Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors.

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 µM kainic acid (KA) for 1-24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced (45)Ca(2+) uptake. The maximum effect (70-80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and (45)Ca(2+) uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4.

Targeting of monomer/misfolded SOD1 as a therapeutic strategy for amyotrophic lateral sclerosis.

There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1(G37R) and SOD1(G93A) mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1(G37R) mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.

Distinct biochemical signatures characterize peripherin isoform expression in both traumatic neuronal injury and motor neuron disease.

Peripherin is a type III intermediate filament protein that is up-regulated during neuronal injury and is a major component of pathological inclusions found within degenerating motor neurons of patients with amyotrophic lateral sclerosis (ALS). The relationship between these inclusions and their protein constituents remains largely unknown. We have previously shown that peripherin expression is characterized by tissue-specific, intra-isoform associations that contribute to filament structure; changes to the normal isoform expression pattern is associated with malformed filaments and intracellular inclusions. Here, we profile peripherin isoform expression and ratio changes in traumatic neuronal injury, transgenic mouse models of motor neuron disease, and ALS. Extensive western blot analyses of Triton X-100 soluble and insoluble fractions of neuronal tissue from these conditions revealed significant changes in peripherin isoform content which could be differentiated by electrophoretic banding patterns to produce distinct peripherin biochemical signatures. Significantly, we found that the pattern of peripherin expression in ALS most closely approximates that of peripherin over-expressing mice, but differs with regard to inter-individual variations in isoform-specific expression. Overall, these results provide important insights into complex post-transcriptional processes that may underlie a continuum between peripherin-mediated neuronal repair and its role in the pathogenesis of motor neuron disease.

Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form.

Amyotrophic lateral sclerosis (ALS) is a conformational disease in which misfolding and aggregation of proteins such as SOD1 (familial ALS) and TDP-43 (sporadic ALS) are central features. The conformations adopted by such proteins within motor neurons in affected patients are not well known. We have developed a novel conformation-specific antibody (USOD) targeted against SOD1 residues 42-48 that specifically recognizes SOD1 in which the beta barrel is unfolded. Use of this antibody, in conjunction with the previously described SEDI antibody that recognizes the SOD1 dimer interface, allows a detailed investigation of the in vivo conformation of SOD1 at the residue-specific level. USOD and SEDI immunohistochemistry of spinal cord sections from ALS cases resulting from SOD1 mutations (A4V and DeltaG27/P28) shows that inclusions within remaining motor neurons contain SOD1 with both an unfolded beta barrel and a disrupted dimer interface. Misfolded SOD1 can also be immunoprecipitated from spinal cord extracts of these cases using USOD. However, in ten cases of sporadic ALS, misfolded SOD1 is not detected by either immunohistochemistry or immunoprecipitation. Using the amyloid-specific dyes, Congo Red and Thioflavin S, we find that SOD1-positive inclusions in familial ALS, as well as TDP-43- and ubiquitin-positive inclusions in sporadic ALS, contain non-amyloid protein deposits. We conclude that SOD1 misfolding is not a feature of sporadic ALS, and that both SOD1-ALS and sporadic ALS, rather than being amyloid diseases, are conformational diseases that involve amorphous aggregation of misfolded protein. This knowledge will provide new insights into subcellular events that cause misfolding, aggregation and toxicity.

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues.

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.

Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis.

OBJECTIVE: In familial amyotrophic lateral sclerosis (fALS) harboring superoxide dismutase (SOD1) mutations (fALS1), SOD1 toxicity has been linked to its propensity to misfold and aggregate. It has recently been proposed that misfolded SOD1 may be causative of all types of ALS, including sporadic cases (sALS). In the present study, we have used a specific antibody to test for the presence of monomer/misfolded SOD1 in sALS. METHODS: Sections from lumbar spinal cords of 5 fALS1 cases, 13 sALS cases, and 1 non-SOD1 fALS case were labeled immunocytochemically using SOD1-exposed-dimer-interface (SEDI) antibody, which we have previously validated as being specific for pathological monomer/misfolded forms of SOD1. RESULTS: Monomer/misfolded SOD1 was detected with SEDI antibody in all 5 of the fALS1 cases, localizing predominantly to hyaline conglomerate inclusions, a specific pathological feature of fALS1. In contrast, monomer/misfolded SOD1 was not detected in any of the 13 sALS cases or in the non-SOD1 fALS cases. These results were confirmed by immunoprecipitation. INTERPRETATION: Although SEDI antibody does not necessarily label all misfolded forms of SOD1, these findings show a distinct difference between fALS1 and sALS, and do not support that monomer/misfolded SOD1 is a common disease entity linking all types of ALS. This is important to our understanding of ALS disease pathogenesis and to considerations of the applicability of using therapeutics that target misfolded SOD1 to non-SOD1-related cases. Ann Neurol 2009;66:75-80.

Cadmium exposure induces mitochondria-dependent apoptosis in oligodendrocytes.

Cadmium toxicity has been associated with learning disabilities and Parkinsonian symptoms in humans. We have previously shown that cultured oligodendrocytes are directly damaged by cadmium exposure. Here, we characterized the molecular mechanisms underlying cadmium-induced cell death in oligodendrocyte progenitors (OLP). Cadmium caused a concentration-dependent decrease in cell viability as assessed by mitochondrial dehydrogenase activity and by the cellular release of lactate dehydrogenase (LDH). A short exposure (1h) to cadmium (25-100 microM), followed by several hours of recovery, produced a predominant apoptotic mechanism of cell death, involving the mitochondrial intrinsic pathway, as evidenced by nuclear condensation, DNA fragmentation, bax integration into the outer mitochondrial membrane, cytochrome c release, and activation of caspases-9 and -3. Pretreatment of OLPs with the pan-caspase inhibitor, zVAD-fmk, prevented caspase-3 activation but only slightly reduced cell death 11h after cadmium exposure and failed to prevent cadmium-induced bax insertion into the mitochondrial membrane. In contrast, the anti-oxidant N-acetyl cysteine blocked caspase-3 activation and significantly protected OLPs from cadmium-induced cell death. Continuous exposure (18-48 h) of OLPs to low micromolar concentrations (0.001-25 microM) of cadmium significantly decreased mitochondrial metabolic activity, increased LDH leakage starting at 5 microM and maximally activated caspase-3. These results suggest that cadmium induces OLP cell death mainly by apoptosis, and at higher concentrations or with prolonged exposure to the heavy metal there is an increase in cytoplasmic membrane damage, an index of necrosis. More importantly, transient exposure to cadmium is sufficient to damage OLPs and could in principle impair myelination in the neonate.

Protection of p27(Kip1) mRNA by quaking RNA binding proteins promotes oligodendrocyte differentiation.

The quaking (Qk) locus expresses a family of RNA binding proteins, and the expression of several alternatively spliced isoforms coincides with the development of oligodendrocytes and the onset of myelination. Quaking viable (Qk(v)) mice harboring an autosomal recessive mutation in this locus have uncompacted myelin in the central nervous system owing to the inability of oligodendrocytes to properly mature. Here we show that the expression of two QKI isoforms, absent from oligodendrocytes of Qk(v) mice, induces cell cycle arrest of primary rat oligodendrocyte progenitor cells and differentiation into oligodendrocytes. Injection of retroviruses expressing QKI into the telencephalon of mouse embryos induced differentiation and migration of multipotential neural progenitor cells into mature oligodendrocytes localized in the corpus callosum. The mRNA encoding the cyclin-dependent kinase (CDK)-inhibitor p27(Kip1) was bound and stabilized by QKI, leading to an increased accumulation of p27(Kip1) protein in oligodendrocytes. Our findings demonstrate that QKI is upstream of p27(Kip1) during oligodendrocyte differentiation.

Developmental differences in HO-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3.

The molecular mechanisms underlying H(2)O(2)-induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H(2)O(2), the antioxidant, N-acetyl-L-cysteine, blocked toxicity at both stages of development. Differentiated oligodendrocytes contained more glutathione than did progenitors and were less susceptible to decreases in glutathione concentration induced by H(2)O(2) stress. As free radicals have been considered to serve as second messengers, we examined the effect of H(2)O(2) on activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) 1/2 and p38. H(2)O(2) caused a time- and concentration-dependent increase in MAPK phosphorylation, an effect that was totally blocked by N-acetyl-L-cysteine. Further exploration of potential mechanisms involved in oligodendrocyte cell death showed that H(2)O(2) treatment caused DNA condensation and fragmentation at both stages of development, whereas caspase 3 activation and poly (ADP-ribose) polymerase cleavage were significantly increased only in oligodendrocyte progenitors. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, blocked DNA fragmentation in progenitors and produced a small but significant level of protection from H(2)O(2) toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H(2)O(2)-induced death most significantly in oligodendrocytes. The poly (ADP-ribose) polymerase inhibitor, PJ34, reduced H(2)O(2)-induced toxicity on its own but was most effective when combined with benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or PD169316. The finding that molecular mechanisms conferring resistance to reactive oxygen species toxicity are regulated during oligodendrocyte differentiation may be of importance in designing therapies for certain neurological diseases affecting white matter.

Cytotoxicity of the E(2)-isoprostane 15-E(2t)-IsoP on oligodendrocyte progenitors.

Oxidant stress plays a significant role in the pathogenesis of periventricular leukomalacia (PVL). Isoprostanes (IsoPs) are bioactive products of lipid peroxidation abundantly generated during hypoxic-ischemic injuries. Because loss of oligodendrocytes (OLs) occurs early in PVL, we hypothesized that IsoPs could induce progenitor OL death. 15-E(2t)-IsoP but not 15-F(2t)-IsoP elicited a concentration-dependent death of progenitor OLs by oncosis and not by apoptosis, but exerted minimal effects on mature OLs. 15-E(2t)-IsoP-induced cytotoxicity could not be explained by its conversion into cyclopentenones, because PGA(2) was hardly cytotoxic. On the other hand, thromboxane A(2) (TxA(2)) synthase inhibitor CGS12970 and cyclooxygenase inhibitor ibuprofen attenuated 15-E(2t)-IsoP-induced cytotoxicity. Susceptibility of progenitor OLs was independent of TxA(2) receptor (TP) expression, which was far less in progenitor than in mature OLs. However, TxA(2) synthase was detected in precursor but not in mature OLs, and TxA(2) mimetic U46619 induced hydroperoxides generation and progenitor OL death. The glutathione synthesis enhancer N-acetylcysteine prevented 15-E(2t)-IsoP-induced progenitor cell death. Depletion of glutathione in mature OLs with buthionine sulfoximine rendered them susceptible to cytotoxicity of 15-E(2t)-IsoP. These novel data implicate 15-E(2t)-IsoP as a product of oxidative stress that may contribute in the genesis of PVL.

Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure.

1 Oligodendrocytes, the myelin forming cells in the CNS, express muscarinic acetylcholine receptors (mAChR), primarily M3, coupled to various signal transduction pathways. 2 In the present study we have investigated whether mAChR undergo functional agonist-induced regulation in cultured oligodendrocyte progenitors and differentiated oligodendrocytes. 3 The muscarinic agonist, carbachol (CCh) caused a time-dependent desensitization of phosphoinositide (PI) hydrolysis, and the internalization and down-regulation of receptors. Short-time desensitization (5 min) of PI hydrolysis occurred without receptor internalization and reached 54% by 1 h. The same treatment decreased cell surface receptors labelled with the non-permeable ligand [(3)H]-NMS by 47%, while total receptor density ([(3)H]-scopolamine binding) decreased by 30%. Longer CCh treatment down-regulated receptors by 70% and desensitized the PI response by 80%. 4 Although protein kinase C (PKC) activation desensitized mAChR, CCh-mediated desensitization was independent of PKC. 5 Inhibition of receptor endocytosis by low temperature during the pre-stimulation period or in the presence of hyperosmotic sucrose (0.5 M) blocked desensitization, receptor internalization and down-regulation. 6 Recovery of surface mAChR and their functional activity following down-regulation was slow, returning to control levels by 24 h after agonist removal. In progenitor cells, dose-response curves for CCh-mediated PI hydrolysis and c-fos mRNA expression showed that newly synthesized mAChR were supersensitive after recovery. 7 Overall, the present results provide evidence of functional agonist-mediated mAChR regulation in brain oligodendroglial cells.

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.