Proliferative reactive gliosis is compatible with glial metabolic support and neuronal function
© Vázquez-Chona et al; licensee BioMed Central Ltd. 2011
Received: 13 June 2011
Accepted: 10 October 2011
Published: 10 October 2011
The response of mammalian glial cells to chronic degeneration and trauma is hypothesized to be incompatible with support of neuronal function in the central nervous system (CNS) and retina. To test this hypothesis, we developed an inducible model of proliferative reactive gliosis in the absence of degenerative stimuli by genetically inactivating the cyclin-dependent kinase inhibitor p27 Kip1 (p27 or Cdkn1b) in the adult mouse and determined the outcome on retinal structure and function.
p27-deficient Müller glia reentered the cell cycle, underwent aberrant migration, and enhanced their expression of intermediate filament proteins, all of which are characteristics of Müller glia in a reactive state. Surprisingly, neuroglial interactions, retinal electrophysiology, and visual acuity were normal.
The benign outcome of proliferative reactive Müller gliosis suggests that reactive glia display context-dependent, graded and dynamic phenotypes and that reactivity in itself is not necessarily detrimental to neuronal function.
Keywordsreactive gliosis retinal degeneration neuronal degeneration GFAP Müller glia p27Kip1 CDKN1B intermediate filaments
In response to neural pathologies, glia display reactive properties associated with wound healing including cellular hypertrophy, proliferation, migration and cytokine release [1–4]. In mammalian CNS and retina, reactive glia contribute to neural tissue repair [5–10] but also to neural dysfunction, scar formation, abberant neural rewiring, and vascular remodeling [1–3, 11, 12], ultimately exacerbating neuronal degenerations [11, 13]. Defining the components of reactive gliosis that are detrimental to neuronal survival and tissue integrity is an important goal but difficult to achieve. Animal models of reactive gliosis also induce neuronal cell death, microglial reactivity, inflammatory responses or tissue damage [1, 3, 4, 14]. An alternate approach to explore glial reactivity and neuronal metabolism, physiology and function is to develop genetically inducible models of reactivity in the absence of gross degenerative cues.
Two hallmarks of reactive glia are proliferation and enhanced intermediate filament expression. Both are associated with opposing properties: neuroprotection and degeneration. Experimental models and gene inactivation studies implicate upregulation of intermediate filament expression in the formation of hypertrophic glial processes. Glial hypertrophy helps maintain the structural integrity of the CNS by filling the space where neurons die and by restoring damaged protective barriers [9, 15, 16]. However, intermediate filaments are abundant in glial scars which are known to impede axonal regeneration [17, 18]. Chronic upregulation of intermediate filament expression is also correlated with glial metabolic dysfunction and altered neuronal electrophysiology [12, 19–21]. The role of glial proliferation is similarly perplexing. Genetic ablation of proliferating glia worsens neurodegeneration [5, 6] while pharmacological inhibition of glial proliferation enhances neuronal survival and function [14, 22]. Given these complexities, more precise dissections of the links between glial reactivity and progressive neurodegeneration are needed.
The cyclin-dependent kinase inhibitor p27 is one such link. It is expressed in many adult glial populations including Schwann cells, cortical astrocytes, spinal cord astrocytes, oligodendrocytes, and retinal Müller glia [23–27]. In germline p27-deficient mice (p27 -/- ), adult glia can display hallmarks of reactive gliosis [24–26, 28]. In the wild-type retina, quiescent Müller glia normally do not express the intermediate filament glial fibrillary acidic protein (GFAP), but Müller glia in p27 -/- mice express high levels of GFAP and in some instances migrate into the subretinal space [24, 26]. This behavior is enhanced by the combinatorial inactivation of p27 and the cyclin-dependent kinase inhibitor p19 Ink4d . Müller glial reactivity and abnormal retinal electrophysiology in p27 -/- mice may partly arise from developmental dysregulation as p27 is critical for neural development and glial differentiation [24, 26, 30–33]. Even so, CNS and retinal trauma models support a role for p27 in maintaining mature glial cells in a quiescent, supportive state. After acute trauma, cortical astrocytes, spinal cord astrocytes and retinal Müller glia downregulate p27, upregulate GFAP and re-enter the cell cycle [14, 25, 27, 34]. Thus p27 appears to be a negative regulator of two classic indices of reactive glia: GFAP upregulation and proliferation. This implies that selective inactivation of p27 could trigger neural remodeling and reprogramming defects in an otherwise normal milieu.
To modulate discrete reactivity indices in the absence of other degenerative stimuli, we induced intermediate filament GFAP upregulation, migration, and proliferation in adult Müller glia by inactivating p27 using a tamoxifen-regulated, Cre-loxP system [35, 36]. This approach bypassed the developmental requirement for p27 [24, 26] as well as the complexities and broad effects of experimentally induced degeneration [1, 3, 4, 14]. To address the significance of enhanced discrete reactivity on neuronal survival and function, we surveyed metabolism, retinal electrophysiology, and visual acuity. Contrary to our expectations, proliferative and GFAP-expressing Müller glia did not significantly impair retinal metabolism, electrophysiology, or visual function. Thus, our genetic model and the p27 pathway offer a new platform to explore how environmental factors involved in neuronal cell stress, microglial activation, inflammatory responses, or blood barrier damage contribute to the transition of resident glia from a supportive to detrimental state.
Inducible model of p27 deficiency in adult mice
p27-deficient Müller glia upregulate intermediate filaments
p27-deficient Müller glia reenter the cell cycle
Müller glia do not proliferate under normal conditions in part due to the presence of cell cycle inhibitors such as p27 [24, 25]. After conditional p27 inactivation, however, cells reentered the cell cycle as indicated by the upregulation of proliferative markers PCNA, pHH3, and MCM6 (Figure 2D and Additional file 2) as well as bromodeoxyuridine (BrdU) labeling (Figure 2E and 2F). BrdU injections followed by a six week chase revealed that BrdU colocalized with Müller glial markers SOX9 and glutamine synthetase (GLUL, Figure 2F) and not with photoreceptor or bipolar markers (Recoverin, OTX2, PKC-alpha). While the number of SOX9+-GLUL+ nuclei increased by 15.8% (p <0.00003) the number of GLUL+ stalks at the inner plexus layer decreased by 6.1% (p < 0.006) (Figure 2D). Increased GLUL+ soma density with decreased stalk density was confirmed with ultrathin radial and oblique sections suggesting that proliferative Müller glia can retract their stalks before dividing and neither they or their daughters regrow stalks after dividing (Additional file 3). These data indicate that p27 inactivation in Müller glia was sufficient to induce cell cycle entry with a low level of proliferation.
SOX9 and GLUL immunoreactivity also revealed that p27-deficient Müller glia displaced their nuclei toward the photoreceptor layer (Figure 2F and Additional file 3). The displacement of Müller glial nuclei occured one week after the start of p27 inactivation and the ectopic location was irreversible. It is unclear whether the nuclear displacement reflects migration of Müller glia or simply interkinetic nuclear migration. Evidence supporting migratory-like behavior is the presence of focal and limited extension of GFAP+ Müller glial endfeet into the photoreceptor segments (Additional file 4, arrows); but unlike p27 -/- retina [24, 26], p27 L-/L- retina displayed continuous outer limiting membrane as seen by the Müller glia microvilli marker CD44 (Additional file 4). In sum, induced p27 deficiency results in adult Müller glia adopting classic indices of proliferative reactive gliosis: intermediate filament upregulation, cell cycle entry, and migratory-like behavior.
Reactive p27L-/L- Müller glia provide homeostatic metabolic support
Normal electrophysiology in retinas with reactive p27L-/L- Müller glia
Normal visual acuity and function in mice with reactive p27L-/L- Müller glia
p27 is a negative regulator of proliferative Müller glial reactivity
The hypothesis that p27 is a key modulator of glial plasticity is supported by our finding that inducing p27-deficiency is sufficient to promote proliferative Müller reactive gliosis in adult retina [24–27, 34]. As a cell cycle inhibitor, p27 modulates glial proliferation and consequently p27 can modulate the potential of glial cells to regenerate neural tissue and to form scars . In mammalian retinal degenerations, Müller glia may fail to re-enter the mitotic cycle because of persistent p27 expression . In contrast, Müller glia and astrocytes adjacent to traumatic injuries downregulate p27 resulting in glial proliferation that contributes to scar formation . The absence of neurogenesis and scar formation after our conditional p27 inactivation suggests that while decreased p27 activity modulates glial proliferation, transitioning to a neurogenic state or scar forming phenotype must be determined by additional signaling mechanisms. Indeed several groups found that addition of neurogenic factors are required to guide proliferating glia into a neurogenic state [24, 25, 48, 49]. Our conditional p27 inactivation also yielded nonproliferative phenotypes including Müller glial nuclear migration, cytoplasmic process extension, and increased intermediate filament content. These phenotypes are consistent with data suggesting that the impact of p27 activity extends beyond cell cycle regulation, possibly by modulating transcription, cell fate, cell migration, or cytoskeletal dynamics [30, 31]. The issue of whether the nonproliferative changes are the direct result of p27 deficiency or a secondary response to cell cycle reentry will be addressed in future studies; however, it is clear that p27 levels have broad effects on the outcome of glial reactivity even in the absence of degenerative cues. Consequently the p27 pathway represents a prime target to facilitate glial-based regeneration and to modulate glial scar formation.
Reactive gliosis displays context-dependent, graded and dynamic phenotypes
Transient GFAP expression in Müller glia is compatible with neuronal metabolism and function
Upregulation of the intermediate filament GFAP is arguably the most extensively described hallmark of reactive glia, yet it remains unclear whether this property is beneficial or detrimental to neuronal function and survival. The negative view regarding GFAP elevation stems from the correlation between decreased metabolic support and neuronal dysfunction with increased GFAP expression in the retina and brain [11, 13, 19, 20, 41]. For example, retinal detachment upregulates GFAP expression and concomitantly results in loss of GLUL expression and extensive derangement of glial and neuronal metabolite profiles . Genetic targeting of intermediate filament expression levels (inactivation or overexpression) [16, 50] and our inducible model of reactive gliosis reveal a complex role for intermediate filaments on the physiology and pathology of glial cells. While mice lacking intermediate filaments show no developmental or motor functional deficits, they display compromised blood-brain barrier, enhanced hippocampal long-term potentiation, decreased cerebellar long-term depression, white matter pathologies and demyelination [8, 9, 55, 56]. In disease models, the absence of intermediate filaments exacerbates traumatic and toxic injury, autoimmune response, stroke, and scrapie prion infection by reducing glial hypertrophy and scarring, and compromising the ability of glial cells to osmoregulate, transport glutamate, and repair the blood-brain barrier [7–10, 56]. Positive outcomes of reducing glial hypertrophy and scarring in mice lacking intermediate filaments include enhanced adult neurogenesis, axon regeneration, and neural graft survival and integration [17, 57, 17, 25]. However, the extent to which increased regeneration potential improves functional recovery in intermediate filament deficient mice is controversial [9, 10, 17]. These findings have raised the question that intermediate filament upregulation plays a beneficial role in the acute stage, but prolonged upregulation interferes with neuronal survival and regeneration . Supporting this view is the finding that mice transgenic for human GFAP accumulate high levels of GFAP leading to glial hypertrophy, intracytoplasmic aggregates, stress response, oxidative stress, microglial activation and neuronal dysfunction [58, 59]. In contrast, transient GFAP upregulation in p27 L-/L- Müller glia was compatible with glial function (osmoregulation, transmitter recyling, radical and retinoid metabolism), neuronal transmission, and visual function. Taken together, data from mice with null, transgenic, and transient GFAP expression suggest that intermediate filament upregulation facilates the cytoarchitectural remodeling necessary for glial cells to protect the integrity of the tissue, limit the lesion site and modulate basic neuroprotective function of glial cells; but prolonged and robust intermediate filament expression can lead to glial dysfunction.
Proliferative Müller reactive gliosis is insufficient to induce glial dysfunction, scar formation or neurogenesis
In mammals, glial cell proliferation localizes to areas of severe tissue damage after trauma, ischemia, infection, autoimmune response, or fast degenerative disease [5, 6, 60]. In these cases, proliferative gliosis can contribute to the formation of scars, which are hypothesized to impair neuronal function, block axonal regeneration, and interfere with tissue grafts and integration of transplanted cells [17, 18]. The contribution of glial proliferation to scar formation was confirmed when proliferating astrocytes were genetically ablated after traumatic brain and spinal cord injury resulting in decreased scar formation. However, there was also impaired functional recovery that correlated with enhanced spread and persistence of inflammatory cells, failure to repair the blood-brain barrier, enhanced tissue damage, neuronal loss, and demyelination [5, 6, 60]. The negative outcomes from ablating proliferative astrocytes argue for the need to define the individual components of reactive gliosis that are detrimental or beneficial to neuronal function and survival. In our study, inducible Müller glial proliferation, intermediate filament upregulation and migration did not result in scar formation, glial dysfunction or neuronal deficits. The benign impact of inducible proliferative reactive gliosis in our model might be explained by the relatively low level of proliferation, but a more likely explanation is the absence of other changes associated with reactivity such as hypertrophy, decreased potassium clearance, and loss of homeostatic and metabolic enzymes [4, 11, 21, 41]. Consequently, the extent to which neuronal cell stress, microglial reactivity, inflammatory response, blood barrier damage, or neuronal cell death contribute to the transition from supportive glia to detrimental glia needs to be determined (Figure 6B).
Reactive gliosis is a ubiquitous but poorly understood hallmark of CNS and retinal pathologies. Despite the extensive characterization and manipulation of reactive gliosis, several questions remain unresolved. What regulates reactive gliosis? Is the response binary or graded? Is the response detrimental or beneficial to neuronal function and survival? Our inducible model of p27 inactivation exhibited three indices of glial reactivity--proliferation, intermediate filament upregulation, and migratory-like behavior--and allowed us to evaluate their intrinsic impact on retinal neuron function and survival (Figure 6A). Our metabolic, electrophysiological, and visual behavior data argue that proliferative reactive gliosis is compatible with neuronal metabolism and function, and that the final detrimental outcome of glial plasticity during degeneration or injury is determined by additional factors (Figure 6B). In combination with genetic, pharmacological or disease model approaches, the inducible model of proliferative reactive gliosis based on conditional p27 inactivation will be a powerful tool to dissect the factors that induce glial dysfunction or neurogenesis.
p27LoxP mice (coisogenic to 129S4) were bred to CAGG:: CreER™ mice (Jax Stock #4453) and backcrossed 10 generations to 129S4 to generate a tamoxifen inducible p27 knockout mutation (p27 L/L ;CreER™) [35, 36]. The promoter driving Cre-ER™ expression is a chimeric promoter of the cytomegalovirus immediate-early enhancer and chicken beta-actin promoter/enhancer (CAGG) known to drive a widespread expression . Genotyping was performed by PCR as previously described [35, 36]. Tamoxifen-treated controls are listed in Figure 1C. This research protocol was approved by the Institutional Animal Care and Use Committee of the University of Utah, and the Fred Hutchinson Cancer Research Center, and conforms to the standards in The Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.
Tamoxifen (Sigma) was dissolved in peanut oil (Sigma) to a concentration of 5 mg·ml-1. 50 μg of Tamoxifen per gram of body weight (gbw) was administered to 2-month-old mice by single daily intraperitoneal injections for 5 consecutive days. All experimental and control (referred to as tamoxifen control) mice were exposed to the identical tamoxifen treatment regimen.
BrdU (Sigma) was dissolved in 0.1 M phosphate buffered saline, pH 7.4., to a concentration of 10 mg·ml-1. 100 μg BrdU per gbw was administered by single daily intraperitoneal injections for 3 consecutive days.
Tissue samples were prepared, processed and analyzed as described previously . Table in Additional file 5 lists the antibodies used for this study. Sections were collected at the level of the optic nerve to make more direct comparisons between experimental and tamoxifen-control retinas (Additional file 1). Averages from biological replicates were pooled together to determine the total average and standard deviation. P-values were determined with unpaired Student's t tests.
Computational molecular phenotyping (CMP)
Tissues were fixed as previously described for electron microscopy (omitting osmium) and serially sectioned at 200 nm onto 12-well HTC Cel-Line slides (Erie Scientific) ([12, 21, 42, 62], http://prometheus.med.utah.edu/~marclab/protocols.html). Additional file 5 lists the antibodies compatible with CMP. IgG binding was visualized with silver-intensification of 1.4 nm gold granules coupled to species-specific secondary IgGs (Nanoprobes, Yaphank, NY), captured as 8-bit high-resolution (221 nm/pixel) images, mosaicked into large arrays and registered using IR-tweak software http://www.sci.utah.edu/~koshevoy/research/. CMP classification (K-means clustering and histogram analysis) was performed as previously described [42, 62]. Monochrome images are density mapped and rgb images are intensity mapped (see ). All images were prepared in Adobe Photoshop® CS3 (Adobe Systems Inc., San Jose CA).
Mouse electroretinogram (ERG)
Full-field corneal ERGs were recorded with the universal testing and electrophysiological system UTAS E-3000 (LKC Technologies, Inc., Gaithersburg, MD). Dark adaptated mice were anesthetized by intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). Before recording, pupils were dilated with 2.5% phenylephrine (Akorn, Inc., Decatur, IL). Light flash intensities for scotopic ERGs ranged from -3.7 to 2.8 log cd·m-2. Light flash intensities for photopic ERGs ranged from -0.8 to 2.9 log cd·m-2 under rod saturating background light of 1.48 log cd·m-2. Six or fewer flashes were averaged for each intensity level with increasing flash intervals as increasing intensity. Scotopic and photopic ERG responses of p27 L-/L- mice and age-matched tamoxifen control mice were analyzed with two-way ANOVA.
Visual acuity test
Optomotor tracking response to moving gratings was measured using a virtual optomotor system (Opto-Motry; CerebralMechanics, Lethbridge, Alberta, Canada) as previously described . The virtual cylinder was rotated at a constant speed (12°/s). On each trial an experimenter judged whether the mouse made tracking movements with its head and neck to follow the drifting grating. The spatial frequency threshold, the point at which animals no longer tracked, was obtained by incrementally increasing the spatial frequency of the grating at 100% contrast. Thresholds through each eye were measured separately by reversing the rotation of the cylinder .
Acknowledgements and funding
Presented at 13th Annual Vision Research Conference, Fort Lauderdale, Florida, 2010; at 14th International Symposium on Retinal Degenerations, Quebec, Canada, 2010; and at the 19th Meeting of the International Society for Eye Research. We thank Anna Clark (University of Utah) and David Woessner (University of Utah) for reviewing the manuscript and Jack Saari (University of Washington) for generously providing the RLBP1/CRALBP antibody. This work was supported by funding from the National Institutes of Health (to EML [R01 EY013760], to REM [R01 EY02576, R01 EY015128, P30 EY014800], to FRVC [T32 HD07491], to WB [R01 EY08123, R01 EY019298]); from Research to Prevent Blindness (to EML [Sybil B. Harrington Scholar Award] and to the John A. Moran Eye Center [unrestricted funding]); from Fight for Sight (to FRVC and to WDF); from International Retinal Research Foundation (to FRVC); from Knights Templar Eye Foundation (to FRVC); and from Foundation Fighting Blindness (to WB [BR-CMM-0709-0483-UUT]).
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