Neuroprotective Efficacy of a Nanomicellar Complex of Carnosine and Lipoic Acid in a Rat Model of Rotenone-Induced Parkinson’s Disease

Round 1

Reviewer 1 Report

The paper by Dr. Olga Kulikova describes a nanomicellar complex of carnosine and lipoic acid has neuroprotective effect on rotenone-induced Parkinson’s disease rat model. Carnosine and lipoic acid were antioxidant effect and some studies reported they attenuate oxidative stress, motor activity and hypokinesia severity in Parkinson’s disease. However, the efficacy was limited by susceptibility to hydrolysis. Therefore the authors carried the compound with nanomicellar to improve solubility in water and bioavailability. The study might shed light on therapy of parkinson's Disease but some of the methods need to be clarified.

 

1.     The data of food intake should be included in figure or supplemental figure.

2.     Please define abbreviation of MPTP, HPLC/ED, LPO and TH-.

3.     Please correct alphabet in figure 6 legend.

 

Author Response

The authors extend their thanks to the Reviewer for their time and attention to the present manuscript. We are grateful for the favorable opinion expressed by the Reviewer and their suggestions, and have amended the text in accordance with their suggestions.  

1. We added a reference to supplementary table S1 in the body of the manuscript. This table contains the data of food intake.
2. Where applicable, we introduced the expansion of the abbreviations MPTP, HPLC/ED, LPO, and TH.
3. We corrected the legend to Figure 6.

Reviewer 2 Report

This is an interesting paper which outlines a study  which outlines the evidence of neuroprotection elicited by carnosine and ALA treatment of a rotenone induced  rat model of Parkinson`s disease (PD).

I just have a few comments/questions for the authors which are outlined below.

1. In the introduction to the paper I could find any information that confirmed the ability of the nanoparticle containing carnosine or ALA to cross the blood brain barrier to access the CNS which I think is pertinent to this study.

2. In this study it appears that the rats received simultaneous injections of both rotenone and CLA nanomicellar complex. Would there be any possibility that the CLA complex could interfere with the binding of rotenone to the CoQ10 binding site and hence appear to induce some therapeutic benefit? Did the authors do any in vitro studies prior to the animal experiments to ensure that the carnosine, ALA or the CLA nanomicellar complex may interfere with the rotenone induced complex I deficiency?

3. I couldn`t see any biochemical investigations to confirm that the rotenone treatment induced a loss of mitochondrial respiratory chain complex I activity as depending on the degree of inhibition this may dictate oxidative stress or an energy deficit as the principal pathological disease factor in the rat PD model.

4. Why was evidence of lipid peroxidation and antioxidant capacity only studied in the  frontal lobe of the cerebral hemi spheres as PD mainly affects the mid brain and can the authors be certain that the inc in antioxidant capacity is simply a reflection of the amount of exogenous antioxidant present in the cerebral rather any other effects on endogenous antioxidant capacity? In view of the details in the introduction and link between PD and  GSH an assessment of the status of this antioxidant would have been appropriate rather than simply relying on the CL assay.

5. What are the rationales for selecting the therapeutic doses of CLA in the study?

 

Author Response

We would like to thank the Reviewer for the time and effort they invested in reviewing our manuscript, and sincerely appreciate all of their valuable questions and suggestions, which will undoubtedly help us in planning future investigations in this research area. We have strived to answer each question to the best of our knowledge and expertise.

1. In the complex investigated in this study, carnosine is conjugated with lipoic acid via an easily hydrolyzed ionic bond. As such, it is extremely difficult to correctly analyze the permeability of the BBB to this nano micellar complex in its unaltered state – it would only be possible by using traceable labels attached to both of the compounds. It should be noted, that carnosine and lipoic acid both occur endogenously in the human and animal brain. Literature data does indicate that carnosine can pass through the BBB (PMID: 26036009). However, carnosine is easily hydrolyzed within the organism by specific proteases, carnosinases, and can be synthesized within the brain de novo from beta-alanine and histidine. Since the carnosine present in the nano micellar complex can also by hydrolyzed and used to re-synthesize it in vivo, the task of analyzing BBB permeability is further complicated by the necessity to attach two isotope labels to carnosine – one to histidine and one to beta-alanine. While the task of evaluating BBB permeability to the investigated complex is certainly of great importance, due to its complexity, we plan on investigating it in future studies after verifying its neuroprotective properties.

2. In our previous investigations Kulikova, O.I. (PMID: 29601911) we demonstrated the effectiveness of CLA on day 14 and 15 post MPTP administration, which indicates that CLA affects the pathological process induced by MPTP, rather than neutralizing its immediate toxic effect.

We also previously investigated the in vitro effects of CLA on neuron cell culture viability in conditions of rotenone-induced toxicity (unpublished results). Introduction of CLA to the cell medium prevented ROS formation, which was accompanied by an increase in cell viability. This effect can be attributed to protection of the mitochondrial complex I from rotenone toxicity by CLA, or direct binding of CLA by ROS. The molecular mechanisms behind the neuroprotective effects of CLA are of great interest, and we intend to investigate them in future studies. The question of whether CLA can directly interfere with rotenone binding to ubiquinone is outside the scope of this manuscript, and deserves a separate investigation. We are grateful to the Reviewer for asking this question, and will definitely conduct an in vitro investigation addressing it in the future.

3. In T.B. Sherer et al. (PMID: 14645467), it was shown in vitro that rotenone targets the mitochondrial complex I, leading to a decrease in ATP, increased ROS generation, oxidative damage and cell death. Protecting the mitochondrial complex I prevented mitochondrial damage, suppressed oxidative stress, and prevented cell loss. Oxidative stress played a key role in these processes. At the same time, the same authors showed that in vivo prolonged rotenone administration to rats led to damage in the brain, accompanied by a decrease in tyrosine hydroxylase levels and carbonylated protein accumulation. These alterations were observed primarily in the olfactory bulb and midbrain, which are known to affected in Parkinson’s disease.

In Thakur P. (PMID: 24946750), who used the same PD model (subcutaneous administration of 2 mg/kg rotenone to rats) over the course of 5 weeks, rotenone was shown to affect not only the mitochondrial complex I, but also complexes II and IV. They also showed a decrease in antioxidant activity, specifically decreased mitochondrial glutathione and decreased Mn-SOD and  Cu-Zn-SOD activity.

In this study, we limited our investigation to the analysis of the consequences of rotenone-induced oxidative stress, including the evaluation of the overall state of the antioxidant system in the frontal lobes of the brain, alterations in catecholamine metabolism in the striatum, and direct neuroprotective effects of CLA expressed in decreased neuron loss in the substantia nigra. We plan to investigate the mechanism of these neuroprotective effects, including the effects CLA exerts on the electron transport chain of the mitochondria and its indirect antioxidant effects, in the future.

4. We evaluated GSH content in the striatum, but did not observe any differences between intact animals and animals which received rotenone over the course of 18 days. Thus, these results were not included in the manuscript. However, in studies published by other authors where rotenone was injected over the course of 5 weeks, GSH levels decreased significantly (El-Sayed E.K. demonstrated a 60.12% decrease in the midbrain, PMID: 30001633; Thakur P. showed a 40% decrease in the midbrain, PMID: 24946750). We conjecture that the changes in GSH level observed by these authors may be attributed to a longer rotenone administration period in comparison to our study.

Regarding the CL assay, we would like to draw the Reviewer’s attention to the fact that this method facilitates the evaluation of overall antioxidant activity and susceptibility of brain tissue to peroxidation. The frontal lobes were chosen due to the presence of the mesocortical dopaminergic pathway, and the striatum was used to evaluate catecholamine content.

5. Previously, in in vivo models of parkinsonism, we and other groups of scientists determined the range of effective doses of carnosine that prevent the development of pathological processes associated with the development of oxidative stress. The doses range from 50 to 500 mg/kg of body weight, depending on the frequency of administration and the method of administration (for example, when administered with drinking water a higher dose of carnosine is used). The CLA dose of 50 mg/kg was chosen based on the minimum effective dose of carnosine, since the animals received CLA intraperitoneally daily. For lipoic acid, the neuroprotective effect of lower doses (e.g., 35 mg/kg in Andreeva-Gateva P. (PMID: 32024109) and 50 mg/kg in Zaitone, S.A. (PMID: 21958946) in PD modeling has been shown, so we also chose to investigate the CLA dose of 25 mg/kg, which is two times less than the effective carnosine dose. Also in our work Kulikova, O.I. (PMID: 29601911), administration of 100 mg/kg of CLA complex two times, on days 14 and 15 after administration of MPTP, was effective.

Round 2

Reviewer 2 Report

The authors have addressed my comments appropriately and manuscript should now be considered for publication.

Minor changes in syntax required.