This study provides the first evidence that axonal swelling and degeneration persists for years after TBI in humans. These axonal swellings were consistently found to contain accumulations of Aβ and the proteins necessary for its generation, including APP, BACE and PS-1. Although Aβ plaques were found shortly after TBI as has been reported earlier (20
), almost no Aβ plaques were found months to years after injury, despite apparent Aβ production in axons. This finding was a surprise considering that progressive and escalating plaque pathology after injury was anticipated. To the contrary, these data suggest that plaques may actually regress over time after TBI. If so, this process may be mediated via proteolysis of Aβ by neprilysin, for which immunoreactivity was also increased in injured brains. These collective data provide the first evidence that TBI in humans triggers progressive axonal degeneration in association with the anabolism and possible catabolism of Aβ for years after injury. However, the absence of Aβ plaques in long-term survivors of TBI fails to provide a pathologic mechanism that might explain the epidemiological association between TBI and AD.
Axonal injury is one of the most frequently found, and arguably most important, pathological findings following TBI (3
). It is characterized by the presence of axonal swellings, either as discrete bulb formations or as elongated varicosities. As the axon undergoes cytoskeletal disorganization and progressive protein accumulation, secondary axotomy will eventually result with swollen axonal bulbs seen at the axon terminal (34
). The heterogeneous nature of TBI is such that axonal pathology may be attributed to not only the direct traumatic mechanical disruption of axons, but also potentially to secondary acute changes such as raised intracranial pressure or compromised vasculature and resultant hypoxia (12
). However, a far greater number of cases than those evaluated in the present study would be necessary to determine the relative contribution of primary and secondary pathologic processes on the extent of axonal pathology and protein accumulation.
APP and NF (transported by fast and slow axonal transport, respectively) are useful markers of protein accumulation associated with axonal pathology (8
). Using these markers, swollen and disconnected axons were found in virtually all the TBI cases examined. It is generally believed that this traumatic axonal pathology is a relatively acute feature of brain trauma, being completely cleared after a few months following injury (1
). Here it is shown that axonal pathology actually persists for at least 3 years after injury. However, the degree of axonal pathology found within the long-term survival group was generally less extensive than those cases who survived for a shorter duration, as has previously been shown in a pig model of DAI (9
). Why axons should continue to swell and disconnect over such a protracted time course is not clear. The long duration and persistent nature of this pathology suggests that TBI can induce a progressive neurodegenerative process. Thus, there may be two phases of axon degeneration after TBI; (i) an immediate and reactive process of axons undergoing acute degeneration subsequent either to devastating mechanical disruption or secondary pressure/vascular complications; and (ii) a delayed process in axons that, despite remaining relatively intact shortly after injury, are primed to undergo degeneration even years later through unknown mechanisms. It may be that this is a form of wallerian degeneration that has not been described previously.
This chronic axon degeneration is also associated with a patchy loss of myelin staining, potentially related to short- and long-term death of oligodendrocytes that has previously been observed after TBI in humans (42
). Additionally, there appears to a be long-term inflammatory response after TBI, with the identification of macrophages/microglia in brain tissue even years after injury, as has previously been observed (13
). However, it remains unknown whether these phagocytic cells are simply clearing debris resulting from ongoing axonal degeneration or are actively involved in the degenerative process (10
A final step towards axon degeneration in TBI is failed axonal transport, a process that has recently gained momentum as a mechanism for several neurodegenerative diseases, including AD (39
). The current data demonstrate that for TBI, the pathologic accumulation of Aβ caused by impaired axonal transport continues for at least 3 years following injury in humans. As previously shown in swine (9
), long-term axonal accumulation of Aβ in humans was accompanied by the co-accumulation of proteins necessary for its generation, including APP, BACE and PS-1. This is strongly suggestive that the generation of Aβ is intra-axonal. Under normal conditions, APP, BACE and PS-1 are transported as distinct cargoes in axons. Disruption of axonal transport may provide a unique environment whereby pathologic accumulations of these proteins interact resulting in the production of Aβ. Indeed, recent studies have described Aβ production mediated by BACE and PS-1 in the axonal membrane compartment of peripheral nerves (25
). Although these observations are in debate (27
), a more recent study has shown that interruption of axonal transport may stimulate the proteolysis of APP, leading to the development of Aβ plaques (48
A potential intra-axonal location of Aβ metabolism is unclear when considering that Aβ is conventionally thought to be processed via the cell membrane. However, there is accumulating evidence implicating lipid rafts as important sites of Aβ processing within neuron cell bodies (10
). Considering that axons are rich with lipid rafts, this may provide a mechanism for Aβ production completely within the axonal membrane compartment after TBI.
If damaged axons provide an environment conducive to Aβ formation and accumulation, subsequent lysis or degeneration of these axons could result in the expulsion of Aβ into the parenchymal tissue. Indeed, Aβ containing plaque-like structures have been identified in brain injured tissue just hours after trauma, often close to damaged axons (17
). Additionally, elevated Aβ peptide has been identified in the cerebrospinal fluid of TBI cases (11
). Our data suggest that the anabolism of Aβ remains elevated in axons both acutely and up to 3 years after trauma. However, plaque formation appears to be minimal in those cases surviving long term.
Potential long-term regression of Aβ plaques formed acutely after TBI may reflect a change in the balance between Aβ anabolism and catabolism. In terms of catabolism, neprilysin has emerged as a primary endogenous Aβ degrading enzyme (23
) and has increasingly been implicated in the pathogenesis of AD (for review, see (22
)). Neprilysin is a transmembrane glycoprotein whose C-terminal catalytic site lies on the extracellular surface (38
). Here extensive neprilysin immunoreactivity was found in the soma of the majority of long-term survival cases as well as being identifiable in swollen axonal bulbs. Interestingly, these cases comprise the same group that have a virtual absence of Aβ plaques. These findings may reflect a long-term process involving an upregulation of neprilysin that continually clears both intracellular and of extracellular Aβ, thereby promoting plaque regression.
What stimulates neprilysin production is not well understood, although a recent study demonstrated that amyloid intracellular domain, a cytoplasmic fragment generated from PS-1 dependant cleavage of Aβ, acts as a mediator of neprilysin expression at a transcriptional level (32
). Thus the persistent generation of Aβ may trigger a feedback loop that results in its own clearance. However, it is unclear whether this process can occur in response to Aβ production within the axonal compartment.
If neprilysin is playing a role in Aβ clearance after trauma, variations in this mechanism may explain why some TBI patients form plaques while others do not. Further work is required to clarify the exact role neprilysin is playing under post-traumatic conditions and how neprilysin effects Aβ accumulation, not only in relation to TBI, but also how it may contribute to the development of AD. It may be such that aberrations of neprilysin mediated Aβ catabolism combined with TBI as an environmental trigger produces a unique subset of TBI patients who go on to develop AD.
Overall, our data suggest a long-term process of Aβ metabolism initiated by TBI. Ongoing axonal pathology appears to supply all of the essential elements for both the anabolism and catabolism of Aβ. However, the absence of Aβ plaques in the brains of long-term survivors for up to 3 years after TBI does not support the premise that plaque pathology progressively fulminates over time, ultimately resulting in the clinical manifestations of AD. To the contrary, based on the present data, Aβ plaques formed in the initial weeks after injury may actually regress with time. Here, a continuously renewed supply of Aβ in degenerating axons may be kept in check through degradation by endogenous mediators such as neprilysin. Nonetheless, our data do not rule out the possibility that in some individuals with TBI, the balance between Aβ anabolism and catabolism eventually shifts during aging, accounting for the epidemiologic evidence of a link between TBI and AD.