Biology subjects

Denes, A.

Publications and source records attributed to Denes, A..

14 recordsLinked to original sources

bioRxiv2025

Stress-induced switch in small extracellular vesicle secretion: from constitutive 'torn bag mechanism' to exocytosis

The biogenesis of small extracellular vesicles (sEVs) is only partially understood. Our recent findings provide evidence that a newly described sEV secretion pathway, the amphiectosome release and the "torn bag mechanism", is present in all tested cell lines and in mouse liver and kidney. Surprisingly, in in situ fixed steady-state cells, transmission electron microscopy did not reveal the classical exosome secretion route, the sEV release via exocytosis of multivesicular endosomes (MVEs). In the current study, we investigated which parameters influence the activation of the two distinct sEV release mechanisms. Our results show that under stress conditions (such as Ca{superscript 2} ionophore-induced membrane stress or metabolic stress-induced by serum starvation), exocytosis of MVEs is activated, while this process is absent in steady-state conditions. By silencing ATG5 (a key regulator of autophagy) and RAB27a (essential small GTPase for MVE exocytosis), we selectively modulated these two mechanisms. Amphiectosome release depended on both autophagy and ATG5, while exocytosis of MVE was autophagy-independent but RAB27a-dependent. Our findings suggest that sEV release via the "torn bag mechanism" is a general and essential secretion pathway in non-stressed, steady-state mammalian cells, while stress conditions induce the sEV release via MVE exocytosis.

cell biology↗

bioRxiv2024

Characterization of AZ12464237 as a high affinity, non-nucleotide antagonist radioligand for the P2Y12 receptor

The purinergic receptor P2Y12 (P2Y12R) is a well-recognized target for anti-thrombotic agents. This receptor is also expressed in microglia, where it plays a key role in neuroinflammation and microglia activation. To investigate P2Y12R-mediated actions in the central nervous system (CNS), the development of novel brain-penetrant ligands is essential, along with further in vitro studies. A radiolabeled, easily accessible tool compound would significantly advance such drug discovery efforts. Herein, we describe the 3H-labeling of a non-nucleotide P2Y12R antagonist AZ12464237, and its in vitro binding properties to the receptor in membrane preparations form transfected cells, as well as on mouse brain tissues. The radioligand shows high affinity toward both the human and rat P2Y12R in transfected cells, with Kd values of 3.12 {+/-} 0.70 nM (human) and 16.6 {+/-} 3.4 nM (rat), as determined by saturation binding studies. The binding kinetics of [3H]AZ12464237 are rapid with a short target residence time ([~]1 min). We further confirmed the selectivity of the radioligand by performing competitive displacement studies, in which reported P2Y12R ligands and other P2Y receptors ligands were tested for binding against [3H]AZ12464237. Additionally, the radioligand proved useful for in vitro autoradiography studies on mouse brain tissues, although a small amount of off-target binding was observed in P2Y12R knock-out mice. This could be traced to glycogen synthase kinase 3 . Considering the growing interest in P2Y12R as a biomarker for microglia activation, [3H]AZ12464237 represents a promising tool for in vitro studies, including screening assays aimed at identifying novel P2Y12R ligands for CNS applications.

pharmacology and toxicology↗

bioRxiv2024

Cellular and molecular events organizing the assembly of tertiary lymphoid structures in glioblastoma

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates associated with improved prognosis in numerous tumors. To date, their prognostic value in central nervous system cancers and the events underlying their formation remain unclear. Here, we find that TLS correlate with improved survival in glioblastoma patients. Furthermore, combining spatial transcriptomics of human tissues with longitudinal studies in murine glioma, we establish that the assembly of T cell-rich clusters is a prerequisite for the development of canonical TLS, and show that CD4 T cells play a central role in this process. Indeed, we provide evidence that IL7R+CCR7+ Th1 lymphocytes with lymphoid tissue-inducing potential are recruited to TLS nucleation sites, where they can initiate TLS assembly by expressing lymphotoxin {beta}. Our work defines TLS as a potential prognostic factor in glioblastoma and identifies cellular and molecular mechanisms of TLS formation. These findings have broader implications for the development of TLS-inducing therapies for cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/601824v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@78be05org.highwire.dtl.DTLVardef@1037706org.highwire.dtl.DTLVardef@7b64e1org.highwire.dtl.DTLVardef@11bfd5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Multi-night EEG reveals positive association between sleep efficiency and hippocampal subfield volumes in healthy aging

Age-related atrophy of the human hippocampus and the enthorinal cortex starts accelerating at around age 60. Due to the contributions of these regions to many cognitive functions seamlessly used in everyday life, this can heavily impact the lives of elderly people. The hippocampus is not a unitary structure and mechanisms of its age-related decline appear to differentially affect its subfields. Human and animal studies have suggested that altered sleep is associated with hippocampal atrophy. Yet, we know little about subfield specific effects of altered sleep in healthy aging and their effect on cognition. Here, in a sample of 118 older adults (Mage = 63.25 years), we examined the association between highly reliable hippocampal subfield volumetry, sleep measures derived from multi-night recordings of portable electroencephalography and episodic memory. Objective sleep efficiency - but not self-report measures of sleep - was associated with entorhinal cortex volume when controlling for age. Age-related differences in subfield volumes were associated with objective sleep efficiency, but not with self-report measures of sleep. Moreover, older adults characterized by a common multivariate pattern of subfield volumes that contributed to positive sleep- subfield volume associations, showed lower rates of forgetting. Our results showcase the benefit of objective sleep measures in identifying potential contributors of age-related differences in brain-behavior couplings.

neuroscience↗

bioRxiv2023

Infection-induced vascular inflammation in COVID-19 links focal microglial dysfunction with neuropathologies through IL-1/IL-6-related systemic inflammatory states

COVID-19 is associated with diverse neurological abnormalities, which predict poor outcome in patients. However, the mechanisms whereby infection-induced inflammation could affect complex neuropathologies in COVID-19 are unclear. We hypothesized that microglia, the resident immune cells of brain, are centrally involved in this process. To study this, we developed an autopsy platform allowing the integration of molecular anatomy-, protein- and mRNA data sets in post-mortem mirror blocks of brain and peripheral organ samples from COVID-19 cases. Nanoscale microscopy, single-cell RNA sequencing and analysis of inflammatory and metabolic signatures revealed distinct mechanisms of microglial dysfunction associated with cerebral SARS-CoV-2 infection. We observed focal loss of microglial P2Y12R at sites of virus-associated vascular inflammation together with dysregulated microglia-vascular-astrocyte interactions, Cx3Cr1-fractalkine axis deficits and mitochondrial failure in severely affected medullary autonomic nuclei and other brain areas. Microglial dysfunction occurs at sites of excessive synapse- and myelin phagocytosis and loss of glutamatergic terminals. While central and systemic viral load is strongly linked in individual patients, the regionally heterogenous microglial reactivity in the brain correlated with the extent of central and systemic inflammation related to IL-1 / IL-6 via virus-sensing pattern recognition receptors (PRRs) and inflammasome activation pathways. Thus, SARS-CoV-2-induced central and systemic inflammation might lead to a primarily glio-vascular failure in the brain, which could be a common contributor to diverse COVID-19-related neuropathologies.

immunology↗

bioRxiv2022

Rapid microglial phenotype changes modulate neuronal networks and sharp wave-ripple activity in acute slice preparations

Acute brain slices represent a "workhorse" model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue injury, it is unclear how microglia, the main immune cells and damage sensors of the CNS shape tissue integrity ex vivo. To this end, we have studied the mechanisms of microglial phenotype changes and contribution to neuronal network organisation and functioning in acute brain slices. Using a novel ATP- reporter mouse line and microglia reporter mice, we show that acute slice preparation induces rapid, P2Y12 receptor (P2Y12R) dependent dislocation and migration of microglia, paralleled with marked morphological transformations driven by early ATP surges and subsequent ATP flashes. Gradual depolarization of microglia is associated with the downregulation of purinergic P2Y12R and time-dependent changes of microglia-neuron interactions, paralleled by altered numbers of excitatory and inhibitory synapses. Importantly, functional microglia not only modulate synapse sprouting, but the absence of microglia or microglial P2Y12R markedly diminishes the incidence, amplitude, and frequency of sharp wave-ripple activity in hippocampal slices. Collectively, our data suggest that microglia are inherent modulators of complex neuronal networks, and their specific actions are indispensable to maintain neuronal network integrity and activity ex vivo. These findings could facilitate new lines of research resulting in improved ex vivo methodologies and a better understanding of microglia-neuron interactions both in physiological and pathological conditions.

neuroscience↗

bioRxiv2021

Somatic junctions connect microglia and developing neurons

Microglia are the resident immune cells of the brain with multiple homeostatic and regulatory roles. Emerging evidence also highlights the fundamental transformative role of microglia in brain development. While tightly controlled, bi-directional communication between microglia and neuronal progenitors or immature neurons has been postulated, the main sites of interaction and the underlying mechanisms remain elusive. By using correlated light and electron microscopy together with super-resolution imaging, here we provide evidence that microglial processes form specialized nanoscale contacts with the cell bodies of developing and immature neurons throughout embryonic, early postnatal and adult neurogenesis. These early developmental contacts are highly reminiscent to somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. We propose that early developmental formation of somatic purinergic junctions represents an ideal interface for microglia to monitor the status of developing neurons and to direct prenatal, early postnatal and adult neurogenesis.

neuroscience↗

bioRxiv2021

Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

Microglia, the main immunocompetent cells of the brain regulate neuronal function in health and disease, but their contribution to cerebral blood flow (CBF) remained elusive. Here we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. We show that microglia establish direct purinergic contacts with cells in the neurovascular unit that shape cerebral perfusion in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in the barrel cortex after whisker stimulation. We also reveal that hypercapnia, which is associated with acidification, induces microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Furthermore, the absence or dysfunction of microglia markedly impairs adaptation to hypoperfusion via P2Y12R after transient unilateral common carotid artery occlusion, which is also influenced by CX3CR1-mediated actions. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation with broad implications for common neurological diseases.

neuroscience↗

bioRxiv2021

Central inhibition of P2Y12R differentially regulates survival and neuronal loss in MPTP-induced Parkinsonism in mice

Parkinsons disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is exclusively expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that while pharmacological or genetic targeting of P2Y12R previous to disease onset augments acute mortality, these interventions protect against neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulate ROCK and p38 MAPK activity and control cytokine production. Understanding protective and detrimental P2Y12 receptor-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD.

neuroscience↗

bioRxiv2021

Microglial NKCC1 shapes microglial phenotype, cerebral inflammatory responses and brain injury

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to osmotic stress in a cell-autonomous manner via regulating membrane potential and chloride fluxes. In addition, microglial NKCC1 deficiency results in increased expression of the D subunit of volume regulated anion channel (VRAC), NLRP3 inflammasome priming and production of interleukin-1{beta} (IL-1{beta}), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels, whereas systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

neuroscience↗

bioRxiv2020

Global analysis of a cancer model with drug resistance due to Lamarckian induction and microvesicle transfer

Development of resistance to chemotherapy in cancer patients strongly effects the outcome of the treatment. Due to chemotherapeutic agents, resistance can emerge by Darwinian evolution. Besides this, acquired drug resistance may arise via changes in gene expression. A recent discovery in cancer research uncovered a third possibility, indicating that this phenotype conversion can occur through the transfer of microvesicles from resistant to sensitive cells, a mechanism resembling the spread of an infectious agent. We present a model describing the evolution of sensitive and resistant tumour cells considering Darwinian selection, Lamarckian induction and microvesicle transfer. We identify three threshold parameters which determine the existence and stability of the three possible equilibria. Using a simple Dulac function, we give a complete description of the dynamics of the model depending on the three threshold parameters. We demonstrate the possible effects of increasing drug concentration, and characterize the possible bifurcation sequences. Our results show that the presence of microvesicle transfer cannot ruin a therapy that otherwise leads to extinction, however it may doom a partially successful therapy to failure.

cancer biology↗

bioRxiv2019

Microglia monitor and protect neuronal function via specialized somatic purinergic junctions

Microglia are the main immune cells in the brain with emerging roles in brain homeostasis and neurological diseases, while mechanisms underlying microglia-neuron communication remain elusive. Here, we identify a novel site of interaction between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions possess specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria is linked with microglial junction formation, which is rapidly induced in response to neuronal activation and blocked by inhibition of P2Y12-receptors (P2Y12R). Brain injury-induced changes at somatic junctions trigger P2Y12R-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Collectively, our results suggest that microglial processes at these junctions are in ideal position to monitor and protect neuronal functions in both the healthy and injured brain.\n\nOne-sentence summaryNeuronal cell bodies possess specialized, pre-formed sites, through which microglia monitor their status and exert neuroprotection.

neuroscience↗

bioRxiv2017

Mitochondrial Ultrastructure Is Coupled to Synaptic Performance at Axonal Release Sites

VISUAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY\n\nSIGNIFICANCE STATEMENTNeuronal networks are highly dynamic structures: cellular morphology and synaptic strength is changing as information is processed and stored. This means that the demand for mitochondrial supply is changing in space and time in neurons. Since the architecture ultimately determines the output performance of these organelles, we hypothesized that the ultrastructure of axonal mitochondria could well be a major parameter of adjusting their performance to the actual energetic needs of the synapse. Our results - describing a cell-type independent and synapse-specific correlation between mitochondrial ultrastructure, mitochondrial molecular fingerprints and synaptic performance - highlight the importance of activity-dependent ultrastructural plasticity of these organelles in neurons, and suggest an even more prominent role for mitochondria in neuroplasticity, than it was thought previously.\n\nABSTRACTMitochondrial function in neurons is tightly linked with metabolic and signalling mechanisms that ultimately determine neuronal performance. The subcellular distribution of these organelles is dynamically regulated as they are directed to axonal release sites on demand, but whether mitochondrial internal ultrastructure and molecular properties would reflect the actual performance requirements in a synapse-specific manner, remains to be established. Here, we examined performance-determining ultrastructural features of presynaptic mitochondria in GABAergic and glutamatergic axons of mice and human. Using electron-tomography and super-resolution microscopy we found, that these features were coupled to synaptic strength: mitochondria in boutons with high synaptic activity exhibited an ultrastructure optimized for high rate metabolism and contained higher levels of the respiratory chain protein cytochrome-c than mitochondria in boutons with lower activity. The strong, cell-type independent correlation between mitochondrial ultrastructure, molecular fingerprints and synaptic performance suggests that changes in synaptic activity could trigger ultrastructural plasticity of presynaptic mitochondria, likely to adjust their performance to the actual metabolic demand.

neuroscience↗

bioRxiv2017

Synaptic co-transmission of acetylcholine and GABA regulates hippocampal states

The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter, acetylcholine. Yet, using immune-electron tomographic, molecular anatomical, optogenetic and physiological techniques, we find that mouse hippocampal cholinergic terminals invariably establish synapses and their vesicles dock at synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes fast composite postsynaptic potentials, which are mutually cross-regulated by presynaptic auto-receptors and display different short-term plasticity. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. The synaptic nature of the forebrain cholinergic system with differentially regulated, fast, GABAergic and cholinergic co-transmission suggests a hitherto unrecognized level of synaptic control over cortical states. This novel model of hippocampal cholinergic neurotransmission could form the basis for alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders.\n\nSupplementary materials are attached after the main text.

neuroscience↗