Selective dendritic susceptibility to bioenergetic, excitotoxic and redox perturbations in cortical neurons

AbstractNeurodegenerative and neurological disorders are often characterised by pathological changes to dendrites, in advance of neuronal death. Oxidative stress, energy deficits and excitotoxicity are implicated in many such disorders, suggesting a potential vulnerability of dendrites to these situations. Here we have studied dendritic vs. somatic responses of primary cortical neurons to these types of challenges in real-time.Using a genetically encoded indicator of intracellular redox potential (Grx1-roGFP2) we found that, compared to the soma, dendritic regions exhibited more dramatic fluctuations in redox potential in response to sub-lethal ROS exposure, and existed in a basally more oxidised state. We also studied the responses of dendritic and somatic regions to excitotoxic NMDA receptor activity. Both dendritic and somatic regions experienced similar increases in cytoplasmic Ca2+. Interestingly, while mitochondrial Ca2+ uptake and initial mitochondrial depolarisation were similar in both regions, secondary delayed mitochondrial depolarisation was far weaker in dendrites, potentially as a result of less NADH depletion. Despite this, ATP levels were found to fall faster in dendritic regions. Finally we studied the responses of dendritic and somatic regions to energetically demanding action potential burst activity. Burst activity triggered PDH dephosphorylation, increases in oxygen consumption and cellular NADH:NAD ratio. Compared to somatic regions, dendritic regions exhibited a smaller degree of mitochondrial Ca2+ uptake, lower fold-induction of NADH and larger reduction in ATP levels. Collectively, these data reveal that dendritic regions of primary neurons are vulnerable to greater energetic and redox fluctuations than the cell body, which may contribute to disease-associated dendritic damage. This article is part of a Special Issue entitled: 13th European Symposium on Calcium

Investigation into the destructive and adaptive responses of neural cells to stress

Homeostasis within the neuro-glial unit is essential to the longevity of neurons. Conversely, loss of homeostasis, particularly of Ca2+ levels, of redox balance and of ATP, contribute to neuronal loss and dysfunction in many neurodegenerative and neurological disorders. This thesis is centred on better understanding the vulnerability of neurons to stress, as well as adaptive responses to these stresses. Since neurodegenerative conditions associated with Ca2+, redox and bioenergetic dyshomeostasis are often characterised by early dendritic pathology, I first studied dendritic vs. somatic responses of primary cortical neurons to these types of challenges in real-time. Using a wide range of genetically-encoded probes to measure Ca2+, ATP, NADH, glutathione and glutamate, I show that dendrites are selectively vulnerable to oxidative stress, excitotoxicity as well as to metabolic demand induced by action potential (AP) burst activity. However, I provide evidence that neurons undergoing energetically demanding AP burst activity can adjust their metabolic output by increasing mitochondrial NADH production in a manner dependent on the mitochondrial calcium uniporter (MCU), as well as increase their capacity to buffer their intracellular redox balance. Finally, I have studied transcriptional programs in astrocytes triggered by neurons and neuronal activity to better understand adaptive signaling between different cell types in the neuro-glial unit. I developed a novel system combining neurons and astrocytes from closely-related species, followed by RNA-seq and in silico read sorting. I uncovered a program of neuron-induced astrocytic gene expression which drives and maintains astrocytic maturity and neurotransmitter uptake function. In addition I identified a novel form of synapse-to-nucleus signaling, mediated by glutamatergic activity and acutely regulating diverse astrocytic genes involved in astrocyte-neuron metabolic coupling. Of note, neuronal activity co-ordinately induced astrocytic genes involved in astrocyte-to-neuron thyroid hormone signaling, extracellular antioxidant defences, and the astrocyte-neuron lactate shuttle, suggesting that this non cell-autonomous signaling may form part of the homeostatic machinery within the neuro-glial unit

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms

Microglial INPP5D limits plaque formation and glial reactivity in the PSAPP mouse model of Alzheimers disease.

INTRODUCTION: The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimers disease (LOAD). METHODS: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL /PSEN1Δexon9 (PSAPP) mice, we injected 3-month-old Inpp5dfl/fl /Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl /Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. RESULTS: At age 6 months, we found that the percent area of 6E10+ deposits and plaque-associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown. DISCUSSION: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. HIGHLIGHTS: Inpp5d knockdown increases plaque burden and plaque-associated microglia number. Spatial transcriptomics identifies an expanded plaque-specific gene expression profile. Plaque-induced gene expression is altered by Inpp5d knockdown in microglia. Our plaque-associated gene signature overlaps with human Alzheimers disease gene networks

Ca²⁺ influx into cortical neurons modifies the expression of many MCRGs.

<p><b>A)</b> Neurons were treated ± KCl/FPL64176/MK-801 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148164#sec008" target="_blank">methods</a>) for 24h, after which MCRG mRNA expression was analysed by qRT-PCR, normalised to Gapdh, and the % change relative to untreated control cells calculated. *p values (left to right) are 0.0003, 0.029, 0.016, 0.047, 0.0002, 0.007, 0.001, 0.042; alpha = 0.05, Student's one-sample t-test (n = 4 except Mcu, Micu1 (n = 8), Mcub. Micu2 (n = 5) and Micu3 (n = 6)). <b>B)</b> Neurons were treated as in (A) and RNA-seq analysis of transcriptome changes performed, with MCRG fold changes shown here. * Exact p values (left to right) are 0.002, 0.025,0.017, 0.038, 0.008, 0.028, 6.4E-05, 0.003; alpha = 0.05; Student's one-sample t-test (n = 3). <b>C)</b> KCl/FPL-induced changes in expression of a selection of nuclear encoded mitochondrial proteins (components of ETC complexes II-V), analysed by RNA-seq (n = 3).</p

RNA-seq read density coverage of MCRGs in astrocyte-free cortical neurons.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148164#sec008" target="_blank">methods</a> for experimental details. n = 3 biological replicates.</p

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca²⁺ Signaling - Table 1

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca²⁺ Signaling - Table