Phospholipid phosphatase related 1 (PLPPR1) increases cell adhesion through modulation of Rac1 activity.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a six-transmembrane protein that belongs to the family of plasticity-related gene proteins, which is a novel brain-specific subclass of the lipid phosphate phosphatase superfamily. PLPPR1-5 have prominent roles in synapse formation and axonal pathfinding. We found that PLPPR1 overexpression in the mouse neuroblastoma cell line (Neuro2a) results in increase in cell adhesion and reduced cell migration. During migration, these cells leave behind long fibrous looking extensions of the plasma membrane causing a peculiar phenotype. Cells expressing PLPPR1 showed decreased actin turnover and decreased disassembly of focal adhesions. PLPPR1 also reduced active Rac1, and expressing dominant negative Rac1 produced a similar phenotype to overexpression of PLPPR1. The PLPPR1-induced phenotype of long fibers was reversed by introducing constitutively active Rac1. In summary, we show that PLPPR1 decreases active Rac1 levels that leads to cascade of events which increases cell adhesion.

Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents.

The accumulation of chondroitin sulfate proteoglycans (CSPGs) in the glial scar following acute damage to the central nervous system (CNS) limits the regeneration of injured axons. Given the rich diversity of CSPG core proteins and patterns of GAG sulfation, identifying the composition of these CSPGs is essential for understanding their roles in injury and repair. Differential expression of core proteins and sulfation patterns have been characterized in the brain and spinal cord of mice and rats, but a comprehensive study of these changes following optic nerve injury has not yet been performed. Here, we show that the composition of CSPGs in the optic nerve and retina following optic nerve crush (ONC) in mice and rats exhibits an increase in aggrecan, brevican, phosphacan, neurocan and versican, similar to changes following spinal cord injury. We also observe an increase in inhibitory 4-sulfated (4S) GAG chains, which suggests that the persistence of CSPGs in the glial scar opposes the growth of CNS axons, thereby contributing to the failure of regeneration and recovery of function.

Parkin targets NOD2 to regulate astrocyte endoplasmic reticulum stress and inflammation.

Loss of substantia nigra dopaminergic neurons results in Parkinson disease (PD). Degenerative PD usually presents in the seventh decade whereas genetic disorders, including mutations in PARK2, predispose to early onset PD. PARK2 encodes the parkin E3 ubiquitin ligase which confers pleotropic effects on mitochondrial and cellular fidelity and as a mediator of endoplasmic reticulum (ER) stress signaling. Although the majority of studies investigating ameliorative effects of parkin focus on dopaminergic neurons we found that astrocytes are enriched with parkin. Furthermore, astrocytes deficient in parkin display stress-induced elevation of nucleotide-oligomerization domain receptor 2 (NOD2), a cytosolic receptor integrating ER stress and inflammation. Given the neurotropic and immunomodulatory role of astrocytes we reasoned that parkin may regulate astrocyte ER stress and inflammation to control neuronal homeostasis. We show that, in response to ER stress, parkin knockdown astrocytes exhibit exaggerated ER stress, JNK activation and cytokine release, and reduced neurotropic factor expression. In coculture studied we demonstrate that dopaminergic SHSY5Y cells and primary neurons with the presence of parkin depleted astrocytes are more susceptible to ER stress and inflammation-induced apoptosis than wildtype astrocytes. Parkin interacted with, ubiquitylated and diminished NOD2 levels. Additionally, the genetic induction of parkin ameliorated inflammation in NOD2 expressing cells and knockdown of NOD2 in astrocytes suppressed inflammatory defects in parkin deficient astrocytes and concurrently blunted neuronal apoptosis. Collectively these data identify a role for parkin in modulating NOD2 as a regulatory node in astrocytic control of neuronal homeostasis.

Correction: A novel cytoskeletal action of xylosides.

[This corrects the article DOI: 10.1371/journal.pone.0269972.].

Wilson disease mutation pattern with genotype-phenotype correlations from Western India: confirmation of p.C271* as a common Indian mutation and identification of 14 novel mutations.

Wilson disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene, with over 600 mutations described. Identification of mutations has made genetic diagnosis of WD feasible in many countries. The heterogeneity of ATP7B mutants is, however, yet to be identified in the Indian population. We analyzed the mutational pattern of WD in a large region of Western India. We studied patients (n = 52) for ATP7B gene mutations in a cohort of families with WD and also in first-degree relatives (n = 126). All 21 exon-intron boundaries of the WD gene were amplified and directly sequenced. We identified 36 different disease-causing mutations (31 exonic and five intronic splice site variants). Fourteen novel mutations were identified. Exons 2, 8, 13, 14, and 18 accounted for the majority of mutations (86.4%). A previously recognized mutation, p.C271*, and the novel mutation p.E122fs, were the most common mutations with allelic frequencies of 20.2% and 10.6%, respectively. Frequent homozygous mutations (58.9%) and disease severity assessments allowed analysis of genotype-phenotype correlations. Our study significantly adds to the emerging data from other parts of India suggesting that p.C271* may be the most frequent mutation across India, and may harbor a moderate to severely disabling phenotype with limited variability.

A novel cytoskeletal action of xylosides.

Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.

Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture.

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Agonist-induced internalization and desensitization of the apelin receptor.

Apelin acts via the G protein-coupled apelin receptor (APJ) to mediate effects on cardiovascular and fluid homeostasis. G protein-coupled receptor (GPCR) trafficking has an important role in the regulation of receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in apelin-induced trafficking are not well understood. We generated a stable HEK-293 cell line expressing N-terminus HA-tagged mouse (m) APJ, and used a semi-automated imaging protocol to quantitate APJ trafficking and ERK1/2 activation following stimulation with [Pyr1]apelin-13. The mechanisms of [Pyr1]apelin-13-induced internalization and desensitization were explored using dominant-negative mutant (DNM) cDNA constructs of G protein-coupled receptor kinase 2 (GRK2), ß-arrestin1, EPS15 and dynamin. The di-phosphorylated ERK1/2 (ppERK1/2) response to [Pyr1]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr1]apelin-13 stimulation caused internalization of mAPJ via clathrin coated vesicles (CCVs) and also caused a rapid reduction in cell surface and whole cell HA-mAPJ. Our data suggest that upon continuous agonist exposure GRK2-mediated phosphorylation targets APJ to CCVs that are internalized from the cell surface in a ß-arrestin1-independent, EPS15- and dynamin-dependent manner. Internalization does not appear to contribute to the desensitization of APJ-mediated ppERK1/2 activation in these cells.

Cofilin 1 activation prevents the defects in axon elongation and guidance induced by extracellular alpha-synuclein.

Impaired adult neurogenesis and axon traumatic injury participate in the severity of neurodegenerative diseases. Alpha-synuclein, a cytosolic protein involved in Parkinson's disease, may be released from neurons, suggesting a role for excess secreted alpha-synuclein in the onset and spread of the pathology. Here we provide evidence that long term exposure of young neurons to extracellular alpha-synuclein hampers axon elongation and growth cone turning. We show that actin turnover and the rate of movement of actin waves along the axon are altered, due to alpha-synuclein-induced inactivation of cofilin. Upon laser disruption of microfilaments, healing of axons is favored by the increased phosphorylation of cofilin, however, at later time points; the defect in neurite extension prevails, being lost the regulation of cofilin activity. Importantly, overexpression of the active form of cofilin in neurons exposed to alpha-synuclein is able to restore the movement of actin waves, physiological axon elongation and growth cone turning. Our study reveals the molecular basis of alpha-synuclein-driven deficits in growth and migration of newborn neurons, and in elongation and regeneration of adult neurons.

Motility flow and growth-cone navigation analysis during in vitro neuronal development by long-term bright-field imaging.

A long-term live-imaging workstation to follow the development of cultured neurons during the first few days in vitro (DIV) is developed. In order to monitor neuronal polarization and axonal growth by live imaging, we built a micro-incubator system that provides stable temperature, pH, and osmolarity in the culture dish under the microscope, while preserving environment sterility. We are able to image living neurons at 2 DIVs for 48 h with a temporal resolution of one frame for every 2 min. The main features of this system are its ability to adapt to every cell-culture support, to integrate in any optical microscope, because of the relatively small dimensions (9.5×6.5×2.5 cm) and low weight of the system (<200 g), and to monitor the physiological parameters in situ. Moreover, we developed an image-analysis algorithm to quantify the cell motility, in order to characterize its complex temporal-spatial pattern. The algorithm applies morphological image processing operations on the temporal variations occurring in the inspected region of interest. Here, it is used to automatically detect cellular motility in three distinct morphological regions of the neurons: around the soma, along the neurites, and in the growth cone.