In sum, RNA-Seq analysis of motor neurons in transgenic G85R SOD-YFP ALS animals at a presymptomatic timepoint identifies a small degree of change of only a few mRNAs, even though aggregation is already occurring in some motor neuron cell bodies, with surrounding astrogliosis, and there are morphologic NMJ abnormalities. Although it cannot be excluded that one or more of these changes, for example depression of the mRNA for Pla2g4e or other calcium-related genes identified here, could be a primary driver of mutant SOD1-linked disease, it seems more likely that the few changes at the RNA level observed here are secondary responses to the effects of high level expression of the misfolded protein in motor neurons. For example, the elevation of mRNA for the cytosolic molecular chaperone, Hsp110, is likely a stress response to the misfolded cytosolic mutant SOD1. The profile observed here contrasts with the substantial changes in mRNA biogenesis reported in the setting of TDP43 or FUS-linked pathogenesis, where both levels and splicing of many RNAs are affected 
An earlier microarray study of RNA from laser captured motor neurons from presymptomatic G37R SOD1, as well as G85R transgenic mice likewise observed a relatively small number of genes that exhibited altered transcript levels 
. Changes were observed in the D/L serine biosynthesis pathway, but these were specific to G37R. Changes in complement components were observed in both strains. In the present study, we detected these RNAs, but only C1qa differed significantly between G85R and wild-type animals. A microarray study of RNA from laser dissected spinal motor neurons of G93A SOD1 mice at three time points in the development of disease also found a relatively small number of changes relative to non-transgenic litter mates, particularly at later stages of disease 
. No significant commonalities between either of the two previous studies 
and the study here were apparent, except that all found one or more components of the complement system upregulated. We validated this change in C1qa via qRT-PCR, suggesting that this difference might be involved in pathogenesis. It is not clear, however, whether this is a motor neuron derived transcript or a contaminant from inadvertently dissected surrounding activated glia, given that C1q has been implicated in neuroinflammation. Another microarray study, using laser captured motor neurons from cranial nerve nuclei and cervical spinal cord of presymptomatic G93A rats, focused on transcription differences between these regions that might account for the sparing of cranial nerves 3/4 from disease 
. Notably, IGF-II and guanine deaminase RNAs were preferentially expressed in cranial nerve 3/4 motor neurons of G93A rats, but no comparisons were made with expression patterns in wild-type animals.
The previous laser capture studies used early versions of mouse microarrays with only single probes to several genes we have identified here, such as Pla2g4e, making them relatively insensitive to changes in such genes. In addition, these array experiments would not have been able to detect any previously unannotated transcripts such as those we observed here at the 3′-end of Limk1 and Gak, both of which could be of further interest. For example, loss of Limk1 results in the regression of presynaptic motor neuron termini in Drosophila and altered dendritic spines in mice 
. Moreover, Limk1 transcripts are translated locally in dendrites of cultured hippocampal neurons and regulated through the 3′-UTR by miR134 
. The extension of the 3′-UTR we have observed could introduce additional regulatory sequences affecting the maintenance of neuromuscular junctions and/or dendritic spines. The role of Gak (auxilin 2) in neurons is less well defined, but it contains an auxilin-type J-domain and interacts with Hsc70 to support clathrin uncoating and vesicle cycling in non-neuronal tissues, where it is the only auxilin 
. It is upregulated in brains of auxilin knockout mice and supports sufficient uncoating activity to permit some animals to survive 
; in contrast, Gak knockout is an early post-natal lethal 
Given the small number of changes observed here in a mutant SOD1-linked setting, it seems more likely that the primary effect(s) of the mutant SOD1 protein either lie at the level of translation or are post-translational. Post-translational effects, in particular, could involve interaction between the misfolded mutant SOD1 and cellular cytosolic or membrane proteins, which could affect their roles in macromolecular traffic, organellar function, and/or synaptic function. Further translational, morphologic, and biochemical analyses may be able to address how the mutant SOD1 protein drives motor neuron pathology.