Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca2+‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.
CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long‐term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody‐mediated cell adhesion, and neurite outgrowth on HIgM12‐coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates it's in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases.
Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.
The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.
Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia‐bound GCPII mediates the hydrolysis of the neurotransmitter N‐acetylaspartylglutamate (NAAG) into glutamate and N‐acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity associated with enhanced glutamate transmission under pathological conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3–5 of GCPII. This generated a new GCPII KO mice line with no overt differences in standard neurological behavior compared to their wild‐type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addition, GCPII gene KO reduced TBI‐induced deficits in long‐term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathological protection with improved long‐term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors.
Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose‐6‐phosphate dehydrogenase, the rate‐limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time‐ and dose‐dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.
Microglia are the resident macrophages of the central nervous system that survey the microenvironment for signals of injury or infection. The response to such signals induces an inflammatory response involving macrophages derived from both resident microglia and recruited circulating monocytes. Although implicated as contributors to autoimmune‐mediated injury, microglia/ macrophages have recently been shown to be critical for the important central nervous system regenerative process of remyelination. This functional dichotomy may reflect their ability to be polarized along a continuum of activation states including the well‐characterized cytotoxic M1 and regenerative M2 phenotypes. Here, we review the roles of microglia, monocytes and the macrophages which they give rise to in creating lesion environments favourable to remyelination, highlighting the specific roles of M1 and M2 phenotypes and how the pro‐regenerative role of the innate immune system is altered by ageing.
Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.
Prion protein (PrP) plays crucial roles in regulating antioxidant systems to improve cell defenses against cellular stress. Here, we show that the interactions of PrP with the excitatory amino acid transporter 3 (EAAT3), γ‐glutamyl transpeptidase (γ‐GT), and multi‐drug resistance protein 1 (MRP1) in astrocytes and the interaction between PrP and EAAT3 in neurons regulate the astroglial and neuronal metabolism of the antioxidant glutathione. Ablation of PrP in astrocytes and cerebellar neurons leads to dysregulation of EAAT3‐mediated uptake of glutamate and cysteine, which are precursors for the synthesis of glutathione. In PrP‐deficient astrocytes, levels of intracellular glutathione are increased, and under oxidative stress, levels of extracellular glutathione are increased, due to (i) increased glutathione release via MRP1 and (ii) reduced activity of the glutathione‐degrading enzyme γ‐GT. In PrP‐deficient cerebellar neurons, cell death is enhanced under oxidative stress and glutamate excitotoxicity, when compared to wild‐type cerebellar neurons. These results indicate a functional interplay of PrP with EAAT3, MRP1 and γ‐GT in astrocytes and of PrP and EAAT3 in neurons, suggesting that these interactions play an important role in the metabolic cross‐talk between astrocytes and neurons and in protection of neurons by astrocytes from oxidative and glutamate‐induced cytotoxicity.
The WWC1 gene has been genetically associated with human episodic memory performance, and its product KIdney/BRAin protein (KIBRA) has been shown to interact with the atypical protein kinase protein kinase M ζ (PKMζ). Although recently challenged, PKMζ remains a candidate postsynaptic regulator of memory maintenance. Here, we show that PKMζ is subject to rapid proteasomal degradation and that KIBRA is both necessary and sufficient to counteract this process, thus stabilizing the kinase and maintaining its function for a prolonged time. We define the binding sequence on KIBRA, a short amino acid motif near the C‐terminus. Both hippocampal knock‐down of KIBRA in rats and KIBRA knock‐out in mice result in decreased learning and memory performance in spatial memory tasks supporting the notion that KIBRA is a player in episodic memory. Interestingly, decreased memory performance is accompanied by decreased PKMζ protein levels. We speculate that the stabilization of synaptic PKMζ protein levels by KIBRA may be one mechanism by which KIBRA acts in memory maintenance.
Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.
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