Reevaluation of neurodegeneration in lurcher mice: constitutive ion fluxes cause cell death with, not by, autophagy.

The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding delta2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2(Lc)) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the "autophagic cell death" hypothesis. Although autophagy and cell death were induced by the expression of GluD2(Lc) in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2(Lc) or by removal of extracellular Na(+). In addition, although GluD2(Lc) is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)-Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2(Lc) showed decreased ATP levels and increased AMP-activated protein kinase (AMPK) activities in a manner dependent on extracellular Na(+). Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST-Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2(Lc)-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro.

CD38 is critical for social behaviour by regulating oxytocin secretion.

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

Exposure of lentiviral vectors to subneutral pH shifts the tropism from Purkinje cell to Bergmann glia.

Cerebellar Purkinje cells play an important role in cerebellar function; lesions of Purkinje cells result in the disruption of motor coordination and motor learning. Although selective gene delivery to Purkinje cells would be a powerful technique for the study of pathophysiology in the cerebellum, a method for such a delivery has not yet been established. Here we employed human immunodeficiency virus-derived lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein to transduce Purkinje cells and examined factors that critically affect the viral tropism for Purkinje cells. Viral vectors encoding GFP were generated using different protocols, and were then injected into the mouse cerebellum. At 7 days and 2 months post-transduction, the relative proportions of transduced Purkinje cells were determined. Lentiviral vectors harvested from a medium of pH 7.2 preferentially transduced Purkinje cells (about half of the transduced cells). In contrast, when the viral vector was harvested from medium of

The C-terminal juxtamembrane region of the delta 2 glutamate receptor controls its export from the endoplasmic reticulum.

Functions of ionotropic glutamate receptors (iGluRs) are tightly regulated by the intracellular trafficking of receptor proteins. Unlike other iGluRs that are considerably retained in the intracellular component, the delta 2 glutamate receptor (GluR delta 2) is efficiently expressed on the Purkinje cell surface. To understand the trafficking mechanism of iGluRs, we deleted various portions of the C-terminal intracellular domain of GluR delta 2 and analysed the localization of the mutant proteins in heterologous cells and neurons. Biotinylation assays indicated that GluR delta 2 lacking the C-terminal juxtamembrane region of 13 amino acids (region A) was not present on the cell surface. This mutant GluR delta 2 was sensitive to endoglycosidase H, which digests unprocessed high-mannose oligosaccharides on proteins retained in the endoplasmic reticulum (ER) or cis-Golgi. Therefore, we concluded that region A is crucial for the transport of GluR delta 2 beyond the trans-Golgi to the cell surface. Because the immunostaining pattern of GluR delta 2 lacking region A in cultured hippocampal neurons completely overlapped the pattern of fluorescence emitted by ER-resident green fluorescent protein, region A is most likely necessary for GluR delta 2's exit from the ER. Furthermore, this region is essential for the proper intracellular trafficking of GluR delta 2 in Purkinje cells. Region A does not rely on a dihydrophobic motif or positively charged residues to participate in trafficking, but its function is dependent on the juxtamembrane position. Therefore, we propose that GluR delta 2's efficient transport to the cell surface utilizes an unknown but general ER exit mechanism, which probably works in close relation to the membrane of heterologous cells and neurons.

Overexpression of Ca2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia.

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Transfer of NMDAR2 cDNAs increases endogenous NMDAR1 protein and induces expression of functional NMDA receptors in PC12 cells.

The pheochromocytoma cell line (PC12) has been used as a model system for the study of regulation of expression of NMDA receptors. PC12 cells express a substantial amount of NMDAR1 subunit (NR1) mRNA, whereas they express only a small amount of NR1 protein. The level of functional NMDA receptor expression is almost negligible. To test the possibility that NMDAR2 subunits (NR2) control expression of functional NMDA receptors as well as NR1 protein, we transferred NR2A-D cDNAs into PC12 cells using adenovirus vectors. Prominent NMDA receptor-mediated currents were recorded in PC12 cells to which NR2A or NR2B cDNA was delivered without NR1 cDNA. The amplitudes of these responses were similar to those in PC12 cells to which NR1 cDNA was delivered together with NR2A or NR2B cDNA. In cells to which either NR2C or NR2D cDNA alone was delivered, NMDA receptor-mediated currents were also detected, although to a much lesser extent. These results showed that NR2 proteins produced by gene transfer are co-assembled with the endogenous NR1 protein to form functional heteromeric receptors. The delivery of NR2A-D cDNAs also increased the amount of NR1 protein but not that of NR1 mRNA, suggesting that this protein increase is due to post-transcriptional mechanisms. The effects of NR2A-B gene transfer on expression of NR1 protein were much more efficient than those of NR2C-D gene transfer.

Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning.

NMDA receptor-dependent long-term potentiation (LTP) at hippocampal synapses has been considered a crucial component of the cellular basis for learning and memory. This form of LTP occurs in excitatory synapses in both the CA1 area and the dentate gyrus in the hippocampus. However, differential roles of LTP in these areas have not yet been identified. To address this issue, we enhanced the degree of LTP by expressing Ca2+-permeable AMPA receptors at either hippocampal CA1 or dentate gyrus synapses using Sindbis viral vectors (SINs) encoding both green fluorescent proteins and unedited GluR2 (GluR2Q) subunits, and examined their effects on rat spatial learning. The viral vectors were locally injected into the 8-week-old-rat brain in vivo bilaterally. The postsynaptic expression of Ca2+-permeable AMPA receptors enhanced the degree of LTP, and induced NMDA receptor-independent LTP in the presence of the NMDA receptor antagonist in SIN-infected regions in both CA1 and dentate gyrus in hippocampal slice preparations. However, the regional expression of Ca2+-permeable AMPA receptors caused opposite behavioural consequences on the Morris water maze task: rats with SIN-infected CA1 pyramidal cells showed shorter escape latency and better probe test performance, whereas those with SIN-infected dentate gyrus granule cells showed impaired performance. Thus, it was demonstrated that CA1 and dentate gyrus synapses play different functional roles in spatial learning despite their similar mechanism for LTP induction.

Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

Postsynaptic expression of a new calcium pathway in hippocampal CA3 neurons and its influence on mossy fiber long-term potentiation.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is induced by postsynaptic Ca(2+) influx via NMDA receptors (NMDARs). However, this synaptic plasticity occurs independently of NMDARs when Ca(2+)-permeable AMPA receptors (AMPARs) are expressed at postsynaptic sites using various genetic techniques, indicating that an increase in Ca(2+) level at critical postsynaptic sites, regardless of its entry pathway, triggers the induction of LTP at CA1 synapses. In contrast, NMDARs are sparsely distributed on mossy fiber (MF) synapses in CA3 hippocampal neurons, and most evidence favors the presynaptic mechanism for LTP induction, although some reports suggested a postsynaptic mechanism. In this study, we examined whether Ca(2+) influx through the newly produced postsynaptic receptors during high-frequency stimulation affects the induction of MF LTP. For this purpose, we expressed Ca(2+)-permeable AMPARs in CA3 pyramidal neurons by Sindbis viral-mediated gene transfer of the unedited form of the glutamate receptor 2 (GluR2Q) subunit, as a new pathway for postsynaptic Ca(2+) entry, in rat hippocampal organotypic cultures. Virally expressed myc-tagged GluR2Q was detected at the complex spines known as the thorny excrescences, which serve as postsynaptic targets for MF synaptic input, on the proximal apical dendrites of CA3 pyramidal cells. Furthermore, endogenous Ca(2+)-impermeable AMPARs at MF synapses were converted into Ca(2+)-permeable receptors by GluR2Q expression. However, the postsynaptic expression of Ca(2+)-permeable AMPARs had no significant influence on the two types of MF LTP induced by different stimulus protocols. These results supported the notion that MF LTP is independent of postsynaptic Ca(2+).