We have already provisionally ruled out reductions in steady-state levels of WldS or levels of NMNAT2 after proteasome inhibition as downstream consequences of such off-target inhibition, given their critical axonal survival and maintenance functions. Interestingly, U0126 has previously been shown to reduce ATP levels in cultured cells resulting in an increased AMP:ATP ratio and activation of AMPK via what appears to be a MEK-independent mechanism. Since NVP-BKM120 PI3K inhibitor declining ATP levels might contribute to the initiation or execution of Wallerian degeneration, a U0126-mediated reduction in ATP could thus account for its effects on preservation of transected axons, although PD184352 may have similar offtarget effects in some cell types. Interestingly, mitochondrial ATP production and Ca2+ buffering have respectively been shown to be enhanced in WldS mice and in transgenic flies expressing WldS. Given the established link between mitochondrial Ca2+ levels and ATP generation, this raises the possibility that the critical off-target effect of U0126 in this study might be to influence mitochondrial Ca2+ homeostasis in some way. However, a recent report suggesting that mitochondria are not required for WldS-mediated axon protection in flies, seems to challenge this idea. Our finding that U0126 does not affect the short-term maintenance and survival of uninjured WldS neurites is in agreement with the previous finding that it only impacts delayed degeneration of severed or otherwise compromised wild-type neurites. Irrespective of any off-target effects of U0126, this clearly indicates that loss of ERK1/2 and ERK5 Y-27632 signaling is not sufficient to induce spontaneous degeneration of intact neurites. Declining ERK1/2 phosphorylation, which appears to precede loss of total ERK1 in transected wild-type neurites, could nevertheless still contribute to the progression of Wallerian degeneration, but the possibility that this is simply an early consequence of the degeneration process itself also cannot be ruled out. We conclude that MEK-ERK signaling, specifically through MEK1/2 or MEK5, is not required for the preservation of transected neurites by WldS or proteasome inhibition. Rather, the widely-used MEK inhibitor U0126 appears to reverse this protection via an as yet unidentified target. Importantly, this study highlights the risk of interpreting results based solely on data obtained with this compound. Reassessment of findings using more selective MEK1/2 and MEK5 inhibitors, such as PD184352 and BIX02189, should be performed as standard and could lead to important new insights into cellular signaling. Human parechovirus, a small, round-structured, non-enveloped virus with a singlestranded and positive-sense RNA genome, belongs to the Picornaviridae. HPeV is structurally similar to other picornaviruses because of its icosahedral symmetry and appearance on electron microscopy. It was first described in 1961 as echoviruses 22 and 23 of the genus Enterovirus on the basis of serology and clinical presentation on identification from an outbreak of diarrhea among children. However, further studies showed that properties of the virus, such as nucleotide sequence in replication and translation elements, differ from other members of the genus Enterovirus. So the virus was re-classified into a new genus, Parechovirus, and the echoviruses 22 and 23 were re-named HPeV1 and HPeV2, respectively. During the past decade, several other HPeV isolates have been reported; to date, we have the full genome sequences for eight HPeV types, HPeV1 to HPeV8, and eight other types, HPeV9 to HPeV16, are known based on their viral protein 1 sequences.