A neurite outgrowth domain of the γ1‐chain of laminin‐1 (RDIAEIIKDI) promotes axon guidance of rat hippocampal neurons, regulates the nuclear movement phase of neuronal migration, and binds to the cellular prion protein (Liesi et al. [ 1995 ] J. Neurosci. Res. 134:447–486; Matsuzawa et al. [ 1998 ] J. Neurosci. Res. 53:114–124; Graner et al. [ 2000 ] Brain Res. Mol. Brain Res. 76:85–92). Using electrophysiology and neuronal culture experiments, we show that this 10 amino acid peptide or its smaller domains induces potassium currents in primary central neurons. Both these currents and the neurotoxicity of high concentrations of the 10 amino acid peptide antigen are prevented by pertussis toxin. The smallest peptide domain capable of inducing both potassium currents and promoting neurite outgrowth of human spinal cord neurons is a tri‐peptide KDI. Our results indicate that KDI may be the biologically active domain of the γ1 laminin, capable of modulating electrical activity and survival of central neurons via a G‐protein coupled mechanism. These results expand the wide variety of functions already reported for the members of the laminin‐gene family. They suggest that biologically active peptide domains of the γ1 laminin may provide tools to promote neuronal regeneration after injuries and to enhance neuronal survival during aging and neuronal degeneration. J Neurosci. Res. 66:1047–1053, 2001. © 2001 Wiley‐Liss, Inc.

We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP gamma-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate. with, but not be responsible for the weaver phenotype. (C) 2000 Wiley-Liss, Inc.

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood, Here we show that BAPTA-AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L-type calcium channel blocker verapamil (Liesi and Wright, 1996), Importantly, BAPTA-AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons, These results indicate that calcium-dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect, We further report that BAPTA-AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol-induced brain damage and neurodegenerative disorders. (C) 1997 Wiley-Liss, Inc.

International Journal of Developmental NeuroscienceVolume 14, Issue S1 p. 59-59 Article 36 The lamines gene family in neural development and repair Päivi Liesi, Päivi LiesiSearch for more papers by this authorMieko Matsuzawa, Mieko MatsuzawaSearch for more papers by this authorJerry Wright, Jerry WrightSearch for more papers by this authorTimo Kauppila, Timo KauppilaSearch for more papers by this author Päivi Liesi, Päivi LiesiSearch for more papers by this authorMieko Matsuzawa, Mieko MatsuzawaSearch for more papers by this authorJerry Wright, Jerry WrightSearch for more papers by this authorTimo Kauppila, Timo KauppilaSearch for more papers by this author First published: July-August 1996 https://doi.org/10.1016/0736-5748(96)80231-0AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume14, IssueS111th Biennial Meeting of the International Society for Developmental NeuroscienceJuly-August 1996Pages 59-59 RelatedInformation

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10-20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.

Zn2+ and ATP are normally present in serum at low levels and are released from some neurons during synaptic activity. In whole-cell recordings, 10 microM Zn2+ potentiated steady-state currents in rat nodose ganglion nerve cells by 18% in 0.5 microM ATP and 120% in 2 microM ATP. Fluctuation analysis of whole-cell suggested the mechanism was increased burst duration. In single-channel recordings, 10 microM Zn2+ did not increase the ATP-activated channel conductance but did increase the mean burst duration of the 35 pS primary open conductance state. These observations indicate that Zn2+ potentiates the steady-state response to extracellular ATP by increasing the burst duration of the channels.

The weaver mouse mutation is a genetic defect of unknown origin that leads to impairment of cerebellar granule neuronal migration and to neuronal cell death. We investigated laminin expression and proteolytic enzyme activity in this migration-deficient mouse mutant in vivo and in vitro to search for a molecular basis for the weaver defect. The weaver cerebellum showed a general increase in immunoreactivity for laminin, for a neurite outgrowth domain of the B2 chain of laminin, and for tissue plasminogen activator compared to the normal animals. Zymographic assays and immunocytochemistry confirmed that tissue plasminogen activator was the proteolytic enzyme synthesized in excess in the weaver mouse cerebellum in vivo. When placed in culture, the weaver granule neurons survived poorly on a laminin substratum, and failed to extend long neurites, unlike the normal cerebellar granule neurons. The cultured weaver granule neurons were proteolytically overactive and secreted excessive amounts of tissue plasminogen activator, which was likely to interfere with their neurite outgrowth potential on a laminin substratum. Indeed, the weaver granule neurons but not the normal neurons degraded laminin from their culture substratum and deposited a neurite outgrowth domain of the B2 chain of laminin onto their surfaces. Electrophysiology showed that the weaver granule neurons had poor resting membrane potentials (-38 V), whereas the normal neurons had normal resting membrane potentials of (-61 V). The resting membrane potentials of the weaver granule neurons were restored to near normal (-59 V) by a protease inhibitor, aprotinin. Aprotinin also rescued the weaver granule neurons from death on a laminin substratum and promoted their neurite outgrowth to the level of the normal animals. These results indicate that increased proteolytic activity accompanied with increased synthesis of laminin, and its B2 chain, distinguish the weaver mutation from the normal animals. These molecular changes may contribute to the impairment of granule neuronal migration and to the neuronal death, characteristic of the weaver mutation.

Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mg2+-free solutions we discovered that N-methyl-D-aspartate (NMDA) channels in excised patches reversibly shifted their open-state probability (P(o)) in a voltage-dependent way, exhibiting approximately 3- to 4-fold greater P(o) at positive potentials than at rest. Changes in P(o) were mainly attributable to shifts in frequency of channel opening. P(o) changed remarkably slowly (2-15 min), explaining the hysteresis of whole-cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in P(o) provides a mechanism by which NMDA channels can substantially increase Ca2+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity.