Therapeutic value of homeoprotein signaling pathways.

Cell signaling based on homeoprotein transfer is a pathway with developmental and physiological functions. For a few transcription factors of this family, primarily ENGRAILED1, ENGRAILED2 and OTX2, their physiological functions have led to therapeutic strategies in animal models of human diseases, including Parkinson's disease, amyotrophic lateral sclerosis, amblyopia and anxiety-related disorders. In mesencephalic dopaminergic neurons which degenerate in Parkinson's disease, ENGRAILED1/2 have cell autonomous activities, but their transducing properties enables their use as therapeutic proteins. In contrast, in spinal alpha-motoneurons, which are lost in amyotrophic lateral sclerosis, ENGRAILED1 is supplied by V1 interneurons. Thus, its use as a therapeutic protein to protect alpha-motoneurons against degeneration mimics its normal non-cell autonomous neurotrophic activity. OTX2, synthesized and secreted by the choroid plexus, is transferred to parvalbumin interneurons and exerts regulatory functions controlling cerebral cortex plasticity. Understanding the latter OTX2 function has led to strategies for manipulating visual acuity and anxiety-like behavior in adult mice. In this review, we describe these cases and what is known about the involved molecular mechanisms. Because the transduction sequences are conserved in most of the few hundred homeoproteins, we argue how this family of molecules constitutes an important reservoir of physiological knowledge, with potential consequences in the search for new therapeutic strategies.

ENGRAILED-1 transcription factor has a paracrine neurotrophic activity on adult spinal α-motoneurons.

Several homeoprotein transcription factors transfer between cells and regulate gene expression, protein translation, and chromatin organization in recipient cells. ENGRAILED-1 is one such homeoprotein expressed in spinal V1 interneurons that synapse on α-motoneurons. Neutralizing extracellular ENGRAILED-1 by expressing a secreted single-chain antibody blocks its capture by spinal motoneurons resulting in α-motoneuron loss and limb weakness. A similar but stronger phenotype is observed in the Engrailed-1 heterozygote mouse, confirming that ENGRAILED-1 exerts a paracrine neurotrophic activity on spinal cord α-motoneurons. Intrathecal injection of ENGRAILED-1 leads to its specific internalization by spinal motoneurons and has long-lasting protective effects against neurodegeneration and weakness. Midbrain dopaminergic neurons express Engrailed-1 and, similarly to spinal cord α-motoneurons, degenerate in the heterozygote. We identify genes expressed in spinal cord motoneurons whose expression changes in mouse Engrailed-1 heterozygote midbrain neurons. Among these, p62/SQSTM1 shows increased expression during aging in spinal cord motoneurons in the Engrailed-1 heterozygote and upon extracellular ENGRAILED-1 neutralization. We conclude that ENGRAILED-1 might regulate motoneuron aging and has non-cell-autonomous neurotrophic activity.

Unconventional Secretion, Gate to Homeoprotein Intercellular Transfer.

Unconventional secretion allows for the secretion of fully mature and biologically active proteins mostly present in the cytoplasm or nucleus. Besides extra vesicle-driven secretion, non-extravesicular pathways also exist that specifically rely on the ability of the secreted proteins to translocate directly across the plasma membrane. This is the case for several homeoproteins, a family of over 300 transcription factors characterized by the structure of their DNA-binding homeodomain. The latter highly conserved homeodomain is necessary and sufficient for secretion, a process that requires PI(4,5)P2 binding, as is the case for FGF2 and HIV Tat unconventional secretion. An important feature of homeoproteins is their ability to cross membranes in both directions and thus to transfer between cells. This confers to homeoproteins their paracrine activity, an essential facet of their physiological functions.

OTX2 stimulates adult retinal ganglion cell regeneration.

Retinal ganglion cell (RGC) axons provide the only link between the light sensitive and photon transducing neural retina and visual centers of the brain. RGC axon degeneration occurs in a number of blinding diseases and the ability to stimulate axon regeneration from surviving ganglion cells could provide the anatomic substrate for restoration of vision. OTX2 is a homeoprotein transcription factor expressed in the retina and previous studies showed that, in response to stress, exogenous OTX2 increases the in vitro and in vivo survival of RGCs. Here we examined and quantified the effects of OTX2 on adult RGC axon regeneration in vitro and in vivo. The results show that exogenous OTX2 stimulates the regrowth of axons from RGCs in cultures of dissociated adult retinal cells and from explants of adult retinal tissue and that RGCs respond directly to OTX2 as regrowth is observed in cultures of purified adult rat RGCs. Importantly, after nerve crush in vivo, we observed a positive effect of OTX2 on the number of regenerating axons up to the optic chiasm within 14 days post crush and a very modest level of acuity absent in control mice. The effect of OTX2 on RGC survival and regeneration is of potential interest for degenerative diseases affecting this cell type. All animal procedures were approved by the local "Comié d'éιthique en expérimentation animale n°59" and authorization n° 00702.01 delivered March 28, 2014 by the French "Ministére de l'enseignement supérieur et de la recherche".

Shuttling Homeoproteins and Their Biological Significance.

The transfer of homeoprotein transcription factors is at the origin of the discovery of Penetratin, one of the first transduction peptides allowing for the addressing of hydrophilic cargoes to the cell cytoplasm and nucleus. Beyond this important technological application, homeoprotein transduction has now been confirmed for more than 150 members of this family, and represents an intriguing mode of signaling for which actual in vivo functions are known for a handful of these proteins. Because homeoproteins are expressed in all eukaryotes, and their intercellular transfer occurs both in plants and animals, it is likely that this signaling activity appeared before the separation between plants, fungi, and animals, and is therefore very ancient. These aspects are discussed in the present review, with an accent placed on evolution and on the comparison of homeoprotein signaling between species belonging to distinct phyla.

Choroid plexus APP regulates adult brain proliferation and animal behavior.

Elevated amyloid precursor protein (APP) expression in the choroid plexus suggests an important role for extracellular APP metabolites such as sAPPα in cerebrospinal fluid. Despite widespread App brain expression, we hypothesized that specifically targeting choroid plexus expression could alter animal physiology. Through various genetic and viral approaches in the adult mouse, we show that choroid plexus APP levels significantly impact proliferation in both subventricular zone and hippocampus dentate gyrus neurogenic niches. Given the role of Aß peptides in Alzheimer disease pathogenesis, we also tested whether favoring the production of Aß in choroid plexus could negatively affect niche functions. After AAV5-mediated long-term expression of human mutated APP specifically in the choroid plexus of adult wild-type mice, we observe reduced niche proliferation, reduced hippocampus APP expression, behavioral defects in reversal learning, and deficits in hippocampal long-term potentiation. Our findings highlight the unique role played by the choroid plexus in regulating brain function and suggest that targeting APP in choroid plexus may provide a means to improve hippocampus function and alleviate disease-related burdens.

Photoreceptor cKO of OTX2 Enhances OTX2 Intercellular Transfer in the Retina and Causes Photophobia.

In the mature mouse retina, Otx2 is expressed in both retinal pigmented epithelium (RPE) and photoreceptor (PR) cells, and Otx2 knock-out (KO) in the RPE alone results in PR degeneration. To study the cell-autonomous function of OTX2 in PRs, we performed PR-specific Otx2 KO (cKO) in adults. As expected, the protein disappears completely from PR nuclei but is still observed in PR inner and outer segments while its level concomitantly decreases in the RPE, suggesting a transfer of OTX2 from RPE to PRs in response to Otx2 ablation in PRs. The ability of OTX2 to transfer from RPE to PRs was verified by viral expression of tagged-OTX2 in the RPE. Transferred OTX2 distributed across the PR cytoplasm, suggesting functions distinct from nuclear transcription regulation. PR-specific Otx2 cKO did not alter the structure of the retina but impaired the translocation of PR arrestin-1 on illumination changes, making mice photophobic. RNA-seq analyses following Otx2 KO revealed downregulation of genes involved in the cytoskeleton that might account for the arrestin-1 translocation defect, and of genes involved in extracellular matrix (ECM) and signaling factors that may participate in the enhanced transfer of OTX2. Interestingly, several RPE-specific OTX2 target genes involved in melanogenesis were downregulated, lending weight to a decrease of OTX2 levels in the RPE following PR-specific Otx2 cKO. Our study reveals a new role of endogenous OTX2 in PR light adaptation and demonstrates the existence of OTX2 transfer from RPE to PR cells, which is increased on PR-specific Otx2 ablation and might participate in PR neuroprotection.

OTX2 Homeoprotein Functions in Adult Choroid Plexus.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

Perturbed DNA methylation by Gadd45b induces chromatin disorganization, DNA strand breaks and dopaminergic neuron death.

Age is a major risk factor for neurodegenerative diseases like Parkinson's disease, but few studies have explored the contribution of key hallmarks of aging, namely DNA methylation changes and heterochromatin destructuration, in the neurodegenerative process. Here, we investigated the consequences of viral overexpression of Gadd45b, a multifactorial protein involved in DNA demethylation, in the mouse midbrain. Gadd45b overexpression induced global and stable changes in DNA methylation, particularly in introns of genes related to neuronal functions, as well as on LINE-1 transposable elements. This was paralleled by disorganized heterochromatin, increased DNA damage, and vulnerability to oxidative stress. LINE-1 de-repression, a potential source of DNA damage, preceded Gadd45b-induced neurodegeneration, whereas prolonged Gadd45b expression deregulated expression of genes related to heterochromatin maintenance, DNA methylation, or Parkinson's disease. Our data indicates that aging-related alterations contribute to dopaminergic neuron degeneration with potential implications for Parkinson's disease.

Non-cell-autonomous OTX2 transcription factor regulates anxiety-related behavior in the mouse.

The OTX2 homeoprotein transcription factor is expressed in the dopaminergic neurons of the ventral tegmental area, which projects to limbic structures controlling complex behaviors. OTX2 is also produced in choroid plexus epithelium, from which it is secreted into cerebrospinal fluid and transferred to limbic structure parvalbumin interneurons. Previously, adult male mice subjected to early-life stress were found susceptible to anxiety-like behaviors, with accompanying OTX2 expression changes in ventral tegmental area or choroid plexus. Here, we investigated the consequences of reduced OTX2 levels in Otx2 heterozygote mice, as well as in Otx2+/AA and scFvOtx2tg/0 mouse models for decreasing OTX2 transfer from choroid plexus to parvalbumin interneurons. Both male and female adult mice show anxiolysis-like phenotypes in all three models. In Otx2 heterozygote mice, we observed no changes in dopaminergic neuron numbers and morphology in ventral tegmental area, nor in their metabolic output and projections to target structures. However, we found reduced expression of parvalbumin in medial prefrontal cortex, which could be rescued in part by adult overexpression of Otx2 specifically in choroid plexus, resulting in increased anxiety-like behavior. Taken together, OTX2 synthesis by the choroid plexus followed by its secretion into the cerebrospinal fluid is an important regulator of anxiety-related phenotypes in the mouse.

Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex.

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Homeoprotein transduction in neurodevelopment and physiopathology.

Homeoproteins were originally identified for embryonic cell-autonomous transcription activity, but they also have non-cell-autonomous activity owing to transfer between cells. This Review discusses transfer mechanisms and focuses on some established functions, such as neurodevelopmental regulation of axon guidance, and postnatal critical periods of brain plasticity that affect sensory processing and cognition. Homeoproteins are present across all eukaryotes, and intercellular transfer occurs in plants and animals. Proposed functions have evolutionary relevance, such as morphogenetic activity and sexual exchange during the mating of unicellular eukaryotes, while others have physiopathological relevance, such as regulation of mood and cognition by influencing brain compartmentalization, connectivity, and plasticity. There are more than 250 known homeoproteins with conserved transfer domains, suggesting that this is a common mode of signal transduction but with many undiscovered functions.

H2O2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum.

Although a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H2O2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H2O2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H2O2 could participate in the patterning of the embryo. We find that perturbations of endogenous H2O2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H2O2 levels and taken up by cells with low H2O2 levels where it leads to increased H2O2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events.

OTX2 Non-Cell Autonomous Activity Regulates Inner Retinal Function.

OTX2 is a homeoprotein transcription factor expressed in photoreceptors and bipolar cells in the retina. OTX2, like many other homeoproteins, transfers between cells and exerts non-cell autonomous effects such as promoting the survival of retinal ganglion cells that do not express the protein. Here we used a genetic approach to target extracellular OTX2 in the retina by conditional expression of a secreted single-chain anti-OTX2 antibody. Compared with control mice, the expression of this antibody by parvalbumin-expressing neurons in the retina is followed by a reduction in visual acuity in 1-month-old mice with no alteration of the retinal structure or cell type number or aspect. The a-waves and b-waves measured by electroretinogram were also indistinguishable from those of control mice, suggesting no functional deficit of photoreceptors and bipolar cells. Mice expressing the OTX2-neutralizing antibody did show a significant doubling in the flicker amplitude and a reduction in oscillatory potential, consistent with a change in inner retinal function. Our results show that interfering in vivo with OTX2 non-cell autonomous activity in the postnatal retina leads to an alteration in inner retinal cell functions and causes a deficit in visual acuity.

Homeoprotein Neuroprotection of Embryonic Neuronal Cells.

Most homeoprotein transcription factors have a highly conserved internalization domain used in intercellular transfer. Internalization of homeoproteins ENGRAILED1 or ENGRAILED2 promotes the survival of adult dopaminergic cells, whereas that of OTX2 protects adult retinal ganglion cells. Here we characterize the in vitro neuroprotective activity of several homeoproteins in response to H2O2 Protection is observed with ENGRAILED1, ENGRAILED2, OTX2, GBX2, and LHX9 on midbrain and striatal embryonic neurons, whereas cell-permeable c-MYC shows no protective effects. Therefore, five homeoproteins belonging to three different classes (ANTENNAPEDIA, PAIRED, and LIM) share the ability to protect embryonic neurons from midbrain and striatum. Because midbrain and striatal neurons do not express the same repertoire of the four proteins, a lack of neuronal specificity together with a general protective activity can be proposed. Interestingly, hEN1 and GBX2 provided protection to primary midbrain astrocytes but not to non-neural cells, including mouse embryo fibroblasts, macrophages or HeLa cells. For the four proteins, protection against cell death correlated with a reduction in the number of H2O2-induced DNA break foci in midbrain and striatal neurons. In conclusion, within the limit of the number of cell types and homeoproteins tested, homeoprotein protection against oxidative stress-induced DNA breaks and death is specific to neurons and astrocytes but shows no homeoprotein or neuronal type specificity.

OTX2 Signals from the Choroid Plexus to Regulate Adult Neurogenesis.

Proliferation and migration during adult neurogenesis are regulated by a microenvironment of signaling molecules originating from local vasculature, from CSF produced by the choroid plexus, and from local supporting cells including astrocytes. Here, we focus on the function of OTX2 homeoprotein transcription factor in the mouse adult ventricular-subventricular zone (V-SVZ), which generates olfactory bulb neurons. We find that OTX2 secreted by choroid plexus is transferred to the supporting cells of the V-SVZ and rostral migratory stream. Deletion of Otx2 in choroid plexus affects neuroblast migration and reduces the number of olfactory bulb newborn neurons. Adult neurogenesis was also decreased by expressing secreted single-chain antibodies to sequester OTX2 in the CSF, demonstrating the importance of non-cell-autonomous OTX2. We show that OTX2 activity modifies extracellular matrix components and signaling molecules produced by supporting astrocytes. Thus, we reveal a multilevel and non-cell-autonomous role of a homeoprotein and reinforce the choroid plexus and astrocytes as key niche compartments affecting adult neurogenesis.

Non-cell Autonomous OTX2 Homeoprotein Regulates Visual Cortex Plasticity Through Gadd45b/g.

The non-cell autonomous transfer of OTX2 homeoprotein transcription factor into juvenile mouse cerebral cortex regulates parvalbumin interneuron maturation and critical period timing. By analyzing gene expression in primary visual cortex of wild-type and Otx2+/GFP mice at plastic and nonplastic ages, we identified several putative genes implicated in Otx2-dependent visual cortex plasticity for ocular dominance. Cortical OTX2 infusion in juvenile mice induced Gadd45b/g expression through direct regulation of transcription. Intriguingly, a reverse effect was found in the adult, where reducing cortical OTX2 resulted in Gadd45b/g upregulation. Viral expression of Gadd45b in adult visual cortex directly induced ocular dominance plasticity with concomitant changes in MeCP2 foci within parvalbumin interneurons and in methylation states of several plasticity gene promoters, suggesting epigenetic regulation. This interaction provides a molecular mechanism for OTX2 to trigger critical period plasticity yet suppress adult plasticity.

Perineuronal nets in brain physiology and disease.

Perineuronal nets (PNNs) in the brain are condensed glycosaminoglycan-rich extracellular matrix structures with heterogeneous composition yet specific organization. They typically assemble around a subset of fast-spiking interneurons that are implicated in learning and memory. Owing to their unique structural organization, PNNs have neuroprotective capacities but also participate in signal transduction and in controlling neuronal activity and plasticity. In this review, we define PNN structure in detail and describe its various biochemical and physiological functions. We further discuss the role of PNNs in brain disorders such as schizophrenia, bipolar disorder, Alzheimer disease and addictions. Lastly, we describe therapeutic approaches that target PNNs to alter brain physiology and counter brain dysfunction.