Vápníková signalizace u gliových buněk v progresi Alzheimerovy choroby

Alzheimerova choroba je neudegenerativní onemocněmí postihující celou centrální nervovou soustavu včetně gliových buněk. Mechanismy tohoto onemocnění stále nejsou zcela objasněny, přesto současný výzkum naznačuje, že spolu se známými charakteristickými znaky Alzheimerovy choroby, jako je hromadění amyloidu β a hyperfosforylovaného tau, by důležitým rysem jak v neuronech, tak v gliových buňkách, především v astrocytech a mikrogliích, mohla být dysregulace vnitrobuněčné vápníkové homeostáze. Gliové buňky hrají důležitou roli jak ve zdravém mozku, tak během progrese Alzheimerovy choroby. Jejich hlavní funkce, jako například podpora neuronů a udržování synapsí, jsou během této nemoci narušeny. Současný výzkum naznačuje, že narušená vápníková signalizace gliových buněk vyvolaná během Alzheimerovy choroby, by eventuálně mohla podporovat nesprávnou činnost těchto buněk a zvýšit jejich zánětlivou reakci, tudíž ovlivňovat neurony a zůsobit poškození mozku. Je pravděpodobné, že probíhající zánětlivá reakce a zhoršená vápníková signalizace se navzájem ovlivňují a následně urychlují progresi Alzheimerovy choroby.Alzheimer's disease (AD) is a neurodegenerative disorder affecting the entire central nervous system including glial cells. The mechanisms of this disease are not yet entirely clear, although recent studies suggest that among the known hallmarks of AD, such as accumulation of amyloid β and hyperphosphorylated tau, dysregulation of intracellular calcium homeostasis is proposed to be a significant feature both in neurons and glial cells, namely astrocytes and microglia. Glial cells play an important role both in healthy brain and during AD progression. Their major functions, such as supporting neurons or maintaining synapses, are impaired during this disease. Recent findings suggest that aberrant glial calcium signaling activated during AD, could possibly promote the malfunction of these cells and increase their inflammatory response, thus affecting neurons and causing brain damage. It is likely, that the ongoing inflammation and the impaired calcium signaling affect one another, consequently enhancing the progression of AD.Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

Glutamátové receptory NG2 gliových buněk: genové profilování a funkční změny po ischemickém poškození mozku

Glutamát je hlavním excitačním neuropřenašečem v mozku savců a jeho přenos je zodpovědný za vyšší mozkové funkce, jako jsou učení, pamět či kognice. Účinek glutamátu je zprostředkován různými typy glutamátových receptorů, jejichž vlastnosti byly doposud studovány převážně v neuronech. Glutamátové receptory jsou ovšem exprimovány také v NG2 gliových buňkách, nicméně jejich role, jak ve zdravém tak i v poškozeném mozku, není zcela známá. Cílem této práce bylo objasnit složení a funkce těchto receptorů u NG2 gliových buněk za fyziologických podmínek a po fokální cerebrální ischemii. K tomuto účelu jsme použili transgenní myši, ve kterých jsou NG2 gliové buňky značeny pomocí fluorescenčního proteinu, což nám umožnilo jejich přesnou identifikaci. Analýza expresního profilu glutamátových receptorů v NG2 gliových buňkách byla provedena pomocí metody RT-qPCR na úrovni jedné buňky. Dále byla použita imunohistochemie a fluorescenční metoda zobrazování intracelulárních hladin vápenatých iontů (calcium imaging) pro detekci glutamátových receptorů na úrovni proteinu a charakterizaci jejich funkčních vlastností.Glutamate is the main excitatory neurotransmitter in the mammalian brain and its transmission is responsible for higher brain functions, such as learning, memory and cognition. Glutamate action is mediated by a variety of glutamate receptors, though their properties were until now studied predominantly in neurons. Glutamate receptors are expressed also in NG2-glia, however their role under physiological conditions as well as in pathological states of the central nervous system is not fully understood. The aim of this work is to elucidate the presence, composition and function of these receptors in NG2-glia under physiological conditions and following focal cerebral ischemia. For this purpose we used transgenic mice, in which NG2-glia are labeled by a fluorescent protein for their precise identification. To analyze the expression pattern of glutamate receptors in NG2-glia we employed single-cell RT-qPCR. Furthermore, we used calcium imaging to characterize their functional properties.Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

GDNF Increases Inhibitory Synaptic Drive on Principal Neurons in the Hippocampus via Activation of the Ret Pathway

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach

Glutamate receptors in NG2-glial cells: gene profiling and functional changes after ischemic brain injury

Glutamate is the main excitatory neurotransmitter in the mammalian brain and its transmission is responsible for higher brain functions, such as learning, memory and cognition. Glutamate action is mediated by a variety of glutamate receptors, though their properties were until now studied predominantly in neurons. Glutamate receptors are expressed also in NG2-glia, however their role under physiological conditions as well as in pathological states of the central nervous system is not fully understood. The aim of this work is to elucidate the presence, composition and function of these receptors in NG2-glia under physiological conditions and following focal cerebral ischemia. For this purpose we used transgenic mice, in which NG2-glia are labeled by a fluorescent protein for their precise identification. To analyze the expression pattern of glutamate receptors in NG2-glia we employed single-cell RT-qPCR. Furthermore, we used calcium imaging to characterize their functional properties

Calcium signalling in glial cells in progress of Alzheimer disease

Alzheimer's disease (AD) is a neurodegenerative disorder affecting the entire central nervous system including glial cells. The mechanisms of this disease are not yet entirely clear, although recent studies suggest that among the known hallmarks of AD, such as accumulation of amyloid β and hyperphosphorylated tau, dysregulation of intracellular calcium homeostasis is proposed to be a significant feature both in neurons and glial cells, namely astrocytes and microglia. Glial cells play an important role both in healthy brain and during AD progression. Their major functions, such as supporting neurons or maintaining synapses, are impaired during this disease. Recent findings suggest that aberrant glial calcium signaling activated during AD, could possibly promote the malfunction of these cells and increase their inflammatory response, thus affecting neurons and causing brain damage. It is likely, that the ongoing inflammation and the impaired calcium signaling affect one another, consequently enhancing the progression of AD

Human stem cell‐derived gabaergic interneurons establish efferent synapses onto host neurons in rat epileptic hippocampus and inhibit spontaneous recurrent seizures

Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacolog-ical treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ‐aminobutyric acid (GABA) could be used to counteract the imbalance between ex-citation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid‐induced status epilepticus. Using whole‐cell patch‐clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic inter-neurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof‐of‐concept that hESC‐derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons

Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks

Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons