Early mitochondrial abnormalities in hippocampal neurons cultured from Fmr1 pre-mutation mouse model

Abstract

Pre-mutation CGG repeat expansions (55-200 CGG repeats; pre-CGG) within the fragile-X mental retardation 1 (FMR1) gene cause fragile-X-associated tremor/ataxia syndrome in humans. Defects in neuronal morphology, early migration, and electrophysiological activity have been described despite appreciable expression of fragile-X mental retardation protein (FMRP) in a pre-CGG knock-in (KI) mouse model. The triggers that initiate and promote pre-CGG neuronal dysfunction are not understood. The absence of FMRP in a Drosophila model of fragile-X syndrome was shown to increase axonal transport of mitochondria. In this study, we show that dissociated hippocampal neuronal culture from pre-CGG KI mice (average 170 CGG repeats) express 42.6% of the FMRP levels and 3.8-fold higher Fmr1 mRNA than that measured in wild-type neurons at 4 days in vitro. Pre-CGG hippocampal neurons show abnormalities in the number, mobility, and metabolic function of mitochondria at this early stage of differentiation. Pre-CGG hippocampal neurites contained significantly fewer mitochondria and greatly reduced mitochondria mobility. In addition, pre-CGG neurons had higher rates of basal oxygen consumption and proton leak. We conclude that deficits in mitochondrial trafficking and metabolic function occur despite the presence of appreciable FMRP expression and may contribute to the early pathophysiology in pre-CGG carriers and to the risk of developing clinical fragile-X-associated tremor/ataxia syndrome.

Figure 1. Premutation cultures express higher levels of Fmr1 mRNAs with decreased FMRP proteins compared with WT paired cultures

(A) Representative western blot in paired cultures of WT and preCGG hippocampal neurons at 4 DIV. (B) Quantification of FMRP expression level relative to β-actin in paired WT and preCGG neuronal cultures at 4 DIV. Data were pooled from two independent cultures. (C) Fmr 1 mRNA comparison between WT and preCGG paired neuronal cultures at 4 DIV. Data were pooled from two independent cultures days performed in duplicate. **, p<0.01, preCGG vs. WT.

Figure 2. Decreased mitochondrial density in premutation hippocampal neurites

(A,B) Representative images of WT and preCGG 4 DIV hippocampal neurons labeled with Mitotracker dye. Below whole cell images are higher magnification images of proximal (within 25µm of soma) and distal (farther than 25µm from soma) neurites. (C) Number of mitochondria was decreased in preCGG proximal neurites by 25% (3.67±0.32 preCGG vs. 4.88±0.29 WT, p=0.01), WT n=98 neurites from 53 cells, preCGG n=53 neurites from 33 cells. (D) The number of mitochondria in distal neurites showed a trend toward fewer mitochondria in preCGG neurites, but this was not statistically significant (1.68±0.25 preCGG vs. 2.12±0.32 WT, p=0.338). WT n=66 neurites from 49 cells, preCGG n=41 neurites from 24 cells. Scale bars represent 5µm.

Figure 3. Decreased mitochondrial motility in premutation neurons

(A, B) Representative images of WT and preCGG 4 DIV hippocampal neurons labeled with Mitotracker dye. Below whole cell images are higher magnification time lapse images of mitochondrial movement in neurites. Labeled examples are shown of immobile (white arrowhead) as well as anterogradely (green arrowhead) and retrogradely (blue arrowhead) moving mitochondria. (C) Number of mobile mitochondria was decreased in preCGG neurons by 48% compared to WT (1.21±0.15 preCGG vs. 2.36±0.15 WT, p<0.01). (D) Number of immobile mitochondria was not significantly different between preCGG and WT neurons (1.91±0.17 preCGG vs. 1.76±0.12 WT, p=0.47). (E) Number of highly mobile mitochondria, was decreased in preCGG neurons by 66% compared to WT (0.16±0.04 preCGG vs.0.47±0.06 WT, p<0.01) (F) The direction of travel of highly mobile mitochondria was not significantly different between preCGG and WT neurons. Both showed similar percentage of mitochondria moving anterograde (ANTR), retrograde (RETR), or both directions (Both). ANTR: 0.20±0.09 preCGG vs. 0.23±0.04 WT, p=0.693. RETR: 0.74±0.09 preCGG vs. 0.63±0.05 WT, p=0.276. BOTH: 0.06±0.03 preCGG vs. 0.13±0.03 WT, p=0.239. WT n=98 neurites from 53 cells, preCGG n=53 neurites from 33 cells. Scale bars represent 5µm.

Figure 4. Bioenergetics in WT and preCGG neurons

(A) Time-response relationships for oxygen consumption before and after addition of oligomycin (1µM), FCCP (1µM) and rotenone (1µM) in both genotypes. The non-mitochondrial OCR determined by addition of rotenone (1µM) was subtracted. (B) Quantification of basal, ATP production, proton leak, and maximal OCR in WT and preCGG 4DIV hippocampal neurons. In preCGG neurons, the basal OCR was 23% higher (16.71±0.40 pmole/min/µg protein, n=36) compared to WT neurons (13.58±0.68 pmole/min/µg protein, n=30, p<0.01). ATP production in preCGG neurons was 17% higher (12.27±0.29 pmole/min/µg protein, n=30) than observed in WT neurons (10.48±0.48 pmole/min/µg protein, n=36, p<0.01). Proton leak was 43% higher (4.44±0.15 pmole/min/µg protein, n=30) compared to WT neurons (3.1±0.28 pmole/min/µg protein, n=36, p<0.01). Maximal OCR was 17% higher in preCGG neurons (14.67±0.69 pmole/min/µg protein, n=30) than observed for WT neurons (12.53±0.58 pmole/min/µg protein, n=36, p<0.05).