Chemokine-like Orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms.

During animal development, neurons often form exuberant or inappropriate axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. Glial cells act as predominant phagocytes during neuronal remodeling and degeneration, and crucial signaling pathways between neurons and glia are necessary for the execution of phagocytosis. Chemokine-like mushroom body neuron-secreted Orion is essential for astrocyte infiltration into the γ axon bundle leading to γ axon pruning. Here, we show a role of Orion in debris engulfment and phagocytosis in Drosophila. Interestingly, Orion is involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role of Orion in eliminating both peptidergic vCrz+ and PDF-Tri neurons via additional phagocytic glial cells like cortex and/or ensheathing glia. Our results suggest that Orion is essential for phagocytic activation of astrocytes, cortex and ensheathing glia, and point to Orion as a trigger of glial infiltration, engulfment and phagocytosis.

Translational and Posttranslational Dynamics in a Model Peptidergic System.

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus, whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses to neuronal stimulation are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganization of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and are hyperphosphorylated following stimulation. By multiomic integration of transcriptome and proteomic data, we identify changes to proteins present in afferent inputs to this nucleus.

The Role of PGC-1α-Mediated Mitochondrial Biogenesis in Neurons.

Neurons are highly dependent on mitochondrial ATP production and Ca2+ buffering. Neurons have unique compartmentalized anatomy and energy requirements, and each compartment requires continuously renewed mitochondria to maintain neuronal survival and activity. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a key factor in the regulation of mitochondrial biogenesis. It is widely accepted that mitochondria are synthesized in the cell body and transported via axons to the distal end. However, axonal mitochondrial biogenesis is necessary to maintain axonal bioenergy supply and mitochondrial density due to limitations in mitochondrial axonal transport rate and mitochondrial protein lifespan. In addition, impaired mitochondrial biogenesis leading to inadequate energy supply and neuronal damage has been observed in neurological disorders. In this review, we focus on the sites where mitochondrial biogenesis occurs in neurons and the mechanisms by which it maintains axonal mitochondrial density. Finally, we summarize several neurological disorders in which mitochondrial biogenesis is affected.

Selection for early shoot vigour in wheat increases root hair length but reduces epidermal cell size of roots and leaves.

Six cycles of recurrent selection for early shoot vigour in wheat resulted in significant increases in leaf width and shoot biomass. Here, in replicated controlled-environment studies, the effect of early shoot vigour on root biomass, rhizosheath size, root hair length, and cell size in the roots and leaves was examined across different cycles of selection. Increased shoot vigour was associated with greater root biomass, larger rhizosheath size, and longer root hairs. Our findings demonstrate that rhizosheath size was a reliable surrogate for root hair length in this germplasm. Examination of the root epidermis revealed that the 'cell body' of the trichoblasts (hair-forming cells) and the atrichoblasts (non-hair-forming cells) decreased in size as shoot vigour increased. Therefore, in higher vigour germplasm, longer root hairs emerged from smaller trichoblasts so that total trichoblast volume (root hair plus cell body) was generally similar regardless of shoot vigour. Similarly, the sizes of the four main cell types on the leaf epidermis became progressively smaller as shoot vigour increased, which also increased stomatal density. The relationship between shoot vigour and root traits is considered, and the potential contribution of below-ground root traits to performance and competitiveness of high vigour germplasm is discussed.

PDK1 Regulates the Maintenance of Cell Body and the Development of Dendrites of Purkinje Cells by pS6 and PKCγ.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a critical role in the development of mammalian brain. Here, we investigated the role of PDK1 in Purkinje cells (PCs) by generating the PDK1-conditional knock-out mice (cKO) through crossing PV-cre or Pcp2-cre mice with Pdk1fl/fl mice. The male mice were used in the behavioral testing, and the other experiments were performed on mice of both sexes. These PDK1-cKO mice displayed decreased cerebellar size and impaired motor balance and coordination. By the electrophysiological recording, we observed the reduced spontaneous firing of PCs from the cerebellar slices of the PDK1-cKO mice. Moreover, the cell body size of PCs in the PDK1-cKO mice was time dependently reduced compared with that in the control mice. And the morphologic complexity of PCs was also decreased after PDK1 deletion. These effects may have contributed to the reduction of the rpS6 (reduced ribosomal protein S6) phosphorylation and the PKCγ expression in PDK1-cKO mice since the upregulation of pS6 by treatment of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1, the agonist of mTOR1, partly rescued the reduction in the cell body size of the PCs, and the delivery of recombinant adeno-associated virus-PKCγ through cerebellar injection rescued the reduced complexity of the dendritic arbor in PDK1-cKO mice. Together, our data suggest that PDK1, by regulating rpS6 phosphorylation and PKCγ expression, controls the cell body maintenance and the dendritic development in PCs and is critical for cerebellar motor coordination.SIGNIFICANCE STATEMENT Here, we show the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in Purkinje cells (PCs). The ablation of PDK1 in PCs resulted in a reduction of cell body size, and dendritic complexity and abnormal spontaneous firing, which attributes to the motor defects in PDK1-conditional knock-out (cKO) mice. Moreover, the ribosomal protein S6 (rpS6) phosphorylation and the expression of PKCγ are downregulated after the ablation of PDK1. Additionally, upregulation of rpS6 phosphorylation by3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1 partly rescued the reduction in cell body size of PCs, and the overexpression of PKCγ in PDK1-KO PCs rescued the reduction in the dendritic complexity. These findings indicate that PDK1 contributes to the maintenance of the cell body and the dendritic development of PCs by regulating rpS6 phosphorylation and PKCγ expression.

Mapping complex cell morphology in the grey matter with double diffusion encoding MR: A simulation study.

This paper investigates the impact of cell body (namely soma) size and branching of cellular projections on diffusion MR imaging (dMRI) and spectroscopy (dMRS) signals for both standard single diffusion encoding (SDE) and more advanced double diffusion encoding (DDE) measurements using numerical simulations. The aim is to investigate the ability of dMRI/dMRS to characterize the complex morphology of brain cells focusing on these two distinctive features of brain grey matter. To this end, we employ a recently developed computational framework to create three dimensional meshes of neuron-like structures for Monte Carlo simulations, using diffusion coefficients typical of water and brain metabolites. Modelling the cellular structure as realistically connected spherical soma and cylindrical cellular projections, we cover a wide range of combinations of sphere radii and branching order of cellular projections, characteristic of various grey matter cells. We assess the impact of spherical soma size and branching order on the b-value dependence of the SDE signal as well as the time dependence of the mean diffusivity (MD) and mean kurtosis (MK). Moreover, we also assess the impact of spherical soma size and branching order on the angular modulation of DDE signal at different mixing times, together with the mixing time dependence of the apparent microscopic anisotropy (µA), a promising contrast derived from DDE measurements. The SDE results show that spherical soma size has a measurable impact on both the b-value dependence of the SDE signal and the MD and MK diffusion time dependence for both water and metabolites. On the other hand, we show that branching order has little impact on either, especially for water. In contrast, the DDE results show that spherical soma size has a measurable impact on the DDE signal's angular modulation at short mixing times and the branching order of cellular projections significantly impacts the mixing time dependence of the DDE signal's angular modulation as well as of the derived µA, for both water and metabolites. Our results confirm that SDE based techniques may be sensitive to spherical soma size, and most importantly, show for the first time that DDE measurements may be more sensitive to the dendritic tree complexity (as parametrized by the branching order of cellular projections), paving the way for new ways of characterizing grey matter morphology, non-invasively using dMRS and potentially dMRI.

Mitochondrial Biogenesis in Neurons: How and Where.

Neurons rely mostly on mitochondria for the production of ATP and Ca2+ homeostasis. As sub-compartmentalized cells, they have different pools of mitochondria in each compartment that are maintained by a constant mitochondrial turnover. It is assumed that most mitochondria are generated in the cell body and then travel to the synapse to exert their functions. Once damaged, mitochondria have to travel back to the cell body for degradation. However, in long cells, like motor neurons, this constant travel back and forth is not an energetically favourable process, thus mitochondrial biogenesis must also occur at the periphery. Ca2+ and ATP levels are the main triggers for mitochondrial biogenesis in the cell body, in a mechanism dependent on the Peroxisome-proliferator-activated γ co-activator-1α-nuclear respiration factors 1 and 2-mitochondrial transcription factor A (PGC-1α-NRF-1/2-TFAM) pathway. However, even though of extreme importance, very little is known about the mechanisms promoting mitochondrial biogenesis away from the cell body. In this review, we bring forward the evoked mechanisms that are at play for mitochondrial biogenesis in the cell body and periphery. Moreover, we postulate that mitochondrial biogenesis may vary locally within the same neuron, and we build upon the hypotheses that, in the periphery, local protein synthesis is responsible for giving all the machinery required for mitochondria to replicate themselves.

Ischemic Cerebral Endothelial Cell-Derived Exosomes Promote Axonal Growth.

BACKGROUND AND PURPOSE: Cerebral endothelial cells (CECs) and axons of neurons interact to maintain vascular and neuronal homeostasis and axonal remodeling in normal and ischemic brain, respectively. However, the role of exosomes in the interaction of CECs and axons in brain under normal conditions and after stroke is unknown. METHODS: Exosomes were isolated from CECs of nonischemic rats and is chemic rats (nCEC-exos and isCEC-exos), respectively. A multicompartmental cell culture system was used to separate axons from neuronal cell bodies. RESULTS: Axonal application of nCEC-exos promotes axonal growth of cortical neurons, whereas isCEC-exos further enhance axonal growth than nCEC-exos. Ultrastructural analysis revealed that CEC-exos applied into distal axons were internalized by axons and reached to their parent somata. Bioinformatic analysis revealed that both nCEC-exos and isCEC-exos contain abundant mature miRNAs; however, isCEC-exos exhibit more robust elevation of select miRNAs than nCEC-exos. Mechanistically, axonal application of nCEC-exos and isCEC-exos significantly elevated miRNAs and reduced proteins in distal axons and their parent somata that are involved in inhibiting axonal outgrowth. Blockage of axonal transport suppressed isCEC-exo-altered miRNAs and proteins in somata but not in distal axons. CONCLUSIONS: nCEC-exos and isCEC-exos facilitate axonal growth by altering miRNAs and their target protein profiles in recipient neurons.

Epithelial cell behaviours during neurosensory organ formation.

Perception of the environment in vertebrates relies on a variety of neurosensory mini-organs. These organs develop via a multi-step process that includes placode induction, cell differentiation, patterning and innervation. Ultimately, cells derived from one or more different tissues assemble to form a specific mini-organ that exhibits a particular structure and function. The initial building blocks of these organs are epithelial cells that undergo rearrangements and interact with neighbouring tissues, such as neural crest-derived mesenchymal cells and sensory neurons, to construct a functional sensory organ. In recent years, advances in in vivo imaging methods have allowed direct observation of these epithelial cells, showing that they can be displaced within the epithelium itself via several modes. This Review focuses on the diversity of epithelial cell behaviours that are involved in the formation of small neurosensory organs, using the examples of dental placodes, hair follicles, taste buds, lung neuroendocrine cells and zebrafish lateral line neuromasts to highlight both well-established and newly described modes of epithelial cell motility.

The osmotic demyelination syndrome: the resilience of thalamic neurons is verified with transmission electron microscopy.

The development of a murine model of osmotic demyelinating syndrome (ODS) allowed to study changes incurred in extrapontine zones of the CNS and featured neuron and glial cell changes in the relay thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei before, during and after ODS induction, and characterized without immune response. There, the neuron Wallerian-type deteriorations were verified with fine structure modifications of the neuron cell body, including some nucleus topology and its nucleolus changes. Morphologic analyses showed a transient stoppage of transcriptional activities while myelinated axons in the surrounding neuropil incurred diverse damages, previously reported. Even though the regional thalamus myelin deterioration was clearly recognized with light microscopy 248 h after osmotic recovery of ODS, ultrastructure analyses demonstrated that, at that time, the same damaged parenchyma regions contained nerve cell bodies that have already reactivated nucleus transcriptions and neuroplasm translations because peculiar accumulations of fibro-granular materials, similar to those detected in restored ODS astrocytes, were revealed in these restructuring nerve cell bodies. Their aspects suggested to be accumulations of ribonucleoproteins. The findings suggested that progressive neural function's recovery in the murine model could imitate some aspects of human ODS recovery cases.

The neurochemistry of peripheral nerve regeneration.

Peripheral nerve injuries (PNIs) can be most disabling, resulting in the loss of sensitivity, motor function and autonomic control in the involved anatomical segment. Although injured peripheral nerves are capable of regeneration, sub-optimal recovery of function is seen even with the best reconstruction. Distal axonal degeneration is an unavoidable consequence of PNI. There are currently few strategies aimed to maintain the distal pathway and/or target fidelity during regeneration across the zone of injury. The current state of the art approaches have been focussed on the site of nerve injury and not on their distal muscular targets or representative proximal cell bodies or central cortical regions. This is a comprehensive literature review of the neurochemistry of peripheral nerve regeneration and a state of the art analysis of experimental compounds (inorganic and organic agents) with demonstrated neurotherapeutic efficacy in improving cell body and neuron survival, reducing scar formation and maximising overall nerve regeneration.

Spatio-temporal characteristics of metabotropic glutamate receptor 5 traffic at or near the plasma membrane in astrocytes.

Astrocytes can sense extracellular glutamate and respond to it by elevating their intracellular Ca(2+) levels via the activation of G-protein coupled receptors, such as metabotropic glutamate receptor 5 (mGluR5), which, during early postnatal development, is the primary receptor responsible for glutamatergic signaling in astrocytes. However, the detailed spatio-temporal characteristics of mGluR5 traffic at or near the plasma membrane of astrocytes are not well understood. To address this issue, we expressed recombinant fluorescent protein chimera of mGluR5 and used total internal reflection fluorescence microscopy on rat visual cortical astrocytes in culture. We used astrocytes lacking major processes, otherwise posing as a diffusion barrier, to infer into the general dynamics of this receptor. We found that plasmalemmal mGluR5 clusters in distinct areas, the size, and initial spatio-temporal level of occupancy of which dictated mGluR5 trafficking characteristics upon glutamate stimulation. These findings will be valuable in the interpretation of point-to-point information transfer and volume transmission between astrocytes and neurons, as well as that of paracrine signaling within astrocytic networks.

N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells.

BACKGROUND: Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells. METHODS: Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed. RESULTS: Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties. CONCLUSIONS: Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties. GENERAL SIGNIFICANCE: Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.

Age-related alterations in human cortical microstructure across the lifespan: Insights from high-gradient diffusion MRI.

The human brain undergoes age-related microstructural alterations across the lifespan. Soma and Neurite Density Imaging (SANDI), a novel biophysical model of diffusion MRI, provides estimates of cell body (soma) radius and density, and neurite density in gray matter. The goal of this cross-sectional study was to assess the sensitivity of high-gradient diffusion MRI toward age-related alterations in cortical microstructure across the adult lifespan using SANDI. Seventy-two cognitively unimpaired healthy subjects (ages 19-85 years; 40 females) were scanned on the 3T Connectome MRI scanner with a maximum gradient strength of 300mT/m using a multi-shell diffusion MRI protocol incorporating 8 b-values and diffusion time of 19 ms. Intra-soma signal fraction obtained from SANDI model-fitting to the data was strongly correlated with age in all major cortical lobes (r = -0.69 to -0.60, FDR-p < 0.001). Intra-soma signal fraction (r = 0.48-0.63, FDR-p < 0.001) and soma radius (r = 0.28-0.40, FDR-p < 0.04) were significantly correlated with cortical volume in the prefrontal cortex, frontal, parietal, and temporal lobes. The strength of the relationship between SANDI metrics and age was greater than or comparable to the relationship between cortical volume and age across the cortical regions, particularly in the occipital lobe and anterior cingulate gyrus. In contrast to the SANDI metrics, all associations between diffusion tensor imaging (DTI) and diffusion kurtosis imaging metrics and age were low to moderate. These results suggest that high-gradient diffusion MRI may be more sensitive to underlying substrates of neurodegeneration in the aging brain than DTI and traditional macroscopic measures of neurodegeneration such as cortical volume and thickness.

Single-Cell Resolution Optogenetics Via Expression of Soma-Targeted Rhodopsins.

Optogenetics allows control of neural activity in genetically targeted neuron populations by light. Optogenetic control of individual neurons in neural circuits would enable powerful, causal investigations of neural connectivity and function at single-cell level and provide insights into how neural circuits operate. Such single-cell resolution optogenetics in neuron populations requires precise sculpting of light and subcellular targeting of optogenetic molecules. Here we describe a group of methods for single-cell resolution optogenetics in neuron cultures, in mouse brain slices, and in mouse cortex in-vivo, via patterned light and soma-targeted optogenetic molecules.

Central nervous system diseases related to pathological microglial phagocytosis.

Microglia are important phagocytes of the central nervous system (CNS). They play an important role in protecting the CNS by clearing necrotic tissue and apoptotic cells in many CNS diseases. However, recent studies have found that microglia can phagocytose parts of neurons excessively, such as the neuronal cell body, synapse, or myelin sheaths, before or after the onset of CNS diseases, leading to aggravated injury and impaired tissue repair. Meanwhile, reduced phagocytosis of synapses and myelin results in abnormal circuit connections and inhibition of remyelination, respectively. Previous studies focused primarily on the positive effects of microglia phagocytosis, whereas only a few studies have focused on the negative effects. In this review, we use the term "pathological microglial phagocytosis" to refer to excessive or reduced phagocytosis by microglia that leads to structural or functional abnormalities in target cells and brain tissue. The classification of pathological microglial phagocytosis, the composition, and activation of related signaling pathways, as well as the process of pathological phagocytosis in various kinds of CNS diseases, are described in this review. We hypothesize that pathological microglial phagocytosis leads to aggravation of tissue damage and negative functional outcome. For example, excessive microglial phagocytosis of synapses can be observed in Alzheimer's disease and schizophrenia, leading to significant synapse loss and memory impairment. In Parkinson's disease, ischemic stroke, and traumatic brain injury, excessive microglial phagocytosis of neuronal cell bodies causes impaired gray matter recovery and sensory dysfunction. We therefore believe that more studies should focus on the mechanism of pathological microglial phagocytosis and activation to uncover potential targets of therapeutic intervention.

Characterization of the small RNA transcriptomes of cell protrusions and cell bodies of highly metastatic hepatocellular carcinoma cells via RNA sequencing.

Increasing evidence suggest that hepatocellular carcinoma (HCC) HCCLM3 cells initially develop pseudopodia when they metastasize, and microRNAs (miRNAs/miRs) and circular RNAs (circRNAs) have been demonstrated to serve important roles in the development, progression and metastasis of cancer. The present study aimed to isolate the cell bodies (CBs) and cell protrusions (CPs) from HCCLM3 cells, and screen the miRNAs and circRNAs associated with HCC infiltration and metastasis in CBs and CPs. The Boyden chamber assay has been confirmed to effectively isolate the CBs and CPs from HCCLM3 cells via observation of microtubule immunofluorescence, DAPI staining and nuclear protein H3 western blotting. Following high-throughput sequencing of the successfully isolated CBs and CPs, 64 pairs of miRNAs, including 23 pairs of upregulated genes and 41 pairs of downregulated genes, and 260 sets of circRNAs, including 127 upregulated genes and 133 downregulated genes, were significantly differentially expressed, using the following criteria: HP/HB ratio, fold change ≥|1.5|, P<0.05). PCR analysis verified that changes in the expression levels of hsa-let-7a-5p, hsa-let-7c-3p, hsa-miR-30c-5p, hsa_circ_0059580, hsa_circ_0067475, hsa_circ_0002100 and hsa_circ_00072309 were consistent with the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze the functions and roles of the differentially expressed miRNAs and circRNAs. The interaction maps between miRNAs and circRNAs were constructed, and signaling pathway maps were analyzed to determine the molecular mechanism and regulation of the differentially expressed miRNAs and circRNAs. Taken together, the results of the present study suggest that the Boyden chamber assay can be used to effectively isolate the somatic CBs and CPs of HCC, which can be used to screen the miRNAs and circRNAs associated with invasion and metastasis of HCC.

Measurement of the Cell-Body Rotation of Leptospira.

Leptospira spp. swim in liquid and crawl on surfaces with two periplasmic flagella. The periplasmic flagella attach to the protoplasmic cylinder via basal rotary motors (flagellar motors) and transform the ends of the cell body into spiral or hook shape. The rotations of the periplasmic flagella are thought to gyrate the cell body and rotate the protoplasmic cylinder for propelling the cell; however, the motility mechanism has not been fully elucidated. Since the motility is a critical virulence factor for pathogenic leptospires, the kinematic insight is valuable to understand the mechanism of infection. This chapter describes microscopic methodologies to measure the motility of Leptospira, focusing on rotation of the helical cell body.

FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury.

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron, with alterations in associated transcription factors detected both locally and remote to the lesion. Stress-induced nuclear translocation of the transcription factor forkhead class box O3a (FOXO3a) was initially linked to activation of apoptotic genes in many neuronal subtypes. However, a more complex role of FOXO3a has been suggested in the injury response of sensory neurons, with the injured neuron expressing less FOXO3a. To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression, the temporal impact of chronic unilateral L4-6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral, contralateral or remote to injury relative to naïve controls was examined. In naïve neurons, high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide, a marker of the nociceptive subpopulation. One hour post-injury, an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1, 2 and 4 days, with levels increasing toward pre-injury levels by 1 week post-injury. A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury. Nuclear levels of FOXO3a peaked at 1 day, decreased by 4 days, then increased by 1 week post-injury, a response mirrored in C4 dorsal root ganglion neurons remote to injury. This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts, a response we recently reported for another stress transcription factor, Luman/CREB3. The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective. Finally, the broader systemic changes support the existence of stress/injury-induced humeral factor(s) influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons. Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board (protocol #19920164).

A Novel Role for Polycystin-2 (Pkd2) in P. tetraurelia as a Probable Mg2+ Channel Necessary for Mg2+-Induced Behavior.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.