amyotrophic lateral sclerosis; post-translational modiﬁcation; SUMO; excitotoxicity
Dysregulation of glutamate handling ensuing down regulation of expression and activity levels of the astroglial gluta-mate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis(ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artiﬁcially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.
The paper was severely critiqued on several grounds. Some of the items that featured in the discussion include:
- They talk about the nuclear localisation of carboxy terminus of EAAT2, through immunoflourescence studies, in-vitro and in-vivo in the form of puncta reported overlapping with the DAPI (nuclear) staining. However, the figures do not really support it; the intensity of EAAT2 (ABR556) fluorescence in the nucleus was too less to reach to an emphatic conclusion.
- It was suggested that it would have been nice if along with/instead of staining isolated nuclei specifically, they could produce some electron microscopic images depicting the nuclear localisation of the CTE-SUMO1 fragment with the PML-NB.
- In the third figure of the article they represent the localisation of EAAT2 in the nucleus of the transgenic (SOD1) astrocytes. However, the GFAP staining also seems to become more nuclear rather than cytoplasmic! Since GFAP is expressed pan-cytoplasmically in the glia and not in the nucleus, the results seem to be inappropriately extrapolated.
- Experiments regarding the impairment of axonal growth convey little inference and way too much confidence; since in a culture, differentiating an axon among the neurites becomes an issue.
- The role of upregulated Netrin in adult astrocytes, as reported with their microarray and Q-real time PCR data, was strongly debated in the discussion.
- While they report previously that SOD1 mutations trigger the caspase 3 activation that degrades EAAT2 in two fragments viz. truncated EAAT2 and Carboxy terminus of EAAT2- which is sumoylated and targetted to the PML- NB; the fact that SOD1 mutations are not the universal feature of ALS makes their conclusive linkage to a central pathogenic mechanism seem rather hasty. Perhaps autopsy studies on human spinal cord tissues would have validated their claim.
It was suggested that a summary of the lacunae be sent to the editor.