神经元内蛋白分区化合成和降解

佚名

(a deconstruction of the growth cone) and turning toward netrin-1 (a positive response). A role for local protein synthesis in growth toward an attractant is consistent with the idea that local synthesis is important for extension of the growth cone. What role new protein synthesis plays in growth cone collapse remains to be established, but the opposite responses to netrin-1 indicate that particular extracellular signals can be “translated” in different ways.

A somewhat different role for local protein synthesis was suggested by studies that revealed adaptation of Xenopus spinal growth cones to the chemoattraction produced by gradients of netrin-1 and BDNF (Ming et al., 2002). The main finding was that attractive turning of the growth cone toward a netrin-1 or BDNF source was attenuated by the presence of background concentrations of either chemoattractant in the bath. This “desensitization” disappeared within 30 min after removing netrin-1 from the bath, and resensitization was blocked by protein synthesis inhibitors.

A still different role for local protein synthesis in axonal growth cones is suggested by the work of Brittis et al. (2002), who document the existence of a mechanism that could allow the local synthesis of membrane receptors for axon guidance molecules. Spinal commissural axons grow initially toward the midline floor plate in response to attractive guidance cues. After crossing the midline, the axons lose their responsiveness to midline attractants and gain responsiveness to a new set of guidance cues so that the axons grow longitudinally toward the brain (Stein and Tessier-Lavigne, 2001). One possible explanation for the change in responsiveness is a local synthesis of receptors for guidance molecules just after growing commissural axons reach the midline. Evidence for such a mechanism came from studies of the expression of an EphA2 receptor-GFP reporter construct in commissural axons. EphA2 is one of several proteins that are expressed selectively on the distal segments of axons of commissural neurons after they have crossed the midline. Commissural neurons transfected with constructs made up of the 3′UTR of EphA2 and a fluorescent reporter protein (GFP) exhibited protein expression in cell bodies, but not in proximal segments of the axons prior to midline crossing. In contrast, the protein was expressed at high levels in the distal segments of the axons that had extended beyond the midline.

These results reveal a mechanism that could allow the local synthesis of receptors for guidance cues. One can imagine a scenario in which one set of receptors is expressed until growing axons reach intermediate point A; signals from point A then trigger the translation of mRNAs for a different set of receptors that are critical to guiding the axon to the next intermediate station. It should be emphasized, however, that the study involved an exogenously transfected reporter that had the 3′UTR from EphA receptor mRNA, and did not directly demonstrate the presence of EphA receptor mRNA in growing axons. It also remains to be established whether there is functional ER and Golgi in axonal growth cones. A key proof of principle would be to show that disrupting local synthesis would disrupt axon guidance.

Interesting new evidence indicates that axonal transport of mRNA may be reinitiated during axonal regeneration. Regenerating axons of adult dorsal root ganglion cells and spinal motoneurons contain ribosomal proteins, translation initiation factors, and rRNA (Zheng et al., 2001). Axons of dorsal root ganglion cells that have been induced to regenerate by a conditioning lesion also contain mRNAs for actin and neurofilament protein, and blocking protein synthesis within these axons causes growth cone retraction. Together, these findings suggest that local protein synthesis is a critical factor in successful axon regeneration.

Protein Degradation
For the reasons mentioned earlier, the idea of local protein synthesis has great appeal as a mechanism to regulate synaptic function and plasticity. It follows that any cellular process that regulates protein availability is of potential importance for synaptic function. Stepping on the heels of the flurry of local protein synthesis studies come several recent studies implicating the ubiquitin-proteasome pathway in the control of synaptic development and plasticity. The degradation of proteins via the ubiquitin-proteasome system requires three basic steps: the recognition of the target protein via specific signals, the tagging of the target protein with a ubiquitin chain, and the delivery of the target protein to the 26S proteasome, a protein complex that degrades the ubiquitinated proteins. The ubiquitination of target proteins is a highly regulated process; the basic biology of the ubiquitin-proteasome pathway is described by Ciechanover and Brundin (2003)(this issue of Neuron).

Degradation Machinery
The machinery required to carry out ubiquitin-dependent proteolysis includes the ubiquitin-conjugating enzymes (E1, E2, and E3), ubiquitin, and the 26S proteasome, which is formed by the coassembly of a 20S proteasome (the catalytic component) and 19S cap (the regulatory component). The initial targeting of the substrates to the proteasome is probably accomplished through the recognition of the polyubiquitin chain by the non-ATPase subunits of the 19S cap. The machinery responsible for ubiquitin-dependent degradation has been detected at mature and developing synapses. Ubiquitinated proteins have been detected in synaptic fractions from adult rat brains (Chapman et al., 1994). In addition, immunostaining experiments with antibodies against ubiquitin, and the α or β subunit of the proteasome, show the presence of these proteins in hippocampal dendrites near synapses (G.N. Patrick et al., 2003, Soc. Neurosci., abstract), in retinal growth cones (Campbell and Holt, 2001), and in Drosophila presynaptic terminals (Speese et al., 2003).

Degradation in Axon Guidance and Pruning
As discussed above, it has been shown that local protein synthesis is required for some axon guidance decisions. Recent studies indicate that protein degradation is important for both axon guidance and pruning. For example, proteasome inhibitors block the chemotropic responses to netrin-1 and growth cone collapse in response to LPA (Campbell and Holt, 2001). In addition, netrin-1 and LPA can induce the rapid accumulation of ubiquitin-protein conjugates in growth cones. Once they reach the target area, many axons undergo pruning to adjust their synaptic contacts. In Drosophila, the pruning of the γ neuron axonal projections requires protein degradation; mutations of either a ubiquitin-activating enzyme or proteasome subunits prevent normal pruning (Watts et al., 2003). Identifying the axonal targets for degradation is the obvious next step.

Proteasome Regulation of Synaptic Form, Function, and Plasticity
Ubiquitin-dependent processes are also clearly important in synapse formation and regulation. For example, the fat facets (faf) gene in Drosophila, which codes for a deubiquitinating enzyme (DUB), is involved in synaptic development in Drosophila(DiAntonio et al., 2001). In a screen for genes whose overexpression leads to synaptic growth abnormalities, DiAntonio et al. identified faf as a candidate regulator of synapses. Targeted overexpression of faf in Drosophila results in an increase in synaptic size, synaptic area, and the number of synaptic branches. Interestingly, both the miniature and evoked excitatory junctional potentials (EJP) are markedly decreased despite the increased size of the synapse. There is also a decrease in the frequency of miniature EJP; taken together, these data indicate a defect in neurotransmitter release and suggest that the target for ubiquitination resides in the presynaptic terminal.

A recent study has suggested a potential presynaptic substrate. In Drosophila, Broadie and colleagues identified the synaptic vesicle priming protein DUNC-13 as a substrate for ubiquitin-mediated degradation (Speese et al., 2003). In addition, they localize both a ubiquitin-conjugating enzyme and the proteasome to presynaptic terminals; using a fluorescent reporter, they observed rapid local degradation in the nerve ending. The pharmacological blockade of proteasome activity led to an increase in DUNC-13 levels as well as an increase in synaptic strength at the nerve-muscle synapse.

One of the earliest demonstrations of the connection between the ubiquitin proteasome and synaptic plasticity came from studies of synaptic facilitation at sensory-motor synapses in Aplysia. The ubiquitin-proteasome pathway is responsible for the decrease in the level of regulatory subunits of the cAMP-dependent protein kinase, permitting the enhanced catalytic activity that is in part responsible for facilitation (Hegde et al., 1993). In addition, injection of proteasome inhibitors into sensory neurons can prevent synaptic facilitation (Chain et al., 1999). A different story emerges, however, from a more recent study (Zhao et al., 2003). In this study, bath application or injection of proteasome inhibitors increased basal synaptic strength and enhanced, rather than prevented, the synaptic facilitation elicited by serotonin treatment (Zhao et al., 2003). These latter data together with the Speese et al. study (Speese et al., 2003) suggest a different function for the proteasome, one in which proteasome activity functions to inhibit plasticity, rather than facilitate it.

Studies of E6-AP, the gene responsible for the human disease Angelman's syndrome, have supported the idea that proteasomal protein degradation is important for both synaptic and behavioral plasticity. E6-AP is a ubiquitin ligase (E3) that is required, together with the papillomavirus E6 oncoprotein, for the ubiquitination and degradation

上一页 [1] [2] [3] [4] [5] [6] 下一页