Journal of Molecular Neuroscience

, Volume 34, Issue 2, pp 131–139

Gephyrin Alterations Due to Protein Accumulation Stress are Reduced by the Lysosomal Modulator Z-Phe-Ala-Diazomethylketone

Article

Abstract

Inhibitory neurotransmission is important for brain function and requires specific transmitter receptors that are organized in synaptic domains. Gephyrin is a cytoskeletal organization protein that binds tubulin and plays an important role in clustering and organizing select inhibitory neurotransmitter receptors. Here, we tested if gephyrin is altered by protein accumulation stress that is common in age-related neurodegenerative disorders. For this, we used the hippocampal slice model that has been shown to exhibit chloroquine (CQN)-induced protein accumulation, microtubule destabilization, transport failure, and declines in excitatory neurotransmitter receptors and their responses. In addition to the decreases in excitatory receptor subunits and other glutamatergic markers, we found that gephyrin isoforms were reduced across the CQN treatment period. Associated with this decline in gephyrin levels was the production of three gephyrin breakdown products (GBDPs) of 30, 38, and 48 kDa. The induced effects on gephyrin were tested for evidence of recovery through enhancement of lysosomal function that is known to promote protein clearance and microtubule integrity. Using the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK), gephyrin levels were completely restored in correspondence with the recovery of excitatory glutamatergic components. In addition, GBDPs were significantly reduced after the 2-day PADK treatment, to levels that were at or below those measured in control cultures. These findings suggest that receptor-clustering mechanisms for inhibitory synapses are compromised during protein accumulation events. They also indicate that a lysosomal enhancement strategy can protect gephyrin integrity, which may be vital for the balance between inhibitory and excitatory signaling during age-related diseases.

Keywords

Gephyrin isoforms Gephyrin breakdown products Lysosomal enhancement Lysosomal modulation PADK 

References

  1. Bahr, B. A. (1995). Long-term hippocampal slices: A model system for investigating synaptic mechanisms and pathological processes. Journal of Neuroscience Research, 42, 294–305.PubMedCrossRefGoogle Scholar
  2. Bahr, B. A. (2003). Dysfunction and activation of the lysosomal system: Implications for and against Alzheimer’s disease. In E. M. Welsh (Ed.), Focus on Alzheimer's disease research (pp. 115–150). Hauppauge, NY: Nova Science.Google Scholar
  3. Bahr, B. A., Abai, B., Gall, C. M., Vanderklish, P. W., Hoffman, K. B., Lynch, G. (1994). Induction of β-amyloid-containing polypeptides in hippocampus: Evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin. Experimental Neurology, 129, 81–94.PubMedCrossRefGoogle Scholar
  4. Bahr, B. A., & Bendiske, J. (2002). The neuropathogenic contributions of lysosomal dysfunction. Journal of Neurochemistry, 83, 481–489.PubMedCrossRefGoogle Scholar
  5. Bahr, B. A., Hoffman, K. B., Kessler, M., Hennegriff, M., Park, G. Y., Yamamoto, R. S. et al. (1996). Distinct distributions of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 MR antigen (GR53) in brain tissue. Neuroscience, 74, 707–721.PubMedCrossRefGoogle Scholar
  6. Bahr, B. A., Kessler, M., Rivera, S., Vanderklish, P. W., Hall, R. A., Mutneja, M. S. et al. (1995). Stable maintenance of glutamate receptors and other synaptic components in long-term hippocampal slices. Hippocampus, 5, 425–439.PubMedCrossRefGoogle Scholar
  7. Bendiske, J., & Bahr, B. A. (2003). Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis-an approach for slowing Alzheimer disease? Journal of Neuropathology and Experimental Neurology, 62, 481–489.Google Scholar
  8. Bendiske, J., Caba, E., Brown, Q. B., & Bahr, B. A. (2002). Intracellular deposition, microtubule destabilization, and transport failure: an “early” pathogenic cascade leading to synaptic decline. Journal of Neuropathology and Experimental Neurology, 61, 640–650.PubMedGoogle Scholar
  9. Bonde, C., Noraberg, J., Noer, H., & Zimmer, J. (2005). Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures. Neuroscience, 136(3), 779–794.PubMedCrossRefGoogle Scholar
  10. Butler, D., Bendiske, J., Michaelis, M. L., Karanian, D. A., & Bahr, B. A. (2007). Microtubule-stabilizing agent prevents protein accumulation-induced loss of synaptic markers. European Journal of Pharmacology, 562, 20–27.PubMedCrossRefGoogle Scholar
  11. Butler, D., Brown, Q. B., Chin, D. J., Batey, L., Karim, S., Mutneja, M. S. et al. (2005). Cellular responses to protein accumulation involve autophagy and lysosomal enzyme activation. Rejuvenation Research, 8, 227–237.PubMedCrossRefGoogle Scholar
  12. Butler, D., Nixon, R. A., & Bahr, B. A. (2006). Potential compensatory responses through autophagic lysosomal pathways in neurodegenerative diseases. Autophagy, 2, 234–237.PubMedGoogle Scholar
  13. Caba, E., & Bahr, B. A. (2004). Biphasic activation of NF-κB in the excitotoxic hippocampus. Acta Neuropathologica, 108, 173–182.PubMedCrossRefGoogle Scholar
  14. Caporaso, G. L., Gandy, S. E., Buxbaum, J. D., & Greengard, P. (1992). Chloroquine inhibits intracellular degradation but not secretion of Alzheimer/A4 amyloid precursor protein. Proceedings of the National Academy of Sciences of the United States of America, 89, 2252–2256.PubMedCrossRefGoogle Scholar
  15. Caspary, D. M., Schatleman, T. A., & Hughes, L. F. (2005). Age-related changes in the inhibitory response properties of dorsal cochlear nucleus output neurons: Role of inhibitory inputs. Journal of Neuroscience, 25, 10952–10959.PubMedCrossRefGoogle Scholar
  16. Charrier, C., Ehrensperger, M. V., Dahan, M., Lèvi, S., & Triller, A. (2006). Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane. Journal of Neuroscience, 26, 8502–8511.PubMedCrossRefGoogle Scholar
  17. Coleman, P., Federoff, H., & Kurlan, R. (2004). A focus on the synapse for neuroprotection in Alzheimer disease and other dementias. Neurology, 63, 1155–1162.PubMedGoogle Scholar
  18. Collins, P. R., Stack, C. M., O’Neill, S. M., Doyle, S., Ryan, T., Brennan, G. P. et al. (2004). Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: Propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. Journal of Biological Chemistry, 279, 17038–17046.PubMedCrossRefGoogle Scholar
  19. Craig, A. M., Banker, G., Chang, W., McGrath, M. E., & Serpinskaya, A. S. (1996). Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. Journal of Neuroscience, 16, 3166–3177.PubMedGoogle Scholar
  20. Craig, A. M., & Boudin, H. (2001). Molecular heterogeneity of central synapses: afferent and target regulation. Nature Neuroscience, 4, 569–578.PubMedCrossRefGoogle Scholar
  21. Danglot, L., Triller, A., & Bessis, A. (2003). Association of gephyrin with synaptic and extrasynaptic GABAA receptors varies during development in cultured hippocampal neurons. Molecular and Cellular Neurosciences, 23, 264–278.PubMedCrossRefGoogle Scholar
  22. Essrich, C., Lorez, M., Benson, J. A., Fritschy, J. M., & Luscher, B. (1998). Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nature Neuroscience, 1, 563–571.PubMedCrossRefGoogle Scholar
  23. Fremeau, R. T. Jr., Troyer, M. D., Pahner, I., Nygaard, G. O., Tran, C. H., Reimer, R. J. et al. (2001). The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron, 31, 247–260.PubMedCrossRefGoogle Scholar
  24. Fuhrmann, J. C., Kins, S., Rostaing, P., El Far, O., Kirsch, J., Sheng, M. et al. (2002). Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes. Journal of Neuroscience, 22(13), 5393–5402, Jul 1.Google Scholar
  25. Graf, R. A., & Kater, S. B. (1998). Inhibitory neuronal activity can compensate for adverse effects of beta-amyloid in hippocampal neurons. Brain Research, 786, 1558–1569.CrossRefGoogle Scholar
  26. Hanus, C., Vannier, C., & Triller, A. (2004). Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate. Journal of Neuroscience, 24(5), 1119–1128 (Feb 4).PubMedCrossRefGoogle Scholar
  27. Heinonen, O., Soininen, H., Sorvari, H., Kosunen, O., Paljarvi, L., Koivisto, E. et al. (1995). Loss of synaptophysin-like immunoreactivity in the hippocampal formation is an early phenomenon in Alzheimer’s disease. Neuroscience, 54, 375z-384.CrossRefGoogle Scholar
  28. Honer, W. G., Dickson, D. W., Gleeson, J., & Davies, P. (1992). Regional synaptic pathology in Alzheimer’s disease. Neurobiology of Aging, 13, 375–382.PubMedCrossRefGoogle Scholar
  29. Jacob, T. C., Bogdanov, Y. D., Magnus, C., Saliba, R. S., Kittler, J. T., Haydon, P. G. et al. (2005). Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. Journal of Neuroscience, 25, 10469–10478.PubMedCrossRefGoogle Scholar
  30. Karanian, D. A., Brown, Q. B., Makriyannis, A., & Bahr, B. A. (2005). Blocking cannabinoid activation of FAK and ERK1/2 compromises synaptic integrity in hippocampus. European Journal of Pharmacology, 508, 47–56.PubMedCrossRefGoogle Scholar
  31. Karanian, D. A., Brown, Q. B., Makriyannis, A., Kosten, T. A., & Bahr, B. A. (2005). Dual modulation of endocannabinoid transport and fatty acid amide hydrolase protects against excitotoxicity. Journal of Neuroscience, 25, 7813–7820.PubMedCrossRefGoogle Scholar
  32. Kawasaki, B. T., Hoffman, K. B., Yamamoto, R. S., & Bahr, B. A. (1997). Variants of the receptor/channel clustering molecule gephyrin in brain: Distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain. Journal of Neuroscience Research, 49, 381–388.PubMedCrossRefGoogle Scholar
  33. Kim, E. Y., Schrader, N., Smolinsky, B., Bedet, C., Vannier, C., Schwarz, G., et al. (2006). Deciphering the structural framework of glycine receptor anchoring by gephyrin. EMBO Journal, 25, 1385–1395.PubMedCrossRefGoogle Scholar
  34. Kirsch, J., & Betz, H. (1993). Widespread expression of gephyrin, a putative glycine receptor-tubulin linker protein, in rat brain. Brain Research, 621, 301–310.PubMedCrossRefGoogle Scholar
  35. Kirsch, J., Wolters, I., Triller, A., Betz, H. (1993). Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons. Nature, 366, 745–748.PubMedCrossRefGoogle Scholar
  36. Kneussel, M., & Betz, H. (2000). Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends in Neurosciences, 23, 429–435.PubMedCrossRefGoogle Scholar
  37. Kneussel, M., Brandstatter, J. H., Laube, B., Stahl, S., Muller, U., & Betz, H. (1999). Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice. Journal of Neuroscience, 19, 9289–9297.PubMedGoogle Scholar
  38. Legendre, P. (2001). The glycinergic inhibitory synapse. Cellular and Molecular Life Sciences, 58, 760–793.PubMedCrossRefGoogle Scholar
  39. Lévi, S., Logan, S. M., Tovar, K. R., & Craig, A. M. (2004). Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons. Journal of Neuroscience, 24, 207–217.PubMedCrossRefGoogle Scholar
  40. Li, X., Serwanski, D. R., Miralles, C. P., Bahr, B. A., & De Blas, A. L. (2007). Two pools of Triton X-100-insoluble GABAA receptors are present in the brain, one associated to lipid rafts and another one to the postsynaptic GABAergic complex. Journal of Neurochemistry, 102, 1329–1345.PubMedCrossRefGoogle Scholar
  41. Masliah, E. (1995). Mechanisms of synaptic dysfunction in Alzheimer’s disease. Histology and Histopathology, 10, 509–519.PubMedGoogle Scholar
  42. Meier, J., De Chaldee, M., Triller, A., Vannier, C. (2000). Functional heterogeneity of gephyrins. Molecular and Cellular Neurosciences, 16, 566–577.PubMedCrossRefGoogle Scholar
  43. Mielke, J. G., Murphy, M. P., Maritz, J., Bengualid, K. M., & Ivy, G. O. (1997). Chloroquine administration in mice increases β-amyloid immunoreactivity and attenuates kainate-induced blood-brain barrier dysfunction. Neuroscience Letters, 227, 169–172.PubMedCrossRefGoogle Scholar
  44. Mizukami, K., Ikonomovic, M. D., Grayson, D. R., Rubin, R. T., Warde, D., Sheffield, R. et al. (1997). Immunohistochemical study of GABAA receptor β2/3 subunits in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes. Experimental Neurology, 147, 333–345.PubMedCrossRefGoogle Scholar
  45. Niewiadomska, G., Baksalerska-Pazera, M., & Riedel, G. (2006). Cytoskeletal transport in the aging brain: focus on the cholinergic system. Reviews in the Neurosciences, 17(6), 581–618.PubMedGoogle Scholar
  46. Nixon, R. A. (2000). A protease activation cascade" in the pathogenesis of Alzheimer’s disease. Annals of the New York Academy of Sciences, 924, 117–131.PubMedCrossRefGoogle Scholar
  47. Oyama, F., Murakami, N., & Ihara, Y. (1998). Chloroquine myopathy suggests that tau is degraded in lysosomes: Implication for the formation of paired helical filaments in Alzheimer’s disease. Neuroscience Research, 31, 1–8.PubMedCrossRefGoogle Scholar
  48. Poe, B. H., Linville, C., & Brunson-Bechtold, J. (2001). Age-related decline of presumptive inhibitory synapses in the sensimotor cortex as revealed by the physical dissector. Journal of Comparative Neurology, 439, 65–72.Google Scholar
  49. Prior, P., Schmitt, B., Grenningloh, G., Pribilla, I., Multhaup, G., Beyreuther, K. et al. (1992). Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron, 8, 1161–1170.PubMedCrossRefGoogle Scholar
  50. Ramming, M., Kins, S., Werner, N., Hermann, A., Betz, H., & Kirsch, J. (2000). Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins. Proceedings of the National Academy of Sciences of the United States of America, 97(18), 10266–10271.PubMedCrossRefGoogle Scholar
  51. Rissman, R. A., Mishizen-Eberz, A. J., Carter, T. L., Wolfe, B. B., De Blas, A. L., Miralles, C. P. et al. (2003). Biochemical analysis of GABAA receptor subunits α1, α5, β1, β2 in the hippocampus of patients with Alzheimer’s disease neuropathology. Neuroscience, 120, 695–704.PubMedCrossRefGoogle Scholar
  52. Rowland, A. M., Richmond, J. E., Olsen, J. G., Hall, D. H., Bamber, B. A. (2006). Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans. Journal of Neuroscience, 26, 1711–1720.PubMedCrossRefGoogle Scholar
  53. Sabatini, D. M., Barrow, R. K., Blackshaw, S., Burnett, P. E., Lai, M. M., Field, M. E. et al. (1999). Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling. Science, 284, 1161–1164.PubMedCrossRefGoogle Scholar
  54. Sassoé-Pognetto, M., Kirsch, J., Grunert, U., Greferath, U., Fritschy, J. M., Mohler, H. et al. (1995). Colocalization of gephyrin and GABAA-receptor subunits in the rat retina. Journal of Comparative Neurology, 357, 1–14.PubMedCrossRefGoogle Scholar
  55. Scornik, O. A. (1984). Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells. Journal of Cellular Physiology, 121, 257–262.PubMedCrossRefGoogle Scholar
  56. Scheff, S. W., Price, D. A., Schmitt, F. A., & Mufson, E. J. (2006). Hippocampal synaptic loss in early Alzheimer’s disease and mild cognitive impairment. Neurobiology of Aging, 27, 1372–1384.PubMedCrossRefGoogle Scholar
  57. Stokin, G. B., Lillo, C., Falzone, T. L., Brusch, R. G., Rockenstein, E., Mount, S. L. et al. (2005). Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease. Science, 307, 1282–1288.PubMedCrossRefGoogle Scholar
  58. Studler, B., Fritschy, J.-M., & Brunig, I. (2002). GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro. Neuroscience, 114, 123–133.PubMedCrossRefGoogle Scholar
  59. Studler, B., Sidler, C., & Fritschy, J. M. (2005). Differential regulation of GABA(A) receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures. Journal of Comparative Neurology, 484, 344–355.PubMedCrossRefGoogle Scholar
  60. Takauchi, S., & Miyoshi, K. (1995). Cytoskeletal changes in rat cortical neurons induced by long-term intraventricular infusion of leupeptin. Acta Neuropathologica, 89, 8–16.PubMedCrossRefGoogle Scholar
  61. Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R. et al. (1991). Physical basis of cognitive alterations in Alzheimer’s disease: Synapse loss is the major correlate of cognitive impairment. Annals of Neurology, 30, 572–580.PubMedCrossRefGoogle Scholar
  62. Tomita, S., Adesnik, H., Sekiguchi, M., Zhang, W., Wada, K., Howe, J. R. et al. (2005). Stargazin modulates AMPA receptor gating and trafficking by distinct domains. Nature, 435, 1052–1058.PubMedCrossRefGoogle Scholar
  63. van Zundert, B., Albarran, F. A., & Aguayo, L. G. (2000). Effects of chronic ethanol treatment on gamma-aminobutyric acid(A) and glycine receptors in mouse glycinergic spinal neurons. Journal of Pharmacology and Experimental Therapeutics, 295, 423–429.PubMedGoogle Scholar
  64. Vornov, J. J., Tasker, R. C., & Coyle, J. T. (1994). Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture. Stroke, 25, 457–464.PubMedGoogle Scholar
  65. Waldvogel, H. J., Baer, K., Snell, R. G., During, M. J., Faull, R. L. M., & Rees, M. I. (2003). Distribution of gephyrin in the human brain: an immunohistochemical analysis. Neuroscience, 116, 145–156.PubMedCrossRefGoogle Scholar
  66. Wheal, H. V., Chen, Y., Mitchell, S. M., Maerz, W., Wieland, H., van Rossum, D. et al. (1998). Molecular mechanisms that underlie structural and functional changes at the postsynaptic membrane during synaptic plasticity. Progress in Neurobiology, 55, 611–640.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesUniversity of ConnecticutStorrsUSA
  2. 2.The Neurosciences ProgramUniversity of ConnecticutStorrsUSA
  3. 3.Synaptic Dynamics, Inc.FarmingtonUSA

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