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Impairment of learning and memory is a significant pathological feature of many neurodegenerative diseases including FTLD-TDP. Appropriate regulation and fine tuning of spinogenesis of the dendrites, which is an integral part of the learning/memory program of the mammalian brain, are essential for the normal function of the hippocampal neurons. TDP-43 is a nucleic acid-binding protein implicated in multi-cellular functions and in the pathogenesis of a range of neurodegenerative diseases including FTLD-TDP and ALS. We have combined the use of single-cell dye injection, shRNA knockdown, plasmid rescue, immunofluorescence staining, Western blot analysis and patch clamp electrophysiological measurement of primary mouse hippocampal neurons in culture to study the functional role of TDP-43 in mammalian spinogenesis. We found that depletion of TDP-43 leads to an increase in the number of protrusions/spines as well as the percentage of matured spines among the protrusions. Significantly, the knockdown of TDP-43 also increases the level of Rac1 and its activated form GTP-Rac1, a known positive regulator of spinogenesis. Clustering of the AMPA receptors on the dendritic surface and neuronal firing are also induced by depletion of TDP-43. Furthermore, use of an inhibitor of Rac1 activation negatively regulated spinogenesis of control hippocampal neurons as well as TDP-43-depleted hippocampal neurons. Mechanistically, RT-PCR assay and cycloheximide chase experiments have indicated that increases in Rac1 protein upon TDP-43 depletion is regulated at the translational level. These data together establish that TDP-43 is an upstream regulator of spinogenesis in part through its action on the Rac1 → GTP-Rac1 → AMPAR pathway. This study provides the first evidence connecting TDP-43 with the GTP-Rac1 → AMPAR regulatory pathway of spinogenesis. It establishes that mis-metabolism of TDP-43, as occurs in neurodegenerative diseases with TDP-43 proteinopathies, e.g., FTLD-TDP, would alter its homeostatic cellular concentration, thus leading to impairment of hippocampal plasticity.

Nociceptin activation of ORL1 (opioid receptor-like 1 receptor) has been shown to antagonize l receptor-mediated analgesia at the supraspinal level. ORL1 and l-opioid receptor (lR) are co-expressed in several subpopulations of CNS neurons involved in regulating pain transmission. The amino acid sequence of ORL1 also shares a high degree of homology with that of l receptor. Thus, it is hypothesized that ORL1 and lR interact to form the heterodimer and that ORL1/lR heterodimerization may be one molecular basis for ORL1-mediated antiopioid effects in the brain. To test this hypothesis, myc-tagged ORL1 and HA-tagged lR are co-expressed in human embryonic kidney (HEK) Grisel et al. 1996;Mogil et al. 1996;Calo et al. 1998;Wang et al. 1999). It has also been reported that intracerebroventricular administration of [Nphe 1 ]nociceptin(1-13)NH2, a selective ORL1 antagonist, potentiates l receptor agonist-induced analgesia and produces a naloxone-resistant antinociceptive effect (Calo et al. 2000a). These results suggest that the functional antagonism of l receptor-mediated analgesia by ORL1 is tonically active at the supraspinal level.A growing number of biochemical, biophysical and functional investigations demonstrated that heterodimerization of distinct G protein-coupled receptors occurs in the plasma membrane and that pharmacological and signaling Received April 20, 2004; revised manuscript received August 13, 2004; accepted October 6, 2004. Address correspondence and reprint requests to Hung-Li Wang, Department of Physiology, Chang Gung University School of Medicine, Kwei-San, Tao-Yuan, Taiwan. E-mail: hlwns@mail.cgu.edu.twAbbreviations used: AT1-R, type 1 angiotensin II receptor; B2-R, type 2 bradykinin receptor; CHO, Chinese hamster ovary; DA, dopamine; DAMGO, [D-Ala 2 ,N-methyl-Phe 4 ,Gly-ol 5 ]enkephalin; GRK2, G proteincoupled receptor kinase 2; HEK, human embryonic kidney; MAPK, mitogen-activated protein kinase; lR, l-opioid receptor; ORL1, opioid receptor-like 1 receptor; PAG, periaqueductal gray; PBS, phosphatebuffered saline; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

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