Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

Reviewer #1 (Public review):

Summary:

This study investigated the role of transcriptional and translational controls of gene expression in dorsal root ganglia and lumbar spinal cord in neuropathic pain in mice. Using ribosome profiling (Ribo-seq) and translating ribosome affinity purification (TRAP), they show changes in transcriptomic and translational gene expression at the peripheral and central levels rapidly after nerve injury. While translational changes in gene expression remained elevated for more than two months in both DRGs and the spinal cord, transcriptomic regulation was absent in the spinal cord long after the onset of neuropathy. Disrupting mRNA translation in dorsal horn neurons using antisense oligonucleotides reduced mechanical withdrawal threshold and facial expression of pain. Using fluorescent noncanonical amino acid tagging (FUNCAT), the authors further show that de novo protein expression primarily occurs in inhibitory neurons in the superficial dorsal horn after nerve injury. Accordingly, a selective increase in translational control of gene expression in spinal inhibitory neurons, or a subset of mainly inhibitory neurons expressing parvalbumin (PV), using transgenic mice, led to a decrease in the excitability of PV neurons and mechanical allodynia. In contrast, decreasing the translational control of spinal PV neurons prevented the alteration of the electrophysiological properties of the PV cells induced by nerve injury.

Strengths:

This is a well-written article that uncovers a previously unappreciated role of gene expression control in PV neurons, which seems to play an important part in the loss of inhibitory control of spinal circuits typically seen after peripheral nerve injury. The conclusions are generally well supported by the data.

Weaknesses:

The study would benefit from further clarifications in the methods section and a deeper analysis of gene expression changes in mRNA expression and ribosomal footprint observed after nerve injury.

Antisense oligonucleotides used to reduce translation by disrupting eIF4E expression were administered i.c.v. It is unknown if the authors controlled for locomotor deficits, which might add confounds in the interpretation of behavioral results. A more local route should have been preferable to avoid targeting brain regions, which could potentially affect behavior.

Only female mice were used for Ribo-Seq, TRAP, FUNCAT, and electrophysiology, but both sexes were used for behavior experiments.

The conditional KO of 4E-BP1 using transgenic animals should be total in the targeted cells. However, only a partial reduction is reported in Figure S2 in GAD2, PV, Vglut2, or Tac1 cells. Again, proper methods for quantification of fluorescence in these experiments are lacking.

The elegant knockdown of eIF4E using AAV-mediated shRNAmir shows a recovery of the electrophysiological intrinsic properties of PV neurons after injury. It is unclear if such manipulation would be sufficient to reverse mechanical allodynia in vivo.

Reviewer #2 (Public review):

Summary:

I reviewed the manuscript titled "Translational Control in the Spinal Cord Regulates Gene Expression and Pain Hypersensitivity in the Chronic Phase of Neuropathic Pain." This manuscript compares transcription and translation in the spinal cord during the acute and chronic phases of neuropathic pain induced by surgical nerve injury. The authors chose to focus their investigation on translation in the chronic phase due to its greater impact on gene expression in the spinal cord compared to transcription.

(1) The study is significant because the molecular mechanisms underlying chronic pain remain elusive. The role of translational regulation in the spinal cord has not been investigated in neuroplasticity and chronic pain mouse models. The manuscript is innovative and technically robust. The authors employed several cutting-edge techniques such as Rio-seq, TRAP-seq, slice electrophysiology, and viral approaches. Despite the technical complexity, the manuscript is well-written. The authors demonstrated that inhibition of eIF4E alleviates pain hypersensitivity, that de novo protein synthesis is more pronounced in inhibitory interneurons, and that manipulating mTOR-eIF4E pathways alters mechanical sensitivity and neuroplasticity.

(2) Strengths: innovation (conceptual and technical levels), data support the conclusions.

Weakness:

Confusion about the sex of the animals. It is unclear whether eIF4E ASO affects translation and which cells. It is not determined that modulating translation in PV+ neurons impacts neuropathic pain behaviors.

Reviewer #3 (Public review):

Summary:

This study provides evidence for translational changes in inhibitory spinal dorsal horn neurons following chronic nerve injury. Gene expression changes have been widely studied in the context of pain induction and provided key insights into the adaptation of the nervous system in the early phases of chronic pain. Whereas this is interesting biologically, most patients will arrive in the clinic beyond the acute phase of their injury, thus limiting the translational relevance of these studies. Recent studies have extended this work to highlight the difference between acute and chronic pain states, potentially explaining the cascading factors leading to chronic pain, and hopefully how to prevent this in vulnerable populations. The present study suggests that translational changes within spinal inhibitory populations could underlie long-term chronic pain, leading to decreased inhibition and heightened pain thresholds.

Strengths:

The approaches used and the broad outcomes of the manuscript are interesting and could be an exciting development in the field. The authors are using approaches more common in molecular biology and extending these into neuroscientific research, getting into the detail of how pathology could impact gene expression differentially across the course of an injury. This could open up new areas of research to selectively target not only defined populations but additionally help alleviate pain symptoms once an injury has already reached the maintenance phase. There is an opportunity to delve into what must be a very large data set and learn more about what genes are differentially translated and how this could affect circuit function.

Weaknesses:

Whereas the authors approach a key question in pain chronicity, the manuscript falls a little short of providing any conclusive data.

The manuscript was in some areas very difficult to follow. Terminology was not always consistent or clear, and the flow of the manuscript could use some attention to highlight key areas. Whereas the overall message is clear in the summary, this would not necessarily be the case when reading the manuscript alone.

The study claims to show that translational control mechanisms in the spinal cord play a role in mediating neuropathic pain hypersensitivity, but the studies presented do not fully support this statement. The authors instead provide some correlation between translation and behavioural reflex excitability (namely vfh and Hargreaves).

It is difficult to fully interpret the work, as there are a number of inconsistencies, namely the range of timings pre- and post-injury, lack of controls for manipulations, the use of shmiRNA versus lineage deletions, and lack of detailed somatosensory testing. It is not completely clear how this work could be translatable as is, without a deeper understanding of how translational control affects circuit function and whether all of this is necessarily bad for the system, or whether this is a positive homeostatic adaptation to the hyperexcitability of the circuit following injury.

A large portion of the work is focussed on showing an inhibitory-selective change in translation following chronic nerve injury. The evidence for this is however lacking. Statistics to show that translational effects are restricted to inhibitory subpopulations are inadequate. The author's choice of transgenic lines is not clear and seems to rely on availability rather than hypothesis.