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\DOI{10.5802/crbiol.169}
\datereceived{2024-09-27}
\daterevised{2024-11-05}
\dateaccepted{2024-11-08}
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\title{Axon regeneration: an issue of translation}

\alttitle{R\'{e}g\'{e}n\'{e}ration axonale : une question de traduction}

\author{\firstname{Julia} \lastname{Schaeffer}\CDRorcid{0000-0002-1282-5793}}
\address{Univ. Grenoble Alpes, Inserm, U1216, CHU Grenoble Alpes,
Grenoble Institut Neurosciences, Grenoble, France}
\curraddr[J. Schaeffer]{Aix Marseille Univ, CNRS, IBDM,
Marseille, France}
\email[J. Schaeffer]{julia.schaeffer@univ-amu.fr}

\author{\firstname{Stephane} \lastname{Belin}\CDRorcid{0000-0001-7074-6885}\IsCorresp}
\addressSameAs{1}{Univ. Grenoble Alpes, Inserm, U1216, CHU Grenoble
Alpes, Grenoble Institut Neurosciences, Grenoble, France}
\email[S. Belin]{stephane.belin@inserm.fr}

\thanks{Agence Nationale pour la Recherche (ANR-18-CE16-0007,
ANR-21-CE16-0008-02), Fondation pour la Recherche M\'{e}dicale,
UNADEV/AVISAN}

\thanks{\textbf{Note.} Julia Schaeffer received the Grandes Avanc\'ees en
Biologie 2024 award.}

\begin{abstract}
In the mammalian central nervous system (CNS), adult neurons fail to
regenerate spontaneously upon axon injury, which leads to a permanent
and irreversible loss of neuronal functions. For more than 15 years,
much effort was invested to unlock axon regrowth programs based on
extensive transcriptomic characterization. However, it is now well
described that mRNA and protein levels correlate only partially in
cells, and that the transcription process (from DNA to mRNA) may not
directly reflect protein expression. Conversely, the translation
process (from mRNA to protein) provides an additional layer of gene
regulation. This aspect has been overlooked in CNS regeneration. In
this review, we discuss the limitations of transcriptomic approaches to
promote CNS regeneration and we provide the rationale to investigate
translational regulation in this context, and notably the regulatory
role of the translational complex. Finally, we summarize our and
others' recent findings showing how variations in the translational
complex composition regulate selective (mRNA-specific) translation,
thereby controlling CNS axon regrowth.
\end{abstract}

\begin{altabstract}
Dans le syst\`{e}me nerveux central (SNC) des mammif\`{e}res, les
neurones adultes ne se r\'{e}g\'{e}n\`{e}rent pas spontan\'{e}ment
apr\`{e}s une l\'{e}sion de leur axone, ce qui entra\^{i}ne une perte
permanente et irr\'{e}versible des fonctions neuronales. Pendant plus
de 15 ans, de nombreux efforts ont \'{e}t\'{e} d\'{e}ploy\'{e}s pour
d\'{e}bloquer les programmes de r\'{e}g\'{e}n\'{e}ration des axones sur
la base d'une caract\'{e}risation transcriptomique approfondie.
Cependant, il est maintenant bien d\'{e}crit que les niveaux d'ARNm et
de prot\'{e}ines ne sont que partiellement corr\'{e}l\'{e}s dans les
cellules, et que le processus de transcription (de l'ADN \`{a} l'ARNm)
peut ne pas refl\'{e}ter directement l'expression des prot\'{e}ines.
Inversement, le processus de traduction (de l'ARNm \`{a} la
prot\'{e}ine) ajoute un niveau suppl\'{e}mentaire de r\'{e}gulation des
g\`{e}nes. Cet aspect a \'{e}t\'{e} n\'{e}glig\'{e} dans la
r\'{e}g\'{e}n\'{e}ration du SNC. Dans cette revue, nous discutons des
limites des approches transcriptomiques pour promouvoir la
r\'{e}g\'{e}n\'{e}ration du SNC et nous expliquons pourquoi il est
n\'{e}cessaire d'\'{e}tudier la r\'{e}gulation traductionnelle dans ce
contexte, en pr\'{e}sentant notamment le r\^{o}le r\'{e}gulateur du
complexe traductionnel. Enfin, nous r\'{e}sumons nos d\'{e}couvertes
r\'{e}centes et celles d'autres chercheurs, qui montrent comment les
variations de la composition du complexe traductionnel r\'{e}gulent la
traduction s\'{e}lective (sp\'{e}cifique \`{a} l'ARNm), contr\^{o}lant
ainsi la r\'{e}g\'{e}n\'{e}ration des axones du SNC.
\end{altabstract}

\keywords{\kwd{Axon regeneration} 
\kwd{Translation regulation} 
\kwd{Selective translation} 
\kwd{Translational complex} 
\kwd{Retinal ganglion cells} 
\kwd{Optic nerve injury}}

\altkeywords{\kwd{R\'{e}g\'{e}n\'{e}ration axonale} 
\kwd{R\'{e}gulation traductionnelle} 
\kwd{Traduction s\'{e}lective} 
\kwd{Complexe traductionnel} 
\kwd{Cellules ganglionnaires de la r\'{e}tine} 
\kwd{L\'{e}sion du nerf optique}}

\maketitle

\twocolumngrid

\end{noXML}

\section{CNS regeneration: an unmet need}

In adult mammals, central nervous system (CNS) neurons fail to
regenerate their axon spontaneously, in contrast to the ones from the
peripheral nervous system (PNS). This has major consequences for
patients affected by neurological disorders, whether chronic
(neurodegenerative diseases) or acute (traumatic brain or spinal cord
injuries), as this leads to permanent and irreversible loss of motor,
sensory and/or cognitive functions. To date, no treatment is available,
making CNS repair one of the biggest challenges in Neuroscience
research.

{At the cellular level, when a CNS axon is lesioned, its distal part
degenerates while the proximal forms a retraction bulb, a structure
unable to bear axon growth. Among the reasons for this regeneration
failure, the extracellular environment becomes growth-inhibitory upon
injury, due to the release of growth-repulsive factors such as
myelin-associated molecules or components of the glial
border}~\cite{Schwab2004,SilverMiller2004,YiuHe2006}. {Yet, modulation
of these inhibitory factors is not sufficient to promote robust
regeneration}~\cite{Leeetal2010}, {so researchers focused on the
intrinsic growth properties of neurons themselves. Indeed, over the
course of development, CNS neurons progressively lose the capacity to
grow an axon}~\cite{HeJin2016}. {Moreover, the injury itself further
impairs the regrowth properties of adult CNS
neurons}~\cite{Belinetal2015}, {which correlates with the \mbox{intrinsic}
downregulation of pro-growth molecular pathways. Over the past 15
years, axon regrowth of injured CNS neurons has been achieved through
experimental activation or co-activation of these pathways, for example
mTOR (mechanistic target of rapamycin)}~\cite{Parketal2008},
{JAK/STAT}~\cite{Smithetal2009}, {Klf (Kr\"{u}ppel-like
factors)}~\cite{Mooreetal2009} {or c-myc}~\cite{Belinetal2015}.

{Many of these breakthrough studies are based on the adult mouse visual
system, as it is part of the CNS.} {It is composed of the eyes, the
optic nerves and different brain nuclei that integrate and process
visual information. The axon lesion is induced mechanically by crushing
the optic nerve. This injury primarily affects retinal ganglion cells
(RGCs), which are the only retinal neurons projecting their axon to
form the optic nerve and connecting the eye to the brain}
(Figure~\ref{fig1}).\looseness=-1

\begin{figure*}
\vspace*{2pt}
\includegraphics{fig01}
\caption{\label{fig1}(A) Section of an adult retina showing specific
labeling of RGCs with specific expression of GFP under Thy1 promoter
and anti-RBPMS antibody. GCL: ganglion cell layer; INL: inner nuclear
layer; ONL: outer nuclear layer. From~\cite{Schaefferetal2020}. (B) Schematic of
optic nerve injury model. In normal conditions, RGCs project their axon
via the optic nerve to the brain. After optic nerve injury, axons
degenerate distally and fail to regrow. (C) Summary of molecular
mechanisms regulating the neuronal injury response and the intrinsic
regenerative properties of adult CNS neurons.}
\end{figure*}

{However, despite great advances in the field, many caveats remain.
First, not all neurons are responsive to such molecular manipulations,
partly owing to the heterogeneity in neuronal subpopulations. In the
adult retina, more than 30 subpopulations of RGCs have been
characterized, based on morphology, molecular markers, dendritic
arborization, primary projections}~\cite{Seabrooketal2017}, {and more recently
transcriptional programs that condition RGC regeneration and survival
after injury}~\cite{Jacobietal2022,Tianetal2022}. {Yet, modulation of such transcriptional
programs is not sufficient to promote robust regeneration, as it
remains unclear how they are integrated in the different
subpopulations. Even the most robust models of regeneration enable no
more than 20\% RGCs to regrow, which might complicate to move to the
next step: functional reconnection.}

{Second, regrowing RGCs fail to reach their proper target. Therefore,
no functional circuit has been obtained yet}~\cite{Pernetetal2013,Belinetal2015,Wangetal2020}. {This is
due to the disorganized regrowth of regenerating axons, which do not
resume their initial trajectory and cannot reinnervate their correct
brain target}~\cite{PernetSchwab2014,CrairMason2016}. {In this regard, our recent work has
provided insight into the possibility and relevance of axon guidance
modulation during regeneration, in order to reroute regrowing axons
onto the correct path}~\cite{Vilallongueetal2022} {and into the correct brain
target}~\cite{Delpechetal2024}.

{Third, the molecular pathways identified to promote adult regrowth,
such as mTOR, JAK/STAT and c-myc, have potential side effects. These
pathways activate multiple cellular and molecular processes. This
explains how axon regrowth can be obtained in the adult CNS, but this
also accounts for the potential oncogenicity of such manipulations.
Therefore, experimental strategies to promote axon\break regeneration are
strong proof-of-concepts, but their application as a therapy has to be
considered with caution. This is the case for the tumor suppressor
Pten, whose inhibition in specific neurons in mice does not seem to
cause detrimental effects in the long-term}~\cite{GutillaSteward2016}, {but whose wide
spectrum of targets may actually cause profound cell changes, and whose
manipulation should be considered at multiple levels (gene expression,
functional dosage, phosphatase activity, post-translational
modifications, etc.)}~\cite{McLoughlinetal2018,Lietal2024}. 
\looseness=-1

{Nonetheless current strategies
remain a valuable experimental paradigm to elucidate the molecular
mechanisms that control CNS regrowth capacity. Elucidating such
mechanisms is fundamental to trigger and sustain axon regrowth, and
ultimately address all subsequent steps of circuit repair that are just
starting to be looked at, e.g., axon guidance in the mature brain as in
our recent proof-of-concept studies}~\cite{Vilallongueetal2022,Schaefferetal2023a,Delpechetal2024}. {So looking at
these models will help understand how such pathways operate to promote
axon regrowth and also neuron survival (as these two processes may be
independent}~\cite{PernetDiPolo2006,Watkinsetal2013,Norsworthyetal2017}), {and refine what are the downstream
targets that are necessary and sufficient for regeneration. Therefore,
there is a critical need to find out other molecular mechanisms
controlling axon regeneration in order to achieve full circuit
formation and functional\break recovery.}

\section{Translational regulation at play to control gene expression}

{Until now, most studies have focused on transcriptional regulation to
unlock adult CNS regenerative properties. In the adult visual system,
pro-growth transcriptional programs were highlighted thanks to
high-throughput screening, via RNA microarrays}~\cite{Sunetal2011} {and
RNA-sequencing}~\cite{Norsworthyetal2017}, {and more recently via
single-cell analysis of
RGCs}~\cite{Tranetal2019,Jacobietal2022,Tianetal2022}. {Another layer
of gene regulation lies in epigenetic mechanisms at play to control
expression of regeneration-associated genes (RAG). Indeed, regenerative
transcriptional programs are governed by a modification of chromatin
accessibility of RAG regulatory regions in response to injury, for
example via regulation of histone acetylation
status}~\cite{PalmisanoDiGiovanni2018}.

{Transcriptomic analysis can stamp different types of cells according
to detailed mRNA expression. This is the case in the retina, where
high-resolution single-cell analyses have discriminated several dozens
of neurons, during development and throughout
adulthood}~\cite{Rheaumeetal2018,Clarketal2019,Laboissonniereetal2019,Shekharetal2022}. 
{Yet, in many cases, transcriptome
characterization does not account for functional or phenotypical
properties of distinct subpopulations---and conversely
indistinguishable subpopulations may actually harbor distinct
transcriptomes}~\cite{Scalaetal2021}. {This phenomenon is even more complex in the
case of pathological or traumatic situations. Regarding axon injury and
regeneration, modulation of transcriptional programs has been
insufficient to recapitulate axon regrowth and neuronal circuit
reconnection. So transcriptomic approaches are insufficient to predict
major gene regulation processes in injured adult neurons.}

{In contrast, the process of mRNA translation into proteins---the
functional readout of gene expression in cells---has been overlooked in
the context of CNS injury and axon regeneration. While the activation
of mTOR}~\cite{Parketal2008,LiuSabatini2020} {and c-myc}~\cite{vanRiggelenetal2010,Belinetal2015} 
{pathways enhances protein
synthesis at a global level---directly or indirectly---their exact
contribution to translational control in this context remains unknown.}

{In fact, it is now well described that mRNA and protein levels
correlate only partially in cells}~\cite{KomiliSilver2008,Maieretal2009,VogelMarcotte2012,Liuetal2016}\break {and in many
cases, the transcriptomic characterization of a cell does not reflect
the actual protein expression at a given stage. While translation was
long described as an invariable process, evidence has recently emerged
that pools of mRNAs are not evenly translated into proteins.
Translation regulation mechanisms include differential activity of
translation-associated factors}~\cite{GebauerHentze2004}, {selective retention or
degradation of mRNAs}~\cite{GebauerHentze2004}, {and more recently variations in the
composition of the translational complex itself}~\cite{GenuthBarna2018}.

\section{Evidence for a specialized ribosome: focus on the translational complex}

{In eukaryotic cells, the translational complex is primarily composed
of the ribosome, a macromolecular complex formed of about 80 ribosomal
proteins and 4 ribosomal RNAs (rRNAs), which assemble in a tightly
ordered process called ribogenesis. Polypeptide chain extension
operates via screening of a given mRNA by the ribosome and translation
of codon \mbox{sequences} by transfer RNAs into amino acids. This process also
involves translation-associated factors that control the initiation,
elongation and termination steps}~\cite{SonenbergHinnebusch2009}
(Figure~\ref{fig2}).

\begin{figure*}
\vspace*{2pt}
\includegraphics{fig02}
\caption{\label{fig2}Schematic representation of gene expression
control in cells: the genetic information flow goes from DNA to mRNA
(transcription), then from mRNA to protein (translation). While long
thought to be a passive process, translation is emerging as an active
regulator in the control of gene expression. In particular, in
pathological contexts, but also for cell specification, function and
external stimulus-induced response (phenotypic outcome), the
translational complex exhibits variations in its composition. These
variations are associated with a specialization of its translational
function. eIF, eEF and eRF: eukaryotic initiation, elongation and
release factor; RBP: RNA-binding protein; RPL and RPS: ribosomal
protein of the large and small subunits; rRNA: ribosomal RNA; CH3:
methylation; ${\psi}$: pseudourydylation.}
\vspace*{2pt}
\end{figure*}

{While long thought to be invariable in its composition, the
translational complex actually exhibits variations in its components
and composition, and through them actively regulates the translation
process in cells. One fundamental illustration lies in the hundreds of
$2'$-O methylation and pseudourydylation modification sites in rRNAs.
Work from Diaz and colleagues shed light on how dysregulation of these
modifications contributes to tumorigenesis by altering translation
fidelity and mRNA-specific translation}~\cite{Belinetal2009,Marceletal2013,Marceletal2015}. {Another
illustration lies in modifications of ribosomal proteins (RPs): either
RP post-translational modifications or RP stoichiometry in ribosomes.
These observations came from the study of so-called
``ribosomopathies'', where somatic point mutations affect an RP or a
ribogenesis factor. This leads to alterations in ribosome homeostasis
and/or impaired translational regulation, and results in pathogenic
outcomes, e.g., Diamond Blackfan anemia but also cancer
development}~\cite{MillsGreen2017}.

{In physiological conditions, transcriptomic studies highlighted the
existence of RP paralogs that confer tissue specificity and compensate
their canonical counterparts. Examples include the ribosomal
protein~L3-like (Rpl3l), which is specific to the skeletal muscle and
controls myotube formation}~\cite{Chaillouetal2016}; {and the ribosomal
protein~L10-like (Rpl10l), which is specific to the testis and controls
male meiotic transition}~\cite{Jiangetal2017}. {These paralogs are thought to
replace their canonical counterparts in the ribosome, supporting
further that the composition of the translational complex itself
controls the translational landscape in tissue specification,
development and\break function.}

{These studies pioneered the concept of ``ribosome specialization'', a
new and active field of research. Recently, RP heterogeneity in the
composition of purified ribosomes was evidenced using quantitative
proteomics and linked to a specialized function as a translation
regulator}~\cite{Shietal2017}. {Bulk proteomic analysis of various adult mouse
tissues and organs also supports this inter-tissue heterogeneity at the
ribosome-incorporated RP level}~\cite{Alkanetal2022,Lietal2022,Brunchaultetal2024}. {Interestingly, in
neurons, several pieces of evidence point towards an in situ remodeling
of ribosomes by ribosome integration of newly locally synthesized RP in
distal compartments of the neuron}~\cite{Shigeokaetal2019,Fuscoetal2021}. {This suggests an\break
additional layer of control of the translational complex composition in
subcellular compartments.}

{Finally, an additional level of heterogeneity comes from the
translation-associated factors themselves. While the canonical
translation factors are known to regulate the translation process at a
global level}~\cite{GebauerHentze2004}, {recent evidence is brought towards a role in
selective (mRNA-specific) translation. For example, in Shh
gradient-dependent patterning of the embryo, the initiation
factor~Eif3c controls Shh signaling through selective translation of}
{\textit{Ptch1}} {mRNA, a negative regulator of Shh}~\cite{Fujiietal2021}.

{In addition, in different contexts, some non-canonical translation
factors have been evidenced to (i)~interact with the ribosome, and 
(ii)~modify its translational activity, at the global level but also in a
selective (specific mRNA-dependent) way. Examples include FMRP, shown
to translationally repress target mRNAs linked to synaptic
transmission}~\cite{Darnelletal2011}; {and Rack1, shown to increase specific
autophagy-related mRNA translation}~\cite{Kimetal2017}. {These observations make
such associated factors integral components of the translational
complex and contributors to its heterogeneity and specialization in
cellular function. Recently, Simsek and colleagues provided an
exhaustive ``ribo-interactome'' in mouse embryonic stem cells. They
identified more than 300 ribosome-associated proteins susceptible to
customize the ribosome, thereby influencing gene expression in specific
physiological and pathological conditions}~\cite{Simseketal2017}.

\section{Translational regulation in CNS axon regeneration}

Our recent work shed light on translational regulation as an essential
process of CNS axon regrowth: we deciphered this process from the
molecular mechanisms to the subsequent in vivo implications regarding
axon regeneration. Using the gold-standard optic nerve injury model, we
demonstrated that axon regrowth of adult RGCs is controlled by the
selective translation of a specific pool of mRNAs critical for axon
regeneration---instead of global \mbox{translation,} i.e., even protein
synthesis in the cell~\cite{Schaefferetal2023b}. This mechanism is regulated by the
differential association of specific factors to ribosomes in response
to a pathological situation, in this case RGC axon injury. In turn,
this modulates the regeneration-associated translational landscape in
RGCs, independently of transcriptional regulation~\cite{Schaefferetal2023b}. This study
is based on the specific example of the protein Huntingtin (HTT),
identified as a hub of the neuronal injury response~\cite{Belinetal2015,Poplawskietal2020}, which
is quite unexpected since Huntingtin is better known for its
neurodegenerative role in Huntington's disease when the \textit{htt}
gene is affected by a CAG-expansion mutation. In contrast, HTT's
physiological roles remain elusive.

In the adult retina, HTT is downregulated in RGCs upon optic nerve
injury. This downregulation correlates with the decrease in the
regenerative potential of injured adult RGCs, supporting a
pro-regenerative role for HTT. To test this hypothesis, we adopted the
strategy to delete HTT expression specifically in RGCs and analyze
its impact on regeneration. As the wild-type RGCs do not regenerate, we
deleted HTT in a context where axon regeneration has been\break enhanced. To
this end, we used the mTOR activation through deletion of the mTOR
inhibitor Pten, within RGCs. Strikingly, HTT deletion in adult RGCs
completely switches off axon regeneration induced by mTOR activation,
meaning that HTT is essential for adult axon regrowth after injury
(Figure~\ref{fig3}A).

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig03}
%\vspace*{-3pt}
\caption{\label{fig3}(A) Translational regulation is at play to control
CNS axon regeneration. In regenerative conditions (mTOR activation),
HTT expression remains stable upon injury. HTT interacts with ribosomes
and regulates the translation of specific pro-regenerative mRNAs. When
HTT is deleted, the translational complex is no longer customized with
HTT. This modifies the pool of mRNAs recruited for translation and
switches neurons to a regrowth failure state. (B) Comparative analysis
of transcriptome and translatome in HTT deletion {{versus}}
{control conditions. Each dot represents an mRNA. The $x$-coordinate is
its variation in transcript abundance (more or less transcribed when
HTT is deleted). The $y$-coordinate is its variation in ribosome
association (more or less translated when HTT is deleted). Orange dots
represent the mRNAs that are selectively regulated at the translational
level, with no variation of their transcriptional regulation. Among
these orange mRNAs is} {\textit{Tox2}}, {a pro-regenerative
translational target of HTT. Data from}~\cite{Schaefferetal2023b}.}
\vspace*{3pt}
\end{figure*}

Starting from an unbiased analysis of HTT's interactants, we showed
that HTT interacts strongly with ribosomes, which suggests a role for
HTT in the translation process. In vitro and in vivo new protein
synthesis assays revealed that HTT is dispensable for global
translation. In contrast, combined transcriptome and translatome
analyses showed that, in the absence of HTT, a subset of mRNAs is
differentially translated, while their total transcript level is
unchanged. We focused on one such selective translational target of
HTT, \textit{Tox2}, to demonstrate that (i)~HTT controls~\textit{Tox2}
translation in neurons, and (ii)~the translation of \textit{Tox2} mRNA
into protein is necessary for axon regeneration. This study elucidates,
in the context of adult CNS injury, how ribosome association of
specific factors, such as HTT, controls adult regrowth via
translational regulation of a key subset of mRNAs~\cite{Schaefferetal2023b}
(Figure~\ref{fig3}A--B).

Further evidence has been recently brought for the active role of the
translational complex in the regulation of the translational landscape
during axon regeneration. First, our work and others' deciphered the
essential role of kinases of the RSK family in the phosphorylation of
the translation elongation factor~eEF2 and of the ribosomal
protein~RPS6~\cite{Maoetal2022,Decourtetal2023}. These studies demonstrate that RSK-mediated
phosphorylation state is determinant for axon regeneration by
controlling the translational landscape of specific mRNAs. Second,
manipulation of one specific RP can also trigger RGC axon regrowth in
the injured optic nerve, as shown for RPL7 and its isoform~RPL7a whose
transcripts are developmentally downregulated~\cite{Xingetal2023}. While this
latter result further supports the role of the translational complex
specialization in CNS regenerative response, it remains to be
determined (i)~whether experimentally overexpressed RPs integrate into
functional ribosomes, and (ii)~whether this confers an active
translational regulatory role to the translational complex.

\section{Perspectives}

{While long studied under the transcriptomic and epigenetic angles, the
intrinsic regenerative capacity of the CNS depends on an additional
layer of gene expression control: translational control through
regulation of the translational complex. Such a mechanism has been
exemplified by our recent work showing that HTT customizes ribosomes
to regulate selective translation of regeneration-associated mRNAs in
injured CNS neurons}~\cite{Schaefferetal2023b}. {It is now more and
more understood that transcriptomics alone cannot predict protein
expression, not only through this active regulatory role of the
translational complex, but also via accumulation and degradation
\mbox{mechanisms} of individual mRNAs}~\cite{GebauerHentze2004}. {In this
context, proteomic profiling should bring essential information
to give a complete picture of how gene expression is regulated and how
transcription, translation and proteostasis are integrated to control a
phenotypic outcome, in this case CNS neuron injury response and axon
regeneration. However, capturing the protein expression profiles in a
single neuronal population, and} {{a fortiori}} {in single cells,
remains a technical challenge capped by the current sensitivity of mass
spectrometry-based proteomic analysis, as proteins cannot be amplified
like nucleic acids. Yet, some studies could detect thousands of
proteins in a single cell and pair transcriptomic and proteomic
data}~\cite{Jiangetal2023}, {owing to major advances in shotgun
proteomics such as SCoPE-MS}~\cite{Budniketal2018} {and
PiSPA}~\cite{Wangetal2024}. {Such paired profiling provides highly
valuable information on the regulation of gene expression at multiple
levels. This can also apply to subcellular compartments, as performed
recently in growth cones of developing projection
neurons}~\cite{Poulopoulosetal2019}. {Today, translatomics and ribosome
footprint profiling (deep-sequencing of ribosome-protected
fragments)}~\cite{Ingoliaetal2009} {offer a relatively accessible way
to bridge transcription and protein synthesis levels, while effort will
continue to improve the sensitivity of proteomic profiling.}

{Our findings are in line with a translational regulatory role for HTT.
Indeed, in Huntington's disease, HTT mutation was found to alter the
translational process by impairing the activity of translation factors
such as 4E-BP}~\cite{Creus-Muncunilletal2019} {or by impacting ribosome stalling}~\cite{Eshraghietal2021}.
{Interestingly, we found a role in selective translation, while studies
of mutant HTT show alterations in global translation. Thus, more
investigation will be needed to decipher (i)~which subdomain of HTT
interacts with ribosomes (and in particular whether it is the
N-terminal domain where the mutant poly-glutamine expansion is
located); and (ii)~whether HTT mutation affects its translational role
directly or indirectly.}

{Whether such a mechanism of translational control applies to other
neuron subtypes is a next question to address. In support of this
hypothesis, we found that HTT is required for axon regeneration of
dorsal root ganglion (DRG) neurons in the PNS upon sciatic nerve
injury}~\cite{Schaefferetal2023b}, {and the work from others highlighted HTT as a key
hub of regeneration of corticospinal tract motor neurons induced by
neural progenitor cell grafting in the lesioned spinal cord}~\cite{Poplawskietal2020}.
{Importantly, our work showed a correspondence in HTT-mediated control
of the translational landscape between mouse embryonic fibroblasts and
injured RGCs}~\cite{Schaefferetal2023b}, {suggesting a shared mechanism in a wide
variety of cell types and developmental stages. In fact, HTT is
ubiquitously expressed throughout multiple brain regions}~\cite{MarquesSousaHumbert2013},
{and more generally in multiple tissues and at various developmental
stages}~\cite{SaudouHumbert2016}. {Additionally, studies of HTT in several model
organisms advocate for a conserved role of its pathogenicity (induced
by the mutated poly-glutamine expansion domain) and of its
physiological functions, although these are still incompletely
understood}~\cite{SchulteLittleton2011,SaudouHumbert2016,Brouillet2022}. 
{So it is possible that HTT's role as a
translation-associated factor is a common cell biology feature, but we
cannot exclude that pools of translationally regulated mRNAs are
actually specific to a given cell type, developmental stage or
physiopathological condition, which will be worth investigating in the\break
future.}

{What controls the selectivity of the translational complex to
translate specific mRNAs? Some cis-regulatory elements identified in
the sequence and/or structure of transcripts account for this
selectivity. They include internal ribosome entry sites (IRES) in the
$5'$ untranslated region (UTR), upstream open reading frames (uORFs), $5'$
terminal oligopyrimidine ($5'$ TOP) and miRNA-binding
sequences}~\cite{XueBarna2015,Olivetoetal2017,ChenTarn2019,Cockmanetal2020}.
{In CNS regeneration, it remains to be
determined whether HTT translational targets contain such elements, and
more generally whether there are consensus mechanisms of selective
recruitment and translation of regeneration-associated mRNAs.}\looseness=-1

{To investigate further the composition of the translational complex, an
unbiased approach would be to determine with proteomics the
ribo-interactome in neurons in response to axon injury, as recently
done in mouse embryonic stem cells}~\cite{Simseketal2017}. {While working from
neurons is quite challenging (low starting amount of proteins),
advances in proteomic sensitivity should enable this characterization
in the near future.}

Finally, the translational regulation angle offers new perspectives in
terms of therapeutic \mbox{design} for CNS repair. Our study led to the
identification of HTT as a pro-regenerative factor, as well as its
several translational targets whose experimental manipulation can
promote CNS axon {regrowth.} Future work will focus on the modulation of
translation-associated factors to regulate regeneration-associated mRNA
translation.\looseness=-1

{To conclude, translational regulation is emerging as critical in the
control of developmental programs, cell type specification, stress
response}~\cite{ShiBarna2015,GenuthBarna2018,Guo2018}, {and now axon regrowth in the injured
CNS}~\cite{NawabiBelin2025}. {Uncovering how the translational complex is
customized in CNS neurons and how it contributes to translational
regulation stands as a building step to fill the gap between gene
expression and axon regrowth.}

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

The work described in this review and their authors were supported by
grants from the Agence Nationale pour la Recherche (ANR-18-CE16-0007
and ANR-21-CE16-0008-02),  the Fondation pour la Recherche M\'{e}dicale
and UNADEV/AVISAN.

\CDRGrant[ANR]{ANR-18-CE16-0007}
\CDRGrant[ANR]{ANR-21-CE16-0008-02}

\section*{Acknowledgements}

We thank all the authors involved in the work described in this review
and the staff of Grenoble Institut Neurosciences facilities.

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