Retrograde Activation of the Extrinsic Apoptotic Pathway in Spinal-Projecting Neurons after a Complete Spinal Cord Injury in Lampreys

Abstract

Spinal cord injury (SCI) is a devastating condition that leads to permanent disability because injured axons do not regenerate across the trauma zone to reconnect to their targets. A prerequisite for axonal regeneration will be the prevention of retrograde degeneration that could lead to neuronal death. However, the specific molecular mechanisms of axotomy-induced degeneration of spinal-projecting neurons have not been elucidated yet. In lampreys, SCI induces the apoptotic death of identifiable descending neurons that are “bad regenerators/poor survivors” after SCI. Here, we investigated the apoptotic process activated in identifiable descending neurons of lampreys after SCI. For this, we studied caspase activation by using fluorochrome-labeled inhibitors of caspases, the degeneration of spinal-projecting neurons using Fluro-Jade C staining, and the involvement of the intrinsic apoptotic pathway by means of cytochrome c and Vα double immunofluorescence. Our results provide evidence that, after SCI, bad-regenerating spinal cord-projecting neurons slowly degenerate and that the extrinsic pathway of apoptosis is involved in this process. Experiments using the microtubule stabilizer Taxol showed that caspase-8 signaling is retrogradely transported by microtubules from the site of axotomy to the neuronal soma. Preventing the activation of this process could be an important therapeutic approach after SCI in mammals.

Role of Caspase-8 and Fas in Cell Death After Spinal Cord Injury

“…Some of these neurons are classified as “good regenerators” (i.e., they regenerate their axon more than 55% of the times) and others are considered “bad regenerators” (i.e., they regenerate their axon less than 30% of the times; Figure 1C ; Jacobs et al, 1997 ). In recent years, mounting evidence has confirmed that descending neurons of lampreys known to be “bad regenerators” suffer a process of delayed death and are also “poor survivors” after a complete SCI (Figure 1C ; Shifman et al, 2008 ; Barreiro-Iglesias and Shifman, 2012 , 2015 ; Busch and Morgan, 2012 ; Hu et al, 2013 , 2017 ; Zhang et al, 2014 ; Barreiro-Iglesias, 2015 ; Fogerson et al, 2016 ; Barreiro-Iglesias et al, 2017 ). The occurrence of cell death in a subset of identifiable descending neurons after SCI in lampreys was confirmed based on the disappearance of Nissl staining (Figure 1C ), the loss of neurofilament expression, the absence of labeling when using retrograde tracers (Shifman et al, 2008 ), and the early staining of these neurons with Fluoro-Jade C (Busch and Morgan, 2012 ; Barreiro-Iglesias et al, 2017 ), which is a marker for degenerating neurons.…”

“…In recent years, mounting evidence has confirmed that descending neurons of lampreys known to be “bad regenerators” suffer a process of delayed death and are also “poor survivors” after a complete SCI (Figure 1C ; Shifman et al, 2008 ; Barreiro-Iglesias and Shifman, 2012 , 2015 ; Busch and Morgan, 2012 ; Hu et al, 2013 , 2017 ; Zhang et al, 2014 ; Barreiro-Iglesias, 2015 ; Fogerson et al, 2016 ; Barreiro-Iglesias et al, 2017 ). The occurrence of cell death in a subset of identifiable descending neurons after SCI in lampreys was confirmed based on the disappearance of Nissl staining (Figure 1C ), the loss of neurofilament expression, the absence of labeling when using retrograde tracers (Shifman et al, 2008 ), and the early staining of these neurons with Fluoro-Jade C (Busch and Morgan, 2012 ; Barreiro-Iglesias et al, 2017 ), which is a marker for degenerating neurons. In addition, the appearance of TUNEL staining (Shifman et al, 2008 ; Hu et al, 2013 ) and activated caspases (Figure 1C ; Barreiro-Iglesias and Shifman, 2012 , 2015 ; Hu et al, 2013 ; Barreiro-Iglesias et al, 2017 ) in the soma of axotomized descending neurons suggests that their death after SCI is apoptotic.…”

“…The occurrence of cell death in a subset of identifiable descending neurons after SCI in lampreys was confirmed based on the disappearance of Nissl staining (Figure 1C ), the loss of neurofilament expression, the absence of labeling when using retrograde tracers (Shifman et al, 2008 ), and the early staining of these neurons with Fluoro-Jade C (Busch and Morgan, 2012 ; Barreiro-Iglesias et al, 2017 ), which is a marker for degenerating neurons. In addition, the appearance of TUNEL staining (Shifman et al, 2008 ; Hu et al, 2013 ) and activated caspases (Figure 1C ; Barreiro-Iglesias and Shifman, 2012 , 2015 ; Hu et al, 2013 ; Barreiro-Iglesias et al, 2017 ) in the soma of axotomized descending neurons suggests that their death after SCI is apoptotic. The detection of activated caspase-8 in the first 2 weeks after the injury (Figure 1B ; Barreiro-Iglesias and Shifman, 2012 ; Barreiro-Iglesias et al, 2017 ) and the lack of cytochrome-c release from mitochondria (Barreiro-Iglesias et al, 2017 ) indicates that the extrinsic apoptotic pathway is activated in descending neurons of lampreys after SCI.…”

“…The survival ability is expressed as the percentage of times that a given neuron shows Nissl staining 1 year after a complete SCI (from Shifman et al, 2008 ). The bottom graph shows a significant correlation between the level of activated caspases (fluorescence intensity, FI) 2 wpl (from Barreiro-Iglesias et al, 2017 ) and the regenerative (from Jacobs et al, 1997 ) and survival abilities (from Shifman et al, 2008 ) of identifiable descending neurons. P -values of Pearson correlation are 0.0044 (Barreiro-Iglesias et al, 2017 ) and 0.0122, respectively.…”

“…Several studies have shown the activation of caspase-8 in intrinsic SC cells following SCI in rodents (Casha et al, 2001 , 2005 ; Keane et al, 2001 ; Takagi et al, 2003 ; Cantarella et al, 2010 ; Chen et al, 2011 ). More recently, work in lampreys has also shown that caspase-8 is retrogradely activated in identifiable descending brain neurons after SCI (Barreiro-Iglesias and Shifman, 2012 , 2015 ; Barreiro-Iglesias et al, 2017 ). Here, we review our current knowledge on the role of caspase-8 in cell death after SCI.…”