Introduction

Recently, the MRI-visible gene expression reporter potential for the metal ion transporter Zip14 delivered by adeno-associated viruses (AAV-Zip14) was demonstrated¹. Focal signal enhancement in T1 weighted MRI was observed in neuron specific Zip14 expressing areas of the mouse brain, including at the injection site and at anterograde projection sites. Excitingly, these results were obtained without needing to supplement the animal with manganese (Mn²⁺) which Zip14 is known to transport effectively^2,3. However, performing Mn²⁺-enhanced MRI (MEMRI) by supplementing with systemic Mn²⁺ may increase sensitivity as well as add to evidence that the contrast is produced by Mn²⁺ transport into Zip14 expressing cells.

Methods

Schematic experimental design is shown in Figure 1a. The Synapsin promoter, the gene coding for Zip14, and a histology-visible tag were packaged into AAVs and injected into the primary somatosensory area, barrel cortex (S1BC, n = 6). MRI were performed using conventional T1 weighted MPRAGE protocol immediately after and 1 week after injection of AAV-Zip14. Two weeks after AAV-Zip14 injection, 100 mM MnCl₂•4H₂O was delivered intraperitoneally at least 20 hours before MEMRI experiments (64 mg/kg) (dashed vertical lines in all figures). Due to global relaxation rate changes in the brain due to systemic Mn²⁺ uptake, a MEMRI-optimized MPRAGE protocol was implemented.

For MEMRI timecourse experiments, a separate cohort of mice were injected with AAV-Zip14 (n = 3) and a control cohort were injected with AAV expressing channelrhodopsin (AAV-ChR2, n = 3) into the S1BC. MnCl₂ was injected intraperitoneally, and imaging was performed at least two weeks after AAV injection using a T1-weighted GRE protocol at various timepoints up to one week after MnCl₂ injection.

Results

Injection of AAV-Zip14 into the S1BC produced signal enhancement in the S1BC and VPM without needing to supplement with MnCl₂ as previously described¹. No enhancement was apparent in the S1BC (magenta arrow) and the VPM (cyan arrow) immediately after injection. Cortical lamina-specific enhancement was apparent in the S1BC and moderate enhancement was apparent in the VPM due to anterograde transport as soon as one week after AAV-Zip14 injection.

Imaging performed one day after MnCl₂ injection, using the same imaging protocol, showed improved contrast between normal appearing gray matter and the Zip14 expressing S1BC and VPM (Figure 1c). No enhancement of M1 immediately after and one week after injection of AAV-Zip14 into the S1BC was apparent (yellow arrow). MEMRI performed two weeks after AAV-Zip14 and one day after systemic MnCl₂ injections revealed previously undetected, moderate M1 enhancement (Figure 1d).

Optimization of the MPRAGE protocol to account for brain-wide longitudinal relaxation rate changes due to MnCl₂ supplementation substantially improved contrast between Zip14 expressing areas and surrounding normal tissue (Figure 2a). Relative enhancement of injected (white bars) with respect to contralateral control (gray bars) S1BC is shown in Figure 2b. There were no significant differences in signal intensity apparent immediately after injection of AAV-Zip14. The injected cortex was significantly hyperintense one week after AAV-Zip14 injection and at the two week post injection timepoint using MEMRI. Optimization of the MEMRI protocol led to improved contrast compared to the routine protocol. There was an approximately four-fold improvement. The same trends were observed for the enhancement of the VPM ipsilateral to the injected S1 due to anterograde transport of Zip14 from the injected S1BC (Figure 2c). No enhancement was apparent immediately after injection of AAV-Zip14 into the S1BC. Significant enhancement was apparent in the ipsilateral VPM one week after AAV-Zip14 injection. Similar magnitude, significant enhancement was apparent using MEMRI at the two week injection timepoint. Optimization of the MEMRI protocol approximately doubled the apparent enhancement of the ipsilateral VPM.

Zip14 expression alters Mn²⁺ uptake and sequestration dynamics (Figure 3). Enhancement due to Zip14 expression was detected using T1-weighted GRE two weeks after AAV-Zip14 into the S1BC before administration of MnCl₂ (Figure 3b, Pre). No enhancement over baseline was apparent two weeks after AAV-ChR2 injection. After administration of MnCl₂, the Zip14 expressing S1BC enhanced above neighboring normal-appearing gray matter significantly more than ChR2 expressing areas. It was apparent both visually and quantitatively that the Zip14 expressing areas remained hyperintense for at least a week after MnCl­₂ administration.

Discussion and Conclusion

MEMRI can improve in vivo sensitivity for Zip14 expression. It is not necessary to introduce systemic Mn²⁺ to robustly detect AAV-Zip14 enhancement at the injection site. However, improved contrast-to-noise afforded by MEMRI improves detection of anterograde sites. Considering that these sites are enhancing due to Zip14 expression in only a small volume fraction occupied by the processes of the expressing neuron, the extra sensitivity should enable measurement of connectivity, structural plasticity, and neurodegeneration in vivo.

Acknowledgements

This work was supported by the Intramural Research program of the National Institutes of Neurological Disorders and Stroke (NINDS), National Institutes of Health. We thank Raymond Fields in the NINDS Viral Production Core Facility for virus production

References

1. Rallapalli, H., McCall, E. C., Bouraoud, N. & Koretsky, A. P. MRI detection of gene expression in the mammalian brain using the Manganese transporter Zip14. in Proceedings of the ISMRM (2023).

2. Steimle, B. L., Smith, F. M. & Kosman, D. J. The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels. Journal of Biological Chemistry 294, 19197–19208 (2019).

3. McCabe, S., Limesand, K. & Zhao, N. Recent progress toward understanding the role of ZIP14 in regulating systemic manganese homeostasis. Comput Struct Biotechnol J 21, 2332–2338 (2023).