Reversal of cell, circuit and seizure phenotypes in a mouse model of DNM1 epileptic encephalopathy.

Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene (DNM1) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.

Phosphatidylinositol 4-kinase IIα is a glycogen synthase kinase 3-regulated interaction hub for activity-dependent bulk endocytosis.

Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3ß (GSK3ß) activity. We reveal the GSK3ß substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3ß site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3ß-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Synaptophysin controls synaptobrevin-II retrieval via a cryptic C-terminal interaction site.

The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.

Metformin Accelerates Glycolytic Lactate Production in Cultured Primary Cerebellar Granule Neurons.

Metformin is the most frequently used drug for the treatment of type-II diabetes. As metformin has been reported to cross the blood-brain barrier, brain cells will encounter this drug. To test whether metformin may affect the metabolism of neurons, we exposed cultured rat cerebellar granule neurons to metformin. Treatment with metformin caused a time- and concentration-dependent increase in glycolytic lactate release from viable neurons as demonstrated by the three-to fivefold increase in extracellular lactate concentration determined after exposure to metformin. Half-maximal stimulation of lactate production was found after incubation of neurons for 4 h with around 2 mM or for 24 h with around 0.5 mM metformin. Neuronal cell viability was not affected by millimolar concentrations of metformin during acute incubations in the hour range nor during prolonged incubations, although alterations in cell morphology were observed during treatment with 10 mM metformin for days. The acute stimulation of neuronal lactate release by metformin was persistent upon removal of metformin from the medium and was not affected by the presence of modulators of adenosine monophosphate activated kinase activity. In contrast, rabeprazole, an inhibitor of the organic cation transporter 3, completely prevented metformin-mediated stimulation of neuronal lactate production. In summary, the data presented identify metformin as a potent stimulator of glycolytic lactate production in viable cultured neurons and suggest that organic cation transporter 3 mediates the uptake of metformin into neurons.

The malleable brain: plasticity of neural circuits and behavior - a review from students to students.

One of the most intriguing features of the brain is its ability to be malleable, allowing it to adapt continually to changes in the environment. Specific neuronal activity patterns drive long-lasting increases or decreases in the strength of synaptic connections, referred to as long-term potentiation and long-term depression, respectively. Such phenomena have been described in a variety of model organisms, which are used to study molecular, structural, and functional aspects of synaptic plasticity. This review originated from the first International Society for Neurochemistry (ISN) and Journal of Neurochemistry (JNC) Flagship School held in Alpbach, Austria (Sep 2016), and will use its curriculum and discussions as a framework to review some of the current knowledge in the field of synaptic plasticity. First, we describe the role of plasticity during development and the persistent changes of neural circuitry occurring when sensory input is altered during critical developmental stages. We then outline the signaling cascades resulting in the synthesis of new plasticity-related proteins, which ultimately enable sustained changes in synaptic strength. Going beyond the traditional understanding of synaptic plasticity conceptualized by long-term potentiation and long-term depression, we discuss system-wide modifications and recently unveiled homeostatic mechanisms, such as synaptic scaling. Finally, we describe the neural circuits and synaptic plasticity mechanisms driving associative memory and motor learning. Evidence summarized in this review provides a current view of synaptic plasticity in its various forms, offers new insights into the underlying mechanisms and behavioral relevance, and provides directions for future research in the field of synaptic plasticity. Read the Editorial Highlight for this article on page 788. Cover Image for this issue: doi: 10.1111/jnc.13815.

Metabolism of Mannose in Cultured Primary Rat Neurons.

Glucose is the main peripheral substrate for energy production in the brain. However, as other hexoses are present in blood and cerebrospinal fluid, we have investigated whether neurons have the potential to metabolize, in addition to glucose, also the hexoses mannose, fructose or galactose. Incubation of primary cerebellar granule neurons in the absence of glucose caused severe cell toxicity within 24 h, which could not be prevented by application of galactose or fructose, while the cells remained viable during incubation in the presence of either mannose or glucose. In addition, cultured neurons produced substantial and almost identical amounts of lactate after exposure to either glucose or mannose, while lactate production was low in the presence of fructose and hardly detectable during incubations without hexoses or with galactose as carbon source. Determination of the KM values of hexokinase in lysates of cultured neurons for the hexoses revealed values in the micromolar range for mannose (32 ± 2 µM) and glucose (59 ± 10 µM) and in the millimolar range for fructose (4.4 ± 2.3 mM), demonstrating that mannose is efficiently phosphorylated by neuronal hexokinase. Finally, cultured neurons contained reasonable specific activity of the enzyme phosphomannose isomerase, which is required for isomerization of the hexokinase product mannose-6-phosphate into the glycolysis intermediate fructose-6-phosphate. These data demonstrate that cultured cerebellar granule neurons have the potential and express the required enzymes to efficiently metabolize mannose, while galactose and fructose serve at best poorly as extracellular carbon sources for neurons.

The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U-13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc.

The Antidiabetic Drug Metformin Stimulates Glycolytic Lactate Production in Cultured Primary Rat Astrocytes.

Metformin is the most frequently used drug for the treatment of type 2 diabetes in humans. However, only little is known about effects of metformin on brain metabolism. To investigate potential metabolic consequences of an exposure of brain cells to metformin, we incubated rat astrocyte-rich primary cultures with this compound. Metformin in concentrations of up to 30 mM did not acutely compromise the viability of astrocytes, but caused a time- and concentration-dependent increase in cellular glucose consumption and lactate production. For acute incubations in the hour range, the presence of 10 mM metformin doubled the glycolytic flux, while already 1 mM metformin doubled glycolytic flux during incubation for 24 h. In addition to metformin, also other guanidino compounds increased astrocytic lactate production. After 4 h of incubation, half-maximal stimulation of glycolysis was observed for metformin, guanidine and phenformin at concentrations of around 3 mM, 3 mM and 30 µM, respectively. The acute stimulation of glycolytic lactate production by metformin was persistent after removal of extracellular metformin and was also observed, if glucose was absent from the incubation medium or replaced by other hexoses. The metformin-induced stimulation of glycolytic flux was not prevented by compound C, an inhibitor of AMP-dependent protein kinase, nor was it additive to the stimulation of glycolytic flux caused by respiratory chain inhibitors. These data demonstrate that the antidiabetic drug metformin has the potential to strongly activate glycolytic lactate production in brain astrocytes.

Intra- or extra-exosomal secretion of HDGF isoforms: the extraordinary function of the HDGF-A N-terminal peptide.

Hepatoma-derived growth factor (HDGF) is a protein with diverse intracellular functions. Moreover, after non-conventional secretion, extracellular HDGF is able to influence different signaling pathways, leading for example to induction of processes like epithelial-mesenchymal transition (EMT) and cell migration. Intriguingly, in recent proteome studies, HDGF was also found secreted by special microvesicles called exosomes. Recently, we demonstrated the existence of two new HDGF isoforms (B and C). These isoforms are involved in different cellular processes than HDGF-A. Along this line, in the present study we discovered that full length HDGF-A clearly is located inside of exosomes, whereas the isoforms HDGF-B and HDGF-C are found exclusively on the outer surface. Furthermore, while HDGF-B and HDGF-C seem to use exosomes mediated pathway exclusively, HDGF-A was found also as unbound protein in the conditioned media. The new finding of an intra- or extra-exosomal localisation of protein splice variants opens a fascinating new perspective concerning functional diversity of HDGF isoforms. Dysregulation of HDGF expression during cancer development and tumor progression is a commonly known fact. With our new findings, unraveling the potential functional impact according to physiological versus pathophysiologically altered levels and compositions of intra- and extra-exosomal HDGF has to be addressed in future studies.

The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23.

Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA) cycle, we incubated primary rat astrocyte cultures with [U-13C]glucose in the absence or the presence of 100 µM T23 for 2 h and analyzed the 13C metabolite pattern. These incubation conditions did not compromise cell viability and confirmed that the presence of T23 doubled glycolytic lactate production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of 13C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly demonstrate that, in addition to glycolysis, also the mitochondrial TCA cycle is strongly accelerated after exposure of astrocytes to T23, suggesting that a protein tyrosine kinase may be involved in the regulation of the TCA cycle in astrocytes.

The Protein Tyrosine Kinase Inhibitor Tyrphostin 23 Strongly Accelerates Glycolytic Lactate Production in Cultured Primary Astrocytes.

Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases. To investigate potential acute effects of T23 on the viability and the glucose metabolism of brain cells, we exposed cultured primary rat astrocytes to T23 for up to 4 h. While the viability and the morphology of the cultured astrocytes were not acutely affected by the presence of T23 in concentrations of up to 300 µM, this compound caused a rapid, time- and concentration-dependent increase in glucose consumption and lactate release. Maximal effects on glycolytic flux were found for incubations with 100 µM T23 for 2 h which doubled both glucose consumption and lactate production. The stimulation of glycolytic flux by T23 was reversible, completely abolished upon removal of the compound and not found in presence of other known inhibitors of endocytosis. Structurally related compounds such as tyrphostin 25 and catechol or modulators of AMP kinase activity did neither affect the basal nor the T23-stimulated lactate production by astrocytes. In contrast, the presence of the phosphatase inhibitor vanadate completely abolished the stimulation by T23 of astrocytic lactate production in a concentration-dependent manner. These data suggest that T23-sensitive phosphorylation/dephosphorylation events are involved in the regulation of astrocytic glycolysis.

Effects of Long-Term Rice Bran Extract Supplementation on Survival, Cognition and Brain Mitochondrial Function in Aged NMRI Mice.

Aging represents a major risk factor for the development of neurodegenerative diseases like Alzheimer's disease (AD). As mitochondrial dysfunction plays an important role in brain aging and occurs early in the development of AD, the prevention of mitochondrial dysfunction might help to slow brain aging and the development of neurodegenerative diseases. Rice bran extract (RBE) contains high concentrations of vitamin E congeners and γ-oryzanol. We have previously shown that RBE increased mitochondrial function and protected from mitochondrial dysfunction in vitro and in short-term in vivo feeding studies. To mimic the use of RBE as food additive, we have now investigated the effects of a long-term (6 months) feeding of RBE on survival, behavior and brain mitochondrial function in aged NMRI mice. RBE administration significantly increased survival and performance of aged NMRI mice in the passive avoidance and Y-maze test. Brain mitochondrial dysfunction found in aged mice was ameliorated after RBE administration. Furthermore, data from mRNA and protein expression studies revealed an up-regulation of mitochondrial proteins in RBE-fed mice, suggesting an increase in mitochondrial content which is mediated by a peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)-dependent mechanism. Our findings suggest that a long-term treatment with a nutraceutical containing RBE could be useful for slowing down brain aging and thereby delaying or even preventing AD.

Glutathione-Dependent Detoxification Processes in Astrocytes.

Astrocytes have a pivotal role in brain as partners of neurons in homeostatic and metabolic processes. Astrocytes also protect other types of brain cells against the toxicity of reactive oxygen species and are considered as first line of defence against the toxic potential of xenobiotics. A key component in many of the astrocytic detoxification processes is the tripeptide glutathione (GSH) which serves as electron donor in the GSH peroxidase-catalyzed reduction of peroxides. In addition, GSH is substrate in the detoxification of xenobiotics and endogenous compounds by GSH-S-transferases which generate GSH conjugates that are efficiently exported from the cells by multidrug resistance proteins. Moreover, GSH reacts with the reactive endogenous carbonyls methylglyoxal and formaldehyde to intermediates which are substrates of detoxifying enzymes. In this article we will review the current knowledge on the GSH metabolism of astrocytes with a special emphasis on GSH-dependent detoxification processes.

Arsenate stimulates glutathione export from viable cultured rat cerebellar granule neurons.

Arsenate is an environmental pollutant which contaminates the drinking water of millions of people worldwide. Numerous in vitro studies have investigated the toxicity of arsenate for a large number of different cell types. However, despite the known neurotoxic potential of arsenicals, little is known so far about the consequences of an exposure of neurons to arsenate. To investigate acute effects of arsenate on the viability and the glutathione (GSH) metabolism of neurons, we have exposed primary rat cerebellar granule neuron cultures to arsenate. Incubation of neurons for up to 6 h with arsenate in concentrations of up to 10 mM did not acutely compromise the cell viability, although the cells accumulated substantial amounts of arsenate. However, exposure to arsenate caused a time- and concentration-dependent increase in the export of GSH from viable neurons with significant effects observed for arsenate in concentrations above 0.3 mM. The arsenate-induced stimulation of GSH export was abolished upon removal of arsenate and completely prevented by MK571, an inhibitor of the multidrug resistance protein 1. These results demonstrate that arsenate is not acutely toxic to neurons but can affect the neuronal GSH metabolism by stimulating GSH export.