher mtDNAcn. (D) Treated cells contained more MTCO2 mRNA. (D) Treated cells contained more MTND3 mRNA. (F) Chloramphenicol treatment reduced the basal OCR and blunted the response to ATP synthase inhibition and mitochondrial membrane permeabilization. (G) Chloramphenicol treatment increased the basal ECAR. (H) Treated cells had a lower COX V_max activity. (I) CS V_max activities were comparable. Statistical testing reflects two‐tailed Student's t‐tests except for the V_max data (H, I), which used Mann‐Whitney U tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. COX, cytochrome c oxidase; CS, citrate synthase; ECAR, extracellular acidification rate; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; OCR, oxygen consumption rate; p+, with mtDNA; V_max, maximal velocity. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 2

Effect of chloramphenicol on SH‐SY5Y ρ+ cell nuclear‐encoded, mitochondria‐associated genes, and proteins. (A) Chloramphenicol‐treated cells contained less COX4 protein. (B) COX4 mRNA levels were comparable. (C) Treated cells contained less NDUFB8 protein. (D) NDUFB8 mRNA was reduced in the treated cells. (E) VDAC1 protein levels were comparable. (F) PPARGC1A mRNA was higher in the treated cells. (G) COX10 mRNA was unchanged. Statistical testing reflects two‐tailed Student's t‐tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. COX, cytochrome c oxidase; p+, with mitochondrial DNA. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 3

Chloramphenicol alters SH‐SY5Y ρ+ cell AD‐pertinent biology. (A) Chloramphenicol‐treated cells contained more APOE mRNA. (B) APP mRNA levels were comparable. (C) Treated cells contained more APP protein. (D) Treated cells contained more lower band APP, less upper band APP, and an increased lower band to upper band APP ratio. (E) BACE1 protein levels were comparable. (F) Treated cells contained less MAPT mRNA. (G) Western blot total tau protein levels were comparable. (H) Treated cells contained more ser9‐phosphorylated GSK‐3β. (I) SNCA mRNA levels were comparable. Statistical testing reflects two‐tailed Student's t‐tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. AD, Alzheimer's disease; APOE, apolipoprotein E; APP, amyloid precursor protein; p+, with mitochondrial DNA; OCR, oxygen consumption rate;. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

FIGURE 4

Chloramphenicol‐induced changes in SH‐SY5Y ρ+ cells are not observed in chloramphenicol‐treated SH‐SY5Y ρ0 cells. (A) NDUFB8 mRNA levels were comparable between chloramphenicol treated and untreated SH‐SY5Y ρ0 cells. (B) APOE mRNA levels were comparable. (C) Total APP protein levels were comparable. (D) Ser9‐phosphorylated GSK‐3β levels were comparable. (E) Whereas chloramphenicol‐treated ρ+ cells contained less COX4 protein than their untreated controls, chloramphenicol‐treated SH‐SY5Y ρ0 cells contained more COX4 protein than their untreated controls. (F) OCRs were essentially undetectable and non‐responsive to both ATP synthase inhibition and mitochondrial membrane permeabilization. (G) ECARs were comparable. Statistical testing reflects two‐tailed Student's t‐tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. APOE, apolipoprotein E; APP, amyloid precursor protein; COX, cytochrome c oxidase; ECAR, extracellular acidification rate; p+, with mitochondrial DNA; ρ0, without mitochondrial DNA. ns = not significant, *p ≤ 0.05.

FIGURE 5

Chloramphenicol inhibits iPSC‐derived neuron mtDNA transcript translation, induces compensatory mtDNA copy number and transcription changes, and alters bioenergetic flux and enzyme function. (A) Chloramphenicol untreated and treated neurons appeared equally viable. (B) Treated neurons contained less MT‐CO2 and MT‐ND3 protein. (C) Treated neurons had a higher mtDNAcn. (D) Treated neurons contained more MTCO2 mRNA. (E) Treated neurons contained more MTND3 mRNA. (F) Treated neurons contained more MTCO1 mRNA. (G) Treated neurons contained more MTND1 mRNA. (H) Chloramphenicol treatment increased the basal OCR. (I) Although the basal ECARs were comparable, following ATP synthase inhibition the ECAR was higher in the chloramphenicol‐treated neurons. (J) Treated neurons had a lower COX V_max activity. (K) The CS V_max activity was higher in the treated neurons. Statistical testing reflects two‐tailed Student's t‐tests except for the V_max data (J, K), which used Mann‐Whitney U tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. COX, cytochrome c oxidase; CS, citrate synthase; ECAR, extracellular acidification rate; iPSC, induced pluripotent stem cell; mtDNA, mitochondrial DNA; OCR, oxygen consumption rate; V_max, maximal velocity. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 6

Effect of chloramphenicol on iPSC‐derived neuron nuclear‐encoded, mitochondria‐associated genes and proteins. (A) Chloramphenicol‐treated neurons contained less COX4 protein. (B) Treated neurons contained more COX4 mRNA. (C) Treated neurons contained less NDUFB8 protein. (D) Treated neurons contained more NDUFB8 mRNA. (E) Treated neurons contained more VDAC1 protein. (F) PPARGC1A mRNA was higher in the treated neurons. (G) COX10 mRNA was higher in the treated neurons. Statistical testing reflects two‐tailed Student's t‐tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. COX, cytochrome c oxidase; iPSC, induced pluripotent stem cell. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 7

Chloramphenicol alters iPSC‐derived neuron AD‐pertinent biology. (A) Chloramphenicol‐treated neurons contained more APOE mRNA. (B) An apoE ELISA showed the amount of apoE protein was higher in the media of the treated neurons. (C) The amount of apoE protein was higher in the treated neuron lysates. On western blot this manifested as an increase in the full‐length protein, and as an increase in an ∼12 kD band that reportedly represents a C‐terminal apoE fragment. The fragment to full length ratio was higher in the treated neurons. (D) The APP mRNA level was higher in the treated neurons. (E) Treated neurons contained more APP protein, but unlike what was observed with the SH‐SY5Y ρ+ cells the neuronlower band to upper band APP ratios were comparable. (F) BACE1 protein levels were higher in the treated neurons. (G) An ELISA for Aβ42 showed the amount of Aβ42 protein was higher in the media of the treated neurons. (H) Treated cells contained more MAPT mRNA. (I) Western blot total tau and serine 404‐phosphorylated tau protein levels were higher in the treated neurons. (J) Treated neurons contained more serine 9‐phosphorylated GSK‐3β. (K) SNCA mRNA levels were higher in the treated neurons. (L) The treated neurons contained more α‐synuclein protein. Statistical testing reflects two‐tailed Student's t‐tests. Error bars represent SEM. −/+ indicates with or without chloramphenicol treatment. Aβ, beta amyloid; AD, Alzheimer's disease; apoE/APOE, apolipoprotein E; APP, amyloid precursor protein; ELISA, enzyme‐linked immunoassay; iPSC, induced pluripotent stem cell; p+, with mitochondrial DNA. ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

FIGURE 8

Inverse correlation between apoE and TOMM20 protein levels in iPSC‐derived neurons. (A) Representative confocal images of chloramphenicol‐treated and untreated neurons stained with an N‐terminus region apoE antibody (green), TOMM20 antibody (red), and DAPI. (B) In the untreated condition, individual neurons showed an inverse relationship between apoE and TOMM20 protein levels; the Pearson correlation coefficient was −0.7626. (C) In the treated condition, individual neurons showed an inverse relationship between apoE and TOMM20 protein levels; the Pearson correlation coefficient was −0.7111. In (B) and (C), the solid line represents the linear regression, while the dashed lines represent the 95% confidence intervals. apoE, apolipoprotein E; DAPI, 4′,6‐diamidino‐2‐phenylindole; iPSC, induced pluripotent stem cell.