Synaptic Region Research Articles

Cellular Nanointerface of Vertical Nanostructures: Impact of Size‐Modulated Nanopillar Arrays on Neuronal Morphology, Maturation, and Synapse Formation

Investigating the cellular mechanisms facilitated by highly ordered nanostructures in vitro neuronal networks is crucial for advancing biomedical research. However, the intricate effects of these nanostructures on cellular behavior remain unclear. Herein, we explore how variations in nanopillar (NP) array dimensions, defined by the spacing‐to‐diameter (S/D) ratio, influence cortical neuronal responses—including cellular morphology, mechanosensing, neuronal maturation, and synapse formation. NP arrays with a low S/D ratio accelerate neurite protrusion and enhance neuronal maturation. These topography‐driven changes are attributed to the degree of cell confinement on NPs, which can be visualized using focused ion beam scanning microscopy. Furthermore, the synaptic density in cortical neurons declines as the S/D ratio decreases, with neurons preferring to form synapses along the NPs. By utilizing correlative light and electron microscopy, the spatial organization of these synapses is mapped relative to the NP geometries, revealing specialized synaptic regions with remarkable clarity. This approach to designing high‐aspect‐ratio nanostructures with various S/D ratios holds broad applications in materials engineering, offering insights into nervous system engineering and neural‐interfacing bioelectronics.

High-Speed Atomic Force Microscopy Reveals Fluctuations and Dimer Splitting of the N-Terminal Domain of GluA2 Ionotropic Glutamate Receptor-Auxiliary Subunit Complex.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which are crucial for AMPAR clustering at synaptic sites. However, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit complexed with TARP γ2 in lipid environments. HS-AFM videos of GluA2-γ2 in the resting and activated/open states revealed fluctuations in NTD dimers. Conversely, in the desensitized/closed state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we observed individual NTD dimers transitioning into monomers, with extended monomeric states in the activated/open state. Molecular dynamics simulations provided further support, confirming the energetic stability of the monomeric NTD states within lipids. This NTD-dimer splitting resulted in subunit exchange between the receptors and increased the number of interaction sites with synaptic protein neuronal pentraxin 1 (NP1). Moreover, our HS-AFM studies revealed that NP1 forms a ring-shaped octamer through N-terminal disulfide bonds and binds to the tip of the NTD. These findings suggest a molecular mechanism in which NP1, upon forming an octamer, is secreted into the synaptic region and binds to the tip of the GluA2 NTD, thereby bridging and clustering multiple AMPARs. Thus, our findings illuminate the critical role of NTD dynamics in the synaptic clustering of AMPARs and contribute valuable insights into the fundamental processes of synaptic transmission.

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc γ RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

A zebrafish gephyrinb mutant distinguishes synaptic and enzymatic functions of Gephyrin

Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.

The Drosophila blood–brain barrier invades the nervous system in a GPCR-dependent manner

The blood–brain barrier (BBB) represents a crucial interface between the circulatory system and the brain. In Drosophila melanogaster, the BBB is composed of perineurial and subperineurial glial cells. The perineurial glial cells are small mitotically active cells forming the outermost layer of the nervous system and are engaged in nutrient uptake. The subperineurial glial cells form occluding septate junctions to prevent paracellular diffusion of macromolecules into the nervous system. To address whether the subperineurial glia just form a simple barrier or whether they establish specific contacts with both the perineurial glial cells and inner central nervous system (CNS) cells, we undertook a detailed morphological analysis. Using genetically encoded markers alongside with high-resolution laser scanning confocal microscopy and transmission electron microscopy, we identified thin cell processes extending into the perineurial layer and into the CNS cortex. Interestingly, long cell processes were observed reaching the glia ensheathing the neuropil of the central brain. GFP reconstitution experiments highlighted multiple regions of membrane contacts between subperineurial and ensheathing glia. Furthermore, we identify the G-protein-coupled receptor (GPCR) Moody as negative regulator of the growth of subperineurial cell processes. Loss of moody triggered a massive overgrowth of subperineurial cell processes into the CNS cortex and, moreover, affected the polarized localization of the xenobiotic transporter Mdr65. Finally, we found that GPCR signaling, but not septate junction formation, is responsible for controlling membrane overgrowth. Our findings support the notion that the Drosophila BBB is able to bridge the communication gap between circulation and synaptic regions of the brain by long cell processes.

Single Nuclear Analyses Reveal Compromised Integrity of Neuromuscular Junction Myonuclei Following Joint Injury

The neuromuscular junction is an essential structure that links electrical signals from the nervous system to contraction of skeletal muscle. The post synaptic region of a muscle cell is maintained by a subset of specialized myonuclei responsible for clustering of post synaptic structural proteins and acetylcholine receptors. Disruptions in the transcriptional program of these myonuclei may result in ineffective myofiber recruitment, slowed force generation, and muscle weakness. We have shown that traumatic joint injury results in protracted deficits in muscle strength and power, but the peripheral mechanistic underpinnings remain unclear. Phenotypically, muscle pathology occurring after traumatic joint injury resembles that of neuromuscular destabilization, yet molecularly, this remains uninvestigated. Therefore, we hypothesize that disruption of neuromuscular regulatory gene programs and destabilization of the neuromuscular junction following joint injury will result in rapid muscle functional deficits. Human muscle biopsies were obtained from the vastus lateralis of anterior cruciate ligament (ACL) injured and healthy limbs of 26 participants. Injured biopsies were taken before and 7 days following reconstruction surgery. Bulk tissue RNA-seq analysis was performed on all participants and single-nucleus RNAseq (snRNAseq) was performed on a subset of participants. Briefly, for snRNAseq, muscle was dounce homogenized, filtered, and nuclei isolated via fluorescent activated cell sorting. Following sequencing, CellRanger output files containing count matrices were filtered, and analyzed using Partek Flow and Seurat. Biopsy sections were processed for histochemistry to analyze fiber size and motor end plate innervation. All participants underwent strength testing on a biodex to assess isometric quadriceps torque and rate of torque development. RNA sequencing displayed >3 fold upregulation of CHRNA1 (P< 0.001) after injury alone and >18 fold upregulation 7 days following reconstruction surgery (P< 0.001). This was accompanied by a host of other neuromuscular regulatory genes including HDAC4 (P< 0.001), GDNF (P< 0.001), and RUNX1 (P< 0.001). Single nucleus sequencing revealed a complete disappearance of neuromuscular junction regulatory myonuclei following reconstruction surgery coupled with delocalization of acetylcholine receptor expression. These findings were confirmed with fluorescent in situ hybridization of CHRNA1 RNA on biopsy sections. Functionally, CHRNA1 RNA expression strongly correlates with deficits in rate of torque development following injury (R^2 = 0.85, P< 0.001). Our results display rapid remodeling of the postsynaptic neuromuscular junction following joint injury that underscores sustained deficits in strength and power. Interventions targeting the preservation of peripheral innervation may be warranted to enhance outcomes following joint injury. R01AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beneficial versus Detrimental Effects of Complement-Microglial Interactions in Alzheimer's Disease.

Research indicates that brain-region-specific synapse loss and dysfunction are early hallmarks and stronger neurobiological correlates of cognitive decline in Alzheimer's disease (AD) than amyloid plaque and neurofibrillary tangle counts or neuronal loss. Even though the precise mechanisms underlying increased synaptic pruning in AD are still unknown, it has been confirmed that dysregulation of the balance between complement activation and inhibition is a crucial driver of its pathology. The complement includes three distinct activation mechanisms, with the activation products C3a and C5a, potent inflammatory effectors, and a membrane attack complex (MAC) leading to cell lysis. Besides pro-inflammatory cytokines, the dysregulated complement proteins released by activated microglia bind to amyloid β at the synaptic regions and cause the microglia to engulf the synapses. Additionally, research indicating that microglia-removed synapses are not always degenerating and that suppression of synaptic engulfment can repair cognitive deficits points to an essential opportunity for intervention that can prevent the loss of intact synapses. In this study, we focus on the latest research on the role and mechanisms of complement-mediated microglial synaptic pruning at different stages of AD to find the right targets that could interfere with complement dysregulation and be relevant for therapeutic intervention at the early stages of the disease.

Membrane Heteroreceptor Complexes as Second-Order Protein Modulators: A Novel Integrative Mechanism through Allosteric Receptor-Receptor Interactions.

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR-D2R, GABAA-D5R, and FGFR1-5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor-receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor-receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor-receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000-2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor-receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor-receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson's disease.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

Aberrant DJ‐1 expression underlies L‐type calcium channel hypoactivity in dendrites in Alzheimer’s disease (AD) and tuberous sclerosis complex (TSC)

AbstractBackgroundL‐type voltage‐dependent Ca2+ channels (L‐VGCC) dysfunction is implicated in several neurological and psychiatric diseases. While a popular therapeutic target, it is unknown if molecular mechanisms leading to disrupted L‐VGCC across neurodegenerative disorders is conserved. Importantly, L‐VGCC integrate synaptic signals to facilitate a plethora of cellular mechanisms; however, mechanisms that regulate L‐VGCC channel density and subcellular compartmentalization are understudied.MethodThe following methods were used: bioinformatics; RNA immunoprecipitation; Surface Sensing of Translation–Proximity Ligation Assay (SUnSET‐PLA); calcium imaging, immunocytochemistry; Western blots, dissociated hippocampal neurons and acute hippocampal slices from APP/PS1 mouse mode of AD and Tsc1 conditionally knockout mice; Postmortem human prefrontal cortex tissue.ResultHerein, we report that in disease models with overactive mammalian target of rapamycin complex 1 (mTORC1) signaling (or mTORopathies), deficits in dendritic L‐VGCC activity are associated with increased expression of the RNA‐binding protein (RBP) DJ‐1. DJ‐1 binds the mRNA coding for the alpha and auxiliary Ca2+ channel subunits CaV1.2 and alpha2delta2 (α2δ2), and represses their mRNA translation, only in the disease states, specifically preclinical models of Alzheimer’s disease (AD) and tuberous sclerosis complex (TSC). In agreement, DJ‐1 mediated repression of CaV1.2/α2δ2 protein synthesis in dendrites is exaggerated in mouse models of AD and TSC, resulting in deficits in dendritic L‐VGCC calcium activity. These preclinical findings are recapitulated in the synaptic and dendritic regions of prefrontal cortices of humans diagnosed with AD.ConclusionDiscovery of DJ‐1 regulated L‐VGCC activity in dendrites in TSC and AD provides a new signaling pathway that can be targeted in clinical mTORopathies.

Physiological and pathological 3R and 4R tau expression in marmoset brain

AbstractBackgroundCommon marmosets (Callithrix jacchus) have recently emerged as an important model for studying aging and neurodegenerative disorders, including Alzheimer’s disease (AD). Amyloid‐β deposits in the cerebral cortex of marmosets present naturally with aging, which has now been widely reported. However, the role of tau in the marmoset brain has been understudied. The purpose of the present studies was to comprehensively examine 3R and 4R tau isoform expression in the marmoset brain.MethodsThe mRNA expression in the marmoset brain was confirmed by RT‐PCR. The total mRNA was isolated from frozen tissue of various age groups and reverse transcribed to cDNA, which was used for RT‐PCR. The sequence of each fragment was confirmed by the Sanger sequencing method. The tau protein expression in Sarkosyl soluble and insoluble fractions was examined by western blot using tau‐specific antibodies, and expression levels of 3R/4R were quantified by Dot blot. Synaptic tau expression was analyzed from crude synaptosome extractions of the frontal cortex. Pathological tau aggregates in cryo‐slices of a 10‐year‐old marmoset brain were visualized by immunohistochemistry.Results3R and 4R tau were expressed in the marmoset brain, with 3R tau predominately expressed in neonatal brains and 4R tau in adult/aged brains. 3R and 4R tau were also localized in synaptic regions of the frontal cortex. The Sarkosyl soluble tau was phosphorylated on T181, T217, and T231 sites with evidence of oligomers. Insoluble tau was phosphorylated on S396/S404 and S202/S205 sites and contained high molecular weight aggregates comprising 3R and 4R tau. Moreover, a 10‐year‐old marmoset brain case study demonstrated a paired helical filament structure in the hippocampus and intracellular 3R and 4R tau accumulation.ConclusionsOur results reveal physiological and pathological 3R/4R tau distributions in the frontal and temporal cortex of the marmoset brain, confirming the presence of 3R tau and AD‐like pathological tau in aging marmosets. These data support the utility of common marmosets as appropriate models for studying AD and neurodegenerative disorders.

Unveiling the nongenomic actions of thyroid hormones in adult mammalian brain: The legacy of Mary B. Dratman.

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brain-region specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NO-dependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study.

Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.

Enhanced presynaptic mitochondrial energy production is required for memory formation

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer.

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

Abstract 6439: Actin remodeling, inhibitory ligand polarization and small vesicle recruitment hinder effective anti-tumor immunity by shaping the tumor cell side of the immune synapse

Abstract Cancer immune evasion is a major obstacle to effective anticancer immunotherapies. Recently, we unveiled a mechanism crucial in cancer escape from natural killer (NK)-mediated killing. The identified process is characterized by a rapid and massive accumulation of actin filaments at the cancer cell side of the immunological synapse (IS). We termed this process “actin response” (AR) and discovered that its inhibition is sufficient to restore cancer cell susceptibility to NK-mediated killing in vitro. Importantly, the AR is conserved across a wide range of malignancies, highlighting the broad translational potential of targeting this pathway. In this study, we investigated the molecular mechanisms underlying AR-mediated cancer cell immune evasion. Cancer-NK cell conjugates were analyzed by imaging flow cytometry (IFC) to investigate the polarization of inhibitory and activating ligands to the cancer cell side of the IS relative to the AR. We found that the AR is associated with and necessary for polarization of several inhibitory ligands. For instance, HLA-A,-B,-C molecules, which act as potent inhibitors of NK cells, were 2-fold increased at the IS of cancer cells with an AR when compared to cancer cells without an AR. Using confocal microscopy, we found that such polarization of inhibitory molecules is associated with the loss of MTOC and lytic granule polarization in conjugated human primary NK cells. In addition, correlative light and electron microscopy analysis revealed the presence of small vesicles (100 nm in size) and multivesicular bodies (MVBs) in the synaptic region of cancer cells with an AR. Quantification of vesicle markers at the IFC revealed a significant enrichment of CD63+/CD9+/CD81+ vesicles in the synaptic region of cancer cells with an AR. Interestingly, ultrastructure analysis showed that the AR is largely made of long actin-rich protrusions projecting into and altering the morphology of the synaptic cleft. Moreover, these protrusions were decorated with inhibitory molecules, such as PD-L1, and CD63. We identified a novel, highly conserved immune escape mechanism that exploits the fast remodeling of the actin cytoskeleton in cancer cells to induce the polarization of inhibitory ligands along with small tumor vesicles towards the IS. These findings suggest that actin remodeling in tumor cells at the IS actively promotes immune evasion. Further insights on the molecular and cellular mechanisms underpinning this pathway, such as the identification of selective targeting of linker proteins between the actin cytoskeleton and inhibitory ligands or small vesicles, represent a promising therapeutic strategy to improve the efficacy of anticancer immunotherapies. Nevertheless, additional research is needed in order to evaluate the translational potential of targeting the AR in a clinical setting. Citation Format: Andrea Michela Biolato, Liza Filali, Céline Hoffmann, Felix Kleine-Borgmann, Elena Ockfen, Max Krecke, Michel Mittelbronn, Clément Thomas. Actin remodeling, inhibitory ligand polarization and small vesicle recruitment hinder effective anti-tumor immunity by shaping the tumor cell side of the immune synapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6439.

Compartment specific mitochondrial dysfunction in Drosophila knock-in model of ALS reversed by altered gene expression of OXPHOS subunits and pro-fission factor Drp1

Amyotrophic Lateral Sclerosis (ALS) is a fatal multisystem neurodegenerative disease, characterized by a loss in motor function. ALS is genetically diverse, with mutations in genes ranging from those regulating RNA metabolism, like TAR DNA-binding protein (TDP-43) and Fused in sarcoma (FUS), to those that act to maintain cellular redox homeostasis, like superoxide dismutase 1 (SOD1). Although varied in genetic origin, pathogenic and clinical commonalities are clearly evident between cases of ALS. Defects in mitochondria is one such common pathology, thought to occur prior to, rather than as a consequence of symptom onset, making these organelles a promising therapeutic target for ALS, as well as other neurodegenerative diseases. Depending on the homeostatic needs of neurons throughout life, mitochondria are normally shuttled to different subcellular compartments to regulate metabolite and energy production, lipid metabolism, and buffer calcium. While originally considered a motor neuron disease due to the dramatic loss in motor function accompanied by motor neuron cell death in ALS patients, many studies have now implicated non-motor neurons and glial cells alike. Defects in non-motor neuron cell types often preceed motor neuron death suggesting their dysfunction may initiate and/or facilitate the decline in motor neuron health. Here, we investigate mitochondria in a Drosophila Sod1 knock-in model of ALS. In depth, in vivo, examination reveals mitochondrial dysfunction evident prior to onset of motor neuron degeneration. Genetically encoded redox biosensors identify a general disruption in the electron transport chain (ETC). Compartment specific abnormalities in mitochondrial morphology is observed in diseased sensory neurons, accompanied by no apparent defects in the axonal transport machinery, but instead an increase in mitophagy in synaptic regions. The decrease in networked mitochondria at the synapse is reversed upon downregulation of the pro-fission factor Drp1. Furthermore, altered expression of specific OXPHOS subunits reverses ALS-associated defects in mitochondrial morphology and function.

The influence of astrocytic leaflet motility on ionic signalling and homeostasis at active synapses

Astrocytes display a highly complex, spongiform morphology, with their fine terminal processes (leaflets) exercising dynamic degrees of synaptic coverage, from touching and surrounding the synapse to being retracted from the synaptic region. In this paper, a computational model is used to reveal the effect of the astrocyte-synapse spatial relationship on ionic homeostasis. Specifically, our model predicts that varying degrees of astrocyte leaflet coverage influences concentrations of K+, Na+ and Ca2+, and results show that leaflet motility strongly influences Ca2+ uptake, as well as glutamate and K+ to a lesser extent. Furthermore, this paper highlights that an astrocytic leaflet that is in proximity to the synaptic cleft loses the ability to form a Ca2+ microdomain, whereas when the leaflet is remote from the synaptic cleft, a Ca2+ microdomain can form. This may have implications for Ca2+-dependent leaflet motility.

Macrophages Promote Repair of Inner Hair Cell Ribbon Synapses following Noise-Induced Cochlear Synaptopathy.

Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX3CR1 GFP/+ mice of both sexes led to robust elimination of resident macrophages (∼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1 day (d) post noise exposure of 93 or 90 dB SPL for 2 hours, the degree of hearing loss and synapse loss were comparable in the presence and absence of macrophages. At 30 d after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages, such synaptic repair was significantly reduced. Remarkably, on cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response thresholds and reduced auditory brainstem response Peak 1 amplitudes showed limited recovery in the absence of macrophages but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remain to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy.SIGNIFICANCE STATEMENT The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost because of noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information, leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells, such as macrophages in synaptic repair, that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss, and associated perceptual anomalies.

In vivo synaptic density loss correlates with impaired functional and related structural connectivity in Alzheimer’s disease

Synapse loss has been considered as a major pathological change in Alzheimer’s disease (AD). It remains unclear about whether and how synapse loss relates to functional and structural connectivity dysfunction in AD. We measured synaptic vesicle glycoprotein 2 A (SV2A) binding using 18F-SynVesT-1 PET to evaluate synaptic alterations in 33 participants with AD, 31 with mild cognitive impairment (MCI), and 30 controls. We examined the correlation between synaptic density and cognitive function. Functional MRI was performed to analyze functional connectivity in lower synaptic density regions. We tracked the white matter tracts between impaired functional connectivity regions using Diffusion MRI. In AD group, lower synaptic density in bilateral cortex and hippocampus was found when compared with controls. The synaptic density changes in right insular cortex and bilateral caudal middle frontal gyrus (MFG) were correlated with cognitive decline. Among them, right MFG synaptic density was positively associated with right MFG - bilateral superior frontal gyrus (SFG) functional connectivity. AD had lower probability of tract (POT) between right MFG and SFG than controls, which was significantly associated with global cognition. These findings provide evidence supporting synapse loss contributes to functional and related structural connectivity alterations underlying cognitive impairment of AD.