The presence of protein aggregates in the nervous system is associated with various pathological conditions, yet their contribution to disease mechanisms is poorly understood. One type of aggregate, the aggresome, accumulates misfolded proteins destined for degradation by the ubiquitin-proteasome pathway. Peripheral myelin protein 22 (PMP22) is a short-lived Schwann cell (SC) protein that forms aggresomes when the proteasome is inhibited or the protein is overexpressed. Duplication, deletion, or point mutations in PMP22 are associated with a host of demyelinating peripheral neuropathies, suggesting that, for normal SC cell function, the levels of PMP22 must be tightly regulated. Therefore, we speculate that mutant, misfolded PMP22 might overload the proteasome and promote aggresome formation. To test this, sciatic nerves of Trembler J (TrJ) neuropathy mice carrying a leucine-to-proline mutation in PMP22 were studied. In TrJ neuropathy nerves, PMP22 has an extended half-life and forms aggresome-like structures that are surrounded by molecular chaperones and lysosomes. On the basis of these characteristics, we hypothesized that PMP22 aggresomes are transitory, linking the proteasomal and lysosomal protein degradation pathways. Here we show that Schwann cells have the ability to eliminate aggresomes by a mechanism that is enhanced when autophagy is activated and is primarily prevented when autophagy is inhibited. This mechanism of aggresome clearance is not unique to peripheral glia, because L fibroblasts were also capable of removing aggresomes. Our results provide evidence for the involvement of the proteasome pathway in TrJ neuropathy and for the role of autophagy in the clearance of aggresomes.

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The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Peripheral myelin protein 22 (PMP22) is a hereditary neuropathy-linked, short-lived molecule that forms aggresomes when the proteasome is inhibited or the protein is mutated. We previously showed that the removal of pre-existing PMP22 aggregates is assisted by autophagy. Here we examined whether the accumulation of such aggregates could be suppressed by experimental induction of autophagy and/or chaperones. Enhancement of autophagy during proteasome inhibition hinders protein aggregate formation and correlates with a reduction in accumulated proteasome substrates. Conversely, simultaneous inhibition of autophagy and the proteasome augments the formation of aggregates. An increase of heat shock protein levels by geldanamycin treatment or heat shock preconditioning similarly hampers aggresome formation. The beneficial effects of autophagy and chaperones in preventing the accumulation of misfolded PMP22 are additive and provide a potential avenue for therapeutic approaches in hereditary neuropathies linked to PMP22 mutations.

Accumulation of misfolded proteins and alterations in the ubiquitin-proteasome pathway are associated with various neurodegenerative conditions of the CNS and PNS. Aggregates containing ubiquitin and peripheral myelin protein 22 (PMP22) have been observed in the Trembler J mouse model of Charcot-Marie-Tooth disease type 1A demyelinating neuropathy. In these nerves, the turnover rate of the newly synthesized PMP22 is reduced, suggesting proteasome impairment. Here we show evidence of proteasome impairment in Trembler J neuropathy samples compared with wild-type, as measured by reduced degradation of substrate reporters. Proteasome impairment correlates with increased levels of polyubiquitinated proteins, including PMP22, and the recruitment of E1, 20S and 11S to aggresomes formed either spontaneously due to the Trembler J mutation or upon proteasome inhibition. Furthermore, myelin basic protein, an endogenous Schwann cell proteasome substrate, associates with PMP22 aggregates in affected nerves. Together, our data show that in neuropathy nerves, reduced proteasome activity is coupled with the accumulation of ubiquitinated substrates, and the recruitment of proteasomal pathway constituents to aggregates. These results provide novel insights into the mechanism by which altered degradation of Schwann cell proteins may contribute to the pathogenesis of certain PMP22 neuropathies.

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Western blot analysis (e) reveal that whereas the 30 kDa subunits remains unchanged (arrowheads), levels of the 25 kDa 20Sa and 20Sb subunits (arrows) are reduced in the TrJ, compared with Wt. GAPDH is shown as a constitutive marker. Molecular mass is given on the left, in kDa.

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Here we propose the use of adeno-associated virus (AAV) vectors as a non-invasive vehicle for the nervous system to deliver genes to spinal motoneurons, based on their retrograde transport from muscle. Long-term protein expression in lower cervical motoneurons was achieved after injections of AAV into the triceps, independently of serotypes 1, 2, or 5. Muscle injections of AAV5-neurotrophin 3 (NT3) resulted in a significant increase in the levels of NT3 in the cervical enlargement, compared to those obtained after injections of AAV5-GFP. Following a dorsal lesion at C4/C5, animals injected with AAV5-NT3 made fewer errors (footslips) in the horizontal ladder test compared to those injected with AAV5-GFP. In parallel, the number and length of corticospinal tract (CST) fibers circumventing the injury site were significantly increased in rats injected with AAV5-NT3. Compared to controls, we observed less astrogliosis and less CST axonal retraction and/or enhanced sprouting in animals injected with AAV5-NT3. In sum, we demonstrate here that the delivery of nt3 via retrograde transport of AAV from triceps to cervical motoneurons leads to reduced functional loss and anatomical reorganization of the CST following injury, without introducing additional injury to the spinal cord.