Among signaling pathways that enable the communication between neuron and cells, Hedgehog cascade plays a crucial role in brain formation but also in the adult brain, facilitating cell-cell interactions. Interestingly, Hedgehog signaling is active in glial cells during brain development and in the adult stage, in precursor cells Han et al. Given the fact that brain repair upon injury is critical to ensure brain function, we aimed to investigate if glial cells and in particular astrocytes elicit a signaling reaction that regulates brain response.
Considering that brain repair upon injury may recapitulate aspects of brain formation and thus of brain development, we hypothesize that the Hedgehog signaling pathway, activated during brain formation and in glial precursor cells, might be involved in brain response to injuries. In order to assess this question, we reviewed on one hand the importance of Hedgehog signaling in astrocyte formation and function, and on the other hand, evidences that Hedgehog pathway is implicated in brain response upon injury, through astrocyte activation.
The review is based on international published articles, available on the Pubmed database of the National Center of Biotechnology Information www. This database was chosen because it offers an updated and wide set of journals of Cell Biology, Molecular Biology, Cell Signaling and Biomedical Sciences, main fields of interests in this investigation. The search was made using the keywords ''hedgehog'' and ''brain'' and ''glioma'', from the start dates of the database to the 31th of January of Literature was reviewed in order to test the hypothesis that Hedgehog signaling plays an important role in glial cells and moreover, in astrocyte response upon brain injury.
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Confident and reproducible published information was selected to assess the working hypothesis. Hedgehog pathway: a key signaling for astrocyte development and function.
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Hedgehog HH signaling pathway is a key signaling cascade for the development and patterning of the CNS. Canonical Hedgehog signaling begins with the secretion of the ligand HH. Once synthesized, HH suffers different post-translational modifications that ensure its secretion and signaling properties.
First, the N-terminal signal sequence residues are removed from HH protein. Then, in the Endoplasmic Reticulum ER , palmitate is added to the N-terminal extremity of SHH, increasing the hydrophobicity of the molecule and its secretion by shedding. Although nonpalmitoylated HH has been found to be functional, it has less signaling activity than palmitoylated forms, in vitro and in vivo Guerrero and Kornberg Also in the ER, HH undergoes autoproteolytic cleaveage, generating one N-terminal fragment containing a Hedge domain, linked to cholesterol and that has signaling function.
The fact that non-cholesterolyated HH has decreased signaling capacities and do not exhibit normal distribution in the tissues, points for a role of cholesterol in HH secretion and gradient formation Guerrero and Kornberg Thus, HH lipid modification appears to play an important role for regulated HH secretion and distribution in the tissue. HH signaling in the brain during development and brain injury might then be facilitated upon secretion of lipid modified HH ligand. Evolutionarily, the apparition of Hedgehog proteins may be related with Hedge and Hog domain ancestors.
While the Hedge domain has been found in proteins of Streptomyces albus, Monosiga spp. Hedgehog proteins may have arisen more than million years ago by the combination of Hedge and Hog domains, in the common ancestors of Cnidarians and bilateral organisms Ingham et al. In the brain, SHH has been the consistently found HH isoform in the brain and will be then considered as the HH ligand of interest in the following sections.
Activation of SMO also involves a conformational switch and localization in the primary cilia of vertebrate cells. In primary cilia, a microtubule-based non motile cilium found on most vertebrate cells, SMO interacts with beta-arrestin and Kif3A in the distal tip of the cilia Huangfu and Anderson , Kovacs et al. In humans, there are three zinc finger proteins GLI. While GLI1 act as a transcriptional activator and thus serves as a readout of HH activity, GLI2 can act as activator or repressor and GLI3 can be a weakly activator but mainly a transcription repressor.
GLI protein activity and stability are regulated by posttranscriptional modifications. In the brain, HH signaling is an important morphogen signaling for CNS formation, determining the differentiation of distinct brain areas and cells. At early embryonic stages, SHH is first expressed ventrally in the brain, in the notochord, in the precordal plate and regulates ventral hindbrain, midbrain and forebrain development Ruiz i Altaba et al. In the ventral brain SHH pathway can induce neuron formation, controls the size of the ventral midbrain and the development of the basal ganglia.
SHH is an important factor for cell growth in the brain, being involved in oligodendrocyte formation, in regulating the size of the dorsal brain and in the cortical plate it might affect precursor cells Ruiz i Altaba et al. Besides the role of SHH in determining the differentiation and formation of different brain regions, HH pathway has a crucial function in astrocyte formation, main glial cells in the brain.
HH pathway is a key signaling for astrocyte formation by promoting progenitor differentiation into astrocytes.
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Indeed, progenitor cells expressing SHH contribute to both, neurons and astrocytes production in a caudal area of the brain. However, SHH expressing progenitors suffer a gradual shift from neurogenesis to gliogenesis, generating mainly hypothalamic astrocytes in later development phases Alvarez-Bolado et al. Additionally, HH signaling is involved in astrocyte generation from other progenitor cells in the brain.
In the case of radial astrocytes, HH pathway inhibition, by the absence of primary cilia, an organelle essential for HH signaling in mammals, or the absence of SMO, prevents radial astrocytes development Han et al. In the adult brain, germinal niches that include the ventricular subventricular zone V-SVZ and the subgranular zone SGZ , continue to produce neurons and glial cells.
Finally, HH signaling plays a role in the differentiation and maturation of astrocytes such as adult cerebellar Bergmann glia astrocytes and mouse cerebellar granule cell precursors form the proliferative zone of the external germinal layer Marazziti et al. Once differentiated, SHH signaling, regulated by the combination of GLI transcriptions factors in their activator and inactivator forms, is important for proper astrocyte functions like the release from neurotransmitters and for maintaining the blood brain barrier Alvarez et al.
Furthermore, SHH signaling regulates glutamate and ATP release from astrocytes, and thus is essential for astrocyte metabolism and metabolic support to neurons Okuda et al. SHH signaling is then an important pathway not only for astrocyte generation in the brain but also for astrocyte differentiation and function. If HH signaling is crucial for brain formation and astrocyte generation during development, and in physiological conditions, what is the implication of this signaling upon brain injury?
Brain repair requires astrocyte activation through SHH signaling. Besides giving ionic and metabolic support to neurons, regulating synapse neurotransmission, and regulating blood brain barrier, astrocytes play key roles upon brain injury.
Initially observed in multiple sclerosis specimens, astrocytes that react to CNS changes present a different appearance, and were denominated ''reactive astrocytes''. Reactive astrocytes have both, biochemical and morphological changes, upon brain injury and in pathological conditions.
In addition to hypertrophy and increased expression of Glial Fibrillary Acidic Protein GFAP , reactive astrocytes secrete more cytokines, growth factors and extracellular matrix components Robel and Sontheimer Upon brain injury conditions such as those found in pathological situations, some astrocytes can proliferate and acquire an immature phenotype, that may be related with a progenitor state that can reestablish damaged cells in the brain Robel and Sontheimer Albeit cell migration was thought to be one of the characteristics of reactive astrocytes to reach injury sites, recent investigations in vivo have shown that astrocytes do not migrate towards injury site.
Indeed, astrocyte response in the brain is heterogeneous, with astrocytes that do not change cell morphology, astrocytes that direct their process toward the lesion site and astrocytes that proliferate Bardehle et al. Thus, instead of migration upon acute injury, astrocytes can extend their cytoplasm towards the wound site or proliferate in close proximity to the vascular system of the brain Bardehle et al. Interestingly, in many tissues, injury repair brings out biological processes that recapitulate tissue development and that enable tissue re-formation.
For instance, cell signaling pathways operative during brain development like Sonic Hedgehog pathway, are reactivated upon brain injury as will be outlined below. Given the importance of Sonic Hedgehog pathway for brain development, it is very interesting to point out evidences in vitro and in vivo of the re-activation of this pathway in the adult brain upon tissue injury annex 1. In many cases, astrocyte activation is due to mechanical, chemical or biological injury and this astrogliosis is in part, mediated by SHH signaling figure 1.
Biological agents like Angiostrongylus cantonensis , an important etiologic agent of eosinophilicmeningitis or eosinophilic meningoencephalitis in humans, have been found to induce SHH signaling. Besides brain injury provoked by biological agents, mechanical injury can also elicit a SHH signaling that may be related with tissue repair. In vitro , scratches of monolayer astrocyte cultures produce an increase in SHH production by astrocytes, the loss of astrocyte markers such as GFAP and S, and the expression of Neural stem cells proteins like nestin, Sox2, and CD Experiments in vivo have also proved the activation of SHH signaling in different settings of brain injury and its implication in tissue repair.
Different types of injury appear to elicit different HH signaling and astrocyte responses in vivo. Comparing ischemic lesion, traumatic injury, progressive chronic amyloid plaque deposition, and a noninvasive model of widespread neuronal death, Sirko et al. In this process, astrocytes acquire neural stem cells characters, by SHH signaling cascade, necessary and sufficient for this response in vitro and in vivo Sirko et al. If SHH mediates astrocyte reactivity in invasive injury situations in the brain, induction of this pathway can be associated with tissue repair.
In the brain, one of the critical parameters for cell maintenance is the permanent supply of oxygen. In the absence of appropriate oxygen concentrations, cells can rapidly undergo cell death. Under restriction in blood supplies or ischemia, irreversible brain damage associated with cerebral hypoxia and glucose deprivation can lead to stroke as fast as 5 minutes later at human body temperature.
Interestingly, cerebral hypoxia induces SHH expression on neural progenitor cells and neurons that promote cell proliferation Sims et al.
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Additionally, astrocytes respond to oxygen-glucose deprivation by secreting SHH that promotes the proliferation, migration of microvascular endothelial cells and tube formation in coculture models, in a RhoA and ROCK-dependent manner He et al. In mice experiments, cortical ischemia upregulates SHH expression in neurons, in reactive astrocytes and in nestin-expressing cells in the cortical area near the injury site and the adjacent striatum Jin et al. In these conditions, SHH signaling promotes tissue stability and injury repair. Furthermore, after stroke, SHH treatment reduces behavioral impact on animals, enhancing multiple horizontal movement parameters compared to vehicle treated mice Jin et al.
BBB is formed by capillary endothelial cells, pericytes, and perivascular astrocytes that create a highly selective permeability barrier protecting the neural tissue from variations in blood composition and toxins. SHH produced by astrocytes plays an important role in maintaining BBB integrity, by upregulating the expression of tight junction proteins. In animal ischemia models, increased SHH secretion increases Ang-1 expression in astrocytes and correlates with increased ZO-1 and occluding expression in primary brain microvessel endothelial cells, enhancing tight junction stability and avoiding BBB disruption Xia et al.
Importantly, endothelial brain cells express HH receptor PTCH, and HH pathway has been found to decrease the expression of proinflammatory mediators and to decrease the adhesion and migration of leukocytes, promoting the immune quiescence of BBB endothelial cells, providing a barrier effect Alvarez et al. If the absence or the decrease of oxygen concentration represent a critical situation for neuron and astrocyte survival, the presence of reactive oxygen species can also elicits a stress response mediated by SHH signaling. HH signaling activation on astrocytes enhances AKT phosphorylation, has a pro-survival effect and a protective effect on cocultured neurons Xia et al.
Excess of other molecules in the brain, such as kainic acid, can represent another source of brain injury. Kainic acid is an analog of the excitatory amino acid L-glutamate and can induce neuron death in the central nervous system. In a model of kainic acid neurodegeneration, SHH expression is upregulated in astrocytes, along with increased Gli activity and astrocyte proliferation, independently of the severity of neurodegeneration Pitter et al.
Upon different types of brain injury, Hedgehog signaling and astrocyte activation appear thus to be essential for brain response annex 1. After injury, Hedgehog signal may be secreted by neurons and received by glial cells such as astrocytes figure 1. Thus, in the first steps of cell reaction to brain injury, SHH pathway may represent a paracrine signaling between neurons and astrocytes that elicits tissue repair. Once activated, astrocyte may communicate through SHH signaling to other glial cells, including astrocytes, to orchestrate a coordinated cell response upon injury figure 2.
SHH signaling is an essential pathway for brain patterning and cell differentiation during development. However, studies in adult organisms have highlighted the importance of this signaling pathway in the interplay between neurons and glial cells.
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Among glial cells, astrocytes play a key role for the regulation of metabolism, neurotansmitter clearance, blood brain barrier and synapse maturation, plasticity and elimination, and thus for brain function. Upon injury, astrocytes exhibit specific cell responses that include cell proliferation and activation of stem cell features mediated in part by SHH signaling. Once activated by SHH, astrocytes coordinate tissue repair, regulating astrocyte and neuron survival, the integrity of blood brain barrier and microglia activity. Thus, by regulating astrocyte activity, SHH pathway appears as a key player, in vitro and in vivo , for tissue repair.
If this link is supported by reliant experimental evidence, many questions remain to be solved. On one hand, novel findings should bring to light the importance of SHH in the dynamic communication between neurons and astrocytes, and other glia cells, for efficient tissue response upon injury. On the other hand, it will be necessary to determine if SHH pathway is related with brain repair in acute injury and if upon chronic brain injury, long-term activation of this pathway may contribute to brain diseases.
Finally, it will be of great interest to understand if SHH signaling elicits the activation of other signaling modules in astrocytes and neurons that may compose the signaling reactions that enables brain cell reaction, and if modulating this signaling network may enhance brain repair in the context of brain injury in different neurological diseases including cancer.