mGfap-Cre transgenic lines target astroglia in cerebellum
Glial fibrillary acidic protein (Gfap) is a widely used marker for astroglia. In the most commonly used 2.2 kb human GFAP promoter-driven cre transgenic mouse line (hGFAP-cre
), cre activity turns on in embryonic multipotent neural precursor cells (Zhuo, Theis et al. 2001
) as early as embryonic day 13.5 (E13.5), thereby leading to widespread lacZ reporter expression in neurons and astroglia throughout the brain of hGFAP-cre;Rosa26-LacZ
reporter mouse. In search for a more astroglial lineage restricted transgenic mouse line, we characterized two independent lines (line B6.Cg-Tg (Gfap-cre)73.12Mvs/J
) and line B6.Cg-Tg(Gfap-cre)77.6Mvs/J (mGfap-cre 77.6)
), in which cre recombinase expression was driven by the 15 kb mouse Gfap
gene regulatory sequence. Previous studies using single cell evaluations of reporter gene expression have shown that cre activity in line 73.12 mice was present in postnatal astrocytes throughout the CNS, as well as in adult neural stem/progenitor cells localized in subventricular zone (SVZ) and subgranular zone (SGZ) (Garcia, Doan et al. 2004
; Herrmann, Imura et al. 2008
). In line 77.6, cre recombinase activity was also targeted to postnatal astrocytes throughout the CNS, and a subpopulation of adult neural stem/progenitor cells in SVZ, but to a much lesser extent in SGZ (Gregorian, Nakashima et al. 2009
) and not to non-astrocytes in other CNS regions (unpublished observations of M.V. Sofroniew).
To further examine expression patterns of mGfap
-driven cre recombinase, both transgenic lines were crossed with a reporter line in which expression of β-galactosidase (β-gal) protein was under the regulation of an ubiquitous promoter (Rosa 26) that contained a loxP
-flanked stop sequence (). As expected, while β-gal expression was detected in astrocytes throughout the CNS as well as in small populations of neurons in olfactory bulb and hippocampus that are derived from Gfap-expressing adult neural/stem progenitors, cerebellum was among the most densely labeled brain regions with β-gal expression mostly restricted to astrocytes. In the cerebellum, cre was first activated in line 73.12 at around P4 and was first detected in line 77.6 at around P7. As β-gal staining of postnatal day 35 (P35) mouse cerebella showed, in line 77.6, the vast majority of labeled cells were Bergmann glia and inner granule layer (IGL) astrocytes that were also positive for Aldh1l1, another astroglial marker (Yang, Vidensky et al.
; Cahoy, Emery et al. 2008
) (). No NeuN-positive IGL granule neurons or Calbindin-positive Purkinje neurons expressed β-gal at any time point examined in line 77.6 (). In line 73.12, besides Bergmann glia and IGL astrocytes, there was a very small fraction (1~5%) of granule neurons expressing β-gal, which were possibly derived from postnatal neuronal progenitors that transiently expressed cre (). No Purkinje neurons expressed reporter protein in line 73.12 at any time point examined. Therefore in contrast to the commonly used hGFAP-cre
line, activation of mGfap-cre
was largely restricted to postnatal astrocytes throughout the CNS in both transgenic lines, and in cerebellum, targeting of cre in mGfap-cre
line 77.6 was restricted exclusively to postnatal astroglial cells.
Figure 1 In cerebella mGfap-cre transgenic lines mainly target postnatal astroglial cells. (A) A schematic of lineage tracing using mGfap-cre transgenic mice crossing with Rosa26-LacZ reporter line. (B)– (C) Brightfield immunohistochemistry DAB stainings (more ...)
mGfap-cre; Dicerflox;flox mutant mice developed ataxia, epilepsy and premature death
To investigate the role of Dicer-dependent pathways in the astroglial lineage, Dicerflox/flox
mice (Cobb, Nesterova et al. 2005
) were crossbred with mGfap-cre
line) mice (). mGfap-cre; Dicerflox/+
(hereafter referred to as control) mice were phenotypically indistinguishable from wild-type mice. mGfap-cre; Dicerflox/flox
(hereafter referred to as mutant) mice were normal at birth and born with normal Mendelian ratios. Genomic DNA PCR with whole cerebella at pre-/early symptomatic stage showed a small percentage of deleted Dicerflox
allele, which was consistent with the fact that the cerebellum harbored numerous granule neurons, and that astroglia were not the major population in the cerebellum (). Using in vitro
cultured neurospheres isolated from postnatal SVZ of mutant mice, where Dicer
was looped out in majority of the cells, as a positive control for the genomic PCR, we confirmed that genetic deletion of Dicer floxed alleles did occur in the cerebellum (). During these experiments we noted both proliferation and differentiation deficits in Dicer-deficient SVZ adult progenitor cells that will be of interest for future investigations (unpublished observations of J.Tao and Y.E.Sun).
Figure 2 Dicerlox/lox; mGfap-cre mutant mice developed ataxia. (A) A schematic of crossing mGfap-cre transgenic mice with Dicer lox/lox line. (B) Genomic DNA PCR showed the ratio of Dicerflox allele (390bp), Dicerdel allele (309bp) and wild-type allele (259bp). (more ...)
Mutant mice grew normally for the first five weeks postnatally, but around postnatal week 7–8, they started to exhibit noticeable wobbly, imbalanced gait. Rotarod training and testing at postnatal 5 week showed a slightly weaker trend of performance by mutant mice as compared to their littermate controls, whereas at postnatal week 7, mutant mice displayed severe impairment in motor function and balance (). The footprint assay also confirmed an ataxic walking pattern of mutant mice at week 7 with increased hind paw distances and poor overlaps of front and hind paws (). Moreover, the mutant mice exhibited spontaneous epileptic seizures around postnatal week 8 and 9 characterized by uncontrollable movements, straub tail and maintained opisthotonos, possibly due to the loss of Dicer in hippocampal subgranular zone neural progenitors, hippocampal and/or cortical astrocytes. With rapidly aggravating locomotor problems and prolonged seizures, the mutant mice became immobile and became moribund between postnatal 8–9 weeks. Both 73.12 and 77.6 line mutants exhibited ataxia and epilepsy phenotypes and showed very steep survival curves (). Line 77.6 mutants survived on average 12 days longer than line 73.12 mutants, possibly due to the fact that fewer hippocampal neurons were targeted in line 77.6 compared to line 73.12, thereby leading to a later onset of epilepsy. Mutant mice typically reached the late symptomatic stage (or end-stage) very quickly with an average of 12 days after the onset of symptoms. End-stage mutant mice also showed slight weight loss and poor grooming.
mGfap-cre; Dicerflox;flox mutant mice had cerebellum degeneration
The brains of end-stage mGfap-cre; Dicerflox;flox
mutant mice were grossly normal in size and morphology, but cerebella were severely degenerated with blurred fissures (). We therefore focused our investigation on the cerebellum for the present study. To discriminate whether the degeneration phenotype that we observed was a cerebellar developmental deficit or a degenerative process that occurred after development had completed, we carried out time-course Nissl stainings with P7, P15 and P30 control and mutant littermates. It was evident that mGfap-cre; Dicerflox;flox
mutant mice had structurally normal cerebellar development and cellular layers were established properly by P30 prior to the onset of motor symptoms (). Therefore, in contrast to the early developmental defects of cerebella (e.g. reduction in cerebellar size, loss of granule cells and disrupted cellular layers) caused by the ablation of Gfap positive astrocytes during the first postnatal week using hGfap-HSV-TK
transgenic mice (Delaney, Brenner et al. 1996
), the degeneration process in mature mGfap-cre; Dicerflox;flox
mice was not simply due to a lack of astrocytes at early postnatal developmental stages.
Figure 3 Dicerlox/lox; mGfap-cre mutant mice had normal cerebellar development but around postnatal week 8–9, they had granule cell apoptosis and Purkinje cell dendrite degeneration. (A) Compared to control mice, end-stage mutant mice brains showed cerebellar (more ...)
In end-stage (P62 for 73.12 line and P70 for 77.6 line) mutant cerebella, Nissl staining showed that inner granule layer (IGL) cell density was dramatically decreased although cerebellar layers and lobes developed normally early on (). To determine whether there was any cell loss at the time of the onset of neurological symptoms, symptomatic mutant mice (P55~P60) cerebellar sections were stained with cresyl violet. The presence of massive, dark condensed nuclei in IGL suggested acute synchronized cell death (). Consistent with the Nissl staining results, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in brain sections of symptomatic stage mice showed abundant positive signals throughout the cerebellum IGL, but nowhere else in the mutant brain (). Since granule neurons are the most abundant cells in the IGL, we carried out double labeling with TUNEL and a neuronal marker, NeuN. The >90% co-localization of the two markers implied that the majority of granule neurons underwent cell death (). Electron microscopy analyses also demonstrated the presence of a large number of condensed darkened granule cells in symptomatic mutant mice cerebella (). Besides massive granule cell death, although Purkinje cell bodies remained largely intact until late symptomatic stage, they showed disintegration of dendritic arborizations as indicated by immunostaining of calbindin, which labeled Purkinje cell dendrites (). Purkinje dendritic degeneration was further confirmed by Golgi staining (). Moreover, in Purkinje cell dendrites, we observed prominent vacuole formation, darkened cytoplasm and rounded mitochondria, which were indicative of dark cell degeneration (). The observation that major neuronal populations underwent degeneration while the cell-type deleted of Dicer was Gfap positive astroglia indicates a non-cell autonomous effect.
It is noteworthy that Bergmann glia, a major cerebellar astroglial population deleted of Dicer, remained alive and exhibited thickened, swollen processes (). To investigate whether the morphological change of Bergmann glia is a secondary response to neuronal degeneration or a manifestation of primary astrocytic pathology or both, Gfap immunohistochemical analyses were carried out in sagittal cerebellar sections of mice at various developmental stages. While mutant mice seemed to have normal astroglia morphology and Gfap immunointensity during early postnatal cerebellar development (up to P15), mutant Bergmann glia showed increased Gfap immunoreactivity as early as P30 (pre-symptomatic stage) (). At early symptomatic stage (P42), mutant Bergmann glia processes were associated with elevated levels of Gfap expression, conspicuously thickened, and disorganized compared with the fine, perpendicular to the pia oriented Gfap positive glial processes in control mice (). In contrast, in IGL astrocytes, both Gfap and S100b staining were decreased () at early symptomatic stage (P42) while Aldh1l1 staining pattern did not change. Furthermore, no obvious change in total cell number (by corresponding nuclear staining) was observed in IGL, nor were there any TUNEL positive cells detected at pre-/early symptomatic stages, suggesting that the decrease of Gfap immunoreactivity in IGL astrocytes did not indicate a loss of IGL astroglia, but more likely reflected changes in glial protein expression in Dicer-deficient astrocytes. These results suggest that the marked morphological abnormality of both IGL astrocytes and Bergmann glia proceeds any obvious behavioral impairments or neuronal death, and therefore may represent early or upstream events in the progression of pathogenesis.
Figure 4 Astroglial morphological changes occurred prior to neurological symptoms. (A) Gfap DAB stainings showed no difference at early postnatal stages (P15) and the earliest morphological changes were observed around P30. (B) Bergman glia in mutant cerebella (more ...)
Deletion of Dicer altered astrocytic transcriptome in pre-symptomatic stage cerebella
To reveal the early molecular changes at the pre-symptomatic stage that might be responsible for more severe neuronal symptoms at the end-stage, we performed whole-genome transcription profiling with four biologically independent pairs of control and mutant cerebella at P30. As shown in , 367 genes were up-regulated and 267 genes were down-regulated consistently in all four pairs of mutant cerebella compared to their littermate controls (false discovery rate (FDR) <0.05). To further dissect relative contributions from different cell types of the cerebellum to the dysregulated gene sets, we cross-referenced our dysregulated gene list to previously annotated databases of cell-type specific genes in various cerebellar glial and neuronal cells (Doyle, Dougherty et al. 2008
). This analysis indicated that the majority of dysregulated genes were highly enriched in astrocytic cells (Bergmann glia and astrocytes) while very few neuron-specific genes showed transcriptional changes (). Although we could not exclude the possibility that some dysregulated genes might arise from neuronal cell types, the majority of the transcriptional changes, however, were associated with astrocyte-specific genes. To further reveal the functions of dysregulated genes in mutant cerebellar astrocytes, we next performed gene ontology (GO) analyses. Among the up-regulated genes, which included hallmark genes of reactive astrocytes (Gfap
, Tenanscin C
etc), 148 genes encoded for glycoproteins, especially genes that functioned in extracellular matrix, cell adhesion and calcium ion binding (). A group of chemotaxis genes were also up-regulated although there was no increase in Iba1+ microglia in mutant cerebella until symptomatic stage, suggesting that these up-regulated cytokine/chemokine genes might serve as signals for later microglia activation. In addition, a group of solute carrier family genes was up-regulated significantly although little was known about their functions in the brain. A group of cell cycle related genes were also up-regulated. In contrast, a cohort of important glial-specific functional genes was down-regulated, which were mostly involved in oxidation reduction, glutathione metabolism, chemical homeostasis and glutamate transport (). Also, a group of synaptic genes were down-regulated, which might be due to secondary neuronal transcriptional changes.
Figure 5 Transcription profiling of control versus mutant mice cerebella at pre-symptomatic stage (P30) showed a cohort of astrocytic genes dysregulated. (A) Heatmap representations of relative changes in gene expression (from four pairs of littermate cerebella) (more ...)
The observation that many immature/ reactive astrocyte genes were up-regulated while astrocytic genes related to mature astrocyte functions were down-regulated in response to Dicer
deletion, raised the possibility that Dicer was required for astrocytes to become fully functional during astrocytic maturation process in the first three postnatal weeks. Recent transcriptome analyses of acutely isolated purified astrocytes demonstrate that astrocytes undergo substantial changes in transcriptome from developing (P7–P8) to mature (P17 and after) stages (Cahoy, Emery et al. 2008
), indicating that astrocytes at different developmental stages are associated with distinct genetic programs that may determine their functional maturity. To examine whether dysregulated genes in Dicer
mutant mice (P30) were associated with specific developmental stages of astrocytes, we cross-referenced Dicer-dependent gene expression changes to lists of genes highly expressed in developing/immature and mature postnatal astrocytes respectively. Interestingly, this analysis indicated that 62 genes were both down-regulated in Dicer
mutant and specifically enriched in mature astrocytes, which was significantly higher than that expected by chance (P
, Fisher’s exact test, representative genes listed in ). For example, GLT1
(also known as Slc1a2
), a glial transporter only highly expressed in mature, but not developing/immature astrocytes, was reproducibly down-regulated in absence of Dicer
in astrocytes at P30. BC055107
, one of the genes most strongly up-regulated during astrocyte development and suggested to maintain cells in a quiescent state (Cahoy, Emery et al. 2008
), was also markedly down-regulated in Dicer
mutant. Other genes highly enriched in mature astrocytes and down-regulated in Dicer
mutant were involved in oxidation reduction and cellular homeostasis pathways. Similarly, 54 genes were both up-regulated in Dicer
mutant astrocytes and enriched in developing/immature astrocytes, which was also significantly higher than that expected by chance (P
, Fisher’s extract test, representative genes listed in ). The representative genes were functionally related to cell cycle (Cdc2a
etc) and neural precursor cells (Doublecortin
etc). Conversely, 37 genes enriched in developing astrocytes were also further down-regulated in Dicer
=0.08). Moreover, only 19 genes enriched in mature astrocytes were found to be up-regulated in Dicer
=0.67). Thus, Dicer
-deficient astrocytes at the mature stage seem to acquire a transcription profiling partially reminiscent of immature/developing astrocytes.
Representative genes that are down-regulated in Dicer mutant and enriched in mature astrocytes
Representative genes that are up-regulated in Dicer mutant and enriched in immature astrocytes
Quantitative RT-PCR analyses further confirmed that typical reactive gliosis-related extracellular matrix genes, such as Thrombospondin2
, Tenascin C
(), were up-regulated in Dicer-deficient cerebella. Tgfb3
is a potential glial-derived factor that is up-regulated in reactive astrocytes (Apelt and Schliebs 2001
), which was also up-regulated in mutant cerebella (). By contrast, genes normally expressed in mature astrocytes were down-regulated in Dicer-deficient cerebella. Aqp4
, the principal aquaporin in mammalian brain, was expressed in astrocyte foot processes, glia limitans and ependyma. Down-regulation of Aqp4
may alter water transport, K+
kinetics and neuronal activity in the brain (reviewed in (Tait, Saadoun et al. 2008
, a glutathione S transferase involved in reducing oxidation stress, was also down-regulated. The mRNA level of Dao1
(D-amino acid oxidase
), an astrocytic protein that catalyzes the degradation of D-serine (Park, Shishido et al. 2006
), was significantly down-regulated (). Glial-derived D-serine, together with glutamate, activates NMDA receptor. D-serine level in Dao1
mutant cerebellum is more than 10-fold higher than that in the wild-type (Hamase, Konno et al. 2005
; Panatier, Theodosis et al. 2006
). Moreover, the only two known astrocytic glutamate transporters responsible for taking up excessive glutamate, GLT-1
) and GLAST
) were both significantly down-regulated (). Down-regulation of Dao1
indicated the possibility of excitotoxicity. In addition, Glul
), which converts glutamate to glutamine in astrocytes and has been implicated as molecule potentially involved in epilepsy (Ortinski, Dong et al.
), exhibited a modest decrease (Fold change=0.79, FDR=0.14) in mGfap-cre; Dicerflox/flox
mice at P30 (pre-symptomatic) compared to wild-type littermates.
Reduced expressions of glutamate transporters and impaired glutamate uptake function
To further test the functional outcome of specific Dicer
-deficient astrocytic transcriptome alterations, we focused on examining glutamate transport pathways. Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS that activates cells through AMPA, kainate and/or NMDA receptors. The extracellular concentration of glutamate needs to be kept low to limit tonic activation of the receptors as excessive glutamate receptor activation can easily damage neurons (reviewed in (Sheldon and Robinson 2007
)). In the cerebellum where glutamate is a major neurotransmitter, astrocytes provide effective protection of neurons against glutamate excitotoxicity as they clear up excessive extracellular glutamate by active uptake via glutamate transporters GLT-1and GLAST (Chaudhry, Lehre et al. 1995
; Lehre, Levy et al. 1995
; Rothstein, Dykes-Hoberg et al. 1996
). In the cerebellum, GLAST is highly expressed in Bergman glia and GLT-1 is expressed in both Bergman glia and IGL astrocytes. We thus examined protein levels of GLT-1 and GLAST in the cerebella of mice at both pre-symptomatic and symptomatic stages. Western blot analyses showed ~70% decrease of GLT-1 and ~40% decrease of GLAST at P30 (pre-symptomatic stage). With progression of neurological symptoms, protein levels of both GLT-1 and GLAST continued to decrease and became barely detectable at P60 ().
Figure 6 Astroglial glutamate uptake pathway is severely compromised in mutant cerebella. (A) and (B) Western blots and quantifications demonstrated that GLT-1 protein level was markedly down-regulated at pre-symptomatic stage and continued to decrease until end-stage. (more ...)
To assess the in vivo glutamate transporter function, we quantified the uptake of radioactively labeled glutamate in synaptosomes prepared from control and mutant mice cerebella at early symptomatic stage (P40) and at late symptomatic stage (P60). We found that glutamate uptake capacity was decreased by ~25% in synaptosomes isolated from P40 mutant cerebella and it was further reduced at P60 (). Collectively, this series of experiments indicate that Dicer deletion in cerebellar astroglial cells impairs astrocytic glutamate transport already at the pre-symptomatic stage, which may directly lead to excitotoxicity of neurons at the late-symptomatic stage.
NMDA receptor mediated tonic conductance significantly increased in mutant cerebellar granule neurons
To directly test the hypothesis that the decrease of glial glutamate transporter expression in mutant mice cerebella may cause a tonic increase in the concentration of glutamate in the extracellular space and affect neuronal physiology, we recorded cerebellar granule cells in acute slices of pre- or early- symptomatic stage (P35~p40) mutant mice and their control littermates before the onset of granule cell apoptosis. At a holding potential of +30 mV and in the presence of extracellular Mg2+, application of the NMDA receptor antagonist D-AP5 at a saturating concentration (50 µM) blocked a tonic current that had a mean conductance of 262 ±31 pS/pF in mutant granule cells (N=7) compared to a mean conductance of 51±9 pS/pF in littermate control granule cells (N=7) (). Application of TBOA (100µM), a broad blocker of both glial and neuronal glutamate transporters, further increased the tonic conductance of both control and mutant granule cells to the same level (776±68 pS/pF for control cells, N=7; 633±71 pS/pF for mutant cells, N=8, ). A bootstrap statistical analysis of the TBOA treatment-induced potentiation for the NMDA receptor-mediated conductance calculated showed a significant difference between control and mutant (potentiation fold is about 20 for control and 4 for mutant, ). Collectively, these results suggest that at pre- or early-symptomatic stage, although the extracellular glutamate concentration has not reached the ceiling level when all glutamate transporters were blocked by TBOA, the increased ambient glutamate in mutant cerebella has already altered the basal physiology of granule cells with more than a 5-fold increase in NMDA receptor mediated tonic conductance (N=7 for wild type and mutant respectively, P<0.001, two-tailed t-test).
Figure 7 NMDA receptor mediated tonic conductance significantly increased in mutant cerebellar granule neurons. (A) Two representative traces showed the different amplitudes of NMDA receptor mediated tonic currents. Horizontal bars indicated the application of (more ...)