Synergistic effect of combined GDNF and Amodiaquine attenuates behavioral deficits and protect dopaminergic neurons of Parkinson’s disease animal model through tyrosine kinase Ret receptor

Parkinson’s disease (PD) is one among the most leading neurodegenerative disease after Alzheimer’s disease, with a prevalence of approximately 0.5–1% among those 65–69 years of age. Efforts to vitiate this disease are ongoing, and several treatment modes such as Glial cell line-derived neurotrophic factor (GDNF) have been in place since 1993. Glial cell line derived neurotrophic factor (GDNF) protects, regenerates, and improves the metabolism of substantia nigra pars compacta neurons (SNpc), and it increases the high-affinity dopamine uptake. It has been recently reported that amodiquine could attenuates the behavioral deficits of an animal model of Parkinson’s disease, nevertheless it mechanism is obscure. We sought to demonstrate the mechanism of neuro-protection effect of amodiquine and ascertain its corroborative effect when used togather with GDNF. We show herein that combined therapy (GDNF and amodiaquine) ameliorated behavioral deficits of PD animal models as compared to single-factor treatment. TH positive neurons increased significantly upon combined therapy treatment, and besides, GDNF and amodiaquine interact functionally to protect dopaminergic neurons through the PIK-3/Akt pathway. We also found that combined therapy (GDNF and amodiaquine) mediates its action through a distinct trans-membrane tyrosine kinase Ret receptor by amplifying its effect. Slight elevated aspartate aminotransferase (AST) were noticed in amodiaquine treated groups, alarming the bio-utility. These findings collectively suggest an interplay between GDNF and amodiaquine and co-express to exert neuronal protection hence a promising approach in PD therapy. Despite its undisputed effect on neuro-protection, we report that amodiaquine may not be safe, particularly in translation to human beings' trial settings.

nevertheless, amodiaquine, GDNF, and combined therapy prompted attenuation of these damages. In contemplation of the artistic effect of a single factor or combined treatment, RT PCR and western blotting were performed.

Combined therapy (GDNF and AQ) interact to promote dopaminergic neurons protection
We rendered to investigate whether combined therapy exhibits more effects as compared to the single factor treatment (GDNF or amodiaquine) in the 6-OHDA animal model. Importantly we implored RT-PCR to test if it can protect TH + and DAT + neurons, which are regarded to be dopaminergic neurons markers. DAT positive neurons were remarkably altered upon combined therapy treatment; the delta-delta ct revealed fold changes of 13, 45, and 72 and P values of P = 0.000274, 7.4663E-9, 1.7106E-10 (one way ANOVA followed by Bonferroni post hoc test) for Amodiaquine, GDNF and combined therapy respectively.
Similarly, TH positive neurons were also increased to a significant level upon combined therapy treatment compared to single-factor therapy, i.e., increased by 10 folds compared to 2.8 and 6.5 of AQ and GDNF. We also tested the RET genes as it is considered to be a GDNF receptor. Results show a fold change of 4.9,15 and 21 for AQ, GDNF, and combined therapy treatment, respectively. Collectively, these findings suggest that GDNF and AQ interact functionally to promote dopaminergic neurons protection (Fig. 2).

Combined therapy confers dopaminergic neurons protection in 6-OHDA animal model by restoring TH expression
We further sought to test the effect of combined therapy targeting tyrosine hydroxylase (TH), which is one of the dopaminergic neurons markers. We performed western blotting ( Fig. 3A.) and immunohistochemistry (Fig. 4), incorporating a single factor and combined treatment. By analysis of variance (ANOVA) followed by Bonferroni post hoc results reveals that the number of TH positive neurons was significantly increased in combined therapy compared to single-factor treatment (p is less than 0.005) Fig. 3A. These results suggest that combined therapy is more suitable to halt Parkinson's disease as it rescues degenerative dopaminergic neurons.
GDNF and amodiaquine interact functionally to protect dopaminergic neurons through PIK-3/Akt pathway It was described earlier that once GDNF bind to its receptors, it may induce the activation of mitogen-activated-kinase (MAPK) through phosphorylation of extracellular regulated kinase (ERK), P-38, JUNK and or PI3-K/Akt pathway. Importantly, Akt, which is downstream of PI3-K, has been implicated in neuronal regulation both in the central and peripheral nervous systems. Previous studies, Kim et al. [22] reported that amodiaquine could abolish behavioral deficits in the 6-OHDA animal model, which means amodiaquine may contain similar cellular effects like GDNF. We first tested the role of Akt and its impact on amodiaquine in 6-OHDA Parkinson's disease animal model and whether the combined therapy would dual its effect. Interestingly Akt in the amodiaquine treated group was slightly higher compared to GDNF treated group while the combined therapy group significantly augmented the Akt level in the ventral midbrain of the animal model. These findings collectively suggest an interplay between GDNF and amodiaquine and co-express to exert neuronal protection.
Combined therapy (GDNF and amodiaquine) mediates its action through a distinct transmembrane tyrosine kinase Ret receptor by amplifying its effect Ret, an orphan receptor tyrosine kinase, which is expressed during vertebrae embryogenesis in motor, dopamine, and noradrenaline, has been demonstrated previously that GDNF binds to and induce phosphorylation. Similarly, ret proteins were also found to bind to GDNF and mediate survival and growth responses. There is clear evidence that GDNF is crucial for the development, maintenance, and protection of dopaminergic neurons through tyrosine kinase Ret receptor, whether amodiaquine does the same is perplexing. We first sought to test the expression of kinase receptor-ret, employing RT-PCR on correspondent groups. Interestingly ret was similarly expressed on AQ as the GDNF group and highly expressed upon combined therapy treatment (Fig. 2). Moreover, we further wanted to elucidate the effect of combined therapy on western blotting, and we found that results are more significant compared to the other groups **p < 0.005 versus corresponding control (Fig. 3C).

Combined therapy could not counteract the weight loss
Previous studies reported that GDNF might not be safe as weight loss was observed in clinical trials. Several mechanisms were stipulated on how GDNF causes this. Lapchak [26] reports that GDNF may gain access to ventricular spaces and distribute to hypothalamic nuclei, where it alters neurotransmission to affect food intake and eventually may cause weight loss. Our results proved that there was a subsequent weight reduction today five in both GDNF and combined therapy treatment group (*p < 0.05 students t-test) Fig. 4b. In contrary to the amodiaquine group, the weight loss was not significant after five days of treatment. This indicates that amodiaquine is safe in terms of weight loss, nevertheless cannot attenuate the weight loss effect of GDNF.
Amodiaquine may not be safe for the liver Despite its undisputed effect on neuro-protection, it is, however, necessary to consider its implications when translated to human beings' trials. Previous studies reported that AQ has a deleterious effect, especially to the liver. To elucidate this, ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were determined as described by

Discussion
We have demonstrated that GDNF and amodiaquine enhanced behavioral deficits in 6-OHDA Parkinson's disease animal model; moreover, a combination of it might be superior in the treatment of Parkinson's disease. Previous studies had proved that GDNF alleviates behavioral deficits by exerting neuroprotection to the dopaminergic neurons of the substantia nigra and striatum [3,4,28,31] similarly to amodiaquine [22] on 6-OHDA Parkinson's disease animal model it has proved to attenuate behavioral deficits and enhance its dual functions. Our study, which is the combination of two therapy avenues, is per Oh et al. [37], who combined two gene therapies in the treatment of midbrain dopaminergic neurons toxic insults and found the synergistic action of the two gene therapy.
Furthermore, we quantified the expression of TH and DAT genes in the mRNA level by performing reverse transcriptase RNA in single factor treatment and combined therapy.
Tyrosine hydroxylase and dopamine transporter are one of the dopaminergic phenotype genes, and they get to be reduced in Parkinson's disease [32]. In the current study, the control group showed a significant reduction of TH and DAT, while the experimental groups revealed a substantial increase of TH and DAT. Moreover, by comparing the number of folds, the combined therapy group elicited a higher number of TH and DAT folds with respect to other groups. Concurrently, we performed the western blotting analysis and immunohistochemistry to elucidate whether the effect could be the same on the protein level, in this case employing TH antibody. Combined therapy could elicit higher TH folds and significant positive neurons compared to single-factor treatment. Collectively, our results give proof that combined therapy confers dopaminergic neurons protection in the 6-OHDA animal model by restoring TH expression and, at a particular standpoint, gives an impression that GDNF and AQ interact functionally to promote dopaminergic neurons protection. It is already known that GDNF protects dopaminergic neurons by restoring TH fibers in the injured dopaminergic neurons, and this concurs our results after 6-OHDA animal model being treated with GDNF independently [12,45].
Multiple lines of evidence indicate that PI3-K/Akt mediates the survival effect of GDNF [25]. Its roles had further been explored in the survival of the sympathetic neurons of the superior cervical ganglion [43], spinal motor neurons [24], cerebella granules cells [30], and midbrain dopamine cells [46]. Whether it does the same in 6-OHDA Parkinson's disease animal model was explored in our study. We found that both GDNF and amodiaquine protect dopaminergic neurons through the PIK-3/Akt pathway. Interestingly, when it was given as combined therapy, the Akt folds were increased significantly. Our findings collectively suggest an interplay between GDNF and amodiaquine and co-express to exert neuronal protection. Another emphasis was to investigate whether the tyrosine receptor -ret is involved in the co-expression effects prompted by the combined therapy of GDNF and amodiaquine.
Tyrosine kinase -ret receptor is the member of receptor tyrosine kinase superfamily that form a complex signaling component of Glial cells line-derived neurotrophic factor (GDNF) and its family ligands [15]. It is expressed in dopaminergic neurons, motor neurons, somatic sensory neurons, enteric neurons, sympathetic and parasympathetic neurons during their development and maintenance. Kramer & Liss, [25] reported that GDNF binds to tyrosine kinase receptor-Ret and induce phosphorylation. After it phosphorylates, activate other signaling cascades, including PI3-K/Akt, MAPK (JNK, P38, and ERK5), which will functionally stimulate cell survival, growth, differentiation, and neuritogenesis [25].
Surprisingly, our results show that Ret was also stimulated when we treated the animal with amodiaquine only and was highly stimulated when we combined the two therapy. This could be due to the fact that amodiaquine is an agonist of the nuclear receptor-related 1(Nurr1), an orphan member of the nuclear receptor superfamily which is highly expressed in the developing and adult ventral midbrain [22] and this might be the one added up the dual function.
In this current study, we noticed a dramatic weight loss after the animal was treated with GDNF. Our results comply the previous studies that reported a weight reduction after the subjects (Rodents-[26], monkeys- [39], and human beings- [5,33,47] being treated with GDNF. We thought amodiaquine could mimic the weight reduction effect of GDNF when combined therapy was applied, but that had never been so. This seems to be a physiological effect as postulated in different theories that GDNF may gain access to ventricular spaces and distribute to hypothalamic nuclei where it alters neurotransmission to affect food intake and eventually may cause weight loss [26]. The weight reduction was not statistically significant in the amodiaquine treated group when a comparison was made between day 0 (Before treatment) and day five after treatment.
Despite its undisputed effect on neuro-protection, we report that amodiaquine may not be safe, particularly in translation to human beings' trial settings. In the current study, we have found that the amodiaquine and combined therapy group had a slight elevation of AST. These elevated values are alarming the safety and utilization by human beings. It was reported earlier that amodiaquine might cause severe idiosyncratic drug reactions (IDR) that included hepatotoxicity and agranulocytosis [29,34]. These findings collectively suggest an interplay between GDNF and amodiaquine and co-express to exert neuronal protection hence a promising approach in PD therapy. Amodiaquine safety should not be underated.

6-OHDA lesion
Animals were anesthetized with sodium pentobarbital [6] and placed in the stereotaxic apparatus with the bite bar set at 0 mm. The skull was exposed, and the burr hole was made using a high-speed dental drill. All animals were administered 6-hydroxydopamine to the dural surface) [13]. The sham-operated animals received vehicle only (0.2% ascorbate in 0.9% saline) at the same coordinates. The injection carried out with a 10-ml the needle was removed, as described in the previous studies [2,4].

Elevated body swing test
Body asymmetry conducted, as previously described by Sanberg [40]. Briefly, the animal was first placed into a standard cage on the table for habituation and to attain a neutral position (all four paws on the ground). The animal was held approximately 3 cm from its tail base and elevated above the surface (3 cm) in the vertical axis. A swing recorded whenever the animal moved its head out of the vertical axis to either side and before attempting another rhythm, the animal must return to the vertical position for the next swing to be counted. Oscillations recorded by using a hand counter. The frequency of initial turning of the head or upper body contralateral to the lesioned side was calculated in 20 consecutive trials and normalized, as follows: % contralateral recovery = [1-(lateral turns in 20 trials-10)/10] X 100% [8,17,40]. Body asymmetry was assessed at three different times: (i) 14 days after 6-OHDA lesion, (ii) 28 days after 6-OHDA lesion, and (iii) 2 and 4 weeks after treatment. Animals with left side nigra 6-OHDA lesion will exhibit right side biased swings [40].
Four weeks after surgery, animals with an insufficient number of net rotations (< 1 clockwise rotation/min) [35] were discarded (about five mice). Apomorphine challenge was assessed at three different times: (i) 14 days after 6-OHDA lesion, (ii) 28 days after 6-OHDA lesion, and (iii) 2 and 4 weeks after treatment.

Rotarod
The rotarod was performed as described by Jiang et al. [21,44]. Briefly, the accelerating rotarod apparatus (Insight Scientific Equipments, Ribeirão Preto, SP, Brazil) consists of a grooved metal roller (6 cm in diameter) and separated 9-cm-wide compartments elevated at 16 cm. The spindle speed was increased to 40 rpm over a maximal period of 300 s, and the time spent on the accelerating rotarod and the corresponding rpm were determined.

GDNF, Amodiaquine and combined treatment
In order to reveal the robust effect of the combined therapy, the PD mice model was divided into four groups. The first group was the control, which received vehicle administration; in short, the PD model was injected normal saline (2 ul) through the same coordinates and continuously received an intraperitoneal injection of normal saline (10 ul/g) for 10 days similar to amodiaquine group. The amodiaquine group received a dose as previously described by Kim et al., [22,23] briefly; Amodiaquine (medchemexpress-china) CAS NO: 6398-98-7) was dissolved in 0.9% physiological saline at 4 mg/ ml and administered to mice at 40 mg/kg intraperitoneally, for ten days in total at the interval of 24hrs daily. GDNF group received 2 ul of GDNF at the concentration of 4ug/ul [6] through the same coordinates used to administer 6OHDA (3.0 mm posterior to bregma, 1.3 mm lateral and 4.7 mm ventral to the cranial surface). The combined therapy group received both GDNF and amodiaquine (the same protocol).

Weight
Previous studies have reported that one of the factors noticed in the failure of clinical trials was weight loss [5]. The same observation reported in 2019 [47]. The leading cause of weight loss for patients treated with GDNF is elusive. Some studies suggest that intranigrally -administered GDNF may gain access to ventricular spaces and distribute to hypothalamic nuclei, where it alters neurotransmission to affect food intake and eventually may cause weight loss [19]. A similar effect documented by Lapchak, a study that showed that GDNF distributes from lateral ventricle to fourth through the third ventricle and labels the hypothalamus, and this would indicate that GDNF alters hypothalamic neurotransmission which is necessary for feeding behavior [26]. In the current study, we wonder if amodiaquine administration could mimic or counteract the effect of GDNF on weight loss.

Immunohistochemistry
Under sodium pentobarbital anesthesia, mice were transcardially perfused with 100 ml PBS followed by 150 ml of cold 4% paraformaldehyde in 0.1M phosphate buffer (0.1M PB).
The brains were removed, fixed for 24 h at 4 •C in the same fixative, processed, and embedded in paraffin. Coronal brain sections (5 µm-thick) were cut on a microtome (Leica RM2155, Nussloch, Germany). Parts were deparaffinized in xylene and rehydrated in a gradient of ethanol and distilled water. After being washed three times with 0.01M PBS (for 5 min each), the sections were incubated in H2O2 solution for 5-15 min to block the endogenous peroxidase activity then washed three times with PBS. In order to reduce nonspecific staining, the sections were incubated with goat-serum at room temperature for 10-15mins then serum removed without washing. Incubated with the primary antibody in 37*C for 60 min and washed three times with 0.01M PBS, then sections were incubated with a biotinylated goat anti-rabbit IgG. After three piles of washing with 0.01 M PBS, horseradish enzyme-labeled streptomycin was added to react for 10-15mins at room temp.
After three times wash with 0.01M PBS, the sections were stained for peroxidase reaction by incubation with a mixture of diaminobenzidine (DAB) for 5-10 min at room temperature. This was followed by recoloration by incubating with hematoxylin for 20seconds, then dehydrated, cleared, and mounted with galvanol and were examined under a light microscope.

Western blot
The proteins of the ventral midbrain were prepared according to the previous studies [14,28]. In brief, animals (n = 4 per group) were sacrificed, brains rapidly removed, and the ipsi-and contra-lateral ventral midbrain were dissected and immediately put on dry ice for

RT-PCR
Total RNA was reverse transcribed into cDNA using PrimeScript™, RT Master Mix [37], and used for quantification of mRNAs encoding TH, DAT, and RET. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used as an internal control. The PCR program run at 25 °C for 10 min, then 42 °C for 30 min and 85 °C for 5 min [16]. Results were analyzed with a delta-delta Ct method.

Statistical analysis
All data were shown as mean ± SD. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test and the statistical significance level was set at p < 0.05 for all tests.

Liver hepatotoxicity test
Although amodiaquine was reported to halt Parkinson's disease motor symptoms, we rendered to determine whether the dosage we used is safe to the liver or not. Previous studies reported that AQ is toxic to the liver as it may cause severe idiosyncratic drug reactions (IDR) that included hepatotoxicity and agranulocytosis, necrosis, and presence of inflammatory cells on histopathological analysis [29,34]. In order to attain this, the liver test was done as described in the protocol ("http://www.bio-protocol.org/e931 Vol 3, Iss 19, Oct 05, 2013). Briefly, blood was collected from the orbital sinus with a microhematocrit blood tube (heparinized). The dropper was used to push out the blood in the heparinized blood tube, and about 300 µl of blood was collected in the 1.     Effect of combined therapy on TH, DAT, and RET mRNA. In the 8th week, substantia nigra of left ventral midbrain was dissected out, and cDNA prepared