Human Neuronal Cells Research Articles

Impact of 17-alpha ethinyl estradiol (EE2) and diethyl phthalate (DEP) exposure on microRNAs expression and their target genes in differentiated SH-SY5Y cells

Environmental endocrine disruptor chemicals (EDCs) have raised significant concerns due to their potential adverse effects on human health, particularly on the central nervous system (CNS). This study provides a comparative analysis of the effects of 17-alpha ethinyl estradiol (EE2) and diethyl phthalate (DEP) on neuronal cell proliferation and neurotoxicity. Using differentiated SH-SY5Y human neuronal cells, we evaluated cell viability, microRNA (miRNA) regulation, and RNA expression following exposure to subtoxic concentrations of EE2 and DEP. Our results show that both EDCs downregulated specific miRNAs—miR-18b-5p, miR-200a-3p, and miR-653-5p—affecting key processes such as cell proliferation, survival, and apoptosis. Gene expression analysis revealed the upregulation of EGFR, IGF1R, BTG2, and SH3BP4, implicating these miRNAs in the regulation of the Ras and PI3K/Akt/mTOR pathways. Our findings highlight distinct cellular responses: DEP disrupts PTEN activity, while EE2 enhances phosphorylation within the PI3K/Akt/mTOR pathway, promoting pro-survival and anti-apoptotic signals. This study emphasizes the urgent need for regulatory measures to mitigate the neurotoxic effects of EDCs and offers valuable insights into their molecular impacts on brain health.

Chlorpyrifos Acts as a Positive Modulator and an Agonist of N-Methyl-d-Aspartate (NMDA) Receptors: A Novel Mechanism of Chlorpyrifos-Induced Neurotoxicity.

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Long-term exposure to low levels of CPF is associated with neurodevelopmental and neurodegenerative disorders. The mechanisms leading to these effects are still not fully understood. Normal NMDA receptor (NMDAR) function is essential for neuronal development and higher brain functionality, while its inappropriate stimulation results in neurological deficits. Thus, the current study aimed to investigate the role of NMDARs in CPF-induced neurotoxicity. We show that NMDARs mediate CPF-induced excitotoxicity in differentiated human fetal cortical neuronal ReNcell CX stem cells. In addition, by using two-electrode voltage clamp electrophysiology of Xenopus oocytes expressing NMDARs, we show CPF potentiation of both GluN1-1a/GluN2A (EC50 ≈ 40 nM) and GluN1-1a/GluN2B (EC50 ≈ 55 nM) receptors, as well as reductions (approximately halved) in the NMDA EC50s and direct activation by 10 μM CPF of both receptor types. In silico molecular docking validated CPF's association with NMDARs through relatively high affinity binding (-8.82 kcal/mol) to a modulator site at the GluN1-GluN2A interface of the ligand-binding domains.

Modulation of Glutamatergic Burst Activity by Hydrolysed Arabinoxylan Rice Bran: A Multielectrode Array Study in Human-Induced Pluripotent Stem Cell-Derived Neurones and Astrocytes.

The natural product MGN-3 (Biobran) is a defatted, partially hydrolysed rice bran-derived hemicellulose enzymatically modified with an extract of Lentinus edodes. It has a high proportion of arabinoxylan. It has a protective action against intracerebroventricular streptozotocin-induced murine sporadic Alzheimer's disease and reverses spatial memory deficit in this disease model. The aim was to test the hypothesis that MGN-3 increases glutamatergic burst activity in human neuronal and glial cells by conducting an in vitro multielectrode array-based micro-electrophysiological study in a cultured mixture of human glutamatergic neurones, GABAergic neurones and astrocytes. The effects of MGN-3 at two concentrations, 0.750 g L-1 and 0.375 g L-1, and vehicle (control), on glutamatergic burst activity in a triculture of human-induced pluripotent stem cell (hiPSC)-derived GABAergic neurones, glutamatergic neurones and astrocytes were studied. The change in the number of glutamatergic bursts normalised to the vehicle control was analysed using a normal or Gaussian generalised linear model. This statistical model was highly significant (p = 1.468 × 10-17). Both MGN-3 concentrations were associated with highly significant main effects. These results provide strong evidence to reject the null hypothesis that MGN-3 does not affect glutamatergic burst activity in human neuronal and astrocytic cells. The study's strengths include the novel use of hiPSC-derived neurones and astrocytes and the robust statistical significance of the findings. Limitations include in vitro conditions that may not fully replicate in vivo conditions,potential variability in hiPSC-derived cell preparations, and the need to test other neuronal subtypes or additional doses to assess dose-dependent effects. These should be addressed in future studies.

Impaired Cerebrospinal Fluid Lipoprotein-Mediated Cholesterol Delivery to Neurons in Alzheimer's Disease.

In the central nervous system, apolipoprotein (APO) E-containing high-density lipoprotein (HDL)-like particles mediate the transport of glial-derived cholesterol to neurons, which is essential for neuronal membrane remodeling and maintenance of the myelin sheath. Despite this, the role of HDL-like cholesterol trafficking on Alzheimer's disease (AD) pathogenesis remains poorly understood. We aimed to examine cholesterol transport via HDL-like particles in cerebrospinal fluid (CSF) of AD patients compared to control individuals. Additionally, we explored the ability of reconstituted HDL containing different APOE isoforms to regulate cholesterol transport. We evaluated the capacity of CSF HDL-like particles to facilitate radiolabeled unesterified cholesterol efflux from A172 human glioblastoma astrocytes and to deliver cholesterol to SH-SY5Y human neuronal cells. The HDL-like proteome in the AD and control groups was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Reconstituted HDL nanoparticles were prepared by combining phospholipids and cholesterol with human APOE3 or APOE4, followed by radiolabeling with unesterified cholesterol. Our results showed that cholesterol efflux from astrocytes to CSF were similar between AD patients and controls, both under baseline conditions and after activation of ATP-binding cassette transporters A1 and G1. However, CSF HDL-like particle-mediated neuronal cholesterol uptake was significantly reduced in the AD group. LC-MS/MS analysis identified 775 proteins associated with HDL-like particles in both groups, with no major alterations in proteins linked to cholesterol metabolism. However, 27 proteins involved in non-cholesterol-related processes were differentially expressed. Notably, synthetic reconstituted HDL particles containing APOE4 exhibited reduced capacity to deliver cholesterol to neurons compared to those with APOE3. These findings indicate that CSF HDL-like particles from patients with AD demonstrate impaired cholesterol delivery to neurons. Our study highlights APOE4 as a critical contributor to abnormal neuronal cholesterol uptake in AD pathophysiology.

Cooperative nuclear action of RNA-binding proteins PSF and G3BP2 to sustain neuronal cell viability is decreased in aging and dementia.

Dysfunctional RNA-binding proteins (RBPs) have been implicated in several geriatric diseases, including Alzheimer's disease (AD). However, little is known about the nuclear molecular actions and cooperative functions mediated by RBPs that affect gene regulation in sporadic AD or aging. In the present study, we investigated aging- and AD-associated changes in the expression of PSF and G3BP2, which are representative RBPs associated with sex hormone activity. We determined that both PSF and G3BP2 levels were decreased in aged brains compared to young brains of mice. RNA sequencing (RNA-seq) analysis of human neuronal cells has shown that PSF is responsible for neuron-specific functions and sustains cell viability. In addition, we showed that PSF interacted with G3BP2 in the nucleus and stress granules (SGs) at the protein level. Moreover, PSF-mediated gene regulation at the RNA level correlated with G3BP2. Interestingly, PSF and G3BP2 target genes are associated with AD development. Mechanistically, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis demonstrated that the interaction of RBPs with the pre-mRNA of target genes enhanced post-transcriptional mRNA stability, suggesting a possible role for these RBPs in preserving neuronal cell viability. Notably, in the brains of patients with sporadic AD, decreased expression of PSF and G3BP2 in neurons was observed compared to non-AD patients. Overall, our findings suggest that the cooperative action of PSF and G3BP2 in the nucleus is important for preventing aging and AD development.

Exploring the Neuroprotective Roles of Oxytocin and Estrogen on Human Glial and Neuronal Cells

Exploring the Neuroprotective Roles of Oxytocin and Estrogen on Human Glial and Neuronal Cells

A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model.

The translocator protein 18 kDa (TSPO) is a multifunctional outer mitochondrial membrane protein associated with various aspects of mitochondrial physiology and multiple roles in health and disease. Here, we aimed to analyse the role of TSPO in the regulation of mitochondrial and cellular functions in a human neuronal cell model. We used the CRISPR/Cas9 technology and generated TSPO knockout (KO) and control (CTRL) variants of human-induced pluripotent stem cells (hiPSCs). In a multimodal phenotyping approach, we investigated cellular and mitochondrial functions in neural progenitor cells (NPCs), astrocytes, and neurons differentiated from hiPSC CTRL and TSPO KO cell lines. Our analysis revealed reduced mitochondrial respiration and glycolysis, altered Ca2+ levels in the cytosol and mitochondrial matrix, a depolarised MMP, and increased levels of reactive oxygen species, as well as a reduced cell size. Notably, TSPO deficiency was accompanied by reduced expression of the voltage-dependent anion channel (VDAC). We also observed a reduced TSPO and VDAC expression in cells derived from patients suffering from major depressive disorder (MDD). Considering the modulatory function of TSPO and the similar functional phenotype of cells derived from patients with depression, we discuss a role of TSPO in the etiology or pathology of MDD. In summary, our findings indicate a general impairment of mitochondrial function in TSPO knockout (KO) cells. This deepens our insight into the intricate role of TSPO in a range of physiological and pathological processes.

Identification of FBLL1 as a neuron-specific RNA 2′-O-methyltransferase mediating neuronal differentiation

2'-O-methylation is one of the most prevalent RNA modifications found in different RNA types. However, the identities of enzymes participating in the transfer of methyl groups are not well defined. To date, fibrillarin (FBL) is the only known small nucleolar ribonucleoprotein (snoRNP) 2'-O-methyltransferase. Whether other snoRNP 2'-O-methyltransferases exist and their functions in targeting RNAs to determine cell differentiation and function need to be elucidated. Here, we identify FBL-like protein 1 (FBLL1) as a 2'-O-methyltransferase and find its function in promoting neuronal differentiation. We show that FBLL1 is a key snoRNP complex enzyme that transfers methyl groups to substrate RNAs both in vitro and in vivo. Moreover, FBLL1 exhibits different 2'-O-methyltransferase site selectivity from FBL and tissue-specific distribution. FBLL1 is preferentially expressed in the brain, especially in human neuron cells, and promotes neuronal differentiation through 2'-O-methylation of GAP43 messenger RNA (mRNA). Knockdown of FBLL1, but not FBL, reduced 2'-O-methylation levels in GAP43 mRNA, decreased expression of GAP43 proteins, and eventually repressed neuronal differentiation. Our finding of neuron-specific FBLL1 adds insights into RNA modification in neurobiology and provides clues for understanding 2'-O-methylation in health and disease.

Mechanics of poly-arginine adsorption onto cell membrane by GM1 and their cluster forming: Coarse-grained molecular dynamics study

Ganglioside GM1 is a crucial glycolipid in neuronal cells, playing significant roles in various biological processes such as pathogen recognition, signal transduction, and protein sorting. Poly-arginine, widely used as a template for cell-penetrating peptides design, is highly valuable for molecular cargo delivery and information transmission. Investigating the interaction mechanisms and physicochemical principles of poly-arginine with GM1-containing cell membranes is essential for understanding the functions of specific cellular components and for developing novel drugs. In this study, we employed coarse-grained molecular dynamics simulations to explore the interaction processes between poly-arginine of varying concentrations and polymerization degrees with model membranes of human brain neuronal cells. Our findings reveal that poly-arginines preferentially adsorb onto GM1 and aggregate into clusters on the membrane, primarily driven by electrostatic interactions, which contributes to membrane stabilization. Additionally, poly-arginine shows a higher affinity for membranes with elevated GM1 concentrations, highlighting its potential for targeting specific membrane compositions. These insights are crucial for designing molecular targets and understanding the biophysical mechanisms in lipidomics using high-performance computational simulation methods.

POLG p.A962T Mutation Leads to Neuronal Mitochondrial Dysfunction That is Restored After Mitochondrial Transplantation.

Mutations in DNA polymerase gamma (POLG) are known as the predominant cause of inherited mitochondrial disorders. But how these POLG mutations disturb mitochondrial function remains to be determined. Furthermore, no effective therapy, to date, has been reported for POLG diseases. Using differentiated SH-SY5Y cells, a human neuronal model cell line, the current study investigated whether the novel POLG variant p.A962T impairs mitochondrial function. This involved quantifying mitochondrial DNA (mtDNA) content using PCR and assessing the expression levels of the subunits of complex IV (COXI-IV), a complex I subunit NDUFV1 and Cytochrome C (Cyto C) release using Western blotting. Activities of mitochondrial complex I, II, and IV were measured using colorimetric assays. Mitochondrial membrane potential (delta Psim) and ATP were evaluated using fluorescence assays and luminescent assays, respectively. In addition, we investigated whether mitochondrial transplantation (MT) using Pep-1-conjugated mitochondria could compensate for mitochondrial defects caused by the variant in cells carrying mutant POLG. The results of this study showed that POLG p.A962T mutation resulted in mitochondrial defects, including mitochondrial DNA (mtDNA) depletion, membrane potential (delta Psim) depolarization and adenosine triphosphate (ATP) reduction. Mechanistically, POLG mutation-caused mtDNA depletion led to the loss of mtDNA-encoded subunits of complex I and IV and thus compromised their activities. POLG p.A962T mutation is a pathogenic mutation leading to mitochondrial malfunction and mtDNA depletion in neurons. Cell-penetrating peptide Pep-1-mediated MT treatment compensated for mitochondrial defects induced by these POLG variants, suggesting the therapeutic application of this method in POLG diseases.

Analysis of Valproic Acid-Induced Gene Expression Changes in Human Neuron Cells

Analysis of Valproic Acid-Induced Gene Expression Changes in Human Neuron Cells

Inhibiting mtDNA transcript translation alters Alzheimer's disease-associated biology.

Alzheimer's disease (AD) features changes in mitochondrial structure and function. Investigators debate where to position mitochondrial pathology within the chronology and context of other AD features. To address whether mitochondrial dysfunction alters AD-implicated genes and proteins, we treated SH-SY5Y cells and induced pluripotent stem cell (iPSC)-derived neurons with chloramphenicol, an antibiotic that inhibits mtDNA-generated transcript translation. We characterized adaptive, AD-associated gene, and AD-associated protein responses. SH-SY5Y cells and iPSC neurons responded to mtDNA transcript translation inhibition by increasing mtDNA copy number and transcription. Nuclear-expressed respiratory chain mRNA and protein levels also changed. There were AD-consistent concordant and model-specific changes in amyloid precursor protein, beta amyloid, apolipoprotein E, tau, and α-synuclein biology. Primary mitochondrial dysfunction induces compensatory organelle responses, changes nuclear gene expression, and alters the biology of AD-associated genes and proteins in ways that may recapitulate brain aging and AD molecular phenomena. In AD, mitochondrial dysfunction could represent a disease cause or consequence. We inhibited mitochondrial translation in human neuronal cells and neurons. Mitochondrial and nuclear gene expression shifted in adaptive-consistent patterns. APP, Aβ, APOE, tau, and α-synuclein biology changed in AD-consistent patterns. Mitochondrial stress creates an environment that promotes AD pathology.

Saxitoxin potentiates human neuronal cell death induced by Zika virus while sparing neural progenitors and astrocytes

The Zika virus (ZIKV) epidemic declared in Brazil between 2015 and 2016 was associated with an increased prevalence of severe congenital malformations, including microcephaly. The distribution of microcephaly cases was not uniform across the country, with a disproportionately higher incidence in the Northeast region (NE). Our previous work demonstrated that saxitoxin (STX), a toxin present in the drinking water reservoirs of the NE, exacerbated the damaging effects of ZIKV on the developing brain. We hypothesized that the impact of STX might vary among different neural cell types. While ZIKV infection caused severe damages on astrocytes and neural stem cells (NSCs), the addition of STX did not exacerbate these effects. We observed that neurons subjected to STX exposure were more prone to apoptosis and displayed higher ZIKV infection rate. These findings suggest that STX exacerbates the harmful effects of ZIKV on neurons, thereby providing a plausible explanation for the heightened severity of ZIKV-induced congenital malformations observed in Brazil’s NE. This study highlights the importance of understanding the interactive effects of environmental toxins and infectious pathogens on neural development, with potential implications for public health policies.

PTEN controls alternative splicing of autism spectrum disorder-associated transcripts in primary neurons.

Phosphatase and tensin homologue (PTEN) is the main antagonist of the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signalling pathway and mutated in 10-20% of individuals with autism spectrum disorder (ASD) exhibiting macrocephaly. Hyperactive mTOR signalling is responsible for some aspects during PTEN-ASD progression, e.g. neuronal hypertrophy and -excitability, but PI3K/mTOR-independent processes have additionally been described. There is emerging evidence that PTEN regulates gene transcription, spliceosome formation and pre-mRNA splicing independently of PI3K/mTOR. Altered splicing is a hallmark of brains from individuals with idiopathic and PTEN-ASD, however, molecular mechanisms are yet to be identified. We performed RNA-Seq followed by analysis of altered transcript splicing in Pten-deficient primary cortical mouse neurons, which we compared with published data from PTEN-deficient human neuronal stem cells. This analysis identified that transcripts were globally mis-spliced in a developmentally regulated fashion and cluster in synaptic and gene expression regulatory processes. Strikingly, splicing defects following Pten-deficiency represent a significant number of other known ASD-susceptibility genes. Furthermore, we show that exons with strong 3' splice sites are more frequently mis-spliced under Pten-deficient conditions. Our study indicates that PTEN-ASD is a multifactorial condition involving the dysregulation of other known ASD-susceptibility genes.

NEMF mutations in mice illustrate how Importin-β specific nuclear transport defects recapitulate neurodegenerative disease hallmarks.

Pathological disruption of Nucleocytoplasmic Transport (NCT), such as the mis-localization of nuclear pore complex proteins (Nups), nuclear transport receptors, Ran-GTPase, and RanGAP1, are seen in both animal models and in familial and sporadic forms of amyotrophic lateral sclerosis (ALS), frontal temporal dementia and frontal temporal lobar degeneration (FTD\FTLD), and Alzheimer's and Alzheimer's Related Dementias (AD/ADRD). However, the question of whether these alterations represent a primary cause, or a downstream consequence of disease is unclear, and what upstream factors may account for these defects are unknown. Here, we report four key findings that shed light on the upstream causal role of Importin-β-specific nuclear transport defects in disease onset. First, taking advantage of two novel mouse models of NEMF neurodegeneration (NemfR86S and NemfR487G) that recapitulate many cellular and biochemical aspects of neurodegenerative diseases, we find an Importin-β-specific nuclear import block. Second, we observe cytoplasmic mis-localization and aggregation of multiple proteins implicated in the pathogenesis of ALS/FTD and AD/ADRD, including TDP43, Importin-β, RanGap1, and Ran. These findings are further supported by a pathological interaction between Importin-β and the mutant NEMFR86S protein in cytoplasmic accumulations. Third, we identify similar transcriptional dysregulation in key genes associated with neurodegenerative disease. Lastly, we show that even transient pharmaceutical inhibition of Importin-β in both mouse and human neuronal and non-neuronal cells induces key proteinopathies and transcriptional alterations seen in our mouse models and in neurodegeneration. Our convergent results between mouse and human neuronal and non-neuronal cellular biology provide mechanistic evidence that many of the mis-localized proteins and dysregulated transcriptional events seen in multiple neurodegenerative diseases may in fact arise primarily from a primary upstream defect in Importin- β nuclear import. These findings have critical implications for investigating how sporadic forms of neurodegeneration may arise from presently unidentified genetic and environmental perturbations in Importin-β function.

Therapeutic Effect of Padina arborescens Extract on a Cell System Model for Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are involved in the pathogenesis of Parkinson's disease. The activity of LRRK2 in microglial cells is associated with neuroinflammation, and LRRK2 inhibitors are crucial for alleviating this neuroinflammatory response. α-synuclein contributes to oxidative stress in the dopaminergic neuron and neuroinflammation through Toll-like receptors in microglia. In this study, we investigated the effect of the marine alga Padina arborescens on neuroinflammation by examining LRRK2 activation and the aggregation of α-synuclein. P. arborescens extract inhibits LRRK2 activity in vitro and decreases lipopolysaccharide (LPS)-induced LRRK2 upregulation in BV2, a mouse microglial cell line. Treatment with P. arborescens extract decreased tumor necrosis factor-α (TNF-α) gene expression by LPS through LRRK2 inhibition in BV2. It also attenuated TNF-α gene expression, inducible nitric oxide synthase, and the release of TNF-α and cellular nitric oxide in rat primary microglia. Furthermore, P. arborescens extract prevented rotenone (RTN)-induced oxidative stress in primary rat astrocytes and inhibited α-synuclein fibrilization in an in vitro assay using recombinant α-synuclein and in the differentiated human dopaminergic neuronal cell line SH-SY5Y (dSH). The extract increased lysosomal activity in dSH cells. In addition, P. arborescens extract slightly prolonged the lifespan of Caenorhabditis elegans, which was reduced by RTN treatment.

Mixtures of organic micropollutants exacerbated in vitro neurotoxicity of prymnesins and contributed to aquatic toxicity during a toxic algal bloom

Prymnesins produced by an algal bloom of Prymnesium parvum led to the death of several hundred tons of freshwater fish in the Oder River in summer 2022. We investigated effects on aquatic life and human cell lines from exposure to extracts of contaminated water collected during the fish kill. We detected B-type prymnesins and >120 organic micropollutants. The micropollutants occurred at concentrations that would cause the predicted mixture risk quotient for aquatic life to exceed the acceptable threshold. Extracts of water and filters (biomass and particulates) induced moderate effects in vivo in algae, daphnids and zebrafish embryos but caused high effects in a human neuronal cell line indicating the presence of neurotoxicants. Mixture toxicity modelling demonstrated that the in vitro neurotoxic effects were mainly caused by the detected B-type prymnesins with minor contributions by organic micropollutants. Complex interactions between natural and anthropogenic toxicants may underestimate threats to aquatic ecosystems.

Atmospheric pressure plasma preconditioning reduces oxygen and glucose deprivation induced human neuronal SH-SY5Y cells apoptosis by activating protective autophagy and ROS/AMPK/mTOR pathway

Reactive oxygen species (ROS)/reactive nitrogen species (RNS) exert a “double edged” effect on the occurrence and development of ischemic stroke. We previously indicate that atmospheric pressure plasma (APP) shows a neuroprotective effect in vitro based on the ROS/RNS generations. However, the mechanism is still unknown. In this work, SH-SY5Y cells were treated with oxygen and glucose deprivation (OGD) injuries for stimulating the ischemic stroke pathological injury process. A helium APP was used for SH-SY5Y cell treatment for evaluating the neuroprotective impacts of APP preconditioning against OGD injuries with the optimized parameters. During the preconditioning, APP significantly raised the extracellular and intracellular ROS/RNS production. As a result, APP preconditioning increased SH-SY5Y cell autophagy by elevating LC3-II/LC3-I ratio and autophagosome formation. Meanwhile, APP preconditioning reduced cell apoptosis caused by OGD with the increased APP treatment time, which was abolished by pretreatment with autophagy inhibitor 3-methyladenine (3-MA). The ROS scavenger N-acetyl-L-cysteine (NAC) alone or combined with NO scavenger carboxy-PTIO abolished the APP preconditioning induced SH-SY5Y autophagy and the cytoprotection, whereas the NO scavenger alone did not. In addition, we observed the elevated phosphorylation of AMP-activated protein kinase (AMPK) and decreased phosphorylation of mammalian target of rapamycin (mTOR) in APP treated SH-SY5Y cells. This effect was attenuated by AMPK inhibitor Compound C (CC), the ROS scavenger NAC and autophagy inhibitor 3-MA. Furthermore, the cytoprotective effect of APP was preliminarily confirmed in the rats of middle cerebral artery occlusion (MCAO) model. Results showed that APP inhalation by rats during MCAO process could improve neurological functions, reduce cell apoptosis in brain tissues and decrease cerebral infarct volume. Our data suggested that ROS produced by APP preconditioning played a vital role in the neuroprotective effect of SH-SY5Y cells against OGD injuries by activating autophagy and ROS/AMPK/mTOR pathway.

Novel CDKL5 targets identified in human iPSC-derived neurons

CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.

Synthesis of copper complexes for potential use in the diagnosis of Alzheimer's disease

Background: Since there is currently no cure for Alzheimer's disease (AD), it is important to develop methods that could contribute to its diagnosis and propose new therapeutic approaches for its treatment. Positron emission tomography (PET) is a medical imaging technique that can be used to diagnose Alzheimer's disease, as some radiotracers have been developed to detect amyloid plaques, one of the hallmarks of the disease. However, the radiotracers already used for the diagnosis of AD have a low half-life, which limits their use over the time and requires the presence of a cyclotron close to the examination site. The use of copper complexes could be a good alternative for the development of new radiotracers, since they can be used in PET imaging and have a longer half-life than the other radiotracers already used for AD diagnosis. Aim of the study: Design and synthesize copper complexes that could be used as PET radiotracers able to cross the blood brain barrier and detecting amyloid plaques.Material and methods: The first objective was to design a series of new copper complexes with the capacity to target amyloid plaques inside the brain and contribute to the rational synthesis of such complexes. To this end, theoretical models were used to predict the ability of the complex to cross the blood-brain barrier (BBB) which separates brain from de blood and is main gatekeeper for drugs entering the brain. The models employed consider the physicochemical properties of the molecules. Based on these models, one of the molecules was synthesized in ten steps. The key step in the synthetic strategy was the coupling of a monopicolinate-N-alkylated cyclam-based ligand with a moiety capable of recognizing Aβ plaques via a Buchwald-Hartwig coupling reaction. Potentiometric, spectrophotometric and electron paramagnetic resonance spectroscopic studies were performed to determine the structure and thermodynamic stability of the complex. In addition, the ability to target amyloid plaques was evaluated using brain sections from Alzheimer's disease patients and the cytotoxicity was evaluated using human neuronal cells.Results: A first complex has been designed and synthesized. The physicochemical studies performed indicate that this complex seem to be thermodynamically stable. In addition, cytotoxicity tests on human neuronal cells indicate low toxicity and labeling tests on brain sections from Alzheimer's patients suggest that the complex is capable of recognizing amyloid plaques. Conclusion: We have developed a novel copper complex that is able to detect recognize amyloid plaques. In the future this molecule could be used in PET and contribute to the diagnosis of Alzheimer's disease. From these results, we also propose to develop a new copper complex for tau PET imaging.