[18F]FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis

Abstract

Background: The cystine/glutamate antiporter (xc-) has been implicated in several neurological disorders and, specifically, in multiple sclerosis (MS) as a mediator of glutamate excitotoxicity and proinflammatory immune responses. We aimed to evaluate an xc-specific positron emission tomography (PET) radiotracer, (4S)-4-(3-[¹⁸F]fluoropropyl)-L-glutamate ([¹⁸F]FSPG), for its ability to allow non-invasive monitoring of xc- activity in a mouse model of MS.

Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG_35-55) peptide in complete Freund's adjuvant (CFA) followed by pertussis toxin. Control mice received CFA emulsion and pertussis toxin without MOG peptide, while a separate cohort of naïve mice received no treatment. PET studies were performed to investigate the kinetics and distribution of [¹⁸F]FSPG in naïve, control, pre-symptomatic, and symptomatic EAE mice, compared to ¹⁸F-fluorodeoxyglucose ([¹⁸F]FDG). After final PET scans, each mouse was perfused and radioactivity in dissected tissues was measured using a gamma counter. Central nervous system (CNS) tissues were further analyzed using ex vivo autoradiography or western blot. [¹⁸F]FSPG uptake in human monocytes, and T cells pre- and post-activation was investigated in vitro.

Results: [¹⁸F]FSPG was found to be more sensitive than [¹⁸F]FDG at detecting pathological changes in the spinal cord and brain of EAE mice. Even before clinical signs of disease, a small but significant increase in [¹⁸F]FSPG signal was observed in the spinal cord of EAE mice compared to controls. This increase in PET signal became more pronounced in symptomatic EAE mice and was confirmed by ex vivo biodistribution and autoradiography. Likewise, in the brain of symptomatic EAE mice, [¹⁸F]FSPG uptake was significantly higher than controls, with the largest changes observed in the cerebellum. Western blot analyses of CNS tissues revealed a significant correlation between light chain of xc- (xCT) protein levels, the subunit of xc- credited with its transporter activity, and [¹⁸F]FSPG-PET signal. In vitro [¹⁸F]FSPG uptake studies suggest that both activated monocytes and T cells contribute to the observed in vivo PET signal.

Conclusion: These data highlight the promise of [¹⁸F]FSPG-PET as a technique to provide insights into neuroimmune interactions in MS and the in vivo role of xc- in the development and progression of this disease, thus warranting further investigation.

Keywords: EAE mice; FSPG; Multiple sclerosis; PET; Xc-.

Fig. 1

Ex vivo biodistribution of [¹⁸F]FSPG and [¹⁸F]FDG in EAE and naive mice. Ex vivo data was obtained 110 min after radiotracer injection (n = 5–9, mean %ID/g ± SD) with magnified views of uptake in brain and spinal cord. EAE scores: 2.6 ± 0.9 and 3.2 ± 0.3 for [¹⁸F]FSPG and [¹⁸F]FDG groups respectively. ***p 0.0001–0.001, **p 0.001–0.01

Fig. 2

Elevated [¹⁸F]FSPG-PET signal in EAE mice at early stages of disease. a Disease scores of mice induced with EAE over 18 days. Arrows indicate PET/CT imaging time points and scores are represented as mean score ± SEM. b Representative sagittal PET/CT images of control, pre-symptomatic EAE (score 0), and EAE mice with early stage disease symptoms (score 1.5). Arrows indicate spinal cord uptake. Bl—bladder, Br—brain, H—Haderian glands, I—immunization site, K—kidneys, S—salivary glands. c Representative sagittal PET/CT images of identical brain sections of control, pre-symptomatic EAE (score 0), and symptomatic EAE (score 1.5) mice. Cb—cerebellum, HC—hippocampus, Mb—midbrain, Med—medulla. Dotted lines highlight region containing possible meningeal vessel(s). d Representative PET/CT volume rendering technique (VRT) image of EAE mouse (score 1.5). PET signal outside spinal column and skull was removed for better visualization of [¹⁸F]FSPG uptake in areas of interest

Fig. 3

Quantification of [¹⁸F]FSPG-PET signal in CNS tissues of pre-symptomatic and symptomatic EAE versus control mice. Static PET imaging was performed 75 min post-injection of radiotracer (n = 6–10) and signal was quantified in the a whole spinal cord, b cervical/thoracic spinal cord, c lumbar spinal cord, and d whole brain. ***p 0.0001–0.001, **p 0.001–0.01, *p 0.01–0.05, NS—non-significant

Fig. 4

Significant correlation between xCT protein levels in CNS tissues and in vivo PET signal. a Representative Western blot with actin and NaK-ATPase as loading controls (Ctrl). Correlation between xCT protein levels (determined by densitometric Western analysis) and [¹⁸F]FSPG uptake (determined by PET quantification) in the b cervical/thoracic spinal cord (SC), c lumbar SC, and d brain. ***p 0.0001–0.001, **p 0.001–0.01, *p 0.01–0.05

Fig. 5

[¹⁸F]FSPG uptake in resting and activated primary human immune cells. In vitro accumulation of [¹⁸F]FSPG in isolated primary human T cells and monocytes (n = 5–11, mean %ID/g ± SD). ****p < 0.0001, **p < 0.01