Scientists develop noninvasive way to monitor gene expression dynamics in the brain

By Vijay Kumar MalesuJan 12 2024Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Nature Biotechnology, a group of researchers introduced a non-invasive, sensitive method for monitoring gene expression in the brain using engineered reporters called released markers of activity (RMAs), which can exit the brain into the blood for easy detection.

Background 

Monitoring gene expression in the living brain is crucial for understanding brain function, behavior, and neurological diseases. Traditional methods face challenges due to the brain's complex structure, requiring non-invasive yet sensitive and specific techniques. There are various methods, such as Magnetic Resonance Imaging (MRI), ultrasound imaging, and optical systems, that have limitations like low sensitivity and difficulty in imaging deep brain regions or limitation of multiplexing.

Most studies depend on post-mortem analysis, which is not capable of tracking changes over time in the same animal.  Further research is needed to optimize and validate the use of RMAs across different brain regions and conditions, ensuring their broad applicability and reliability in diverse neurological research contexts.

About the study

Researchers from Rice University conducted different studies on mice with a protocol that was approved by the Institutional Animal Care and Use Committee as part of several experiments. These experiments were done using male and female mice from different strains, which were procured from The Jackson Laboratory and kept under controlled conditions.

The first phase involved plasmid construction. Researchers utilized a specific vector and executed several procedures, including digestion, isolation, amplification, and extraction of deoxyribonucleic acid (DNA). They focused on creating various constructs by inserting different sequences into the plasmid backbone. These constructions included Adeno-Associated Virus-human Synapsin-Ribosomal Mutant A (AAV-hSyn-RMA), with different variations, AAV-Glial Fibrillary Acidic (Gfa)-RMA, AAV-Fos protein (Fos)-RMA, and others, each serving a unique purpose in the research.

For cell culture experiments, the Pheochromocytoma Cell Line 12 (PC-12) was used. These were cultured in controlled environments and later subjected to an in vitro luciferase assay. Similarly, rat cortex-derived astrocytes were cultured and transfected to proceed with the assays. In both scenarios, the bioluminescence produced by the transfected cells was carefully recorded.

The researchers also conducted experiments on protein purification, using Escherichia coli cells for transforming and expressing RMA proteins. These proteins were then subjected to various processes, including centrifugation, lysis, and elution, to achieve the desired purity for further use.

A critical part of the study involved the production of AAV vectors. These were created by transfecting Human Embryonic Kidney 293 cells with SV40 large T-antigen (HEK293T) cells with various plasmids and later harvesting and purifying the virus using a series of complex procedures.

Stereotaxic injections were performed on mice to introduce proteins or AAV into specific brain regions. The injection process was carefully carried out using precise coordinates and controlled conditions.

Various methods were used to assess the effects of the substances introduced. These included blood draws for luciferase assays, serum half-life of RMA proteins, and chemical administration. Furthermore, histological imaging and analysis of mice brains were done to investigate the distribution patterns as well as effects procured from injected substances.

Finally, the researchers applied statistical analysis to their data interpretation. This involved several tests to compare datasets and determine the significance of their findings. The results were carefully documented, and figures were constructed using Adobe Illustrator, illustrating the comprehensive nature of the study.

Study results 

In the study, researchers conducted several experiments to evaluate the effectiveness of RMAs in detecting gene expression in the brain. Initially, RMAs were tested for their ability to cross the blood-brain barrier (BBB). It was achieved by injecting RMA proteins into the caudate putamen of mouse brains and determining plasma concentration.

The results indicated a significant rise in plasma concentration of Gaussia luciferase (Gluc)-RMA, a variant of RMA, which suggested that RMA transportation from the brain to the blood occurred through a fragment crystallizable (Fc)-dependent mechanism. Furthermore, plasma Gluc-RMA levels declined only slightly over a period of time while controls demonstrated pronounced reduction, suggesting that the half-life of this biomarker is several hours long and thus allows it to accumulate in the blood.

The study then explored the in vivo detection of brain gene expression using RMAs. Researchers injected AAV encoding both Gluc-RMA and Green Fluorescent Protein (GFP) under the hSyn promoter into the mouse brain. They observed substantial signal increases in plasma, indicating the ability of RMAs to detect gene expression in vivo. The experiments were conducted in various brain regions like the caudate putamen, Cornu Ammonis 1 (CA1), and substantia nigra.

The findings showed more than 20,000-fold higher signals over baseline in all three regions. Additionally, the signal levels persisted up to the third week, possibly due to plasma Gluc-RMA reaching a steady state. The study also indicated that Gluc-RMA reliably detects approximately 0.001% of neurons in the mouse brain.

In assessing the safe expression levels of RMAs, the study focused on identifying expression levels that maximize benefits while minimizing side effects, particularly immune responses. Gluc-RMA was expressed in the left caudate putamen under the hSyn promoter with varying AAV doses. The results showed significant plasma RMA signals for all doses without significant neuronal loss, suggesting that any potential adverse effects of Gluc-RMA were not sufficient to cause neuronal death even at the highest dose.

The researchers also monitored brain cell-type-specific gene expression. They delivered hSyn-controlled double-floxed Gluc-RMA into Tyrosine Hydroxylase-Cre (Th-Cre) mice, which express Cre in dopaminergic neurons. The study found specific expressions of Gluc-RMA and GFP in Th-positive cells at the local injection site, demonstrating Gluc-RMA's ability to detect brain gene expression in small neuronal cell populations and in a cell-type-specific manner.

Furthermore, the ability of RMAs to track changes in the expression of the immediate early gene Fos, which rapidly expresses upon cellular stimulus or neuronal activity, was investigated. An in vitro model using PC-12 cells transfected with Gluc-RMA controlled under the Fos promoter was used.

The results showed increased expression of Gluc-RMA and Fos upon nerve growth factor induction, with the luminescence signal of the culture media rising significantly within six hours of exposure, suggesting that Gluc-RMA can generate a distinguishable signal output in response to changes in promoter activity.

Lastly, the study explored RMAs for non-invasive measurement of neuronal activity. A double-conditional strategy was employed, linking Gluc-RMA expression to neuronal activity. The study demonstrated multiplexed ratiometric measurements of RMAs and their ability to report on in vivo neuronal activity in specific brain regions. Additionally, Gluc-RMA was used to improve bioluminescence imaging (BLI), showing enhanced signal intensity and correlation with gene expression levels.