Defects in axonal transport are implicated in a range of neurodegenerative diseases, including ALS, Huntington’s Disease, and Alzheimer Disease. Here we describe for the first time a complete mechanism for how axonal transport defects may lead to severe neurodegeneration. This mechanism shows how extracellular signaling from mSOD1-expressing glia acts through neuronal receptors to activate intracellular stress signals causing downstream activation of stress responses in the neuronal nucleus.
We used in-vivo, in-vitro and live cell imaging assays to fully characterize the axonal transport defects in the SOD1G93A model of familial ALS. We also found that mouse models with impaired dynein function, Loa and Tgdynamitin show similar decreased efficiency of retrograde transport but unlike the mSOD1 model develop only mild neurodegeneration. Therefore, the slowing of neurotrophic factor signaling is not sufficient to induce pronounced neuronal loss. Instead, in the mSOD1 model in the early pre-symptomatic stage, there are profound changes in the nature of the cargo being transported by dynein along the axon; there is an overall shift from survival signaling to stress/death factor signaling. Inhibition of retrograde stress signaling is sufficient to delay activation of cellular stress pathways, as assessed by activation of c-Jun, and to rescue motor neurons from mSOD1-induced toxicity. These results support the model that inhibition of retrograde transport efficiency including the slowed transport of neurotrophic factors leads to only slowly progressive and mild neurodegeneration whereas alterations in the nature of the signals being retrogradely transported, from survival to stress, may leads to severe neuronal dysfunction and cell death as seen in ALS (). Still, it is not clear if axonal transport alterations are the cause for the diseases or are secondary symptoms effect of it. This reason/result question is not easy to answer. Because the axonal transport alterations are an early event, it is reasonable to speculate that these changes play a causative role. However, we cannot exclude the possibility that protein aggregation or mitochondrial dysfunction as well as other factors also play contributing roles in disease pathology. Further studies should lead to more comprehensive understanding of the machinery involved in retrograde signaling along the axon.
Here we examined the mechanisms of inhibition of retrograde transport observed in mSOD1 mice. In-vitro
microtubule gliding assays indicate that the dynein motor itself is not impaired. Instead, in-vitro
assays for vesicular motility indicate the effects of mutant SOD1 expression may be either direct or indirect, altering the regulatory balance of vesicle-associated motor proteins. For example expression of mSOD1 on the vesicle may interfere with motor-cargo binding and this may disrupt the motor regulation (Friedman and Vale, 1999
). Furthermore, mSOD1 may also activate or repress one or more signaling pathways that regulate activity of motors in-vivo
through coordination of motor activity. We have shown by live-cell imaging and in-vitro
vesicle assays that there is an increase in bidirectional movement and a reduction in unidirectional, processive movement, which may be interpreted as unregulated motor function. These observations are consistent with the suggested role of signaling molecules such as P-JNK in regulating motor activity (Morfini et al., 2006
; Horiuchi et al., 2007
). JNK interacts with kinesin and the dynein/dynactin complex via JIP and sunday driver (syd) scaffolding proteins (Bowman et al., 2000
; Verhey et al., 2001
; Yano and Chao, 2004
; Cavalli et al., 2005
). JNK is downstream of p75NTR
effectors and may have a role as a stress/death signal that leads to apoptosis. JNK may also be involved in the regulation of p75NTR
cleavage, suggesting the possibility that there is a feedforward loop involved in the local regulation of JNK activation in the axon, leading to slowed transport and thus a further enhancement of localized signaling.
Inhibition of retrograde axonal transport means that vital factors associated with the signaling endosome (Delcroix et al., 2003
), including neurotrophic factors and signaling molecules such as P-Trk, Erk1/2 and Erk5, are not effectively reaching the cell body. Impaired communication between target tissues and the neuron cell body may lead with time to the activation of programmed cell death and slow neurodegeneration. Here for the first time we show that neurotrophin inhibition is not sufficient to activate severe and rapidly progressive degeneration such as that seen in the mSOD1 model, and that activation of retrograde death/stress pathways are likely to be key.
Recent progress has suggested that the motor neuron cell death observed in the mSOD1 model results from a non-cell autonomous process (Lino et al., 2002
; Clement et al., 2003
; Boillee et al., 2006
; Di Giorgio et al., 2007
; Nagai et al., 2007
). In contrast, if expression of mSOD1 is limited to motor neurons only slow axonal degeneration is observed (Jaarsma et al., 2008
). Expression of mSOD1 may either activate or repress signaling pathways controlling cell fate. Glia cells expressing mSOD1 may release factors that will differentially trigger neuronal receptors, leading to alterations in dynein associated cargo and neurodegeneration. Indeed, a critical initiating event for the mechanism outlined above may be the differential activation of receptors like P-Trk and p75NTR
due to the expression of mSOD1 in the surrounding glial cells. We also found activation of caspase- 8 in mSOD1 neurons, as well as an increased association of caspase-8 with dynein. Interestingly, axoplasmic caspase-8 was shown to be transported back to the cell body by the dynein/dynactin complex in a p75NTR
-dependent manner causing cell death, after target removal (bulbectomy) or synaptic instability (Carson et al., 2005
). Furthermore, the Fas/p38 signaling pathway was shown to activate caspase-8 leading to cell death in ALS motor neurons (Raoul et al., 2002
). Thus, the cellular environment contributes significantly to cell death, leading to a change in the balance between survival and death receptors that enhance the progress of neurodegenerative disease as seen in the mSOD1 model.
We used a proteomics approach to examine changes in dynein cargo more broadly, focusing on changes in cellular signaling pathways. Our results suggest that there is a set of signaling factors that are altered, rather than a single pathway that is affected. These observations may explain the limited effects observed when crossing mSOD1 mice with knockouts in pathways that are thought to be important to disease pathology at the cellular level, such as p75 (Küst et al., 2003
) FasL−/− (Petri et al., 2006
) or Bax−/− (Gould et al., 2006
) null mice. In contrast, crossing Loa mice with mSOD1 mice showed some rescue effects both in axonal transport defects and in the life span of the mice (Kieran et al., 2005
). It is possible that in this cross, the mutation in dynein resulted in a delayed arrival of stress/death signals at the cell body, resulting in an extension of life span.
Partitioning of signals into the endosomal compartment may represent a key mechanism contributing to the specificity of a signal transduction pathway (Schenck et al., 2008
). The retrograde transport of the signaling endosomes may also ensure spatial and temporal regulation that controls the fidelity of specific signals. Therefore, the specific balance between positive and negative signaling in time and place may mediate the regulation of cell survival, and a change in this balance toward stress/death signaling may lead to aggressive neuronal degeneration and cell death.