PREreview del Chemogenetic Manipulation of the Subthalamic Nucleus-Substantia Nigra Pars Reticulata Pathway Promotes Recovery in HemiParkinsonian Rat Models

Summary

In this study, researchers modified neurons in the subthalamic nucleus to release GABA using chemogenetic tools, resulting in a recovery of motor control in rats modeling one-sided Parkinson's disease. These preliminary findings provide a treatment that is a less-invasive alternative to conventional deep brain stimulation.

PROS:

Abstract

• The paper clearly states that there are 2 pathways in the basal ganglion and what the indirect pathway does. This effectively establishes the necessary background context. Although not necessary, some readers might benefit from a slightly more in-depth analysis of how the direct and indirect pathways are different.

• Explains which pathway is affected in Parkinson’s disease. This immediately hooks the reader and highlights the significance of the research.

• Clear and quantifiable result. Clearly outlines what the results of the study were (a reduction in post-lesional amphetamine-induced rotations…).

• The flow of the abstract is easy to understand at an undergraduate level, even for someone who has minimal background on the effects of Parkinson’s. However, neuroscience jargon such as GAD65 and 6OHDA is used, which many will be unfamiliar with, so it is important to explain these terms further in the paper.

Introduction

• Clearly includes the demographics of Parkinson’s disease, emphasizing its devastating nature and the need for further studies surrounding it.

• Clinical relevance: This paper emphasizes that PD is a neurodegenerative disease with no current known cure, while also providing sufficient background information on pharmacological treatments involving neurotransmitters that can be used for symptom relief.

• Etiology: clearly explains how PD is caused, the immediate effects of the disease (decrease in the neurotransmitter dopamine in the synaptic cleft), and the pathway of MAO- B degrading dopamine, which promotes glutamate accumulation and results in symptoms manifesting.

• Thoroughly explains what the symptoms of PD are, emphasizing physical limitations and decreases in motor performance.

• This paper provides a great transition from deep-brain stimulation (DBS) as a current therapeutic approach to the direct and indirect motor pathways of the STN and basal ganglia. By mentioning the possible damage and detriment that DBS can cause, it allows the reader to understand that new, safer approaches to counteract PD symptoms are needed (the gap in research).

• Reference to prior studies (e.g., Dr. Luo’s 2002 GAD65 study and the macaque DREADD experiments) provides context for this project regarding the current scientific landscape.

• Thoroughly explains the aim of the research project and how they hope to combine GAD and DREADDs in their project.

  • Use GAD expression to convert excitatory neurons to inhibitory neurons

  • Excite inhibitory STN neurons via excitatory DREADDs to reduce the indirect pathway’s excessive suppression

Results

• Clearly introduced how each co-expression of the AAV’s (Adeno-associated Virus) in the injections was tracked through fluorescent markers.

• Very thorough and transparent about injection usages (60HDA) and its effects on all of the rats (increase in CCW rotations).

• The paper’s strategy and mechanisms were very well constructed and easy to follow along.

• Going over the process of the post-effects of 60HDA, where it increased the CCW rotations in rats, allowing them to train their contralateral lesioned side, so that later on they can utilize both sides more efficiently after recovery, helps the reader understand the process of how the results came to be.

• Results positively show a slight decrease in CCW movement, which shows that the therapeutic method is working, and the utilization of statistics evidently supports it.

• Ended their results very well as their statistics did show that through CNO the GAD65 neurons activated showed a significant decrease in CCW rotations (Before CNO: 1743 and After CNO: 895.3).

Methodology

Plasmid creation and characterization

• The use of a hSyn promoter guarantees that your final product will have neuronal specificity.

• The cassette created is polycistronic and encodes GAD65 and a reporter (eGFP) that is separated by a self-cleaving sequence (P2A), which increases the likelihood of the two proteins being expressed in similar amounts, reducing variability.

• Sequencing confirmation of the constructs mitigates the risk of frame-shifts or mutations in the constructs.

Viral production and purification

• Triple transfection in HEK293T cells is a common method for AAV production and is considered to be reliable.

• Cesium chloride ultracentrifugation produces high-purity AAV preps.

• The viral titers, which are quantified using qPCR targeting ITRs, will be an accurate measure of genome copies present.

Stereotaxic surgeries

• Using the rat brain atlas and confirming coordinates allows for better targeting and more precision.

• Using a slow infusion rate (0.1μL/min.) and allowing for a needle hole time of 10 minutes will minimize backflow and improve uptake of the viral vector.

• Post-operative care of examination of the head and recovery under saline and Rimadyl after surgery supports animal care standards.

Behavioral assays

• Amphetamine-induced rotations are a well-validated assessment of a Parkinsonian model.

• Using each animal as its own control (pre- vs. post-lesion, ±CNO) adds to the rigor of the experiment.

Histological validation

• Immunofluorescence with both GFP/mCherry confirmation provides evidence for expression/activity and the target.

• Using well-characterized antibodies will add to the validity of your assays compared to non-verified antibodies.

Discussion

• The authors link their findings with previous studies, such as the DREADD study.

• The authors appropriately discuss the limitations of sample size and lesion model in detail.

• The authors try their best to provide logical explanations to some unexpected results in the pre-lesion rotational data, such as suggesting an imbalance in basal ganglia circuits and hm3dq only neurons increasing excitability.

Conclusion

• The author carefully and concisely explains how the STN-SNr circuit can be manipulated.

• The authors highlight how minimally invasive gene therapies could mirror DBS in high-risk PD patients.

• There is a very clear distinction between the experimental conditions of treatment with and without CNO. The study emphasizes that behavioral changes only occurred when hm3dq was activated.

Figures

• Figures and data are clearly illustrated and are easy to interpret.

CONS:

Abstract

• The paper could benefit from clearly explaining what the ‘gap in the field’ is. The abstract currently mentions that “the indirect pathway is thought to suppress movements through excessive excitation of inhibitory neurons within the globus pallidus internus and substantia nigra pars reticulata”. It would be beneficial to expand on the repercussions of this and explain how exactly movements are suppressed by adding a phrase or sentence here.

• Abstracts include about 1-2 sentences regarding the methods. Include a short phrase or sentence on how inducing the expression of GAD65 in glutamatergic subthalamic neurons might be a novel approach for the motor circuit.

• A sentence is provided briefly explaining what the findings of previous studies showed. This sentence should be removed, and the information should be expanded greatly in the introduction section. This would include analyzing a method used in a previous study that was to be replicated, or showing a result that another study achieved that requires further work, or has left a gap regarding the co-expression of GABA with glutamate and the effects on the motor system. In its place, the abstract should add another sentence about the methods used or about the results achieved. For example, a sentence on how a therapeutic augmentation of the Parkinsonian phenotype was achieved.

• The abstract ends with “positive effect on motor outcome”. Provide a stronger statement of significance on what the next steps of this research might mean for research into Parkinson's or novel approaches that can result from this (a small phrase on what we can do with this information).

• It states that “a reduction in post-lesional amphetamine-induced rotations” was achieved, but does not quantitatively inform us on the degree of reduction, which is crucial for context. The abstract needs to provide statistical significance.

Introduction

• The aim is almost complete, but it could benefit from an explanation of how restoring the balance of the direct and indirect pathways would recover motor function. As the intended mechanism of action of their methodology is quite complex, more information on how this would work at the molecular level would benefit the reader.

• A hypothesis of the research project would be beneficial to include at the end of the introduction to tell the readers what they expect to achieve with their work. Given this novel approach, this section might also benefit from mentioning research objectives instead of a hypothesis.

• Provide more information on the direct and indirect pathways of the basal ganglia and the pathophysiology surrounding this for more background information.

• Explain in further detail how DREADDS, which are synthetic neurotransmitters, function in comparison to conventional neurotransmitters, and how DREADDS are localized within the basal ganglia pathway. This can provide an additional angle for the reader to understand why excitatory DREADDS can be used to suppress excessive motor activity.

  • An inclusion of a diagram of these pathways would be beneficial to visually understand what pathways this project aims to alter (STN to GPi/SNr). These thorough neurobiological topics need to be explained at a slower pace to provide a thorough understanding of the ‘why’ behind the project and to keep the reader's attention.

Results

• Wasn’t transparent on the description of 6OHDA and could use more elaboration on it to clear confusion, but the mechanisms of it are clearly stated.

• Small sample size. The results would be more reliable if they used a two-tailed z test instead of a t-test, where the sample size is greater than or equal to 30. Would also benefit from testing with a sample without CNO in pre-lesioned rats.

• Small P value of 0.0153=>1.53% when comparing post-lesioned rats with other rats with and without CNO. Could potentially show the unreliability of that data used to support the claim that post-lesioned rats had a higher CW rotation than rats with and without CNO.

Methodology

Plasmid/Viral Construct

• The experimentation only involves the use of hSyn promoter and not a neuron-specific promoter (e.g. CAMKII), which could influence the results if expression in off-target neurons occurs.

• Any potential for activity-induced recombination or rearrangements that may occur in long constructs was not addressed beyond sequencing in one instance.

• The PEG precipitation step before cesium chloride purification may introduce contaminants that impact in vivo immune responses.

Viral Fluorescent Protein Production

• Zoned purification with cesium chloride is effective (removes fats and salts), however delays the use of the viral construct. It can also be toxic to the viral construct and might limit viral infectivity when compared to iodixanol-based methods.

• When quantifying viral stocks at a concentration in gc/mL there is no measure of functional titer (infectious particles versus empty capsids).

Surgical Model 

• The limited sample size of subjects (n=9) limits statistical power.

• Only unilateral injections were performed. The bilateral effect was not evaluated.

• Reliance on retrograde AAV transport assumes uniformity of transport efficiency exists across animals.

• The 6-OHDA lesion model is well-established, but there is notable variance regarding the size and severity of the lesion.

Behavioral Testing 

• Amphetamine-induced rotation is a broad estimate of the Parkinsonian phenotype that may miss subtle motor changes and variability in performance.

• Clozapine can create problems with inference after CNO is nonselectively metabolized. Since clozapine itself has pharmacological effects, these drugs can mask results.

Histology 

• Having only GAD65 (and GABAergic) at the Green Fluorescent Protein (GFP) and dsRED reporters comprises the antibody panels that were used. They did not validate GAD65 expression or function directly.

• While they state that they will be assessing immune or inflammatory markers, none were quantified in the respective and stated manner.

Discussion

• The authors give speculative explanations regarding the therapeutic mechanism.

• The discussion gives much credit to studies with small sample sizes, which has been regarded as a limitation.

• Grammar: “Findings elicited in this study contribute to PD research and neuroscience as a whole.” should be changed to “Findings elicited in this study contribute to PD research and neuroscience as a whole.”

• Concise: “Our study’s findings demonstrated a therapeutic augmentation” repeated twice, can be rephrased to be more concise.

• The paper mentions a “statistically significant reduction” and an “increase,” but is not specific about the p-values or variance, which would make the interpretation more meaningful.

• The release mechanism of GABA is stated to be unknown, and non-vesicular release is mentioned, but the way this impacts STN, STN circuitry, and STN behavior is not explained. Including this would make the findings more impactful

Conclusion

• Again, the authors are overly optimistic about the positive impacts despite the limited sample size.

Figures

• In Figure 2, the images show no quantified data or individual data points

• Figure 3’s legend describes 3 panels: A, B, and C. However, it does not give an interpretation of why this information is statistically significant to the research findings.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.