Neuronal Cell Function Research Articles

Recent Advancements in CNS Acting Drugs: A Step Towards End of Illness

Progressive degeneration in the morphology and functions of neuronal cells leads to multifactorial pathogenesis conditions of oxidative stress, mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity, and neuro-inflammation to mediate heterogeneous types of neurodegenerative diseases, such as Epilepsy, Alzheimer's (AD) and Parkinson's (PD), more prominently among aging populations. In this editorial, complex mechanisms, challenges, and advancements made in the discovery of new neurotherapeutics, as well as designing approaches being adopted to fabricate brain-targeted delivery systems, are discussed.

Multisystem Inflammatory Syndrome in Children and Long COVID: The SARS-CoV-2 Viral Superantigen Hypothesis.

Multisystem inflammatory syndrome in children (MIS-C) is a febrile pediatric inflammatory disease that may develop weeks after initial SARS-CoV-2 infection or exposure. MIS-C involves systemic hyperinflammation and multiorgan involvement, including severe cardiovascular, gastrointestinal (GI) and neurological symptoms. Some clinical attributes of MIS-C—such as persistent fever, rashes, conjunctivitis and oral mucosa changes (red fissured lips and strawberry tongue)—overlap with features of Kawasaki disease (KD). In addition, MIS-C shares striking clinical similarities with toxic shock syndrome (TSS), which is triggered by bacterial superantigens (SAgs). The remarkable similarities between MIS-C and TSS prompted a search for SAg-like structures in the SARS-CoV-2 virus and the discovery of a unique SAg-like motif highly similar to a Staphylococcal enterotoxin B (SEB) fragment in the SARS-CoV-2 spike 1 (S1) glycoprotein. Computational studies suggest that the SAg-like motif has a high affinity for binding T-cell receptors (TCRs) and MHC Class II proteins. Immunosequencing of peripheral blood samples from MIS-C patients revealed a profound expansion of TCR β variable gene 11-2 (TRBV11-2), which correlates with MIS-C severity and serum cytokine levels, consistent with a SAg-triggered immune response. Computational sequence analysis of SARS-CoV-2 spike further identified conserved neurotoxin-like motifs which may alter neuronal cell function and contribute to neurological symptoms in COVID-19 and MIS-C patients. Additionally, autoantibodies are detected during MIS-C, which may indicate development of post-SARS-CoV-2 autoreactive and autoimmune responses. Finally, prolonged persistence of SARS-CoV-2 RNA in the gut, increased gut permeability and elevated levels of circulating S1 have been observed in children with MIS-C. Accordingly, we hypothesize that continuous and prolonged exposure to the viral SAg-like and neurotoxin-like motifs in SARS-CoV-2 spike may promote autoimmunity leading to the development of post-acute COVID-19 syndromes, including MIS-C and long COVID, as well as the neurological complications resulting from SARS-CoV-2 infection.

Enhancement of Neuroglial Extracellular Matrix Formation and Physiological Activity of Dopaminergic Neural Cocultures by Macromolecular Crowding

The neuroglial extracellular matrix (ECM) provides critical support and physiological cues for the proper growth, differentiation, and function of neuronal cells in the brain. However, in most in vitro settings that study neural physiology, cells are grown as monolayers on stiff surfaces that maximize adhesion and proliferation, and, therefore, they lack the physiological cues that ECM in native neuronal tissues provides. Macromolecular crowding (MMC) is a biophysical phenomenon based on the principle of excluded volume that can be harnessed to induce native ECM deposition by cells in culture. Here, we show that MMC using two species of Ficoll with vitamin C supplementation significantly boosts deposition of relevant brain ECM by cultured human astrocytes. Dopaminergic neurons cocultured on this astrocyte–ECM bed prepared under MMC treatment showed longer and denser neuronal extensions, a higher number of pre ad post synaptic contacts, and increased physiological activity, as evidenced by higher frequency calcium oscillation, compared to standard coculture conditions. When the pharmacological activity of various compounds was tested on MMC-treated cocultures, their responses were enhanced, and for apomorphine, a D2-receptor agonist, it was inverted in comparison to control cell culture conditions, thus emulating responses observed in in vivo settings. These results indicate that macromolecular crowding can harness the ECM-building potential of human astrocytes in vitro forming an ultra-flat 3D microenvironment that makes neural cultures more physiological and pharmacological relevant.

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.

APP processing: a biochemical competition influenced by exercise-induced signaling mediators?

Neurodegenerative diseases, such as Alzheimer's disease (AD), are becoming more common in aging our society. One specific neuropathological hallmark of this disease is excessive accumulation of amyloid-β (Aβ) peptides, which can aggregate to form the plaques commonly associated with this disease. These plaques are often observed well before clinical diagnosis of AD. At the cellular level, both the production and aggregation of Aβ peptides in the brain are detrimental to neuronal cell production, survival, and function, as well as often resulting in neuronal dysfunction and death. Exercise and physical activity have been shown to improve overall health, including brain health, and in the last several years there has been evidence to support that exercise may be able to regulate Aβ peptide production in the brain. Exercise promotes the release of a wide array of signaling mediators from various metabolically active tissues and organs in the body. These exercise-induced signaling mediators could be the driving force behind some of the beneficial effects observed in brain with exercise. This review will aim to discuss potential exercise-induced signaling mediators with the capacity to influence various proteins involved in the formation of Aβ peptide production in the brain.

Alzheimer's disease associated AKAP9 I2558M mutation alters posttranslational modification and interactome of tau and cellular functions in CRISPR-edited human neuronal cells.

Alzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR‐Cas9‐engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH‐SY5Y P301L neuron‐like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification‐mass spectrometry revealed that differentially expressed tau‐interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau‐dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.

High-Throughput Sequencing to Investigate lncRNA-circRNA-miRNA-mRNA Networks Underlying the Effects of Beta-Amyloid Peptide and Senescence on Astrocytes.

Astrocytes are widely distributed in the central nervous system and play an essential role in the function of neuronal cells. Associations between astrocytes and Alzheimer’s disease (AD) have been noted, and recent work has implicated circular RNA (circRNA) and long non-coding RNA (lncRNA) in the development of AD. However, few reports have investigated which lncRNA and circRNA are involved in the influence of amyloid beta (Aβ) and senescence on astrocytes. This study therefore examines changes at the transcriptome level to explore the effects of Aβ and senescence on astrocytes. Primary cultured astrocytes were treated with Aβ and cultured for 90 days in vitro, and high-throughput sequencing was performed to identify differentially expressed RNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed genes were associated with the focal adhesion signaling pathway, extracellular matrix receptor signaling pathway, and the extracellular matrix. The protein–protein interaction network was then constructed, and 103 hub genes were screened out; most of these were strongly associated with the expression of the extracellular matrix, extracellular matrix receptor signaling pathway, and focal adhesion. Two competing endogenous RNA networks were constructed based on the selected hub gene and differential RNAs, and we identified multiple competing endogenous RNA regulatory axes that were involved in the effects of Aβ and senescence on astrocytes. This is the first study to explore the molecular regulation mechanism of Aβ and senescence on primary astrocytes from the perspective of the whole transcriptome. In uncovering the signaling pathways and biological processes involved in the effects of Aβ and senescence on astrocytes, this work provides novel insights into the pathogenesis of AD at the level of competing endogenous RNA network regulation.

Cytokine and Chemokine Release from Immortalised Human Myotubes in Culture: Effect of Contractions and Comparison with Isolated Intact Mature Adult Murine Muscle Fibres

Skeletal muscle releases a number of signalling molecules, including immunomodulatory cytokines and chemokines which can act in an autocrine, paracrine and endocrine fashion. For example, neuromuscular integrity is dependent on efficient and appropriate signalling from muscle to nerve and vice versa and when such signalling fails then tissue deterioration occurs. The aim of this study was to characterise the pattern and time course of release of pro‐ and anti‐ inflammatory cytokines and chemokines from cultured immortalised human myotubes at rest and following contractions compared with release from isolated viable intact mouse muscle fibres. Immortalised human myoblasts were fused to mature into myotubes for 7 days (Donna et al, 2003). Alternatively, single muscle fibres were isolated from the flexor digitorum brevis (FDB) muscle of adult C57Bl/6 mice (Pye et al, 2007). Cells and isolated fibres were subjected to electrical field stimulation with trains of biphasic square wave pulses of 2 ms duration for 0.5 s repeated every 5 s at 50 Hz and 30 V per well of fibres for a total stimulation time of 15 min. Media from all samples was analysed for cytokine content at 15 minutes and 24 hours following contractions using a multiplexed cytokine assay (Luminex) and data were standardised to protein content of the well. At 15 minutes following contractions of isolated muscle fibres, a significant increase in a number of cytokines and chemokines were seen in the media, including IL‐5, IL‐6, IL‐10 and G‐CSF although this was not fully replicated in 2D cultured myotubes. At 24 hours following contractions, IL‐6 content was significantly increased in the media of both isolated fibres and 2D cultured myotubes. Thus, data demonstrate some commonality of cytokines/chemokines released but future studies will examine release from more physiological 3D muscle constructs as well as the effects of such release on neuronal cell structure and function.Pye et al, J Physiol. 2007 May 15; 581(Pt 1): 309–318.Donna et al, Mol Cancer Res. 2003 (1) (9) 643‐653.

MicroRNA Alteration, Application as Biomarkers, and Therapeutic Approaches in Neurodegenerative Diseases.

MicroRNAs (miRNAs) are essential post-transcriptional gene regulators involved in various neuronal and non-neuronal cell functions and play a key role in pathological conditions. Numerous studies have demonstrated that miRNAs are dysregulated in major neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington’s disease. Hence, in the present work, we constructed a comprehensive overview of individual microRNA alterations in various models of the above neurodegenerative diseases. We also provided evidence of miRNAs as promising biomarkers for prognostic and diagnostic approaches. In addition, we summarized data from the literature about miRNA-based therapeutic applications via inhibiting or promoting miRNA expression. We finally identified the overlapping miRNA signature across the diseases, including miR-128, miR-140-5p, miR-206, miR-326, and miR-155, associated with multiple etiological cellular mechanisms. However, it remains to be established whether and to what extent miRNA-based therapies could be safely exploited in the future as effective symptomatic or disease-modifying approaches in the different human neurodegenerative disorders.

Gene expression changes in the brain of a Cushing's syndrome mouse model.

Excess glucocorticoid exposure affects emotional and cognitive brain functions. The extreme form, Cushing's syndrome, is adequately modelled in the AdKO2.0 mouse, consequential to adrenocortical hypertrophy and hypercorticosteronemia. We previously reported that the AdKO2.0 mouse brain undergoes volumetric changes that resemble closely those of Cushing's syndrome human patients, as well as changes in expression of glial related marker proteins. In the present work, the expression of genes related to glial and neuronal cell populations and functions was assessed in regions of the anterior brain, hippocampus, amygdala and hypothalamus. Glucocorticoid target genes were consistently regulated, including CRH mRNA suppression in the hypothalamus and induction in amygdala and hippocampus, even if glucocorticoid receptor protein was downregulated. Expression of glial genes was also affected in the AdKO2.0 mouse brain, indicating a different activation status in glial cells. Generic markers for neuronal cell populations, and cellular integrity were only slightly affected. Our findings highlight the vulnerability of glial cell populations to chronic high levels of circulating glucocorticoids.

Colony-stimulating factor 1 receptor signaling in the central nervous system and the potential of its pharmacological inhibitors to halt the progression of neurological disorders.

Colony Stimulating Factor-1 (CSF-1)/Colony Stimulating Factor-1 Receptor (CSF-1R) signaling axis plays an essential role in the development, maintenance, and proliferation of macrophage lineage cells. Within the central nervous system, CSF-1R signaling primarily maintains microglial homeostasis. Microglia, being the resident macrophage and first responder to any neurological insults, plays critical importance in overall health of the human brain. Aberrant and sustained activation of microglia along with continued proliferation and release of neurotoxic proinflammatory cytokines have been reported in various neurological and neurodegenerative diseases. Therefore, halting the neuroinflammatory pathway via targeting microglial proliferation, which depends on CSF-1R signaling, has emerged as a potential therapeutic target for neurological disorders. However, apart from regulating the microglial function, recently it has been discovered that CSF-1R has much broader role in central nervous system. These findings limit the therapeutic utility of CSF-1R inhibitors but also highlight the need for a complete understanding of CSF-1R function within the central nervous system. Moreover, it has been found that selective inhibitors of CSF-1R may be more efficient in avoiding non-specific targeting and associated side effects. Short-term depletion of microglial population in diseased conditions have also been found to be beneficial; however, the dose and therapeutic window for optimum effects may need to be standardized further.This review summarizes the present understanding of CSF-1R function within the central nervous system. We discuss the CSF-1R signaling in the context of microglia function, crosstalk between microglia and astroglia, and regulation of neuronal cell function. We also discuss a few of the neurological disorders with a focus on the utility of CSF-1R inhibitors as potential therapeutic strategy for halting the progression of neurological diseases.

Phenotype and genotype spectrum of variants in guanine nucleotide exchange factor genes in a broad cohort of Iranian patients.

BackgroundGuanine nucleotide exchange factors (GEFs) play pivotal roles in neuronal cell functions by exchanging GDP to GTP nucleotide and activation of GTPases. We aimed to determine the genotype and phenotype spectrum of GEF mutations by collecting data from a large Iranian cohort with intellectual disability (ID) and/or developmental delay (DD).MethodsWe collected data from nine families with 20 patients extracted from Iranian cohort of 640 families with ID and/or DD. Next‐generation sequencing (NGS) was used to identify the causing variants in recruited families. We also compared our clinical and molecular findings with previously reported patients carrying mutations in these GEF genes in the literature published until mid‐2021.ResultsWe identified disease‐causing variants in eight GEF genes including ALS2, IQSEC2, MADD, RAB3GAP1, RAB3GAP2, TRIO, ITSN1, and DENND2A. The major clinical manifestations in 203 previously reported cases along with our 20 patients with disease causing variants in eight GEF genes were as follow; speech disorder (85.2%), ID (81.6%), DD (81.1%), inability to walk (71.3%), facial dysmorphisms features (52.4%), abnormalities in skull morphology (55.6%), hypotonia and muscle weakness (47%), and brain MRI abnormalities (43.4%).ConclusionOur study provides new insights into the genotype and phenotype spectrum of mutations in GEF genes.

Nanonization of Magnoflorine-Encapsulated Novel Chitosan-Collagen Nanocapsules for Neurodegenerative Diseases: In Vitro Evaluation.

Neurodegeneration is one of the most common diseases in the aged population, characterized by the loss in the function of neuronal cells and their ultimate death. One of the common features in the progression of this type of diseases is the oxidative stress. Drugs which are currently being used have been found to show lateral side effects, which is partly due to their inefficiency to cross blood–brain barrier. Nanoencapsulation of bioactive compounds is a profound approach in this direction and has become a method of choice nowadays. This study involved the evaluation of the anti-oxidative properties of magnoflorine (MF), which is an aporphine quaternary alkaloid, and synthesis of MF-loaded chitosan–collagen nanocapsules (MF-CCNc) for its better efficacy as a potent anti-oxidant. Physiochemical characterization of the synthesized nanocapsules was done by using dynamic light scattering and transmission electron microscopy. It revealed that the synthesized nanocapsules are of small size range, as small as 12 ± 2 nm, and are more or less of spherical shape. Sustained release was shown by MF in the in vitro drug release studies. Both MF and MF-CCNc were found to have good anti-oxidant potential with IC50 < 25 μg/mL. No major cytotoxicity was shown by the synthesized nanocapsules on SH-SY5Y cells. In silico anti-acetylcholinesterase (AChE) studies were also done, and they revealed that MF can be a potent inhibitor of AChE.

Preventing cognitive impairment by reducing air pollution

Preventing cognitive impairment by reducing air pollution

Autoregulation of Pax6 in neuronal cells is mediated by Pax6(5a), Pax6(ΔPD), SPARC, and p53.

Pax6, a multifunctional protein and a transcriptional regulator is critical for optimal functioning of neuronal cells. It is known that alternatively spliced Pax6 isoforms and co-expressed interacting proteins mediate cell/tissue specific autoregulation of Pax6, however, underlying mechanism(s) are poorly understood. We used Neuro-2a cells to explore the mechanism of autoregulation of Pax6 in neuronal cells whereas NIH/3T3 cells were used as control. We first studied the transcript expression of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD); and the two co-expressed Pax6-interacting partners: SPARC and p53 in normal and overexpressed conditions, through the semi-quantitative RT-PCR. Further, we used the luciferase reporter assay to study the binding and transactivation of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD) to their respective promoters: P0, P1, and Pα; followed by that of the two co-expressed Pax6-interacting partners: SPARC and p53 to the Pax6-P1 promoter. Expression and distribution of Pax6, Pax6(5a) and Pax6(ΔPD), their binding to Pax6-promoters (P0, P1, and Pα) and transactivation were modulated in transfected Neuro-2a cells. Our results suggest that autoregulation of Pax6 in neuronal cells is driven by a promoter dependent mechanism which is mediated by spliced variants [Pax6(5a) and Pax6(ΔPD)] and interacting proteins (SPARC and p53) of Pax6.

Rapid Generation of Ventral Spinal Cord-like Astrocytes from Human iPSCs for Modeling Non-Cell Autonomous Mechanisms of Lower Motor Neuron Disease.

Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human-induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. Here, we describe the efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord. These induced astrocytes exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal cord features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS.

Neuronal STAT3/HIF-1α/PTRF axis-mediated bioenergetic disturbance exacerbates cerebral ischemia-reperfusion injury via PLA2G4A.

Ischemic stroke is an acute and severe neurological disease with high mortality and disability rates worldwide. Polymerase I and transcript release factor (PTRF) plays a pivotal role in regulating cellular senescence, glucose intolerance, lipid metabolism, and mitochondrial bioenergetics, but its mechanism, characteristics, and functions in neuronal cells following the cerebral ischemia-reperfusion (I/R) injury remain to be determined.Methods: Transcription factor motif analysis, chromatin immunoprecipitation (ChIP), luciferase and co-Immunoprecipitation (co-IP) assays were performed to investigate the mechanisms of PTRF in neuronal cells after I/R injury. Lentiviral-sgRNA against PTRF gene was introduced to HT22 cells, and adeno-associated virus (AAV) encoding a human synapsin (hSyn) promoter-driven construct was transduced a short hairpin RNA (shRNA) against PTRF mRNA in primary neuronal cells and the cortex of the cerebral I/R mice for investigating the role of PTRF in neuronal damage and PLA2G4A change induced by the cerebral I/R injury.Results: Here, we reported that neuronal PTRF was remarkably increased in the cerebral penumbra after I/R injury, and HIF-1α and STAT3 regulated the I/R-dependent expression of PTRF via binding to its promoter in neuronal cells. Moreover, overexpression of neuronal PTRF enhanced the activity and stability of PLA2G4A by decreasing its proteasome-mediated degradation pathway. Subsequently, PTRF promoted reprogramming of lipid metabolism and altered mitochondrial bioenergetics, which could lead to oxidative damage, involving autophagy, lipid peroxidation, and ferroptosis via PLA2G4A in neuronal cells. Furthermore, inhibition of neuronal PTRF/PLA2G4A-axis markedly reduced the neurological deficits, cerebral infarct volumes, and mortality rates in the mice following cerebral I/R injury.Conclusion: Our results thus identify that the STAT3/HIF-1α/PTRF-axis in neurons, aggravating cerebral I/R injury by regulating the activity and stability of PLA2G4A, might be a novel therapeutic target for ischemic stroke.

FAM3A Ameliorates Brain Impairment Induced by Hypoxia–Ischemia in Neonatal Rat

Hypoxia–ischemia (HI) during crucial periods of brain formation can lead to changes in brain morphology, propagation of neuronal stimuli, and permanent neurodevelopmental impairment, which can have profound effects on cognitive function later in life. FAM3A, a subgroup of family with sequence similarity 3 (FAM3) gene family, is ubiquitously expressed in almost all cells. Overexpression of FAM3A has been evidenced to reduce hyperglycemia via the PI3K/Akt signaling pathway and protect mitochondrial function in neuronal HT22 cells. This study aims to evaluate the protective role of FAM3A in HI-induced brain impairment. Experimentally, maternal rats underwent uterine artery bilateral ligation to induce neonatal HI on day 14 of gestation. At 6 weeks of age, cognitive development assessments including NSS, wire grip, and water maze were carried out. The animals were then sacrificed to assess cerebral mitochondrial function as well as levels of FAM3A, TNF-α and IFN-γ. Results suggest that HI significantly reduced FAM3A expression in rat brain tissues, and that overexpression of FAM3A through lentiviral transduction effectively improved cognitive and motor functions in HI rats as reflected by improved NSS evaluation, cerebral water content, limb strength, as well as spatial learning and memory. At the molecular level, overexpression of FAM3A was able to promote ATP production, balance mitochondrial membrane potential, and reduce levels of pro-inflammatory cytokines TNF-α and IFN-γ. We conclude that FAM3A overexpression may have a protective effect on neuron morphology, cerebral mitochondrial as well as cognitive function.Graphical Created with Biorender.com

ACD856: A novel positive allosteric modulator of Trk‐signaling in clinical development for the treatment of Alzheimer’s disease

AbstractBackgroundNeurotrophin pathways, such as those mediated by NGF and BDNF, have in numerous studies been shown to be important for neuronal cell function, communication and cell survival in brain areas vital for cognitive function. Therefore, enhancers of NGF and BDNF signaling through positive allosteric modulation of the TrkA and TrkB receptors may be a valuable new option for the treatment of cognitive decline in various disease states. The aim of this study was to identify and characterize compounds that potently stimulate neurotrophin signaling by binding to the Trk‐receptors and to investigate the effects of such a compound in various preclinical model. Moreover, the aim was also to assess its safety and tolerability in both animals and man.MethodAll binding studies were performed using a Biacore instrument. The effects of ACD856 were investigated in different preclinical in vivo behavioural models to assess its effect on cognitive function. Safety and toxicology studies were subsequently conducted in line with regulatory requirements before a microdosing study in humans was performed to evaluate the pharmacokinetics, safety and tolerability in man.ResultA binding site on the intracellular domain of TrkA was observed for the compound ACD856 using surface plasmon resonance experiments. In an in vitro kinase assay using human full‐length TrkA, ACD856 could significantly increase the apparent maximal velocity (Vmax(app)) of TrkA. ACD856 also demonstrated potent pro‐cognitive effects in vivo, with a favorable PK profile. The compound was shown to be safe and well tolerated in the preclinical toxicological studies. Micodosing results in man showed that ACD856 had a profile suitable for further clinical develoment.ConclusionThe in vitro results suggests that ACD856 binds directly to the TrkA receptor to increase the number of catalytic cycles per timepoint. This mechanism of action is a probably a likely cause for the observed in vivo effects with respect to improved cognitive performance. These data suggest that ACD856 may have a beneficial effect on cognition in patients with cognitive dysfunction and the compound is currently in clinical development for the treatment of cognitive dysfunction in Alzheimer’s disease.

Role of ferroptosis iron-dependent cell death in neurodegenerative processes.

Accumulation or overload of bioavailable redox active metals causes protein overexpression-aggregation, mitochondrial dysfunction, nucleus alterations, inflammation-immune responses and high oxidative stress that directly disrupt normal neuronal cell functions. Significantly, a novel form of iron-dependent regulated cell death process termed ferroptosis was recently identified. Misregulated ferroptosis pathways have been speculated to trigger neurodegeneration through programmed cell death that is distinct from classical mechanisms, although the underlying mechanisms remain only partially understood. We induced ferroptosis (erastin, buthioninesulfoximine and sulfasalazine, ammonium iron(III) citrate) on in vitro cultured neurons. Neuronal responses were characterized by bioassays (ROS, GSH, Iron, DPPP), RNA-seq and confocal microscopy. We induced ferroptosis using compounds that target the main ferroptotic pathways and analyzed the biochemical responses. We selected buthionine-sulfoximine targeting GPX4, erastin targeting system Xc - , and iron (III) citrate targeting iron transporters/trafficking . We dosed neurons with 0-200μM to determine IC50, dose- and time-dependent responses. Notably, the ferroptosis inducers caused a time-dependent increase in cytosolic and lipid ROS, as assessed by microplate assay and fluorescent microscopy using fluorescent probes H2 DCFDA and DPPP, respectively. After 24 h, the increase in ROS was accompanied by cell detachment, cell death and change in morphology. Intracellular glutathione pool suffered depletion, whereas labile iron accumulated over the course of the treatment. ROS accumulation and cell death were suppressed by cotreatment with the iron chelator deferoxamine (100 μM). Furthermore, we performed RNA-seq to determine pathways altered by ferroptosis, and morphological characterization by confocal and electron microscopy. Our data show marked alteration in metabolism of iron, glutathione depletion, ROS overproduction, and marked lipid peroxidation in neurons dosed with ferroptosis inducers, confirming susceptibility of neurons to iron-dependent ferroptosis cell death.