Dynamics In Neurons Research Articles

Dynamics of elongation factor 2 kinase regulation in cortical neurons in response to synaptic activity.

The rapid regulation of cell signaling in response to calcium in neurons is essential for real-time processing of large amounts of information in the brain. A vital regulatory component, and one of the most energy-intensive biochemical processes in cells, is the elongation phase of mRNA translation, which is controlled by the Ca(2+)/CaM-dependent elongation factor 2 kinase (eEF2K). However, little is known about the dynamics of eEF2K regulation in neurons despite its established role in learning and synaptic plasticity. To explore eEF2K dynamics in depth, we stimulated synaptic activity in mouse primary cortical neurons. We find that synaptic activity results in a rapid, but transient, increase in eEF2K activity that is regulated by a combination of AMPA and NMDA-type glutamate receptors and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways. We then used computational modeling to test the hypothesis that considering Ca(2+)-coordinated MEK/ERK, mTORC1, and eEF2k activation is sufficient to describe the observed eEF2K dynamics. Although such a model could partially fit the empirical findings, it also suggested that a crucial positive regulator of eEF2K was also necessary. Through additional modeling and empirical evidence, we demonstrate that AMP kinase (AMPK) is also an important regulator of synaptic activity-driven eEF2K dynamics in neurons. Our combined modeling and experimental findings provide the first evidence that it is necessary to consider the combined interactions of Ca(2+) with MEK/ERK, mTORC1, and AMPK to adequately explain eEF2K regulation in neurons.

Calcium-dependent inactivation of calcium channels in the medial striatum increases at eye opening.

Influx of calcium through voltage-gated calcium channels (VGCCs) is essential for striatal function and plasticity. VGCCs expressed in striatal neurons have varying kinetics, voltage dependences, and densities resulting in heterogeneous subcellular calcium dynamics. One factor that determines the calcium dynamics in striatal medium spiny neurons is inactivation of VGCCs. Aside from voltage-dependent inactivation, VGCCs undergo calcium-dependent inactivation (CDI): inactivating in response to an influx of calcium. CDI is a negative feedback control mechanism; however, its contribution to striatal neuron function is unknown. Furthermore, although the density of VGCC expression changes with development, it is unclear whether CDI changes with development. Because calcium influx through L-type calcium channels is required for striatal synaptic depression, a change in CDI could contribute to age-dependent changes in striatal synaptic plasticity. Here we use whole cell voltage clamp to characterize CDI over developmental stages and across striatal regions. We find that CDI increases at the age of eye opening in the medial striatum but not the lateral striatum. The developmental increase in CDI mostly involves L-type channels, although calcium influx through non-L-type channels contributes to the CDI in both age groups. Agents that enhance protein kinase A (PKA) phosphorylation of calcium channels reduce the magnitude of CDI after eye opening, suggesting that the developmental increase in CDI may be related to a reduction in the phosphorylation state of the L-type calcium channel. These results are the first to show that modifications in striatal neuron properties correlate with changes to sensory input.

Mathematical model to study the nonlinear influence of Dyrk1A in actin polymerization

Event Abstract Back to Event Mathematical model to study the nonlinear influence of Dyrk1A in actin polymerization German A. Patterson1* and Mara Dierssen1 1 Center for Genomic Regulation, Spain Dyrk1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. It is believed that Dyrk1A plays a significant role in signaling pathways regulating cell proliferation and may be involved in neuron development. On the one hand, it has been reported that overexpression of Dyrk1A is sufficient to produce the dendritic alterations, in particular, it was shown that changes in Dyrk1A gene dosage strongly alter the dendritic arborization processes characterized by a reduction of neurite outgrowth [Martinez de Legran et al., 2012]. On the other hand, it was found that cells in Dyrk1A+/- mouse, a model lacking one copy of Dyrk1A, were less branched and less spinous than WT. This results suggest that Dyrk1A is affecting cellular pathways involved in neural development in a way that dosage changes lead to similar alteration regardless the direction of this change [Benavides-Piccione et al., 2005]. Here, we present a simple mathematical model that may explain both alterations observed in Dyrk1A+/- and Ts65Dn. It is based on a model proposed by Park et al., which describes a regulatory mechanism for Neural Wiskott–Aldrich syndrome protein (N-WASP) activity through Dyrk1A phosphorylation and how it regulates the actin filament assembly [Park et al., 2011]. In this work, we present a full characterization of the behavior of the system as a function of different set of parameters. We study how Dyrk1A may affect the dynamics of the system and how Dyrk1A dosage may regulate the actin polymerization. We believe that these findings may shed same light on how Dyrk1A affects cellular processes such as morphogenesis and neuronal differentiation. References R. Benavides-Piccione, M. Dierssen, I. Ballesteros-Yáñez, M. Martínez de Lagrán, M.L. Arbonés, V. Fotaki, J. DeFelipe, G.N. Elston, Alterations in the phenotype of neocortical pyramidal cells in the Dyrk1A+/− mouse, Neurobiology of Disease, Volume 20, Issue 1, October 2005, Pages 115-122, ISSN 0969-9961, doi:10.1016/j.nbd.2005.02.004. M. Martinez de Lagran, R. Benavides-Piccione, I. Ballesteros-Yañez, M. Calvo, M. Morales, C. Fillat, J. DeFelipe, G. J. A. Ramakers, and M. Dierssen, Dyrk1A Influences Neuronal Morphogenesis Through Regulation of Cytoskeletal Dynamics in Mammalian Cortical Neurons, Cereb. Cortex (2012) 22 (12): 2867-2877, doi:10.1093/cercor/bhr362 Joongkyu Park, Jee Young Sung, Joohyun Park, Woo-Joo Song, Sunghoe Chang, and Kwang Chul Chung, Dyrk1A negatively regulates the actin cytoskeleton through threonine phosphorylation of N-WASP, J Cell Sci 2012 125:67-80, doi:10.1242/jcs.086124 Keywords: Cellular Pathway Analysis and Simulation, Morphogenesis, actin polymerization, DYRK1A, NWASP Conference: B·DEBATE | A Dialogue with the Cerebral Cortex: Cortical Function and Interfacing (Workshop), Barcelona, Spain, 29 Apr - 30 Apr, 2015. Presentation Type: Poster Presentation Topic: SESSION 1: Cortical Function and Computations Citation: Patterson GA and Dierssen M (2015). Mathematical model to study the nonlinear influence of Dyrk1A in actin polymerization. Front. Syst. Neurosci. Conference Abstract: B·DEBATE | A Dialogue with the Cerebral Cortex: Cortical Function and Interfacing (Workshop). doi: 10.3389/conf.fnsys.2015.06.00006 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Apr 2015; Published Online: 06 May 2015. * Correspondence: Dr. German A Patterson, Center for Genomic Regulation, Barcelona, Spain, german.patterson@crg.eu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers German A Patterson Mara Dierssen Google German A Patterson Mara Dierssen Google Scholar German A Patterson Mara Dierssen PubMed German A Patterson Mara Dierssen Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Visualization of cyclic nucleotide dynamics in neurons.

The second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks.

Dynamic divisive normalization predicts time-varying value coding in decision-related circuits.

Normalization is a widespread neural computation, mediating divisive gain control in sensory processing and implementing a context-dependent value code in decision-related frontal and parietal cortices. Although decision-making is a dynamic process with complex temporal characteristics, most models of normalization are time-independent and little is known about the dynamic interaction of normalization and choice. Here, we show that a simple differential equation model of normalization explains the characteristic phasic-sustained pattern of cortical decision activity and predicts specific normalization dynamics: value coding during initial transients, time-varying value modulation, and delayed onset of contextual information. Empirically, we observe these predicted dynamics in saccade-related neurons in monkey lateral intraparietal cortex. Furthermore, such models naturally incorporate a time-weighted average of past activity, implementing an intrinsic reference-dependence in value coding. These results suggest that a single network mechanism can explain both transient and sustained decision activity, emphasizing the importance of a dynamic view of normalization in neural coding.

An assay to image neuronal microtubule dynamics in mice.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [13C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [13C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.

Multifractal analysis of information processing in hippocampal neural ensembles during working memory under Δ9-tetrahydrocannabinol administration

BackgroundMultifractal analysis quantifies the time-scale-invariant properties in data by describing the structure of variability over time. By applying this analysis to hippocampal interspike interval sequences recorded during performance of a working memory task, a measure of long-range temporal correlations and multifractal dynamics can reveal single neuron correlates of information processing. New methodWavelet leaders-based multifractal analysis (WLMA) was applied to hippocampal interspike intervals recorded during a working memory task. WLMA can be used to identify neurons likely to exhibit information processing relevant to operation of brain–computer interfaces and nonlinear neuronal models. ResultsNeurons involved in memory processing (“Functional Cell Types” or FCTs) showed a greater degree of multifractal firing properties than neurons without task-relevant firing characteristics. In addition, previously unidentified FCTs were revealed because multifractal analysis suggested further functional classification. The cannabinoid type-1 receptor (CB1R) partial agonist, tetrahydrocannabinol (THC), selectively reduced multifractal dynamics in FCT neurons compared to non-FCT neurons. Comparison with existing methodsWLMA is an objective tool for quantifying the memory-correlated complexity represented by FCTs that reveals additional information compared to classification of FCTs using traditional z-scores to identify neuronal correlates of behavioral events. Conclusionz-Score-based FCT classification provides limited information about the dynamical range of neuronal activity characterized by WLMA. Increased complexity, as measured with multifractal analysis, may be a marker of functional involvement in memory processing. The level of multifractal attributes can be used to differentially emphasize neural signals to improve computational models and algorithms underlying brain–computer interfaces.

Glucose Regulates Mitochondrial Motility via Milton Modification by O-GlcNAc Transferase

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. Here, we report a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. We establish the mitochondrial motor-adaptor protein Milton as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that the GlcNAcylation state of Milton is altered by extracellular glucose and that OGT alters mitochondrial motility in vivo. Our findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.

Comparison of Orientational Tuning and Its Dynamics in Neurons in Different Functional Domains of the Primary Visual Cortex

We report here studies of orientational tuning and its dynamics during generation of responses in neurons located in orientation columns and the centers of orientation pinwheels in the primary visual cortex of the cat. Intrinsic signal optical mapping was used to define the position of the working module on functional and vascular maps of the cortical surface. Both maps were used for targeted implantation of microelectrodes. The dynamics of orientational tuning were studied using a time slice method. Neurons whose preferred orientations were stable during response generation were observed in pinwheel centers and in orientation columns, accounting for 14.5% of cells, along with neurons in which the preferred orientation sequentially shifted by an average of 102 ± 5° (40.8%) and neurons with mixed-type dynamics (44.7%). Neurons with stable tuning in pinwheel centers differed from neurons with unstable tuning by producing greater responses to the preferred orientation, while those in orientation columns were characterized by later onset of responses to all orientations, including the preferred and non-preferred orientations. The orientational qualities of detection in stable neurons in these functional modules were better than those of cells with unstable tuning. Comparison of neurons with the same type of preferred orientation dynamics but different locations showed that stable neurons in pinwheel centers had shorter latent periods and larger responses than neurons in orientation columns. The width and selectivity of orientation tuning in these cells showed no differences. In unstable neurons, the characteristics of orientation tuning and responses to the preferred orientation were independent of location and were essentially identical. The functional significance of neurons with stable and unstable tuning during response generation in pinwheel centers and orientation columns in the primary visual cortex is discussed

Ca2 + regulation of mitochondrial function in neurons

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca2+ signal or to Ca2+ itself. [Na+]i, [Ca2+]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca2+ clearly showed that Ca2+-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca2+ may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca2+ entry in mitochondria through the calcium uniporter (MCU). Two Ca2+-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate–glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate–aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca2+ of 300nM and 3.4μM, respectively. The lack of SCaMC-3 results in a smaller Ca2+-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca2+-dependent responses to all workloads: a reduction of about 65–70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K+-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K+-depolarization, this occurs in spite of the presence of large [Ca2+]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca2+-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca2+ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.

The Armc10/SVH gene: genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation.

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1–6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents Aβ-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against Aβ-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

TGFβ1 downregulates neurite outgrowth, expression of Ca2+ transporters, and mitochondrial dynamics of in vitro cerebellar granule cells

Acute injury to central nervous system (CNS) triggers neurodegenerative processes that can result in serious damage or complete loss of function. After injury, production of transforming growth factor β1 (TGFβ1) increases and initiates creation of a fibrotic scar that prevents normal growth, plasticity, and recovery of damaged neurons. Administration of TGFβ1 antagonists can prevent its pathological effects. To define consequences of increased TGFβ1 release on calcium signaling, neuronal plasticity, excitability, and mitochondrial dynamics in CNS neurons we directly exposed a rat primary culture of cerebellar granule neurons to TGFβ1. We focused on changes in expression of intracellular calcium transporters, especially inositol-1,4,5-trisphosphate receptor (IP3R) type 1, mitochondrial dynamics, and membrane excitability. TGFβ1 significantly decreased the gene and protein expression of inositol-1,4,5-trisphosphate receptor type 1 and the gene expression of additional intracellular Ca transporters such as IP3R2, ryanodine receptor type 1 (RyR1), RyR2, and SERCA2. Altered calcium signaling suppressed neurite outgrowth and significantly decreased the length of the mitochondria and the frequency of mitochondrial fusion. The resting membrane potential of cerebellar granule neurons was hyperpolarized and slow after depolarization of single action potential was suppressed. LY364947, a blocker of TGFβ1 receptor I, prevented these effects, and IP3 receptor blocker 2-aminoethoxydiphenyl borate (2APB) mimicked them. After CNS injury TGFβ1 downregulates intracellular Ca levels and alters Ca signaling within injured neurons. We suggest that in our model TGFβ1 may trigger both neurodegenerative and neuroprotective events through IP3-induced Ca signaling.

MRNA, Live and Unmasked

Visualization of mRNA dynamics in live neurons reveals its release from granules at synapses during neuronal plasticity. [Also see Reports by Buxbaum et al. and Park et al. ]

Regulation of EB1/3 proteins by classical MAPs in neurons

Microtubules (MTs) are key cytoskeletal elements in developing and mature neurons. MT reorganization underlies the morphological changes that occur during neuronal development. Furthermore, MTs contribute to the maintenance of neuronal architecture, enable intracellular transport and act as scaffolds for signaling molecules. Thus, a fine-tuned regulation of MT dynamics and stability is crucial for the correct differentiation and functioning of neurons. Different types of proteins contribute to the regulation of the MT state, such as plus-end tracking proteins (+TIPs), which interact with the plus-ends of growing microtubules, and classical microtubule-associated proteins (MAPs), which bind along the microtubule lattice. Recent evidence indicates that MAPs interplay with End Binding Proteins (EBs), the core +TIPs, in neuronal cells. This might contribute to the orchestrated regulation of MT dynamics in neurons. In this mini-review article, we address recent research on the neuronal crosstalk between EBs and classical MAPs and speculate on its possible functional relevance.

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.

Neuroactive alkaloids that modulate the neuronal nicotinic receptor and provide neuroprotection in an Alzheimer's disease model: the case of Teline monspessulana.

Neuroactive alkaloids that modulate the neuronal nicotinic receptor and provide neuroprotection in an Alzheimer's disease model: the case of Teline monspessulana.

Energetic and Dynamic: How Mitochondria Meet Neuronal Energy Demands

Mitochondria are the power houses of the cell, but unlike the static structures portrayed in textbooks, they are dynamic organelles that move about the cell to deliver energy to locations in need. These organelles fuse with each other then split apart; some appear anchored and others more free to move around, and when damaged they are engulfed by autophagosomes. Together, these processes—mitochondrial trafficking, fusion and fission, and mitophagy—are best described by the term “mitochondrial dynamics”. The molecular machineries behind these events are relatively well known yet the precise dynamics in neurons remains under debate. Neurons pose a peculiar logistical challenge to mitochondria; how do these energy suppliers manage to traffic down long axons to deliver the requisite energy supply to distant parts of the cell? To date, the majority of neuronal mitochondrial dynamics studies have used cultured neurons, Drosophila larvae, zebrafish embryos, with occasional experiments in resting mouse nerves. However, a new study in this issue of PLOS Biology from Marija Sajic and colleagues provides an in vivo look at mitochondrial dynamics along resting and electrically active neurons of live anaesthetized mice.

Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients' neurons

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K+-dependent Na+/Ca2+-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K+-dependent Na+/Ca2+-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca2+ signal dynamics in olfactory and other neurons. Synaptic Ca2+ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca2+ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca2+ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca2+ signalling by activating a signal damping and termination pathway.