MODULATION OF NEURITE BRANCHING BY PROTEIN-PHOSPHORYLATION IN CULTURED RAT HIPPOCAMPAL-NEURONS

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra., J. Avila, MAP2 phosphorylation pa rallels dendrite arborization in hippocampal neurones in culture, Neur oReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive c ross-linker and adjustable spacer in dendritic architecture, FEBS Lett . 295 (1991) 5-9]. The assembly and stability of microtubules, which a re prominent cytoskeletal elements in both axons and dendrites, are re gulated by microtubule-associated proteins, including tau (predominant ly found in axons) and MAP2 (predominantly found in dendrites). The ph osphorylation state of tau and MAP2 modulates their interactions with microtubules. Ln their low-phosphorylation states, tau and MAP2 bind t o microtubules and increase microtubule assembly and/or stability. Inc reased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite a rborization in hippocampal neurones in culture, NeuroReport 4 (1993) 4 12-419] found that protein phosphorylation correlates with neurite bra nching in cultured rat hippocampal neurons, and hypothesized that incr eased protein phosphorylation stimulates neurite branching. To test th is hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatas es. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibi tor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca2+-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of pr otein kinase C, inhibited branching of axons but not dendrites. Treatm ents that would be expected to increase protein phosphorylation, inclu ding inhibitors of protein phosphatases (okadaic acid, cyclosporin A a nd FK506) and stimulators of PKA (S-p-cAMPS) or PKC (phorbol 12-myrist ate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these a gents was tested to confirm their effects on protein phosphorylation i n this preparation. Okadaic acid, FK506 and S-p-cAMPS all increased pr otein phosphorylation; KT5720 and KN62 decreased protein phosphorylati on. On Western blots, the position of MAP2c extracted from cultures ex posed to okadaic acid was slightly shifted toward higher molecular wei ght, suggesting greater phosphorylation, while the position of MAP2c f rom cultures exposed to KT5720 and KN62 was slightly shifted toward lo wer molecular weight, suggesting less phosphorylation. We conclude tha t protein phosphorylation modulates both dendrite branching and axon b ranching, but with differences in sensitivity to phosphorylation and/o r dephosphorylation by specific kinases and phosphatases. (C) 1997 Els evier Science B.V.