Neuronal Cultures Research Articles

Silver bismuth sulfide quantum dot‐based bioelectronics for light‐mediated neuronal stimulation

Aims/Purpose: Nanomaterial based bioelectronics have been effectively developed as optoelectronic bio‐interfaces for neural stimulation and can be fabricated from diverse substances to adapt to cellular environments. In this study, we employed silver bismuth sulfide (AgBiS2) quantum dot (QD)‐based neural interfaces stimulated by near‐infrared light.Methods: Primary neuron isolation and culture on AgBiS2 QD based photovoltaic device and indium tin oxide (ITO) control device was performed. Cell viability was assessed by CTG, MTT, Live/Dead assay and LDH leakage assays. Cellular stress with light stimulation was assessed by measuring intracellular reactive oxygen species. Cell specific biomarkers, NeuN, beta‐III Tubulin and F‐actin, were examined by immunofluorescence staining to demonstrate short‐ and long‐term morphological changes and neural network improvements. Number of neuron count and neurite length measurement were analyzed to compare groups. Under light stimulation (λ = 780 nm), neural dynamics and electrophysiological activity were examined through intracellular calcium flow and patch clamp.Results: Throughout the 14‐day culture period, neurons remained healthy and viable, preserving their characteristics and forming extensive networks with neurite outgrowth on both the device and ITO. Light stimulation was found to have no adverse effects on viability or intracellular stress levels. The device demonstrated successful photostimulation of neurons with calcium release and generating action potential under 780 nm light illumination which is validating its potential to be used as optoelectronic bio‐interfaces for neural applications.Conclusions: Our study holds high potential for the development of QD‐based retinal prosthesis, enabling near infrared light‐controlled activation in vision related diseases.Funding Information: This study was funded by the European Union (ERC, MESHOPTO, 101045289).References Balamur R, Oh JT, Karatum O, Wang Y, Onal A, Kaleli HN, Pehlivan C, Şahin A, Hasanreisoglu M, Konstantatos G, Nizamoglu S. Capacitive and Efficient Near‐Infrared Stimulation of Neurons via an Ultrathin AgBiS2 Nanocrystal Layer. ACS Applied Materials & Interfaces. 2024 May 29

Silver bismuth sulfide quantum dot‐based bioelectronics for light‐mediated neuronal stimulation

Aims/Purpose: Nanomaterial based bioelectronics have been effectively developed as optoelectronic bio‐interfaces for neural stimulation and can be fabricated from diverse substances to adapt to cellular environments. In this study, we employed silver bismuth sulfide (AgBiS2) quantum dot (QD)‐based neural interfaces stimulated by near‐infrared light.Methods: Primary neuron isolation and culture on AgBiS2 QD based photovoltaic device and indium tin oxide (ITO) control device was performed. Cell viability was assessed by CTG, MTT, Live/Dead assay and LDH leakage assays. Cellular stress with light stimulation was assessed by measuring intracellular reactive oxygen species. Cell specific biomarkers, NeuN, beta‐III Tubulin and F‐actin, were examined by immunofluorescence staining to demonstrate short‐ and long‐term morphological changes and neural network improvements. Number of neuron count and neurite length measurement were analyzed to compare groups. Under light stimulation (λ = 780 nm), neural dynamics and electrophysiological activity were examined through intracellular calcium flow and patch clamp.Results: Throughout the 14‐day culture period, neurons remained healthy and viable, preserving their characteristics and forming extensive networks with neurite outgrowth on both the device and ITO. Light stimulation was found to have no adverse effects on viability or intracellular stress levels. The device demonstrated successful photostimulation of neurons with calcium release and generating action potential under 780 nm light illumination which is validating its potential to be used as optoelectronic bio‐interfaces for neural applications.Conclusions: Our study holds high potential for the development of QD‐based retinal prosthesis, enabling near infrared light‐controlled activation in vision related diseases.Funding Information: This study was funded by the European Union (ERC, MESHOPTO, 101045289).References Balamur R, Oh JT, Karatum O, Wang Y, Onal A, Kaleli HN, Pehlivan C, Şahin A, Hasanreisoglu M, Konstantatos G, Nizamoglu S. Capacitive and Efficient Near‐Infrared Stimulation of Neurons via an Ultrathin AgBiS2 Nanocrystal Layer. ACS Applied Materials & Interfaces. 2024 May 29

Oxidative Stress Promotes Axonal Atrophy through Alterations in Microtubules and EB1 Function.

Axons are crucial for transmitting neurochemical signals. As organisms age, the ability of neurons to maintain their axons declines; hence, aged axons are more susceptible to damage or dysfunction. Understanding how aging causes axonal vulnerability is crucial for developing strategies to enhance overall resilience of neurons and prevent neuronal deterioration during aging and in age-related neurodegenerative diseases. Increasing levels of reactive oxygen species (ROS) causes oxidative stress - a hallmark of aging and age-related diseases. Despite this association, a causal relationship between oxidative stress and neuronal aging remains unclear, particularly in how subcellular physiology may be affected by ROS. By using Drosophila-derived primary neuronal cultures and a recently developed in vivo neuronal model of aging, which involves the visualisation of Drosophila medulla neurons, we investigated the interplay between oxidative stress, neuronal aging and the microtubule cytoskeleton. Our results showed that oxidative stress is a key driver of axonal and synaptic decay, as shown by an enhanced appearance of axonal swellings, microtubule alterations (in both axons and synapses) and morphological transformation of axonal terminals during aging. We demonstrated that increasing the levels of ROS sensitises microtubule plus end-binding protein 1 (EB1), leading to microtubule defects that effect neuronal integrity. Furthermore, manipulating EB1 proved to be a valuable therapeutic strategy to prevent aging hallmarks enhanced in conditions of elevated ROS. In summary, we demonstrate a mechanistic pathway linking cellular oxidative stress with changes in the microtubule cytoskeleton leading to axonal deterioration during aging and provide evidence of the therapeutic potential of enhancing microtubule plus-end physiology to improve the resilience of axons.

Rapid neurostimulation at the micron scale with an optically controlled thermal-capture technique.

Precise control of cellular temperature at the microscale is crucial for developing novel neurostimulation techniques. Here, the effect of local heat on the electrophysiological properties of primary neuronal cultures and HEK293 cells at the subcellular level using a cutting-edge micrometer-scale thermal probe, the diamond heater-thermometer (DHT), is studied. A new mode of local heat action on a living cell, thermal-capture mode (TCM), is discovered using the DHT probe. In TCM, the application of a 50 °C temperature step induces a great increase in cellular response, allowing the cell to be thermally captured and depolarized by up to 20 mV. This thermal effect is attributed to local phase changes in the phospholipid membrane, enabling precise and reproducible modulation of cell activity. The TCM is shown to open up new opportunities for thermal cell stimulation. DHT reliably triggers action potentials (APs) in neurons at rates up to 30 Hz, demonstrating the ability to control cell excitability with millisecond and sub-millisecond resolution. AP shape is modulated by local heat as well. The ability to precisely control the AP shape and rate via thermal-capture mode opens new avenues for non-invasive, localized neurostimulation techniques, particularly in controlling neuron excitability.

Combined additive effects of neuronal membrane glycoprotein GPM6a and the intercellular cell adhesion molecule ICAM5 on neuronal morphogenesis.

The mechanisms underlying neuronal development and synaptic formation in the brain depend on intricate cellular and molecular processes. The neuronal membrane glycoprotein GPM6a promotes neurite elongation, filopodia/spine formation, and synapse development, yet its molecular mechanisms remain unknown. Since the extracellular domains of GPM6a (ECs) command its function, we investigated the interaction between ICAM5, the neuronal member of the intercellular adhesion molecule (ICAM) family, and GPM6a's ECs. Our study aimed to explore the functional relationship between GPM6a and ICAM5 in hippocampal culture neurons and cell lines. Immunostaining of 15 days invitro (DIV) neurons revealed significant co-localization between endogenous GPM6a clusters and ICAM5 clusters in the dendritic shaft. These results were further corroborated by overexpressing GPM6a and ICAM5 in N2a cells and hippocampal neurons at 5 DIV. Moreover, results from the co-immunoprecipitations and cell aggregation assays prove the cis and trans interaction between both proteins in GPM6a/ICAM5 overexpressing HEK293 cells. Additionally, GPM6a and ICAM5 overexpression additively enhanced neurite length, the number of neurites in N2a cells, and filopodia formation in 5 DIV neurons, indicating their cooperative role. These findings highlight the dynamic association between GPM6a and ICAM5 during neuronal development, offering insights into their contributions to neurite outgrowth, filopodia formation, and cell-cell interactions.

Expression of c-fos in cortical neuron cultures under dynamic magnetic field is not suppressed by calcium channel blockers

Previously, we developed a dynamic magnetic field (DMF) device using neodymium magnets that induced c-fos expression in cortical neurons, while activity-regulated cytoskeleton-associated protein (Arc), and brain-derived neurotrophic factor (BDNF) remained unaffected. The precise signal transduction pathway for c-fos induction under DMF was unclear. This study aimed to investigate the mechanism of immediate early gene (IEG) induction using calcium channel blockers (CCBs). Six experiments were conducted with cortical neurons, employing an NMDA receptor antagonist and an L-type voltage-dependent calcium channel blocker as CCBs. Neuronal cultures were exposed to DMF, CCBs, or both, and IEG expression (Arc, c-fos, BDNF) was measured through polymerase chain reaction. Results showed a tendency for increased c-fos expression with DMF exposure, which was unaffected by CCBs. In contrast, Arc and BDNF were not induced under DMF exposure but were significantly inhibited by CCBs. These findings suggest that c-fos induction under DMF involves a distinct pathway, potentially relevant to stress resistance and drug discovery.

The effect of ACTH/MSH N-terminal fragment analogs on the anxiety level, pain sensitivity and levels of neurotrophic factors BDNF and VEGF in primary neuronal cultures of rats

ACTH/MSH-like peptides (melanocortins) have a wide range of neurotropic effects, including effects on learning and memory processes, neuroprotection, emotional state and pain sensitivity. Present work is aimed to compare the effects of peptides, the structure of which includes a natural fragment of ACTH and a stabilizing tripeptide PGP. The peptides ACTH4-7PGP (Semax), ACTH6-9PGP и ACTH7-10PGP were used in the work. The effects of these peptides on the exploratory behavior, anxiety level and pain sensitivity of white rats, as well as on the protein levels of the neurotrophic factors BDNF (brain derived neurotrophic factor) and VEGF (vascular endothelial growth factor) in primary neuron cultures were studied. A comparative study of the effects of analogs of different ACTH/MSH fragments revealed both similarities and differences in their neurotropic activity. The peptides structure of which includes a sequence of ACTH4-7 or ACTH6-9 have nootropic, anxiolytic and analgesic activity, and also cause an increase in VEGF levels in the culture of hippocampal neurons. The peptide containing the ACTH7-10 sequence in the structure exhibits anxiolytic activity, increases exploratory behavior, does not affect pain sensitivity and has a stimulating effect on BDNF and VEGF levels in neuronal cultures. The data obtained indicate that different parts of the N-terminal region of the ACTH molecule are responsible for the manifestation of certain neurotropic effects of melanocortins. The results of the study can be used in the development of therapeutics based on natural melanocortins.

Truncated TrkB: The predominant TrkB Isoform in Nociceptors.

Truncated TrkB (TrkBT1), traditionally considered a dominant-negative regulator of full-length TrkB (TrkBTK+), remains poorly understood in peripheral sensory neurons, particularly nociceptors. Furthermore, sensory neuronal TrkB expression and function has been traditionally associated with non-nociceptive neurons, particularly Aδ low-threshold mechanoreceptors. This study challenges prevailing assumptions by demonstrating that TrkBT1 is the predominant TrkB isoform expressed in sensory neurons and plays a functional role in modulating neuronal activity. We demonstrate that TrkBT1 is the predominant isoform expressed in nociceptors, identified by markers such as TRPV1, TRPA1, TRPM8 and 5HT3A, as well as non-nociceptors, while the full-length isoform (TrkBTK+) is restricted to non-nociceptive subpopulation. Functionally, we show that acute application of BDNF induces modest calcium influx in nociceptors and prolonged BDNF exposure significantly potentiates capsaicin-induced calcium influx, an effect blocked by the TrkB-specific antagonist ANA12. Additionally, BDNF also promotes the survival of both nociceptive and non-nociceptive neurons in culture, an effect dependent on TrkBT1 activity. Our data also reveal that ANA12 inhibits BDNF-mediated neuronal sensitization and survival in a concentration-dependent manner, implicating distinct TrkBT1 signaling pathways in these processes. Collectively, our findings redefine TrkBT1 as a functional modulator of nociceptor activity rather than a passive regulator of full-length TrkB. By uncovering its dual roles in nociceptor sensitization and survival, this study provides new insights into the molecular mechanisms of BDNF/TrkB signaling in pain. Future work evaluating the role of TrkBT1 in sensory biology could offer new perspectives on how this receptor contributes to neuronal function and plasticity during chronic pain conditions.

Cathepsin B Modulates Alzheimer's Disease Pathology Through SAPK/JNK Signals Following Administration ofPorphyromonas gingivalis-Derived Outer Membrane Vesicles.

Porphyromonas gingivalis, a consensus periodontal pathogen, is thought to be involved in Alzheimer's disease (AD) progression, and P. gingivalis-derived outer membrane vesicles (PgOMVs) are a key toxic factor in inducing AD pathology. This study aimed to clarify the regulatory mechanism underlying the PgOMV-induced AD-like phenotype. We intraperitoneally injected PgOMVs into the periphery of wild-type and CatB knockout mice for 4 or 8 weeks to assess the effect of CatB on PgOMV-induced AD pathology. Mice were evaluated for cognitive change, tau phosphorylation, microglial activation, neuroinflammation and synapse loss. Microglial and primary neuron culture were prepared to verify the invivo results. CatB deficiency significantly alleviated PgOMV-induced cognitive dysfunction, microglia-mediated neuroinflammation, tau hyperphosphorylation and synapse loss. Subsequent transcriptomic analysis, immunofluorescence and immunoblotting suggested that CatB modulates microglia-mediated neuroinflammation through stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) signals after administration of PgOMVs, which in turn regulates neuronal tau phosphorylation and synapse loss in a SAPK/JNK-dependent manner. Our study unveils a previously unknown role of CatB in regulating PgOMV-induced AD pathology.

Carboxylated Zn-phthalocyanine attenuates brain Aβ in AD model mouse.

The deposition of aggregated amyloid β (Aβ) is considered as a key factor for Alzheimer's Disease (AD). Previously, we demonstrated that a carboxylated Zn-phthalocyanine (ZnPc) inhibits Aβ fibril formation, consequently protects neurons in culture. This study evaluated the effects of ZnPc on pathological changes in an AD mouse model (J20). Nine-month-old J20 mice received weekly intraperitoneal injection of ZnPc (2 and 4 mg/kg) for 12 weeks. Cognitive performance was assessed using Y-maze and open field tests. ZnPc levels in the tissues were evaluated using near-infrared microscopy and spectroscopy. ZnPc accumulated primarily in the liver and kidney. A considerable amount was also detected in brain tissue, where it co-localized with neurons, microglia, and extracellularly deposited Aβ. ZnPc treatment (2 mg/kg) significantly improved cognitive functions of J20 mice. Immunostaining results showed that Aβ was positive intracellularly in neurons, and extracellularly around the vessels and parenchyma in the cortex and hippocampus of PBS-treated J20 mice, which was significantly decreased in ZnPc-treated J20 mice in a dose-dependent manner. Nissl staining demonstrated that neuronal numbers were increased both in the cortex and hippocampus, whereas GFAP-positive astrocytes and Iba-1 positive microglia were decreased by ZnPc treatment. Also, vessel numbers were increased in ZnPc-treated groups. In PBS-treated group, aquaporin 4 immunopositive area extended beyond STL-positive vessels into the parenchyma, which was confined primarily around the vessels in the ZnPc-treated group. Claudin 5 levels were increased in ZnPc-treated group. Therefore, ZnPc can decrease brain Aβ deposition in J20 mice, suggesting it as a potential therapeutic agent for AD.

In vivo changes in zebrafish anesthetic sensitivity in response to the loss of kif5Aa are associated with the alteration of mitochondrial motility.

Anesthetic and sedative drugs are small compounds known to bind to hundreds of proteins. One intriguing binding partner of propofol is the kinesin motor domain, kif5A, a neuronal mitochondrial transport protein. Here, we used zebrafish WT and kif5Aa KO larval behavioral assays to assess anesthetic sensitivity and combined that with zebrafish primary neuronal cell culture to probe for alteration in mitochondrial motility. We found that the loss of kif5Aa increases behavioral sensitivity to propofol and etomidate, with etomidate hypersensitivity greater than propofol. In contrast, kif5Aa KO animals were resistant to the behavioral effects of dexmedetomidine. Finally, WT and kif5Aa KO larvae responded similarly to the behavioral effects of ketamine. Propofol inhibited the anterograde motility of mitochondria in WT zebrafish neurons, while etomidate inhibited mitochondrial motility in both anterograde and retrograde directions; neither drug altered mitochondrial motility in the kif5Aa knockout (KO) neurons. In contrast, dexmedetomidine enhanced retrograde mitochondrial motility in both WT and kif5Aa KO animals. Finally, ketamine had little significant effect on mitochondrial motility in either mutant or WT animals. These data demonstrate that each anesthetic/sedative drug affects the motor protein machinery uniquely and is associated with unique changes in behavior. Understanding how different anesthetic compounds alter neuron motor proteins will be important in defining how anesthetics alter neuronal signaling and energetic dynamics.

Suppression of MT5-MMP Reveals Early Modulation of Alzheimer's Pathogenic Events in Primary Neuronal Cultures of 5xFAD Mice.

We previously reported that membrane-type 5-matrix metalloproteinase (MT5-MMP) deficiency not only reduces pathological hallmarks of Alzheimer's disease (AD) in 5xFAD (Tg) mice in vivo but also impairs interleukin-1 beta (IL-1β)-mediated neuroinflammation and Aβ production in primary Tg immature neural cell cultures after 11 days in vitro. We now investigate the effect of MT5-MMP on incipient pathogenic pathways that are activated in cortical primary cultures at 21-24 days in vitro (DIV), during which time neurons are organized into a functional mature network. Using wild-type (WT), MT5-MMP-/- (MT5-/-), 5xFAD (Tg), and 5xFADxMT5-MMP-/- (TgMT5-/-) mice, we generated primary neuronal cultures that were exposed to IL-1β and/or different proteolytic system inhibitors. We assessed neuroinflammation, APP metabolism, synaptic integrity, and electrophysiological properties using biochemical, imaging and whole-cell patch-clamp approaches. The absence of MT5-MMP impaired the IL-1β-mediated induction of inflammatory genes in TgMT5-/- cells compared to Tg cells. Furthermore, the reduced density of dendritic spines in Tg neurons was also prevented in TgMT5-/- neurons. IL-1β caused a strong decrease in the dendritic spine density of WT neurons, which was prevented in MT5-/- neurons. However, the latter exhibited fewer spines than the WT under untreated conditions. The spontaneous rhythmic firing frequency of the network was increased in MT5-/- neurons, but not in TgMT5-/- neurons, and IL-1β increased this parameter only in Tg neurons. In terms of induced somatic excitability, Tg and TgMT5-/- neurons exhibited lower excitability than WT and MT5-/-, while IL-1β impaired excitability only in non-AD backgrounds. The synaptic strength of miniature global synaptic currents was equivalent in all genotypes but increased dramatically in WT and MT5-/- neurons after IL-1β. MT5-MMP deficiency decreased endogenous and overexpressed C83 and C99 levels but did not affect Aβ levels. C99 appears to be cleared by several pathways, including γ-secretase, the autophagolysosomal system, and also α-secretase, via its conversion to C83. In summary, this study confirms that MT5-MMP is a pivotal factor affecting not only neuroinflammation and APP metabolism but also synaptogenesis and synaptic activity at early stages of the pathology, and reinforces the relevance of targeting MT5-MMP to fight AD.

Impact of c-JUN deficiency on thalamus development in mice and human neural models

Backgroundc-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers, c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development, its impact on nervous system development in higher mammals, especially for some deep structures, for example, thalamus in diencephalon, remains unclear.MethodsTo investigate the influence of c-JUN on early nervous system development, c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs), cerebral organoids (COs), and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining, omics analysis, and loss/gain of function analysis.ResultsAt embryonic day 14.5, c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6+/NESTIN+ exterior layer but lacked a fibers-connected core. Moreover, the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably, in guided ThOs, c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2, WNT8B, MXRA5, HSPG2 and PLAGL1 while 24 genes including MSX1, CYP1B1, LMX1B, NQO1 and COL2A1 were significantly up-regulated.ConclusionOur findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.

Heterogeneous Ribonucleoprotein K Is a Host Regulatory Factor of Chikungunya Virus Replication in Astrocytes.

Chikungunya virus (CHIKV) is an emerging, mosquito-borne arthritic alphavirus increasingly associated with severe neurological sequelae and long-term morbidity. However, there is limited understanding of the crucial host components involved in CHIKV replicase assembly complex formation, and thus virus replication and virulence-determining factors, within the central nervous system (CNS). Furthermore, the majority of CHIKV CNS studies focus on neuronal infection, even though astrocytes represent the main cerebral target. Heterogeneous ribonucleoprotein K (hnRNP K), an RNA-binding protein involved in RNA splicing, trafficking, and translation, is a regulatory component of alphavirus replicase assembly complexes, but has yet to be thoroughly studied in the context of CHIKV infection. We identified the hnRNP K CHIKV viral RNA (vRNA) binding site via sequence alignment and performed site-directed mutagenesis to generate a mutant, ΔhnRNPK-BS1, with disrupted hnRNPK-vRNA binding, as verified through RNA coimmunoprecipitation and RT-qPCR. CHIKV ΔhnRNPK-BS1 demonstrated hampered replication in both NSC-34 neuronal and C8-D1A astrocytic cultures. In astrocytes, disruption of the hnRNPK-vRNA interaction curtailed viral RNA transcription and shut down subgenomic RNA translation. Our study demonstrates that hnRNP K serves as a crucial RNA-binding host factor that regulates CHIKV replication through the modulation of subgenomic RNA translation.

Elastic properties of the cell surface and metabolic profile of an embryonic primary mixed culture of hippocampal neurons under conditions of P2X3 receptor blockade

Elastic properties of the cell surface and metabolic profile of an embryonic primary mixed culture of hippocampal neurons under conditions of P2X3 receptor blockade

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.

Clickable, Thermally Responsive Hydrogels Enabled by Recombinant Spider Silk Protein and Spy Chemistry for Sustained Neurotrophin Delivery.

The ability to deliver protein therapeutics in a minimally invasive, safe, and sustained manner, without resorting to viral delivery systems, will be crucial for treating a wide range of chronic injuries and diseases. Among these challenges, achieving axon regeneration and functional recovery post-injury or disease in the central nervous system remains elusive to most clinical interventions, constantly calling for innovative solutions. Here, a thermally responsive hydrogel system utilizing recombinant spider silk protein (spidroin) is developed. The protein solution undergoes rapid sol-gel transition at an elevated temperature (37°C) following brief sonication. This thermally triggered gelation confers injectability to the system. Leveraging SpyTag/SpyCatcher chemistry, the hydrogel, composed of SpyTag-fusion spidroin, can be functionalized with diverse SpyCatcher-fusion bioactive motifs, such as neurotrophic factors (e.g., ciliary neurotrophic factor) and cell-binding ligands (e.g., laminin), rendering it well-suited for neuronal culturing. More importantly, the intravitreous injection of the protein materials decorated with SpyCatcher-fusion CNTF into the vitreous body after optic nerve injury leads to prolonged JAK/STAT3 signaling, increased neuronal survival, and enhanced axon regeneration. This study illustrates a generalizable material system for injectable and sustained delivery of protein therapeutics for neuroprotection and regeneration, with the potential for extension to other chronic diseases and injuries.

The measles virus matrix F50S mutation from a lethal case of subacute sclerosing panencephalitis promotes receptor-independent neuronal spread.

Subacute sclerosing panencephalitis (SSPE) is a lethal neurological disorder occurring several years after measles. Reconstruction of the evolution of the measles virus (MeV) genome in an SSPE case suggested that the matrix (M) protein mutation M-F50S, when added to other mutations, drove neuropathogenesis. However, whether and how M-F50S would promote spread independently from other mutations was in question. We investigated here the cell specificity of MeV spread in this brain and documented that both neurons and astrocytes were heavily infected. We then generated recombinant MeV with individual mutations in the three proteins of the viral membrane fusion apparatus, M, fusion (F), and hemagglutinin (H). These viruses reached similar titers as the parental wild-type virus, kept the respective mutations upon passage, and infected cells expressing the tissue-specific MeV receptors SLAM and nectin-4 with similar efficiencies. However, after inoculation of receptor-negative neurons and astrocytes differentiated from human induced pluripotent stem cells, only MeV M-F50S spread with moderate efficiency; the parental virus and its derivatives coding for a hyperfusogenic F protein, or for a cytoplasmic tail-mutated H protein, did not spread. When delivered to primary mouse neurons by cell-mediated neurite overlay, MeV M-F50S frequently reached the cell bodies and occasionally formed small infectious centers, while the other MeV reached the cell bodies only sporadically. These results demonstrate that, in neuronal cell cultures, M-F50S can enable receptor-independent spread in the absence of other mutations, and validate the inference that this single amino acid change initiated ubiquitous MeV brain spread.IMPORTANCEMeasles virus (MeV), a non-integrating negative-strand RNA virus, rarely causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. During brain adaptation, the MeV genome acquires multiple mutations reducing the dependence of its membrane fusion apparatus (MFA) from an activating receptor. It was proposed that one of these mutations, matrix protein F50S, drove neuropathogenesis in an SSPE case. We report here that, in two types of neuronal cultures, a recombinant MeV with only this mutation gained receptor-independent spread, whereas viruses expressing MFA proteins with other mutations acquired during brain adaptation did not. Our results validate the inference that M-F50S initiated ubiquitous MeV brain spread resulting in lethal disease. They also prompt studies of the impact of analogous amino acid changes of the M proteins of other nonsegmented negative-strand RNA viruses on their interactions with membrane lipids and cytoskeletal components.

NMDA Receptor-Mediated Ca2+ Flux Attenuated by the NMDA Receptor/TRPM4 Interface Inhibitor Brophenexin.

Transient receptor potential melastatin-4 (TRPM4) forms a complex with N-methyl-D-aspartate receptors (NMDARs) that facilitates NMDAR-mediated neurotoxicity. Here we used pharmacological tools to determine how TRPM4 regulates NMDAR signaling. Brophenexin, a compound that binds to TRPM4 at the NMDAR binding interface, protected hippocampal neurons in culture from NMDA-induced death, consistent with published work. Brophenexin (10 μM) reduced NMDA-evoked whole-cell currents recorded at 22°C by 87% ± 14% with intracellular Ca2+ chelated to prevent TRPM4 activation. Brophenexin inhibited NMDA-evoked currents recorded in Na+-free solution by 87% ± 13%, suggesting that brophenexin and TRPM4 modulate NMDAR function. Incubating cultures in Mg2+-free buffer containing tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, and bicuculline for 30 min inhibited NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) recorded at 22°C by 50% ± 18% and prevented inhibition by brophenexin. In the absence of these inhibitors, brophenexin inhibited the NMDA-evoked response by 51% ± 16%. Treatment with the TRPM4 inhibitor 4-chloro-2-(1-naphthyloxyacetamido)benzoic acid (NBA; 10 μM) increased NMDA-evoked Ca2+ influx by 90% ± 15%. Increasing extracellular NaCl to 237 mM, a treatment that activates TRPM4, inhibited the NMDA-evoked increase in [Ca2+]i by a process that occluded the inhibition produced by brophenexin and was prevented by NBA. In recordings performed at 32°C-34°C, brophenexin inhibited the NMDA-evoked [Ca2+]i response by 42% ± 10% but NBA was without effect. These results are consistent with a model in which TRPM4 interacts with NMDARs to potentiate Ca2+ flux through the NMDAR ion channel and thus provides a potential mechanism for the neuroprotection afforded by NMDAR/TRPM4 interface inhibitors such as brophenexin.

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.