Neural regeneration requires detection of axonal injury by retrograde signaling from the injured site to the cell body in order to induce those genes whose activity is necessary for the initiation, formation, elongation, and guidance of growing axons as well as reformation of specialized contacts with targets. Most growth-associated proteins are induced in the soma and transported orthogradely to the growth cone, but several transcripts are also translated locally in the axon, including Hsp27 (32
). Some growth-associated proteins constitute the actual cytoskeletal molecular machinery necessary for extension of growth cones (31
) while others enable growth cones to interact with guidance cues in the environment to regulate the rate and direction of growth (34
) and may also detect growth inhibitory cues.
While axonal growth after nerve injury in the adult may recapitulate some of the processes used during the development of the nervous system, they are not mechanistically identical (35
). Unlike mammalian CNS neurons, primary sensory and motor neurons can regenerate after axonal injury in the adult, and this provides an opportunity for identifying which injury-induced genes are necessary and the nature of permissive growth environments. These neurons can be primed into an active growth state by a preconditioning peripheral nerve lesion (10
), after which the rate and extent of axon formation is greatly increased as a result of induction by the preinjury of the proteins required for growth. Our data now suggest that Hsp27 plays a major role in enabling axons to grow and in the conditioning effect.
Hsp27 was identified by its capacity to promote survival in heat-stressed cells (37
). In addition to a classic chaperone function, which contributes to its actions after heat shock, Hsp27 interacts with proapoptotic proteins to reduce apoptosis (38
), a function it also subserves in injured adult sensory and motor neurons (16
). Hsp27 regulates actin dynamics and contributes thereby to cell motility, migration, contraction (19
), and, as we now show, axonal elongation. This actin-interacting role involves Hsp27 cycling from its dephosphorylated to its phosphorylated form through p38 MAPK (19
), for which Hsp27 is a major substrate (20
), and our data indicate that inhibition of MK2 is sufficient to block the growth-promoting effects of Hsp27 in vitro and in vivo.
mHsp25 is induced in injured sensory neurons, and its phosphorylated form is present at the tip of filopodia, the sole site of actin monomer addition in growth cones. Therefore, Hsp27 is well positioned anatomically to regulate filopodial cytoskeletal dynamics and facilitate growth cone locomotion during regeneration. We demonstrate that forced expression of hHsp27 in uninjured mouse neurons increased intrinsic axonal growth and that it may also be required for such growth as indicated from mHsp25 knockdown. Our data point to a pivotal role for Hsp27 in increasing the intrinsic growth of injured sensory neurons and show that its expression accelerates the rate of functional reinnervation of injured sensory neurons in damaged peripheral nerves.
Peripheral nerve injury results in a massive alteration in transcription (11
) mediated by activation of several transcription factors, Atf3
, and Sox11
, all of which increase regeneration (13
). These genes also contribute to overcoming the metabolic stress of the axonal injury, promote cell survival by inhibiting apoptosis, and alter membrane excitability and synaptic transmission as well as promoting axon regrowth (13
). What is not clear, though, is whether these functional changes are independently regulated. We found that the genes most highly coassociated with the regulation of Hsp27 are highly enriched for growth and antiapoptotic functions, with few candidate neuropathic pain genes, suggesting that the genes upregulated or downregulated after nerve injury are linked into functionally coherent modules or networks.
Increasing the intrinsic growth status of injured neurons, although necessary for successful regeneration is not by itself sufficient. Neurons also need a permissive growth environment, such as that provided in peripheral nerves by Schwann cells and laminin (4
). However, permissive growth environments also are by themselves not adequate for regeneration, because most unprimed neurons do not have enough intrinsic growth capacity to drive full regeneration (43
). Furthermore, if distal nerve segments lose Schwann cells, as occurs after chronic denervation of distal nerves for many months, this provides limited support for axonal growth, and regeneration is reduced or halted (45
). The extent of motor recovery in the rat reduces over time after chronic denervation, and early nerve repair is required for muscle strength to return (8
). Although this finding aligns with our data, it was assumed to be the effect of decreased axonal regeneration in the nerve distal stump due to Schwann cell atrophy after prolonged denervation (8
), while our data from the plantar muscles of the mouse points to another more specific defect, failure of reinnervation of the motor end plate.
Recovery after injury of peripheral nerves in humans is more limited than in rodents, because the distance to the target is longer and the intrinsic axonal growth rate is slower (48
). Major factors recognized as influencing the outcome of peripheral nerve repair are the degree of injury, the age of the patient, type of nerve, level of injury, and the timing of repair, the last of which is particularly related to our findings. Proximal injuries, such as brachial plexus injuries, show only very limited motor recovery for wrist and finger flexors and none for intrinsic muscles (5
). Delay in surgery of over 6 months significantly reduces the extent of motor recovery, and it is considered clinically fruitless to expect motor recovery if an operation is delayed for more than a year (50
). However, sensation commonly recovers even though the quality may be defective, pointing to a clear difference in functional sensory and motor recovery. Ninety percent of patients with brachial plexus injury, for example, recover protective sensation in their fingers in the absence of any meaningful recovery in thenar muscles (49
). This agrees well with our data, showing substantial sensory but only minimal motor recovery after a SNT injury. We show, in addition, data suggestive of a critical period of 10 to 12 months in patients for recovery of motor function after denervation, while in the mouse we found the cut off for plantar muscles to be 4–6 weeks. Several mechanisms could potentially be responsible for the failure of reinnervation of the motor end plate that we found for plantar muscles in mice after a critical period of 5 to 6 weeks (Figure and Figure A). The first could be a loss or reduction in the intrinsic growth capacity of injured motor neurons over time, such that growth of their axonal tip ceases before they reach the end plate. This seems unlikely, however, since motor neurons appear to be capable of axonal elongation even after prolonged periods of time, provided the milieu is permissive (46
). In addition, we found that motor axons after a transection injury in WT mice eventually do grow in locations immediately adjacent to the end plate, so that they only would need a few microns of additional growth to complete the reinnervation. Furthermore, we found that sensory axons are capable of reinnervating even the most distal skin areas. Another possibility is that Schwann cells in denervated motor nerves become nonpermissive or even repellent to axon growth over time (47
) so that regenerating axons halt when they reach such denervated nerves (53
). Because we found many axons in the most distal muscle nerves in WT mice after a sciatic transection, we think this is unlikely at the time window we are looking at (~8 weeks); the nerves appear to be fully permissive for axonal regeneration, although they may certainly become less permissive at longer periods of denervation (9
). Our hypothesis is that the terminal Schwann cell in the end plate may become nonpermissive for axonal growth after prolonged denervation. Soon after denervation, these specialized cells, which are normally in contact with the axon up to the synaptic cleft, dedifferentiate and elaborate processes that spread away from the synaptic zone (56
) and then fully recover to the uninjured phenotype after reinnervation after crush injuries (57
). The terminal Schwann cell processes extending from denervated end plates act as substrates for axonal growth, assisting both reinnervation and collateral sprouting (58
). However, the number and phenotype of terminal Schwann cell can decrease with prolonged denervation (59
). A lack of terminal Schwann cells, their processes, or of the trophic and guidance molecules they express, such as TGF-β, agrin, or CNTF (58
), may contribute to reduced reinnervation. Because we found terminal Schwann cells still present in the end plate in WT mice with no reinnervation, we concluded that the failure of regrowth is not due to a simple loss of these cells at this duration of denervation. Terminal Schwann cells may begin to produce growth inhibitory molecules like Sema 3A or chondroitin sulfate proteoglycan (61
), which could block axonal growth into this site. Finally, it is well know from developmental studies that the formation of a functional synapse at the NMJ depends on a fine orchestration of signaling pathways between the nerve and the muscle. Regenerating nerve terminals may require a positive trophic signal, such as FGF, GDNF, β1
integrin, or synaptic collagens (63
), from the muscle to complete formation of the end plate, and this may diminish with denervation over extended periods of time. Future experiments are required to tease out why motor axons do not reestablish synaptic contact with muscles after a critical period of denervation.
We conclude that accelerating axonal growth by overexpressing a single gene that increases actin polymerization in growth cones can increase the rate of sensory recovery and substantially restore motor function after a complete transection/resuture of a peripheral nerve. This finding contrasts with the situation in WT mice, in which there is little return of motor function in plantar muscles after this injury, even though their slowly growing motor axons eventually reach within a few microns of the motor end plate. Surprisingly, failure of successful regeneration in this model is not due, therefore, to an absence of distal axonal growth or to the finding by axons of pathways in the distal nerve, as previously assumed (9
), but rather to the failure of formation of the presynaptic component of the NMJ. Success in recovery of motor function may occur in the hHsp27 mice, because the motor axons reach the motor end plate before this structure becomes nonpermissive for reinnervation. The molecular determinants, in muscle or nerve, responsible for preventing synapse reformation after prolonged denervation need to be established as well as whether limited permissive periods for reinnervation are restricted to these peripheral synapses. Nevertheless, based on our mouse data and clinical observations, we suggest that strategies that increase the rate of intrinsic growth in motor neurons may enhance functional recovery in patients after peripheral nerve damage.