In a recent study published in Gene Therapy, researchers utilize the CRISPR-Cas9 system to induce the synthesis of dopamine (DA) in the brains of a rat model for Parkinson’s disease (PD).
Study: CRISPR/sgRNA-directed synergistic activation mediator (SAM) as a therapeutic tool for Parkinson´s disease. Image Credit: Sergey Nivens / Shutterstock.com
Background
The incidence of PD increases with age and is the second most common neurological disease. PD is caused by the degeneration of dopaminergic neurons in the substantia nigra pars compacta, which produces DA to regulate motor control.
The optimal approach for managing PD involves the use of levodopa (L-DOPA), a precursor that facilitates the formation and substitution of DA. Importantly, this treatment has a finite duration of effectiveness, which typically spans about five years.
Astrocytes are critical for the immunological response of the brain in PD, as demonstrated by animal models and post-mortem investigations. Tyrosine hydroxylase (Th) is an enzyme found within astrocytes that is crucial for the production of DA. Previously, the authors of the current study reported that the implantation of astrocytes expression Th successfully increased DA production in the stratium of both rat and non-human primate models of PD.
Clustered regularly interspaced short palindromic repeats associated with Cas (CRISPR-Cas) is a novel technology that has revolutionized gene therapy. Synergistic activation mediator (SAM) is a second-generation CRISPR system that activates gene expression using enzymatically inactive Cas9 (dCas9) and co-transcriptional activators.
The present study examines the effects of endogenous gene activation in stimulating DA production in astrocytes, followed by the implantation of these cells in a rat model of PD. These findings offer a targeted therapeutic approach that can increase the drug-free period of treatment and can be combined with other therapies in advanced cases of PD.
About the study
The researchers analyzed the rat genome to identify potential th small guide ribonucleic acid (sgRNA) sequences that were highly specific and did not align with other areas of the rat genome. This led to the identification of 13 sgRNAs for th gene activation.
The levels of Th messenger ribonucleic acid (mRNA) in C6 cells transfected with the selected Th sgRNAs were assessed using real-time polymerase chain reaction (RT-PCR).
The 6-hydroxydopamine (6-OHDA) lesion stereotaxic surgery is a widely used rat model to study PD. Herein, 6-OHDA is injected directly into the median forebrain bundle (MFB), wherein it destroys dopamine neurons. Control rats underwent the same procedure but were injected with a 0.1% ascorbic acid in saline solution.
The 6-OHDA rats were subsequently implanted with 20,000 astrocytes (AST) or astroycytes expression Th (AST-TH) in both the anterior and posterior portions of the striatum. Both before and after surgery, rats underwent several behavioral tests including amphetamine-induced rotations, cylinder, and inclined beam balance tests to determine whether astrocyte implantation improved behavioral characteristics of hemi-Parksonian rats.
Study findings
Of the 13 th sgRNAs that were identified in the analysis, the researchers discussed the results of TH4 sgRNA, as it achieved the highest levels of Th protein expression. Both C6 and AST-TH cells transfected with TH4 sgRNA constantly released DA into the culture medium, which was not observed in their respective untransfected cells. The expression of TH4 sgRNA was also confirmed through immunofluorescence and Western blot analysis.
Behavioral studies revealed that rats that received primary astrocytes exhibited significantly greater circling behavior as compared to rats that received AST-TH cells. Likewise, rats treated with control astrocytes exhibited significant differences in forelimb placement asymmetry (FPA) in the cylinder test as compared to AST-TH transplanted rats.
The inclined beam balance test, which is used to measure fine motor control and balance, indicated that AST-TH transplanted rats exhibited similar behavioral characteristics to non-Parkinsonian rats.
Immunohistochemical analysis of the rat brains indicated that areas of the striatum that were implanted with AST-TH exhibited increased expression of DA, which is comparable to the lack of DA observed in lesioned areas of the brain. Furthermore, the turnover of DA within AST-TH transplanted brains remained intact, whereas striatum as treated with control astrocytes did not exhibit any turnover of DA. These findings demonstrate that AST-TH improves the rate of DA metabolism in the brain.
As compared to rat brains that were injected with control astroyctes, those implanted with AST-TH exhibited co-localization of Th and glial fibrillary acidic protein (GFAP), the latter of which is a biomarker of neuroinflammation.
Irritable bowel syndrome not linked to higher Parkinson's risk, study finds