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Fulltext Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1) Expression by Orphan Nuclear Receptor Nr4a1

Greenwood MP, Greenwood M, Gillard BT, Chitra Devi R, Murphy D

Front Mol Neurosci, 2017;10:413.
PMID: 29311806 DOI: 10.3389/fnmol.2017.00413

Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5'UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.
Fulltext Can We Use 2,3,5-Triphenyltetrazolium Chloride-Stained Brain Slices for Other Purposes? The Application of Western Blotting

Sanchez-Bezanilla S, Nilsson M, Walker FR, Ong LK

Front Mol Neurosci, 2019;12:181.
PMID: 31417355 DOI: 10.3389/fnmol.2019.00181

2,3,5-Triphenyltetrazolium chloride (TTC) staining is a commonly used method to determine the volume of the cerebral infarction in experimental stroke models. The TTC staining protocol is considered to interfere with downstream analyses, and it is unclear whether TTC-stained brain samples can be used for biochemistry analyses. However, there is evidence indicating that, with proper optimization and handling, TTC-stained brains may remain viable for protein analyses. In the present study, we aimed to rigorously assess whether TTC can reliably be used for western blotting of various markers. In this study, brain samples obtained from C57BL/6 male mice were treated with TTC (TTC+) or left untreated (TTC-) at 1 week after photothrombotic occlusion or sham surgery. Brain regions were dissected into infarct, thalamus, and hippocampus, and proteins were extracted by using radioimmunoprecipitation assay buffer. Protein levels of apoptosis, autophagy, neuronal, glial, vascular, and neurodegenerative-related markers were analyzed by western blotting. Our results showed that TTC+ brains display similar relative changes in most of the markers compared with TTC- brains. In addition, we validated that these analyses can be performed in the infarct as well as other brain regions such as the thalamus and hippocampus. Our findings demonstrate that TTC+ brains are reliable for protein analyses using western blotting. Widespread adoption of this approach will be key to lowering the number of animals used while maximizing data.
Fulltext Conditioned Medium of Human Menstrual Blood-Derived Endometrial Stem Cells Protects Against MPP+-Induced Cytotoxicity in vitro

Li H, Yahaya BH, Ng WH, Yusoff NM, Lin J

Front Mol Neurosci, 2019;12:80.
PMID: 31024252 DOI: 10.3389/fnmol.2019.00080

Mesenchymal stem cells (MSCs) showed the potential to treat Parkinson's disease (PD). However, it is unknown whether the conditioned medium of human menstrual blood-derived endometrial stem cells (MenSCs-CM) has the function to alleviate syndromes of PD. In this study, human neuroblastoma SH-SY5Y cells were exposed to neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) for inducing a range of response characteristics of PD. After culturing this cell model with 24 h/48 h collected MenSCs-CM for different days, cell viability, pro-inflammation cytokines, mitochondrial membrane potential (ΔΨm), oxidative stress, and cell apoptosis were detected. Finally, protein assay was performed to detect 12 kinds of neurotrophic factors inside MenSCs-CM. Our results showed that MPP+ caused SH-SY5Y cell viability reduction as an increasing dose and time dependent manner. MPP+ treatment resulted in inflammation, mitochondrial dysfunction, reactive oxygen species (ROS) production accumulation, and apoptosis of SH-SY5Y at its IC50 concentration. Forty-eight hours-collected MenSCs-CM and culturing with the MPP+-treated SH-SY5Y for 2 days are the optimized condition to increase cell viability. Besides, MenSCs-CM was efficacious against MPP+ induced inflammation, ΔΨm loss, ROS generation, and it could significantly decrease cells numbers in late apoptosis stage. What's more, protein assay showed that MenSCs-CM contained various neuroprotective factors. Our study provided the first evidence that MenSCs-CM has a protective effect on MPP+-induced cytotoxicity in various aspects, and firstly showed that MenSCs can release at least 12 kinds of neurotrophic factors to medium, which may contribute to the protective function of MenSCs-CM to treat PD. This research enlightening that MenSCs-CM is beneficial in the therapy for PD and probably also for other neurodegenerative diseases.
Fulltext Development of brain organoid technology derived from iPSC for the neurodegenerative disease modelling: a glance through

Jusop AS, Thanaskody K, Tye GJ, Dass SA, Wan Kamarul Zaman WS, Nordin F

Front Mol Neurosci, 2023;16:1173433.
PMID: 37602192 DOI: 10.3389/fnmol.2023.1173433

Neurodegenerative diseases are adult-onset neurological conditions that are notoriously difficult to model for drug discovery and development because most models are unable to accurately recapitulate pathology in disease-relevant cells, making it extremely difficult to explore the potential mechanisms underlying neurodegenerative diseases. Therefore, alternative models of human or animal cells have been developed to bridge the gap and allow the impact of new therapeutic strategies to be anticipated more accurately by trying to mimic neuronal and glial cell interactions and many more mechanisms. In tandem with the emergence of human-induced pluripotent stem cells which were first generated in 2007, the accessibility to human-induced pluripotent stem cells (hiPSC) derived from patients can be differentiated into disease-relevant neurons, providing an unrivaled platform for in vitro modeling, drug testing, and therapeutic strategy development. The recent development of three-dimensional (3D) brain organoids derived from iPSCs as the best alternative models for the study of the pathological features of neurodegenerative diseases. This review highlights the overview of current iPSC-based disease modeling and recent advances in the development of iPSC models that incorporate neurodegenerative diseases. In addition, a summary of the existing brain organoid-based disease modeling of Alzheimer's disease was presented. We have also discussed the current methodologies of regional specific brain organoids modeled, its potential applications, emphasizing brain organoids as a promising platform for the modeling of patient-specific diseases, the development of personalized therapies, and contributing to the design of ongoing or future clinical trials on organoid technologies.
Fulltext Potential Roles of microRNAs in the Regulation of Monoamine Oxidase A in the Brain

Higuchi Y, Soga T, Parhar IS

Front Mol Neurosci, 2018;11:339.
PMID: 30271325 DOI: 10.3389/fnmol.2018.00339

Monoamine oxidase A (MAO-A) is an enzyme that regulates the levels of monoamine neurotransmitters, such as serotonin, noradrenaline and dopamine and it has been used as a therapeutic target for depression. However, MAO-A inhibitors, which directly acts on MAO-A protein, have limited use due to their adverse effects. microRNAs (miRNAs) are 18-22 nucleotide long, small non-coding RNAs, which have recently emerged as regulators of protein levels that could potentially be new therapeutic targets for psychiatric disorders. This review article aims to discuss the current status of the treatment for depression with MAO-A inhibitors and the regulatory factors of MAO-A. Further, the review also proposes possible regulatory mechanisms of MAO-A by miRNAs, which leads to better understanding of the pathology of depressive disorders and their potential use as therapeutic agents.
Fulltext High-throughput autoantibody screening identifies differentially abundant autoantibodies in autism spectrum disorder

Mesleh A, Ehtewish H, Lennard K, Abdesselem HB, Al-Shaban F, Decock J, et al.

Front Mol Neurosci, 2023;16:1222506.
PMID: 37908488 DOI: 10.3389/fnmol.2023.1222506

INTRODUCTION: Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by defects in two core domains, social/communication skills and restricted/repetitive behaviors or interests. There is no approved biomarker for ASD diagnosis, and the current diagnostic method is based on clinical manifestation, which tends to vary vastly between the affected individuals due to the heterogeneous nature of ASD. There is emerging evidence that supports the implication of the immune system in ASD, specifically autoimmunity; however, the role of autoantibodies in ASD children is not yet fully understood.
MATERIALS AND METHODS: In this study, we screened serum samples from 93 cases with ASD and 28 healthy controls utilizing high-throughput KoRectly Expressed (KREX) i-Ome protein-array technology. Our goal was to identify autoantibodies with differential expressions in ASD and to gain insights into the biological significance of these autoantibodies in the context of ASD pathogenesis.
RESULT: Our autoantibody expression analysis identified 29 differential autoantibodies in ASD, 4 of which were upregulated and 25 downregulated. Subsequently, gene ontology (GO) and network analysis showed that the proteins of these autoantibodies are expressed in the brain and involved in axonal guidance, chromatin binding, and multiple metabolic pathways. Correlation analysis revealed that these autoantibodies negatively correlate with the age of ASD subjects.
CONCLUSION: This study explored autoantibody reactivity against self-antigens in ASD individuals' serum using a high-throughput assay. The identified autoantibodies were reactive against proteins involved in axonal guidance, synaptic function, amino acid metabolism, fatty acid metabolism, and chromatin binding.
Fulltext MicroRNAs and Target Genes As Biomarkers for the Diagnosis of Early Onset of Parkinson Disease

Arshad AR, Sulaiman SA, Saperi AA, Jamal R, Mohamed Ibrahim N, Abdul Murad NA

Front Mol Neurosci, 2017;10:352.
PMID: 29163029 DOI: 10.3389/fnmol.2017.00352

Among the neurodegenerative disorders, Parkinson's disease (PD) ranks as the second most common disorder with a higher prevalence in individuals aged over 60 years old. Younger individuals may also be affected with PD which is known as early onset PD (EOPD). Despite similarities between the characteristics of EOPD and late onset PD (LODP), EOPD patients experience much longer disease manifestations and poorer quality of life. Although some individuals are more prone to have EOPD due to certain genetic alterations, the molecular mechanisms that differentiate between EOPD and LOPD remains unclear. Recent findings in PD patients revealed that there were differences in the genetic profiles of PD patients compared to healthy controls, as well as between EOPD and LOPD patients. There were variants identified that correlated with the decline of cognitive and motor symptoms as well as non-motor symptoms in PD. There were also specific microRNAs that correlated with PD progression, and since microRNAs have been shown to be involved in the maintenance of neuronal development, mitochondrial dysfunction and oxidative stress, there is a strong possibility that these microRNAs can be potentially used to differentiate between subsets of PD patients. PD is mainly diagnosed at the late stage, when almost majority of the dopaminergic neurons are lost. Therefore, identification of molecular biomarkers for early detection of PD is important. Given that miRNAs are crucial in controlling the gene expression, these regulatory microRNAs and their target genes could be used as biomarkers for early diagnosis of PD. In this article, we discussed the genes involved and their regulatory miRNAs, regarding their roles in PD progression, based on the findings of significantly altered microRNAs in EOPD studies. We also discussed the potential of these miRNAs as molecular biomarkers for early diagnosis.