Combining CRISPR/Cas9 and brain imaging: from genes to molecules to networks

Abstract

Understanding how functional connections between brain regions are arranged and influenced by genes, in the healthy and diseased states, is a major goal in neuroscience. Functional connectivity alterations are linked to molecular changes in several neurodegenerative disorders and could serve as early markers of pathology. Yet, the underlying molecular signatures driving the functional alterations remain largely unknown. Here, we combine CRISPR/Cas9 gene-editing with in vivo positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to investigate the direct link between genes, proteins, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent basis for studying the gene networks relationships. We edited Slc18a2 into the substantia nigra pars compacta of adult rats and used in vivo molecular imaging, behavioral, histological, and biochemical assessments to characterize the CRISPR/Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to inspect the functional brain adaptations, beyond the predicted dopaminergic changes. Further, [11C]flumazenil PET was carried out to investigate the dopamine-GABA interplay. We found a regional increase in postsynaptic dopamine receptor availability, preceded by a reorganization of brain networks that adapt to reduced dopamine transmission states by becoming functionally connected and organized. The hyperconnectivity within and between brain networks spreads from the contralateral thalamus and prefrontal cortical regions to the striata and hippocampi. Additionally, impaired striatal dopamine release reduces GABA-A receptor availability, complementing the increased synchrony and functional connectivity between networks observed at rest. Our study reveals that recruiting different brain networks may be an early response to the dopaminergic dysfunction preceding neuronal cell loss, postsynaptic changes, and motor impairment in neurodegenerative disorders such as Parkinson's disease. We anticipate our combinatorial approach to be a starting point to investigate the impact of specific genes on brain molecular and functional dynamics, aiding in the identification of early neurobiological markers and promising therapeutic interventions.

Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks

“…Data associated with the reported findings are available in the manuscript or SI Appendix . All original PET and fMRI data and codes for the data analysis have been deposited at DRYAD repository: doi: 10.5061/dryad.zw3r228bb , and is publicly available as of the date of publication ( 107 ).…”

“…In several neurological disorders, including epilepsy, major depression, Parkinson’s disease, and Alzheimer’s disease, next-generation sequencing approaches have identified novel receptors, transporters, and ion channels involved in disease initiation and progression. However, their role in pathogenesis is poorly understood ( 43 ). Gene editing combined with in vivo imaging modalities, as developed in the present study, will help to identify the molecular and functional role of these targets and the adaptations following their regional dysfunction on the whole-brain level.…”