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Gene Therapy Jan 2024Parkinson`s disease (PD) is the second most prevalent neurodegenerative disease, and different gene therapy strategies have been used as experimental treatments. As a...
Parkinson`s disease (PD) is the second most prevalent neurodegenerative disease, and different gene therapy strategies have been used as experimental treatments. As a proof-of-concept for the treatment of PD, we used SAM, a CRISPR gene activation system, to activate the endogenous tyrosine hydroxylase gene (th) of astrocytes to produce dopamine (DA) in the striatum of 6-OHDA-lesioned rats. Potential sgRNAs within the rat th promoter region were tested, and the expression of the Th protein was determined in the C6 glial cell line. Employing pseudo-lentivirus, the SAM complex and the selected sgRNA were transferred into cultures of rat astrocytes, and gene expression and Th protein synthesis were ascertained; furthermore, DA release into the culture medium was determined by HPLC. The DA-producing astrocytes were implanted into the striatum of 6-OHDA hemiparkinsonian rats. We observed motor behavior improvement in the lesioned rats that received DA-astrocytes compared to lesioned rats receiving astrocytes that did not produce DA. Our data indicate that the SAM-induced expression of the astrocyte´s endogenous th gene can generate DA-producing astrocytes that effectively reduce the motor asymmetry induced by the lesion.
Topics: Rats; Animals; Parkinson Disease; RNA, Guide, CRISPR-Cas Systems; Oxidopamine; Rats, Sprague-Dawley; Neurodegenerative Diseases; Clustered Regularly Interspaced Short Palindromic Repeats; Dopamine; Corpus Striatum; Tyrosine 3-Monooxygenase; Substantia Nigra
PubMed: 37542151
DOI: 10.1038/s41434-023-00414-0 -
CNS Neuroscience & Therapeutics Feb 2024Tyrosine decarboxylase (TDC) presented in the gut-associated strain Enterococcus faecalis can convert levodopa (L-dopa) into dopamine (DA), and its increased abundance...
AIM
Tyrosine decarboxylase (TDC) presented in the gut-associated strain Enterococcus faecalis can convert levodopa (L-dopa) into dopamine (DA), and its increased abundance would potentially minimize the availability and efficacy of L-dopa. However, the known human decarboxylase inhibitors are ineffective in this bacteria-mediated conversion. This study aims to investigate the inhibition of piperine (PIP) on L-dopa bacterial metabolism and evaluates the synergistic effect of PIP combined with L-dopa on Parkinson's disease (PD).
METHODS
Metagenomics sequencing was adopted to determine the regulation of PIP on rat intestinal microbiota structure, especially on the relative abundance of E. faecalis. Then, the inhibitory effects of PIP on L-dopa conversion and TDC expression of E. faecalis were tested in vitro. We examined the synergetic effect of the combination of L-dopa and PIP on 6-hydroxydopamine (6-OHDA)-lesioned rats and tested the regulations of L-dopa bioavailability and brain DA level by pharmacokinetics study and MALDI-MS imaging. Finally, we evaluated the microbiota-dependent improvement effect of PIP on L-dopa availability using pseudo-germ-free and E. faecalis-transplanted rats.
RESULTS
We found that PIP combined with L-dopa could better ameliorate the move disorders of 6-OHDA-lesioned rats by remarkably improving L-dopa availability and brain DA level than L-dopa alone, which was associated with the effect of PIP on suppressing the bacterial decarboxylation of L-dopa via effectively downregulating the abnormal high abundances of E. faecalis and TDC in 6-OHDA-lesioned rats.
CONCLUSION
Oral administration of L-dopa combined with PIP can improve L-dopa availability and brain DA level in 6-OHDA-lesioned rats by suppressing intestinal bacterial TDC.
Topics: Humans; Rats; Animals; Levodopa; Parkinson Disease; Oxidopamine; Tyrosine Decarboxylase; Gastrointestinal Microbiome; Dopamine; Bacteria; Antiparkinson Agents; Disease Models, Animal; Alkaloids; Piperidines; Benzodioxoles; Polyunsaturated Alkamides
PubMed: 37528534
DOI: 10.1111/cns.14383 -
Molecules (Basel, Switzerland) Jul 2023Parkinson's disease (PD) is an age-related, progressive neurodegenerative disease characterized by the gradual and massive loss of dopaminergic neurons in the substantia...
Parkinson's disease (PD) is an age-related, progressive neurodegenerative disease characterized by the gradual and massive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). We have recently reported that artemisinin, an FDA-approved first-line antimalarial drug, possesses a neuroprotective effect. However, the effects and underlying mechanisms of artemisinin on Parkinson's disease remain to be elucidated. In this study, we investigated the neuroprotective effects of artemisinin on 6-OHDA and MPP in neuronal cells and animal models, as well as the underlying mechanisms. Our results showed that artemisinin significantly attenuated the loss of cell viability, LDH release, elevated levels of reactive oxygen species (ROS), the collapse of the mitochondria trans-membrane potential and cell apoptosis in PC12 cells. Western blot results showed that artemisinin stimulated the phosphorylation of ERK1/2, its upstream signaling proteins c-Raf and MEK and its downstream target CREB in PC12 cells in a time- and concentration-dependent manner. In addition, the protective effect of artemisinin was significantly reduced when the ERK pathway was blocked using the ERK pathway inhibitor PD98059 or when the expression of ERK was knocked down using sgRNA. These results indicate the essential role of ERK in the protective effect of artemisinin. Similar results were obtained in SH-SY5Y cells and primary cultured neurons treated with 6-OHDA, as well as in cellular models of MPP injury. More interestingly, artemisinin attenuated PD-like behavior deficit in mice injected with 6-OHDA evaluated by behavioral tests including swimming test, pole-test, open field exploration and rotarod tests. Moreover, artemisinin also stimulated the phosphorylation of ERK1/2, inhibited apoptosis, and rescued dopaminergic neurons in SNc of these animals. Application of ERK pathway inhibitor PD98059 blocked the protective effect of artemisinin in mice during testing. Taking these results together, it was indicated that artemisinin preserves neuroprotective effects against 6-OHDA and MPP induced injury both in vitro and in vivo by the stimulation of the ERK1/2 signaling pathway. Our findings support the potential therapeutic effect of artemisinin in the prevention and treatment of Parkinson's disease.
Topics: Rats; Humans; Mice; Animals; Parkinson Disease; MAP Kinase Signaling System; Oxidopamine; Neuroprotective Agents; Neuroprotection; Neurodegenerative Diseases; RNA, Guide, CRISPR-Cas Systems; Neuroblastoma; Apoptosis; Artemisinins; Dopaminergic Neurons
PubMed: 37513399
DOI: 10.3390/molecules28145527 -
Neurobiology of Disease Sep 2023L-DOPA-induced dyskinesia (LID) is a frequent adverse side effect of L-DOPA treatment in Parkinson's disease (PD). Understanding the mechanisms underlying the...
L-DOPA-induced dyskinesia (LID) is a frequent adverse side effect of L-DOPA treatment in Parkinson's disease (PD). Understanding the mechanisms underlying the development of these motor disorders is needed to reduce or prevent them. We investigated the role of TrkB receptor in LID, in hemiparkinsonian mice treated by chronic L-DOPA administration. Repeated L-DOPA treatment for 10 days specifically increased full-length TrkB receptor mRNA and protein levels in the dopamine-depleted dorsal striatum (DS) compared to the contralateral non-lesioned DS or to the DS of sham-operated animals. Dopamine depletion alone or acute L-DOPA treatment did not significantly increase TrkB protein levels. In addition to increasing TrkB protein levels, chronic L-DOPA treatment activated the TrkB receptor as evidenced by its increased tyrosine phosphorylation. Using specific agonists for the D1 or D2 receptors, we found that TrkB increase is D1 receptor-dependent. To determine the consequences of these effects, the TrkB gene was selectively deleted in striatal neurons expressing the D1 receptor. Mice with TrkB floxed gene were injected with Cre-expressing adeno-associated viruses or crossed with Drd1-Cre transgenic mice. After unilateral lesion of dopamine neurons in these mice, we found an aggravation of axial LID compared to the control groups. In contrast, no change was found when TrkB deletion was induced in the indirect pathway D2 receptor-expressing neurons. Our study suggests that BDNF/TrkB signaling plays a protective role against the development of LID and that agonists specifically activating TrkB could reduce the severity of LID.
Topics: Mice; Animals; Levodopa; Antiparkinson Agents; Brain-Derived Neurotrophic Factor; Dopamine; Receptor, trkB; Dyskinesia, Drug-Induced; Corpus Striatum; Mice, Transgenic; Dopaminergic Neurons; Receptors, Dopamine D2; Oxidopamine
PubMed: 37495178
DOI: 10.1016/j.nbd.2023.106238 -
Clinics (Sao Paulo, Brazil) 2023The 6-OHDA nigro-striatal lesion model has already been related to disorders in the excitability and synchronicity of neural networks and variation in the expression of...
BACKGROUND
The 6-OHDA nigro-striatal lesion model has already been related to disorders in the excitability and synchronicity of neural networks and variation in the expression of transmembrane proteins that control intra and extracellular ionic concentrations, such as cation-chloride cotransporters (NKCC1 and KCC2) and Na+/K+-ATPase and, also, to the glial proliferation after injury. All these non-synaptic mechanisms have already been related to neuronal injury and hyper-synchronism processes.
OBJECTIVE
The main objective of this study is to verify whether mechanisms not directly related to synaptic neurotransmission could be involved in the modulation of nigrostriatal pathways.
METHODS
Male Wistar rats, 3 months old, were submitted to a unilateral injection of 24 µg of 6-OHDA, in the striatum (n = 8). The animals in the Control group (n = 8) were submitted to the same protocol, with the replacement of 6-OHDA by 0.9% saline. The analysis by optical densitometry was performed to quantify the immunoreactivity intensity of GFAP, NKCC1, KCC2, Na+/K+-ATPase, TH and Cx36.
RESULTS
The 6-OHDA induced lesions in the striatum, were not followed by changes in the expression cation-chloride cotransporters and Na+/K+-ATPase, but with astrocytic reactivity in the lesioned and adjacent regions of the nigrostriatal. Moreover, the dopaminergic degeneration caused by 6-OHDA is followed by changes in the expression of connexin-36.
CONCLUSIONS
The use of the GJ blockers directly along the nigrostriatal pathways to control PD motor symptoms is conjectured. Electrophysiology of the striatum and the substantia nigra, to verify changes in neuronal synchronism, comparing brain slices of control animals and experimental models of PD, is needed.
Topics: Rats; Animals; Male; Parkinson Disease; Oxidopamine; Rats, Wistar; Chlorides; Disease Models, Animal; Adenosine Triphosphatases; Symporters
PubMed: 37480642
DOI: 10.1016/j.clinsp.2023.100242 -
Annals of Anatomy = Anatomischer... Oct 2023Parkinson's disease (PD) is a neurodegenerative disorder that affects primarily the dopaminergic (DAergic) neurons of the mesostriatal system, among other nuclei of the...
BACKGROUND
Parkinson's disease (PD) is a neurodegenerative disorder that affects primarily the dopaminergic (DAergic) neurons of the mesostriatal system, among other nuclei of the brain. Although it is considered an idiopathic disease, oxidative stress is believed to be involved in DAergic neuron death and therefore plays an important role in the onset and development of the disease. RAD9B is a paralog of the RAD9 checkpoint, sharing some similar functions related to DNA damage resistance and apoptosis, as well as the ability to form 9-1-1 heterotrimers with RAD1 and HUS1.
METHODS
In addition to immunohistochemistry, immunofluorescence and Western-blot analysis, we implemented Quantitative RT-PCR and in situ hybridization techniques.
RESULTS
We demonstrated RAD9B expression in rat and human mesencephalic DAergic cells using specific markers. Additionally, we observed significant overexpression of RAD9B mRNA (p<0.01) and protein (p<0.01) in the midbrain 48 h after inducing damage with 150 µg of 6-hydroxydopamine (6-OHDA) injected in a rat model of PD. Regarding protein expression, the increased levels were observed in neurons of the mesostriatal system and returned to normal 5 days post-injury.
CONCLUSIONS
This response to a neurotoxin, known to produce oxidative stress specifically on DAergic neurons indicates the potential importance of RAD9B in this highly vulnerable population to cell death. In this model, RAD9B function appears to provide neuroprotection, as the induced lesion resulted in only mild degeneration. This observation highlights the potential of RAD9B checkpoint protein as a valuable target for future therapeutic interventions aimed at promoting neuroprotection.
Topics: Animals; Humans; Rats; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Oxidative Stress; Oxidopamine; Parkinson Disease
PubMed: 37460044
DOI: 10.1016/j.aanat.2023.152135 -
Clinics (Sao Paulo, Brazil) 2023Although reduced life expectancy in Parkinson's Disease (PD) patients has been related to severe cardiac arrhythmias due to autonomic dysfunctions, its molecular...
AIMS
Although reduced life expectancy in Parkinson's Disease (PD) patients has been related to severe cardiac arrhythmias due to autonomic dysfunctions, its molecular mechanisms remain unclear. To investigate the role of cardiac β-Adrenergic (βAR) and A-Adenosine (AR) receptors in these dysfunctions, the pharmacological effects of stimulation of cardiac βAR (isoproterenol, ISO), in the absence and presence of cardiac βAR (atenolol, AT) or AR (1,3-dipropyl-8-cyclopentyl xanthine, DPCPX) blockade, on the arrhythmias induced by Ischemia/Reperfusion (CIR) in an animal PD model were studied.
METHODS
PD was produced by dopaminergic lesions (confirmed by immunohistochemistry analysis) caused by the injection of 6-hydroxydopamine (6-OHDA, 6 μg) in rat striatum. CIR was produced by a surgical interruption for 10 min followed by reestablishment of blood circulation in the descendent left coronary artery. On the incidence of CIR-Induced Ventricular Arrhythmias (VA), Atrioventricular Block (AVB), and Lethality (LET), evaluated by Electrocardiogram (ECG) analysis, the effects of intravenous treatment with ISO, AT and DPCPX (before CIR) were studied.
RESULTS
VA, AVB and LET incidences were significantly higher in 6-OHDA (83%, 92%, 100%, respectively) than in control rats (58%, 67% and 67%, respectively). ISO treatment significantly reduced these incidences in 6-OHDA (33%, 33% and 42%, respectively) and control rats (25%, 25%, 33%, respectively), indicating that stimulation of cardiac βAR induced cardioprotection. This response was prevented by pretreatment with AT and DPCPX, confirming the involvement of cardiac βAR and AR.
CONCLUSION
Pharmacological modulation of cardiac βAR and AR could be a potential therapeutic strategy to reduce severe arrhythmias and increase life expectancy in PD patients.
Topics: Rats; Animals; Adrenergic Agents; Parkinson Disease; Oxidopamine; Arrhythmias, Cardiac; Receptors, Purinergic P1
PubMed: 37459671
DOI: 10.1016/j.clinsp.2023.100243 -
International Journal of Molecular... Jul 2023Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from...
Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson's disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of and in the 6-OHDA-treated SH-SY5Y cells. Additionally, the inhibitor attenuated the effect of DT on and reduced the cell viability. The DT and activators activated and , and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed.
Topics: Humans; Parkinson Disease; Reactive Oxygen Species; Oxidopamine; Caspase 3; Sirtuin 1; ARNTL Transcription Factors; Cell Line, Tumor; Neuroblastoma; Apoptosis; Neuroprotective Agents
PubMed: 37446264
DOI: 10.3390/ijms241311088 -
PloS One 2023Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and...
Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user's camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 20 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
Topics: Animals; Caenorhabditis elegans; Animals, Genetically Modified; Dopamine; Caenorhabditis elegans Proteins; Oxidopamine; Dopaminergic Neurons
PubMed: 37418455
DOI: 10.1371/journal.pone.0281797 -
Molecular Biology Reports Aug 2023Repetitive transcranial magnetic stimulation (rTMS) has been demonstrated to be effective in Parkinson's disease (PD), but whether rTMS treatment has a relieving effect...
BACKGROUND
Repetitive transcranial magnetic stimulation (rTMS) has been demonstrated to be effective in Parkinson's disease (PD), but whether rTMS treatment has a relieving effect on neuroinflammation remains to be investigated. In this article, we explored the effects of rTMS on forelimb use asymmetry and neuroinflammation-related mechanisms in a 6-hydroxydopamine (6-OHDA)-induced PD rat model.
METHODS AND RESULTS
Rats in the 6-OHDA+rTMS group received 10 Hz rTMS daily for 4 weeks. Behavioral tests (the cylinder test) were performed at the 3rd and 7th weeks after the operation. Astrocyte and microglia activation and protein levels of tyrosine hydroxylase(TH), high-mobility group box 1(HMGB1) and toll-like receptors 4(TLR4) were investigated by immunohistochemistry and Western blot analyses, respectively. After 4 weeks of treatment, forelimb use asymmetry was ameliorated in the 6-OHDA+rTMS group. Consistent with the behavioral tests, rTMS increased TH in the substantia nigra (SN) and the striatum of PD rats. High glial activation and HMGB1/TLR4 expression in the SN and the striatum were observed in the 6-OHDA group, while rTMS alleviated these changes.
CONCLUSIONS
This study showed that rTMS might be a promising method for alleviating neuroinflammation in PD rat models, and the effects might be mediated through the downregulation of the HMGB1/TLR4 pathway.
Topics: Rats; Animals; Parkinson Disease; Rats, Sprague-Dawley; Transcranial Magnetic Stimulation; Oxidopamine; HMGB1 Protein; Toll-Like Receptor 4; Neuroinflammatory Diseases
PubMed: 37328582
DOI: 10.1007/s11033-023-08561-8