Primary Neuronal Cultures Research Articles

Reduced Neurite Arborization in Primary Dopaminergic Neurons in Autism-Like Shank3B-Deficient Mice.

Despite many studies on dopamine changes in autism, specific alterations in midbrain dopamine neurons projecting to the striatum and cortex remain unclear. Mouse models with diverse SH3 domain and ankyrin repeat containing protein 3 (Shank3) deficiencies are used for investigating autistic symptoms and underlying neurobiological mechanisms. SHANK3 belongs to postsynaptic proteins crucial for synapse formation during development, and disruptions in SHANK3 structure could lead to impaired neurite outgrowth and altered dendritic arborization and morphology. Therefore, we aimed to investigate whether Shank3 deficiency (Shank3B) leads to changes in the morphology of primary neuronal cell cultures from dopaminergic brain regions of neonatal mouse pups and whether it results in alterations in synaptic proteins in dopaminergic nerve pathway projection areas (striatum, frontal cortex). Significantly reduced neurite outgrowth was observed in primary dopaminergic neurons from the midbrain and striatum of Shank3-deficient compared to WT mice. A decrease in Synapsin I immunofluorescence signal in the cortical neurons isolated from Shank3-deficient mice was found, although neurite arborization changes were less severe. Importantly, the deficit in the length of the longest neurite was confirmed in primary cortical neurons isolated from Shank3-deficient mice. No changes in the gene expression of synaptic proteins were observed in the striatum and frontal cortex of Shank3-deficient mice, but an altered gene expression profile of dopaminergic receptors was found. These results show structural changes of dopaminergic neurons, which may explain autistic symptomatology in the used model and provide a basis for understanding the long-term development of autistic symptoms.

Neuritogenesis and protective effects activated by Angiotensin 1–7 in astrocytes-neuron interaction

The renin angiotensin system (RAS) has been studied for its effects on various neurological disorders. The identification of functional receptors for Ang-(1–7) and Ang II peptides in astrocytes highlights the physiological modulation and the important role of these cells in the central nervous system. The present study aims to understand the role of RAS peptides, particularly Ang-(1–7) and Ang II, in the secretion of trophic factors by astrocytes and their effects on hippocampal neurons. We used primary cultures of astrocytes and neurons from the hippocampus of either sex neonate of Wistar strain rats. In the present study, we demonstrated that the treatment of astrocytes with Ang-(1–7) acts on the modulation of these cells, inducing reactive astrogliosis, identified through the increase in the expression of GFAP. Furthermore, we obtained a conditioned medium from astrocytes treated with Ang-(1–7), which in addition to promoting the secretion of neurotrophic factors essential for neuronal-glial interactions that are fundamental for neuritogenesis and neuronal survival, showed a neuroprotective effect against glutamatergic excitotoxicity. In turn, Ang II does not exhibit the same effects on astrocyte modulation, exacerbating deleterious effects on brain RAS. Neuron-astrocyte interactions have been shown to be an integral part of the central effects mediated by RAS, and this study has significantly contributed to the understanding of the biochemical mechanisms involved in the functioning of this system.

Regulation of neuronal fate specification and connectivity of the thalamic reticular nucleus by the Ascl1-Isl1 transcriptional cascade

The thalamic reticular nucleus (TRN) is an anatomical and functional hub that modulates the flow of information between the cerebral cortex and thalamus, and its dysfunction has been linked to sensory disturbance and multiple behavioral disorders. Therefore, understanding how TRN neurons differentiate and establish connectivity is crucial to clarify the basics of TRN functions. Here, we showed that the regulatory cascade of the transcription factors Ascl1 and Isl1 promotes the fate of TRN neurons and concomitantly represses the fate of non-TRN prethalamic neurons. Furthermore, we found that this cascade is necessary for the correct development of the two main axonal connections, thalamo-cortical projections and prethalamo-thalamic projections. Notably, the disruption of prethalamo-thalamic axons can cause the pathfinding defects of thalamo-cortical axons in the thalamus. Finally, forced Isl1 expression can rescue disruption of cell fate specification and prethalamo-thalamic projections in in vitro primary cultures of Ascl1-deficient TRN neurons, indicating that Isl1 is an essential mediator of Ascl1 function in TRN development. Together, our findings provide insights into the molecular mechanisms for TRN neuron differentiation and circuit formation.

Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software.

Dendritic spine morphology is associated with the current state of the synapse and neuron, and changes during synaptic plasticity in response to stimulus. At the same time, dendritic spine alterations are reported during various neurodegenerative and neurodevelopmental disorders and other brain states. Accurate and informative analysis of spine shape has an urgent need for studying the synaptic processes and molecular pathways in normal and pathological conditions, and for testing synapto-protective strategies during preclinical studies. Primary neuronal cultures enable high quality imaging of dendritic spines and offer a wide spectrum of accessible experimental manipulations. This article outlines the protocol for isolating, culturing, fluorescent labeling, and imaging of mouse primary hippocampal neurons by three-dimensional (3D) confocal microscopy in a normal state and in conditions of low amyloid toxicity-an in vitro model of Alzheimer's disease. An alternate protocol describes the neuronal morphology analysis using the EGFP expressing neurons in line-M transgenic mouse brain slices. Since the dendritic spines are relatively small structures lying close to the confocal microscope resolution limit, their proper segmentation on the images is challenging. This protocol highlights the image-preprocessing steps, including generation of theoretical point spread function and deconvolution, which enhances resolution and removes noise, thereby enhancing the 3D spine reconstruction results. SpineTool, an open source Python-based script, enables 3D segmentation of dendrites and spines and numerical metric calculation, including key measures, such as spine length, volume, and surface area, with a new feature, the chord length distribution histogram, improving clustering results. SpineTool supports both manual and machine learning spine classification (i.e., mushroom, thin, stubby, filopodia) and automated clustering using k-means and DBSCAN methods. This protocol provides detailed instructions for using SpineTool to analyze and classify dendritic spines in control and experimental groups, enhancing our understanding of spine morphology across different experimental conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Obtaining 3D confocal dendritic spine images of hippocampal neuronal culture in normal state and conditions of low amyloid toxicity Alternate Protocol: Obtaining confocal dendritic spine images of mice hippocampal neurons from fixed brain slices Support Protocol: Post-processing deconvolution of confocal images Basic Protocol 2: Segmentation of dendritic spines with SpineTool Basic Protocol 3: Spine dataset preparation using SpineTool Basic Protocol 4: Clustering of dendritic spines with SpineTool Basic Protocol 5: Machine classification of dendritic spines with SpineTool.

Assessment of the neurotoxicity of monosodium glutamate on neural stem cells and hippocampal neurogenesis in a rodent model

Monosodium glutamate (MSG) is a widely used flavor enhancer in processed foods and valued for its ability to enhance the savory taste known as umami. MSG is classified as non-toxic and recognized as a safe food additive with no specific usage restrictions in many countries. However, neurotoxic studies on MSG have primarily focused on neurons, and the effects of MSG on neural stem cells (NSCs) have not been reported. This study aimed to evaluate the neurotoxic effect of MSG on NSCs and hippocampal neurogenesis in a rodent model. In vitro studies showed that MSG induces cytotoxicity in primary neuron cultures but has no toxic effect on NSCs. Furthermore, in vivo studies on 4-week-old male C57BL/6 mice orally administered MSG and sodium chloride (NaCl) for two weeks revealed that neither MSG nor NaCl induced changes in the expressions of neuronal markers or glutamate receptors in the hippocampus. In addition, no differences in NSC proliferation or survival were detected, and MSG did not adversely affect the neuronal differentiation of NSCs. Moreover, neurobehavioral tests showed that MSG treatment did not impair spatial learning and memory. These findings provide a first assessment of the neurotoxic effects of MSG on NSCs and hippocampal neurogenesis.

Development and validation a novel FEZF2 based fluorescent reporter for corticospinal motor neurons.

Corticospinal motor neurons (CSMNs), also named upper motor neurons, are the giant pyramidal neurons called Betz cells. In mammals, the majority of CSMNs reside within layer V of the primary motor cortex, where they extend long axon bundles named the pyramidal tract into the brainstem and the spinal cord to control voluntary movement. CSMN lesions are implicated in a variety of neurodegenerative disorders, such Amyotrophic Lateral Sclerosis, Primary Lateral Sclerosis and Hereditary Spastic paraplegia. Although FEZF2-CTIP2 genetic axis have been indicated as the cardinal molecular pathway underlying the development of CSMNs, these proteins are transcription factors that are mostly used to label the nuclei of CSMNs in the fixed cells and tissues. Therefore, a fluorescent reporter to mark CSMNs will be invaluable in identifying living CSMNs, including their extended processes, for time-lapse imaging and high-throughput molecular analyses with much more improved specificity. Based on the in-silico analysis, we identified a putative region within the promoter sequence of FEZF2 and assembled it with an indispensable enhancer motif at its downstream of the gene to form a complex promoter that drives the expression of reporter GFP. The plasmid and virus of FEZF2:eGFP reporter constructs were further validated for its use in specifically labeling CSMNs in primary neuronal cultures from the embryonic rat motor cortex, postnatal mouse cortex. This innovative molecular labeling tool has the potential to offer indispensable support in diverse experimental setups, enabling a comprehensive understanding of the susceptibility and specificity of CSMNs in a wide array of neurological disorders.

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in both primary mouse cerebrocortical neuronal cultures and human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H2O2) and 1-methyl-4-phenylpyridinium ion (MPP+). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.

CNSC-13. CROSSTALK BETWEEN SENSORY NEURONAL ACTIVITY AND TUMOR CELLS PROMOTES MALIGNANCY AND CANCER PAIN

Abstract BACKGROUND Nerve infiltration of solid tumors is associated with poor prognosis in multiple cancer types. Understanding the role of neuronal activity in cancer progression offers innovative approaches for treating malignancies and cancer-associated neurological issues such as pain. Pain perception is mediated by the activity of peripheral sensory neurons. Many cancer types induce pain, and sensory innervation could support tumor growth, raising the interesting hypothesis that cancer pain-associated neuronal activity promotes malignancies. Our study aims to test the hypothesis using malignant peripheral nerve sheath tumor (MPNST) as the experimental platform. METHODS in vivo allograft models were established by implanting mouse MPNST cells into the sciatic nerves of male and female mice, followed by performing pain behavior assays (von Frey, Hargreaves, and conditioned place preference tests) and tumor growth measurements. Primary mouse dorsal root ganglion (DRG) sensory neuron cultures were prepared for calcium imaging and neuron-tumor co-culture experiments. RESULTS MPNST tumor-bearing mice exhibited hypersensitivity to noxious and non-noxious stimuli (evoked pain) and ongoing pain, compared to the sham controls. MPNST-conditioned media increased intracellular calcium concentration and ATF3 (indicator for nerve injury/stress) expression in primary sensory neuron culture, suggesting that MPNST tumor cell secretory factors may induce pain by triggering sensory neuron injury/stress responses that increase sensory neuron activity/sensitivity. Reciprocally, we found that stimulation of sensory neuron activity accelerates MPNST tumor growth. The conditioned media from stimulated sensory neurons increased tumor proliferation (EdU assay) in vitro, suggesting that sensory neuron activity-induced paracrine factors increase MPNST cell growth. CONCLUSIONS MPNST is a devastating cancer that grows within peripheral nerves which induce intractable pain. Our findings demonstrate a feedforward cycle where the cancer cells induce sensory neuron hypersensitivity/hyperactivity (pain) which in turn further accelerates tumor growth. Future studies will identify the molecular mechanisms underlying this crosstalk between MPNST and sensory neuron activity.

Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.IMPORTANCEEstablishing models to investigate host-pathogen interactions requires mimicking physiological conditions as closely as possible. One consideration is the oxygen concentration used for in vitro tissue culture experiments. Standard incubators do not regulate oxygen levels, exposing cells to oxygen concentrations of approximately 18%. However, cells within the body are exposed to much lower oxygen concentrations, with physiological oxygen concentrations in the brain being 0.55%-8% oxygen. Here, we describe a model for herpes simplex virus 1 (HSV-1) latent infection using neurons cultured in 5% oxygen. We show that culturing neurons in more physiological oxygen concentrations improves neuronal health to permit long-term studies of virus-cell interactions and the impact on reactivation.

Effects of quercetin-immobilized albumin cerium oxide nanoparticles on glutamate toxicity: in vitro study.

One aspect of glutamate (Glut) toxicity may be the opening of the blood-brain barrier to albumin (Al), which in itself can cause nerve cell death. Quercetin (Q) is a polyphenolic substance and has a neuroprotective effect. Cerium oxide nanoparticles (Ce2O3NPs) are highly interested in biological applications due to their antioxidant properties. The current study aimed to investigate the impact of Q-immobilized Al+Ce2O3NPs in Glut-induced neurotoxicity, mainly focusing on cell viability and neurobiochemical changes. Hydrothermal synthesis and characterization of Q-immobilized Al+Ce2O3NPs were performed. After preparing the primary neuron culture, it was exposed to Glut to induce neurotoxicity. Then, various doses of Ce2O3NP, Al+Ce2O3NP, and Q+Al+Ce2O3NPs (1, 5, 10, and 25 µg/ml) were applied to the wells and incubated for 24 h. Then, cell viability was determined by MTT analysis. Additionally, oxidative stress parameters were measured. When the obtained data were examined, it was shown that cell viability decreased with Glut concentration but significantly increased with Q+Al+Ce2O3NPs treatment. When oxidative stress markers were considered, Glut treatment increased LDH, AChE, and TOS levels, while TAC and GSH levels decreased. However, the trend changed after Q+Al+Ce2O3NPs treatment, suggesting that damaged neurons were protected against oxidative stress. The results of this study indicate that Q+Al+Ce2O3NP can ameliorate Glut-induced neurotoxicity, especially when used at a dose of 25 µg/ml.

Cycloastragenol reduces microglial NLRP3 inflammasome activation in Parkinson's disease models by promoting autophagy and reducing Scrib-driven ROS

BackgroundIn Parkinson's disease (PD), microglial autophagy is crucial for the maintenance of cellular redox homeostasis. Meanwhile, cycloastragenol (CAG), a triterpenoid saponin and the principal active component of Astragalus, reduces the activation of NLRP3 inflammasomes. Nevertheless, the specific molecular mechanisms underlying the CAG-mitigated microglial neuroinflammation remains obscure in PD. PurposeThis study explored the role of CAG in the activation of microglial NLRP3 inflammasome and the mechanisms underlying its therapeutic potential for PD treatment. Study designThe effect of CAG was assessed in α-Syn-induced primary microglia and PD models. MethodsAAV1/2-hsyn-SNCA (A53T) was stereo-injected into the striatum of mice to induce PD models and CAG was orally administered. The mice underwent quantitative 4D proteomics analysis and behavioral assessments. The primary microglia and neuron cultures were analyzed by western blotting, immunofluorescence, transmission electron microscopy, etc. ResultsCAG reduced phagocytosis-induced reactive oxygen species (ROS) by suppressing the microglial Scribble (Scrib) and p22phox expression. Concurrently, CAG enhanced autophagy, promoted α-Syn clearance, and reduced mitochondrial damage. These synergistic effects downregulated NLRP3 inflammasome activation, in turn reducing gasdermin D cleavage, caspase-1 activation, and the release of interleukin-1β and interleukin-18. Further investigation revealed that CAG shielded neurons from α-Syn toxicity, thus attenuating behavioral impairments observed in the mouse PD model. ConclusionCAG mitigates neuroinflammation by inhibiting ROS-induced NLRP3 inflammasome activation in microglia via promoting microglial autophagy and reducing the activity of Scrib-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which signifies a promising alternative approach to PD management.

Magnetic field in the extreme low frequency band protects neuronal and microglia cells from oxygen-glucose deprivation.

Ischemic stroke consists of rapid neural death as a consequence of brain vessel obstruction, followed by damage to the neighboring tissue known as ischemic penumbra. The cerebral tissue in the core of the lesions becomes irreversibly damaged, however, the ischemic penumbra is potentially recoverable during the initial phases after the stroke. Therefore, there is real need for emerging therapeutic strategies to reduce ischemic damage and its spread to the penumbral region. For this reason, we tested the effect of Extreme Low Frequency Electromagnetic Stimulation (ELF-EMS) on in vitro primary neuronal and microglial cultures under oxygen-glucose deprivation (OGD) conditions. ELF-EMS under basal non-OGD conditions did not induce any effect in cell survival. However, ELF-EMS significantly reduced neuronal cell death in OGD conditions and reduced ischemic induced Ca2+ overload. Likewise, ELF-EMS modulated microglia activation and OGD-induced microglia cell death. Hence, this study suggests potential benefits in the application of ELF-EMS to limit ischemic irreversible damages under in vitro stroke conditions, encouraging in vivo preclinical validations of ELF-EMS as a potential therapeutic strategy for ischemic stroke.

Ethanol-activated microglial exosomes induce MCP1 signaling mediated death of stress-regulatory proopiomelanocortin neurons in the developing hypothalamus

BackgroundMicroglia, a type of resident immune cells within the central nervous system, have been implicated in ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined if microglial extracellular vesicles (exosomes) are involved in the ethanol-induced neuronal death of the β-endorphin neuron via secreting elevated levels of the chemokine monocyte chemoattractant protein 1 (MCP1), a key regulator of neuroinflammation.MethodsWe employed an in vitro model, consisting of primary culture of hypothalamic microglia prepared from postnatal day 2 (PND2) rat hypothalami and treated with or without 50 mM ethanol for 24 h, and an in vivo animal model in which microglia were obtained from hypothalami of PND6 rats fed daily with 2.5 mg/kg ethanol or control milk formula for five days prior to use. Exosomes were extracted and characterized with nanosight tracking analysis (NTA), transmission electron microscopy and western blot. Chemokine multiplex immunoassay and ELISA were used for quantitative estimation of MCP1 level. Neurotoxic ability of exosome was tested using primary cultures of β-endorphin neurons and employing nucleosome assay and immunocytochemistry. Elevated plus maze, open field and restraint tests were used to assess anxiety-related behaviors.ResultsEthanol elevated MCP1 levels in microglial exosomes both in vitro and in vivo models. Ethanol-activated microglial exosomes when introduced into primary cultures of β-endorphin neurons, increased cellular levels of MCP1 and the chemokine receptor CCR2 related signaling molecules including inflammatory cytokines and apoptotic genes as well as apoptotic death of β-endorphin neurons. These effects of microglial exosomes on β-endorphin neurons were suppressed by a CCR2 antagonist RS504393. Furthermore, RS504393 when injected in postnatal rats prior to feeding with ethanol it reduced alcohol-induced β-endorphin neuronal death in the hypothalamus. RS504393 also suppressed corticosterone response to stress and anxiety-like behaviors in postnatally alcohol-fed rats during adult period.ConclusionThese data suggest that alcohol exposures during the developmental period elevates MCP1 levels in microglial exosomes that promote MCP1/CCR2 signaling to increase the apoptosis of β-endorphin neurons and resulting in hormonal and behavioral stress responses.

Thioredoxin-1 protein interactions in neuronal survival and neurodegeneration

Neuronal cell death remains the principal pathophysiologic hallmark of neurodegenerative diseases and the main challenge for treatment strategies. Thioredoxin1 (Trx1) is a major cytoplasmic thiol oxidoreductase protein involved in redox signaling, hence a crucial player in maintaining neuronal health. Trx1 levels are notably reduced in neurodegenerative diseases including Alzheimer's and Parkinson's diseases, however, the impact of this decrease on neuronal physiology remains largely unexplored. This is mainly due to the nature of Trx1 redox regulatory role which is afforded by a rapid electron transfer to its oxidized protein substrates. During this reaction, Trx1 forms a transient bond with the oxidized disulfide bond in the substrate. This is a highly fast reaction which makes the identification of Trx1 substrates a technically challenging task. In this project, we utilized a transgenic mouse model expressing a Flag-tagged mutant form of Trx1 that can form stable disulfide bonds with its substrates, hence allowing identification of the Trx1 target proteins. Autophagy is a vital housekeeping process in neurons that is critical for degradation of damaged proteins under oxidative stress conditions and is interrupted in neurodegenerative diseases. Given Trx1's suggested involvement in autophagy, we aimed to identify potential Trx1 substrates following pharmacologic induction of autophagy in primary cortical neurons. Treatment with rapamycin, an autophagy inducer, significantly reduced neurite outgrowth and caused cytoskeletal alterations. Using immunoprecipitation and mass spectrometry, we have identified 77 Trx1 target proteins associated with a wide range of cellular functions including cytoskeletal organization and neurodegenerative diseases. Focusing on neuronal cytoskeleton organization, we identified a novel interaction between Trx1 and RhoB which was confirmed in genetic models of Trx1 downregulation in primary neuronal cultures and HT22 mouse immortalized hippocampal neurons. The applicability of these findings was also tested against the publicly available proteomic data from Alzheimer's patients. Our study uncovers a novel role for Trx1 in regulating neuronal cytoskeleton organization and provides a mechanistic explanation for its multifaceted role in the physiology and pathology of the nervous system, offering new insights into the molecular mechanisms underlying neurodegeneration.

Neuron-selective and activity-dependent splicing of BDNF exon I–IX pre-mRNA

Brain-derived neurotrophic factor (BDNF) is essential for numerous neuronal functions, including learning and memory. The expression of BDNF is regulated by distinctive transcriptional and post-transcriptional mechanisms. The Bdnf gene in mice and rats comprises eight untranslated exons (exons I–VIII) and one exon (exon IX) that contains the pre-proBDNF coding sequence. Multiple splice donor sites on the untranslated exons and a single acceptor site upstream of the coding sequence result in the characteristic exon skipping patterns that generate multiple Bdnf mRNA variants, which are essential for the spatiotemporal regulation of BDNF expression, mRNA localization, mRNA stability, and translational control. However, the regulation of Bdnf pre-mRNA splicing remains unclear. Here, we focused on the splicing of Bdnf exon I–IX pre-mRNA. We first constructed a minigene to evaluate Bdnf exon I–IX pre-mRNA splicing. Compared with Bdnf exon I–IX pre-mRNA splicing in non-neuronal NIH3T3 cells, splicing was preferentially observed in primary cultures of cortical neurons. Additionally, a series of overexpression and knockdown experiments suggested that neuro-oncological ventral antigen (NOVA) 2 is involved in the neuron-selective splicing of Bdnf exon I–IX pre-mRNA. Supporting this finding, endogenous Nova2 mRNA expression was markedly higher in neurons, and a strong correlation between endogenous Bdnf exon I–IX and Nova2 mRNA was observed across several brain regions. Furthermore, Bdnf exon I–IX pre-mRNA splicing was facilitated by Ca2+ signals evoked via L-type voltage-dependent Ca2+ channels. Notably, among the Bdnf pre-mRNA splicing investigated in the current study, neuron-selective and activity-dependent splicing was observed in Bdnf exon I–IX pre-mRNA. In conclusion, Bdnf exon I-IX pre-mRNA splicing is preferentially observed in neurons and is facilitated in an activity-dependent manner. The neuron-selective and activity-dependent splicing of Bdnf exon I–IX pre-mRNA may contribute to the efficient induction of Bdnf exon I–IX expression in neurons.

Early-life cisplatin exposure induces neuroinflammation and chemotherapy-induced neuropathic pain.

Chemotherapy-induced neuropathic pain (CINP) is a common adverse health-related comorbidity that manifests later in life in patients with paediatric cancer. Current analgesia is ineffective, aligning closely with our lack of understanding of CINP. The aim of this study was to investigate how cisplatin induces nerve growth factor (NGF)-mediated neuroinflammation and nociceptor sensitisation. In a rat model of cisplatin-induced survivorship pain, cisplatin induced a neuroinflammatory environment in the dorsal root ganglia (DRG), demonstrated by NGF-positive macrophages infiltrating into the DRG. Cisplatin-treated CD11b- and F4/80-positive macrophages expressed more NGF compared to those treated with vehicle control. Mouse primary DRG sensory neuronal cultures demonstrated enhanced NGF-dependent TRPV1-mediated nociceptor activity after cisplatin treatment. Increased nociceptor activity was also observed when cultured mouse DRG neurons were treated with conditioned medium from cisplatin-activated macrophages. Elevated nociceptor activity was inhibited in a dose-dependent manner by an NGF-neutralising antibody. Intraperitoneal administration of the NGF-neutralising antibody reduced cisplatin-induced mechanical hypersensitivity and aberrant nociceptor intraepidermal nerve fibre density. These findings identify that a monocyte- or macrophage-driven NGF-TrkA pathway is a novel analgesic target for adult survivors of childhood cancer.

Nucleophosmin 1 overexpression enhances neuroprotection by attenuating cellular stress in traumatic brain injury

BackgroundTraumatic Brain Injury (TBI) is a multifaceted injury that can cause a wide range of symptoms and impairments, leading to significant effects on brain function. Nucleophosmin 1 (NPM1), a versatile phosphoprotein located in the nucleolus, is being recognized as a possible controller of cellular stress reactions and could be important in reducing neuro dysfunction caused by TBI. However the critical roles of NPM1 in cellular stress in TBI remains unclear. MethodsWe employed a control cortical impact mouse model and a scratch-induced primary neuronal culture model. Hematoxylin and eosin staining was used to evaluate tissue damage and cellular changes, with NPM1 expression in the cortical area assessed through immunofluorescence staining and Western blot analysis. Neuronal morphology was assessed using Nissl staining. Behavioral assessments were performed to evaluate the impact of NPM1 overexpression on neurobehavioral results in TBI mice. Mitochondrial function was assessed using an Extracellular Flux Analyzer. ResultsFollowing TBI, an increase in NPM1 expression was observed, with a peak at 72 h post-injury. Increased levels of NPM1 resulted in decreased neuronal cell death, as shown by Nissl staining, and lower levels of Caspase 8, APE1, H2AX, and 8-OHDG expression, indicating a reduction in DNA damage. NPM1 overexpression also resulted in improved neurobehavioral outcomes, characterized by decreased neurological deficits and enhanced motor function post-TBI. Additionally, in vitro, scratch-induction experiments revealed that NPM1 overexpression mitigated mitochondrial damage, as evidenced by the downregulation of P53, BCL2, and Cyto C expression levels and improvements in mitochondrial respiratory function. ConclusionThese findings suggest NPM1 as a promising target for developing interventions to alleviate TBI-related cellular stress and promote neuronal survival.

Evaluating deep learning techniques for optimal neurons counting and characterization in complex neuronal cultures.

The counting and characterization of neurons in primary cultures have long been areas of significant scientific interest due to their multifaceted applications, ranging from neuronal viability assessment to the study of neuronal development. Traditional methods, often relying on fluorescence or colorimetric staining and manual segmentation, are time consuming, labor intensive, and prone to error, raising the need for the development of automated and reliable methods. This paper delves into the evaluation of three pivotal deep learning techniques: semantic segmentation, which allows for pixel-level classification and is solely suited for characterization; object detection, which focuses on counting and locating neurons; and instance segmentation, which amalgamates the features of the other two but employing more intricate structures. The goal of this research is to discern what technique or combination of those techniques yields the optimal results for automatic counting and characterization of neurons in images of neuronal cultures. Following rigorous experimentation, we conclude that instance segmentation stands out, providing superior outcomes for both challenges.