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Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10-and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.Extension of neuronal processes is fundamental to establishing the intricate wiring of the nervous system. These processes can bridge vast distances that separate nerve cell bodies from their respective target cells. Electron microscopic analysis of neuronal processes has shown that they are densely packed with parallel arrays of filamentous structures, prominent among which are microtubules (35). Microtubules are present in all eukaryotic cells but especially abundant in neurons where they play an essential role in both the extension and maintenance of neuronal processes (10,40,49), and serve also as tracks for vesicular transport between cell bodies and process tips (37, 47). Thus, to understand how these elongated neuronal processes are formed and how vesicular traffic is targeted within them, it is crucial to determine how the spatial and temporal arrangement of microtubule assembly is controlled. In particular, for further biochemical investigations we would first want to know if microtubule assembly in developing neuronal processes involves a rearrangement of pre-existing microtubules or the assembly of new microtubules.Microtubule assembly has been studied extensively in vitro using neuronal tissue as a source of t.ubulin and associated proteins. Although pure tubulin monomers assemble poorly

STRACTIn newborn rats the levels of nerve growth fa tor (NGF) mRNA (mRNANGF) and NGF receptor mRNA ( A') in the sciatic nerve were 10 and 120 times higher, r pectively, than in adult animals. mRNAL levels decreased st adily from birth, approaching adult levels by the third stnatal week, whereas mRNANUF levels decreased only after th first postnatal week, although also reaching adult levels by the third week. Transection of the adult sciatic nerve resulted in a marked biphasic increase in mRNANGF with time. On the p oximal side of the cut, this increase was confined to the area immediately adjacent to the cut; peripherally, a similar biphasic increase was present in all segments. mRNAL levels were al markedly elevated distal to the transection site, in agreem nt with previous results obtained by immunological transection of the sciatic nerve, local NGF synthesis increases dramatically. Augmented NGF synthesis is observed in all segments distal to the transection site but is confined proximally to those parts of the nerve stump immediately adjacent to the lesion (9). This part of the nerve stump may be considered as a "substitute target" for the axotomized and then regenerating axons of the sympathetic and sensory neurons. Although the NGF concentrations in the proximal nerve stump correspond to those of a densely innervated peripheral target organ (10, 11), the volume ofthe "substitute organ" is too small to fully replace the interrupted supply from the peripheral physiological target tissues. This is apparent from the fact that the NGF levels in the proximal unlesioned part of the sciatic nerve reach only 40% of their normal values (9). Simultaneously with the enhanced synthesis of NGF, the transection of the sciatic nerve leads also to reexpression of NGF receptors by Schwann cells (12), receptors normally seen only in earlier stages of development (13,14).In the present study we asked whether the reexpression of NGF receptors and the enhanced synthesis of NGF by Schwann cells are mediated by a common mechanism. We first followed the developmental changes in the levels of mRNA encoding NGF (mRNANGF) and of mRNA encoding NGF receptor (mRNArec) from birth to adulthood and then compared the time course of the levels of these two mRNAs after sciatic nerve transection both distally and proximally to the lesion. In experiments using a crush injury rather than transection, we studied whether neuronal regeneration brought about the return of mRNANGF and mRNA1 to normal levels. MATERIALS AND METHODSPreparation of Sciatic Nerves. Wistar rats (male or female, 150-200 g) were anesthetized with diethyl ether and the sciatic nerve was cut or crushed at the sciatic notch. After cutting, the distal stump of the nerve was diverted into muscle tissue in order to minimize regrowth of fibers. For crushing of the nerve, forceps were cooled in liquid nitrogen and the crush site was marked by a thread.At various times after nerve injury, animals were killed, the nerves were cut into three segments [B (proximal)

Schwann cells in developing and regenerating peripheral nerves express elevated levels of the neurotrophin receptor p75NTR. Neurotrophins are key mediators of peripheral nervous system myelination. Our results show that myelin formation is inhibited in the absence of functional p75NTR and enhanced by blocking TrkC activity. Moreover, the enhancement of myelin formation by endogenous brain-derived neurotrophic factor is mediated by the p75NTR receptor, whereas TrkC receptors are responsible for neurotrophin-3 inhibition. Thus p75NTR and TrkC receptors have opposite effects on myelination.

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