A role for glia in the progression of Rett syndrome (original) (raw)

Nature. Author manuscript; available in PMC 2012 Jan 30.

Published in final edited form as:

PMCID: PMC3268776

NIHMSID: NIHMS351055

Daniel T. Lioy,1,6,12 Saurabh K. Garg,1,6,12 Caitlin E. Monaghan,1,6,12 Jacob Raber,2,3,6 Kevin D. Foust,7 Brian K. Kaspar,7 Petra G. Hirrlinger,8 Frank Kirchhoff,9,10 John M. Bissonnette,4,5,6 Nurit Ballas,11 and Gail Mandel1,6,12,*

Daniel T. Lioy

1Vollum Institute, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

12Howard Hughes Medical Institute.

Saurabh K. Garg

1Vollum Institute, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

12Howard Hughes Medical Institute.

Caitlin E. Monaghan

1Vollum Institute, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

12Howard Hughes Medical Institute.

Jacob Raber

2Departments of Behavioral Neuroscience and Neurology, Portland, Oregon

3Division of Neuroscience, ONPRC, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

Kevin D. Foust

7Department of Pediatrics, The Ohio State University, Center for Gene Therapy, Nationwide Children's Hospital, Columbus, Ohio

Brian K. Kaspar

7Department of Pediatrics, The Ohio State University, Center for Gene Therapy, Nationwide Children's Hospital, Columbus, Ohio

Petra G. Hirrlinger

8Paul-Flechsig-Institute for Brain Research, Leipzig, Germany

Frank Kirchhoff

9Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Gottingen, Germany

10Institute of Physiology, University of Saarland, Homburg, Germany

John M. Bissonnette

4Department of Cell and Developmental Biology, Portland, Oregon

5Department of Obstetrics and Gynecology, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

Nurit Ballas

11Department of Biochemistry and Cell Biology, State University of New York, Stony Brook

Gail Mandel

1Vollum Institute, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

12Howard Hughes Medical Institute.

1Vollum Institute, Portland, Oregon

2Departments of Behavioral Neuroscience and Neurology, Portland, Oregon

3Division of Neuroscience, ONPRC, Portland, Oregon

4Department of Cell and Developmental Biology, Portland, Oregon

5Department of Obstetrics and Gynecology, Portland, Oregon

6Oregon Health and Science University, Portland, Oregon

7Department of Pediatrics, The Ohio State University, Center for Gene Therapy, Nationwide Children's Hospital, Columbus, Ohio

8Paul-Flechsig-Institute for Brain Research, Leipzig, Germany

9Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Gottingen, Germany

10Institute of Physiology, University of Saarland, Homburg, Germany

11Department of Biochemistry and Cell Biology, State University of New York, Stony Brook

12Howard Hughes Medical Institute.

Supplementary Materials

Supplementary.

GUID: 419974B6-3335-483F-91E2-F2475880214D

Supplementary Methods

All animal studies were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee.

Mice - maintenance, breeding, and genotyping

Mice were group housed with littermates in standard housing on a 12:12 h light:dark cycle. For rescue experiments, hGFAPcreT2 mice were backcrossed for eight generations to the C57BL/6 strain. hGFAPcreT2 mice used for knockout experiments were on a FVB/N/C57BL/6 background. Mecp2Stop (ref. 14) and Mecp2Bird.knockout (Mecp2B.Null) (ref. 3) mice were obtained from Jackson Laboratories and were also on a C57BL/6 background. Mecp2Jaenisch.Flox (Mecp2J.Flox) (ref. 4) mice were obtained from the Mutant Mouse Medical Resource Center at University of California, Davis and were also on a C57BL/6 background. Male hemizygous hGFAPcreT2 mice were crossed to female Mecp2+/Stop mice to yield male and female Mecp2Stop-hGFAPcreT2, Mecp2Stop, Mecp2+/y and hGFAPcreT2 genotypes. The floxed Stop sequence was identified from tail biopsies using the following primers: common 5'-AACAGTGCCAGCTGCTCTTC-3', WT 5'-CTGTATCCTTGGGTCAAGCTG-3', and mutant 5'-GCCAGAGGCCACTTGTGTAG-3'. The hGFAPcreT2 sequence was identified with the following primers: 5'-CAGGTTGGAGAGGAGACGCATCA-3', 5'-CGTTGCATCGACCGGTAATGCAGGC-3'. Note that this primer set is specific for the hGFAPcre locus and does not recognize other cre loci. The Jaenich floxed Mecp2 genotype for astrocyte knockout was identified using primers 5'-CACCACAGAAGTACTATGATC-3' and 5'-CTAGGTAAGAGCTCTTGTTGA-3'.

Tamoxifen (TAM) treatments

TAM (Sigma) was made fresh weekly by dissolving in 90% sunflower seed oil/10% ethanol solution by bath sonication for 20-30 min at 4 °C with intermittent vortexing. Final concentration of TAM was 20 mg ml-1. Three-to-four-week-old mice were injected intraperitoneally with 100 mg kg-1 with TAM or oil once daily for 8 days. Occasionally mice began to show signs of discomfort during TAM treatments (that is, decreased mobility, increased tremors, dehydration, rough coat, or gasping). In that case, the remaining TAM treatments were given once every 3-4 days until all mice received 8 injections. The longest interval over which TAM was given was 15 days. Additional TAM treatments to symptomatic mice did not rescue survival.

Phenotype scoring

Mice were removed from their home cage and placed onto a metal laminar flow hood for observation.

1. Mobility: 0 = as wild type. 1 = reduced movement when compared to wild type, with extended freezing periods or extended delay to movement when first placed on the surface. 2 = lack of spontaneous movement when placed on the surface.

2. Gait: 0 = as wild type. 1 = hindlims spread wider than wild type when ambulating and/or a lowered pelvis when ambulating. 2 = lack of full strides by hind limbs resulting in a dragging of hindquarters.

3. Limb posture: 0 = hindlimbs splay outward when suspended by the tail. 1 = one hindlimb is pulled into the body or forelimbs are stiff and splayed outward without motion. 2 = one hindlimb is pulled into the body and forelimbs are stiff and splayed outward without motion and might form a widened bowl shape or both hindlimbs are pulled into the body with or without abnormal forelimb posture.

4. Tremor: 0 = no tremor. 1 = intermittent mild tremor. 2 = continuous tremor or intermittent violent tremor.

5. General Condition: 0 = Shiny coat, clear and opened eyes, normal body stance. 1 = dull or squinty eyes, dull or ungroomed coat, somewhat hunched stance. 2 = piloerection, hunched stance.

Tissue preparation, immunohistochemistry, cell counts, and neuronal soma measurements

Mice were anaesthetized by intraperitoneal injection of Avertin (2-2-2 Tribromoethanol) and killed by transcardial perfusion of 4% parafomaldehyde in phosphate-buffered saline. Brains were post-fixed overnight and then equilibrated in 30% sucrose overnight at 4 °C. Sagittal sections (40 μm) were cut at -20 °C using a cryostat (Leica) and stored at -20 °C. Sections were immunolabelled overnight at 4 °C using the following primary antibodies: rabbit-MeCP2 (1:400, Covance), mouse-GFAP (1:400, Abcam), chicken-GFAP (1:400, Abcam), mouse-NeuN (1:200, Millipore), goat-somatostatin (1:200, Santa Cruz), rabbit-EGFP (1:100, Millipore), sheep-VGLUT1 (1:200, Abcam). Nissl staining (at either 594 nm or 640 nm) was performed as instructed by the manufacturer (NeuroTrace, Invitrogen).

Appropriate Alexa Fluor secondary antibodies (1:500, Molecular Probes) or Cy5 were used for 1 h at room temperature. DAPI was present in the ProLong Gold Antifade (Invitrogen) mounting reagent. All images were collected on a Zeiss confocal laser scanning LSM 510 microscope and an Olympus confocal laser scanning FW1000 microscope.

MeCP2 expressing cells were identified as follows: nuclei of astrocytes (GFAP+ at 594 nm or 640 nm) and neurons (NeuN+ at 594 nm; somatostatin+ at 594 nm or 640 nm; GFAP- at 640 nm/Nissl+ at 594 nm) were first identified by DAPI staining. Cells with clearly identified nuclei were then assessed for MeCP2 expression by analysing 505 nm signal (excitation: 488 nm) in the nucleus. MeCP2 antibody specificity was previously confirmed11 and re-confirmed by immunostaining and western blot of samples taken from male Mecp2Bird.knockout mice3. Cell counts are expressed as the percentage of total astrocytes or neuronal populations in specific brain regions that are MeCP2+.

Somal diameters of Nissl-stained neurons were determined by averaging the lengths of the long and short axes across the cell body. Long and short axes were perpendicular to each other. Only cells with a clearly visible DAPI-stained nucleus were considered. Every fourth serial section was used. Only after all cell diameters were collected was the genotype of each section revealed to the experimenter.

Golgi staining was performed using the FD Rapid GolgiStaining Kit according to the manufacturer's instructions (FD NeuroTechnologies, catalogue number PK401). Tissue was vibratome sectioned at 200 μm. Hippocampal CA1 pyramidal neuron apical branches were analysed using an inverted bright-field microscope at ×20 magnification by two separate experimenters blind to the genotypes.

Neuronal soma VGLUT1+ puncta were counted in the medulla oblongata under ×63 magnification. Only neuronal somas showing VGLUT1+ staining were considered. The experimenter was blind to the tissue genotypes.

Fluorescence intensity measurements

Cells with clear nuclei were identified by DAPI fluorescence. MeCP2 signal for only these cells were considered. MeCP2 signal for this analysis was not amplified. Rather, a Cy2 secondary antibody was used (collected at 505 nm), directed directly against the primary anti-MeCP2 antibody. All images were captured using an AxioCam HRc (Zeiss) at exactly the same exposure. Raw pixel intensities associated with the DAPI and MeCP2 signals were measured separately in Photoshop. The genotypes of the data were revealed to the experimenter only after all data were collected and analysed.

Fluorescence-activated cell sorting (FACS) and semi-quantitative PCR

Whole brains were dissected from 6-8-week-old mice and tissue was minced in small pieces in pre-cooled dissociation medium (80 mM Na2SO4, 30 mM K2SO4, 0.25 mM CaCl2, 20 mM glucose, 10 mM MgCl2, 0.001% phenol red and 10 mM HEPES pH 7.5). The tissue was dissociated in medium containing 40 U ml-1 papain (Worthington) for 45 min at 37 °C. The tissue was washed twice in dissociation buffer before transferring to deactivation buffer (DMEM, 0.5 mg ml-1 DNase I and 10% FBS). Sequential trituration was carried out using 10-, 5- and 1-ml pipette tips. Debris was allowed to settle for 2 min. Supernatant was filtered through a 40 μm cell strainer before cells were harvested at 1,000 r.p.m. for 10 min at 4 °C and re-suspended in Dulbecco's PBS (DPBS). To fix, cells were treated with 1% formaldehyde for 15 min at 25 °C. Cells were washed twice with DPBS and then permeabilized in buffer (PBS, 0.2% Triton X-100 and 10% FBS) for 30 min at 25 °C. To identify the NeuN+ cells in the preparation, cells were probed with anti-mouse NeuN antibody for 30 min at 25 °C. Preparation was probed with anti-mouse IgG-Alexa-488 secondary antibody (Invitrogen). Cells were washed twice with PBS containing 0.2% Triton X-100 before re-suspending in DPBS. To sort the cells using FACS, cells were again passed through a 40 μm filter and subjected to FACS. The sorted cells were collected at 8,000 r.p.m. for 10 min and genomic DNA was prepared from NeuN+ and NeuN- fractions using the QIAamp DNA kit (Qiagen). Genomic PCR for the Mecp2 locus was carried out using the following oligonucleotides: forward MECP2-U2 5'-GTTCAGAATCAGGGGAGCAGCCC-3' and reverse upexIII-R3 5'-CCTTGGGTCAAGCTGGGGCC-3'. For genomic PCR of the β-actin promoter, the following oligonucleotides were used: forward 5'-CCCAACACACCTAGCAAATTAGAACCAC-3' and reverse 5'-CCTGGATTGAATGGACAGAGAGTCACT-3'. PCR products were analysed on a 1% ethidium-bromide-stained agarose gel.

Plethysmography

Respiratory parameters were determined in a body plethysmograph. Individual unanaesthetized animals were placed in a 65-ml chamber with their head exposed through a close-fitting hole in parafilm. A pneumotachograph was connected to the chamber and a differential pressure transducer (Model PT5A, Grass Instrument). The pressure signal was integrated to give tidal volume. Volume changes were calibrated by injecting known amounts of air into the chamber. The analogue signal from the transducer was amplified, converted to digital, displayed on a monitor, and stored to disk by computer for later analysis. Apnoea was defined as an expiratory time of 1.0 s or greater. Irregularity score was determined from: absolute (T_TOT_n - T_TOT_n + 1)/(T_TOT_n + 1).

Motor activity and anxiety assessment

Motor activity and anxiety tests were carried out at the same time of day (12.00 to 18.00) and in the same dedicated observation room. Mice were placed singly into an observation box, which was akin to a new home cage, for a total of 20 min, or a standard open field box for 20 min with side-viewing and top-viewing cameras (Clever Systems), or an elevated Zero maze for 5 min with top-viewing cameras (Clever Systems). Mice were allowed to acclimatize to the observation box for the first 10 min and the next 10 min of recording was analysed on a Dell computer. Activity traces were acquired in real time using StereoScan Software (Clever Systems). The mice could not see the experimenter during recordings. Mice were never tested in the three arenas on the same day.

Western blot

Mice were killed by decapitation, and brains immediately isolated and homogenized on ice in nuclear lysis buffer containing 2-mercaptoethanol. Lysates were boiled for 5 min and separated on a denaturing 10% acrylamide gel. We used an antibody to MeCP2 as described above and a mouse-tubulin antibody (Sigma). Horseradish peroxidaseconjugated secondary antibodies were used and chemically activated with the Western Lighting Chemiluminescent System (PerkinElmer Life Sciences).

AAV9 production and injections

AAV9 was produced by transient transfection procedures using a double-stranded AAV2-ITR based vector system as previously described19. MeCP2 expression was driven from a chicken-β-actin promoter with CMV enhancer. AAV9 virus was titred by quantitative PCR, and stored as previously described19. MeCP2-AAV9 or empty AAV9 (control-AAV9) was injected via the tail vein at 1 × 1012 viral particles in a volume of 300 μl. Injected mice included symptomatic Mecp2Stop/y, Mecp2B.Null/y, or Mecp2J.Null/y mice between 4 and 8 weeks old.

Statistics

All behaviour tests were analysed using two-way ANOVAs followed, when appropriate (P < 0.05), by Newman-Keuls post-hoc test. Soma size measurements were analysed using unpaired two-tailed _t_-tests. All other morphological measurements were analysed using two-way ANOVAs followed, when appropriate (P < 0.05), by Tukey's post-hoc test. Statistical analyses were performed using PRISM software.

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Abstract

Rett syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl CpG-binding protein 2 (MeCP2)1. Although MeCP2 is expressed in most tissues2, loss of MeCP2 results primarily in neurological symptoms1,3,4. Earlier studies propelled the idea that RTT is due exclusively to loss of MeCP2 function in neurons2,4-10. While defective neurons clearly underlie the aberrant behaviors, we and others showed recently that the loss of MeCP2 from glia negatively influences neurons in a non-cell autonomous fashion11-13. Here, we show that in globally MeCP2-deficient mice, re-expression of MeCP2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter (VGlut1). Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports targeting glia as a strategy for improving the associated symptoms.

Global re-expression of MeCP2 postnatally in MeCP2-deficient mice allows normal longevity, rescues motor behaviors, and improves overall health14. Because expression of MeCP2 from the neuronal tau locus in early development prevents appearance of several RTT-like symptoms9, neurons are likely crucial components in a rescue. However, prior in vitro studies indicate that astrocytic MeCP2 supports normal neuronal morphology11,12. Therefore, we asked whether astrocytes might also play a role in rescuing RTT neuropathology in vivo.

To this end, we crossed mice harboring a tamoxifen (TAM)-inducible cre recombinase transgene driven by the human astrocytic glial fibrillary acidic protein (hGFAP) promotor15 (also see Ref. 16 - 18), with mice containing a cre-excisable transcriptional stop sequence in the endogenous Mecp2 gene (Mecp2Stop)14. The progeny that inherited both alleles are referred to as _Mecp2Stop_-_hGFAP_creT2 mice (Supplemantary Fig. 1a). We determined the efficiency of astrocytic excision in ROSA-reporter15 and _Mecp2Stop/y_-hGFAPcreT2 mice (Supplementary Fig. 1b, c, and d). The percentage of MeCP2+ / GFAP+ astrocytes was extremely high in caudal brain regions15, similar to Mecp2+/y (Fig. 1a and Supplementary Fig. 1e). Re-expression of MeCP2 was not detected in oil-treated _Mecp2Stop/y_-hGFAPcreT2 mice (Supplementary Fig. 2a). Importantly, only a very low percentage (< 5%) of excision in neurons was detected by immunolabeling, PCR analysis of the recombined stop sequence, and single cell immunofluorescence intensity measurements (Fig. 1 and Supplementary Figs. 1f and g and 3). This low percentage did not increase with age (Supplementary Figs. 1g and 4), and MeCP2 re-expression was restricted to brain (Supplementary Fig. 5c). Over-expression of MeCP2 in rescued astrocytes was not observed (Supplementary Fig. 2b).

a, Efficiencies of Mecp2 re-expression. The numbers above the bars indicate total number of cells counted. b, Genomic PCR analysis of non-recombined (Stop; 4.3 kb) and recombined amplicons (1.29 kb) of FACS-sorted NeuN+ and NeuN- cells from the whole brain of a TAM-treated Mecp2Stop/y-hGFAPcreT2 mouse. Genomic DNA was prepared from 500,000 cells per group. The wild-type (1.25 kb) Mecp2 amplicon is indicated. The β-actin promoter amplicon shows that similar amounts of DNA were present in the reactions. c, Fluorescence-intensity histogram derived from individual hippocampal pyramidal neurons in tissue sections. Cy2 immunofluorescence intensities of nuclear MeCP2 protein are indicated above the line; DAPI fluorescence intensities of the same neurons are indicated below the line. ALU, arbitrary linear units. n = 3 mice per genotype and 100 cells per mouse.

To rule out that the small percentage of neurons, in combination with the low constitutive level of MeCP2 in the stop mice (Supplemental Fig. 5b and c), might mediate any behavioral changes we would measure, we systemically injected young male Mecp2Stop/y mice with a suboptimal titer of recombinant MeCP2-AAV9 virus19 or virus lacking MeCP2. This resulted in physiological levels of MeCP2 expression in 2% to 35% of neurons, depending on the brain region (Supplementary Fig. 6a - c). Regardless of genotype, none of the treated mice showed improvement of RTT-like phenotypes compared to the control-AAV9 injected Mecp2-/y mice (Supplementary Fig. 6d - g). Taken together, the results validate the use of the hGFAPcreT2 system for dissecting astrocytic contributions to RTT.

The average lifespan of oil-treated _Mecp2Stop/y_-hGFAPcreT2 and Mecp2Stop/y mice was only three months14, which is prolonged compared to Mecp2-/y mice3,4 (Supplementary Fig. 5a), and likely due to the small amount of MeCP2 protein expressed from the stop locus (Supplementary Fig. 5b and c). In contrast, nine of 11 TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice were alive at 7.5 months, when seven of the nine were sacrificed for further analysis. The longest living mouse was sacrificed at 15 months. The TAM-treated _MeCP2Stop/y_-hGFAPcreT2 mice were also on average 20% larger than oil-treated _MeCP2Stop/y_-hGFAPcreT2 mice (Supplementary Fig. 7a). Using a previously described observational scoring system14, overall health of the TAM-treated male (Supplementary Fig. 7b) and female (Supplementary Fig. 8) mice stabilized, rather than worsened like the oil-treated controls, and TAM treatment of a highly symptomatic _MeCP2Stop/y_-hGFAPcreT2 mouse reversed symptoms to nearly hGFAPcreT2 values (Supplementary Fig. 9, Videos S1 and S2).

MeCP2-deficient mice are hypoactive1,3,4 and show altered measures of anxiety-related behaviors6. In the home cage (Fig. 2a and b) and open field (Fig. 2c), oil-treated _Mecp2Stop/y_-hGFAPcreT2 mice traveled only ~20% the distance, and did so at ~20% the velocity, of hGFAPcreT2 control mice. TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice, however, improved to ~50% the level of hGFAPcreT2 mice in both measures. Similar improvements were observed in an open field test to measure anxiety. The oil-treated _Mecp2Stop/y_-hGFAPcreT2 mice spent only ~20% as much time in the center of the cage as hGFAPcreT2 mice, while TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice again improved to ~50% the level of hGFAPcreT2 mice (Fig. 2d). The ratio of distance traveled in the center square to total distance was the same for all genotypes (data not shown). In the elevated zero and plus mazes, Mecp2-/y mice consistently show decreased anxiety-related behavior20,21. The TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice were more anxious than the oil-treated _Mecp2Stop/y_-hGFAPcreT2 mice in the elevated zero maze, improving up to ~50% the level of the control hGFAPcreT2 mice (Fig. 2e)

a, Representative activity in a home-cage-like setting. Duration interval, 5 min. Scale bars indicate 7 inches. b, Locomotor activity histograms in a home-cage-like setting. Duration interval, 10 min. c, Locomotor activity histograms in an open field. Duration, 20 min. d, Time spent in centre of an open field. e, Time spent in open portions of an elevated zero maze. Mice aged 3-4 months. *P < 0.05, **P < 0.01, ***P < 0.001. All error bars indicate s.e.m. The number of mice analysed is indicated above each bar. b-e, Genotypes as in a.

RTT patients and mouse models have abnormal respiration1,22 (Fig. 3a). By 12 weeks, _Mecp2Stop/y_-hGFAPcreT2 mice had irregularity scores and apnea rates significantly more severe than hGFAPcreT2 controls (Fig. 3a and b and Supplementary Fig. 7c, Traces 1 and 2). In contrast, two months after TAM-treatment, the respiratory pattern in 10 of 12 _Mecp2Stop/y_-hGFAPcreT2 mice were within normal range (Fig. 3b). Two mice followed over the subsequent five-month period maintained a regular breathing patterns (data not shown). In two of three TAM-treated mice, we observed complete reversal to a normal respiratory pattern (Fig. 3a and Supplementary Fig. 7c, Traces 3 and 4). The apneic frequency in the third mouse was reduced but did not completely reverse to control levels (Supplementary Fig. 7c, Trace 5). The improvement in respiration was consistent with efficient re-expression of MeCP2 in GFAP+ astrocytes within the preBötzinger complex of the brainstem, an area implicated in respiratory defects in RTT23 (Supplementry Fig. 7e). Treatment of a Mecp2Stop/y mouse with TAM did not alleviate the irregular breathing or apneic frequency (Supplementary Fig. 7c, Trace 2). Oil-treated female _Mecp2+/Stop_- hGFAPcreT2 mice developed a significant number of apneas beginning at four to six months (Fig. 3c). The apneic breathing was corrected by TAM treatment (Fig. 3c), even in the most severely affected female (Supplementary Fig. 7d).

a, Representative plethysmographic recordings from a female RTT patient (modified from ref. 22) and an Mecp2Stop/y-hGFAPcreT2 mouse and control. The two middle traces are from the same Mecp2Stop/y-hGFAPcreT2 mouse before and 62 days after TAM treatment (Supplementary Fig. 7c, trace 3). b, Respiratory irregularity scores and apnoea rates for male mice. c, Same as in b except for female mice. Mice showing at least 1 apnoea per hour were considered for apnoea rates. All error bars indicate s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. The number of mice analysed is indicated above each bar.

The brains of girls with RTT and affected mice exhibit smaller neuronal somal sizes and reduced dendritic complexity in some regions1,4,24,25. At ~ 3.5 months of age, the somal sizes of neurons in hippocampus, cerebellum, and cortex were still smaller in TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice compared to hGFAPcreT2 controls. At seven months, however, somal size was restored only in brain regions showing astrocytic re-expression of MeCP2 (Fig. 4a). Regarding dendritic complexity, the Mecp2Stop/y and oil- treated _Mecp2Stop/y_-hGFAPcreT2 mice had ~25% fewer total number of apical dendrite branches compared to controls. By 3.5 months of age, however, neurons in TAM-treated _Mecp2Stop/y_-hGFAPcreT2 mice had a normal number of branches and this was sustained with further age (Fig. 4b and c). MeCP2-deficient neurons also show deficits in proteins necessary for excitatory neurotransmission, such as VGlut126,27. We detected ~20% fewer peri-nuclear VGlut1+ puncta in Mecp2Stop/y and oil-treated _Mecp2Stop/y_-hGFAPcreT2 mice compared to controls, but the levels increased to normal by three to four months of age with TAM-treatment of _Mecp2Stop/y_-hGFAPcreT2 mice (Fig. 4d and e). Taken together, the anatomical findings suggest that re-expression of MeCP2 in astrocytes can, through a non-cell autonomous mechanism, positively influence components of the neurotransmission machinery in vivo.

a, Somal diameters of indicated neurons. Control, hGFAPcreT2 + TAM. b, Representative traces of silver-impregnated hippocampal CA1 neurons from male mice aged 3-4 months. c, Number of silver-impregnated CA1 apical branches in male mice. Control, Mecp2+/y + TAM. d, Representative images of Nissl-stained neurons immunolabelled for VGLUT1 from medulla oblongata. Scale bar: 10 μm (b); 2 μm (d). e, Number of VGLUT1+ puncta associated with neuronal cell bodies from the medulla oblongata. All error bars indicate s.e.m. ***P < 0.001. NS, not significant. The number of analysed cells is indicated above each bar.

Our results show that re-expression of MeCP2 in astrocytes ameliorates overt RTT-like phenotypes in mice. To address the complementary question of the consequences of the removal of MeCP2 from astrocytes, we crossed mice with a floxed MeCP2 allele4 to the same hGFAPcreT215 line used for the rescue. Recombination efficiencies throughout the brain were again higher in caudal compared to rostral regions (Supplementary Fig. 10a and b). The knockout progeny displayed some phenotypes shared with null MeCP2 mice, such as smaller body size, clasped hindlimb posture, and irregular breathing (Supplementary Fig. 10c, d, and f), but their lifespans, locomotion (data not shown), and anxiety-related behaviors were all normal (Supplementary Fig. 10e). Further, loss of MeCP2 from astrocytes did not affect the number of CA1 apical dendritic branches (Supplementary Fig. 10g). This suggests that loss of MeCP2 in astrocytes at postnatal day 21 is unable to disrupt the already established hippocampal neuronal circuitry. In contrast, loss from and gain of MeCP2 in astrocytes resulted in a strong non-cell autonomous influence on breathing pattern. Thus, distinct neuronal-glial interactions may underlie hippocampal and hindbrain breathing circuitries.

Our results indicate that RTT involves impairments in both neurons and glia. In familial amyotrophic lateral sclerosis28, neurons and glia are proposed to play different roles in the disease process, with neurons primarily initiating the disease and astrocytes primarily affecting disease progression. Our results are compatible with this model, because removal of MeCP2 just from astrocytes, at postnatal day 21, results in a subtler phenotype than the global null, and re-expression in astrocytes mainly stabilizes symptoms. Along these lines, the appearance of a subset of phenotypes after embryonic removal of MeCP2 from subsets of neurons4,6-10,29 could be explained by causing disease initiation, and prevention of RTT-like phenotypes after MeCP2 re-expression in embryonic neurons9 could be interpreted as preventing disease initiation. None of these studies address whether it could take both MeCP2-deficient neurons and glia to cause disease progression, or whether other non-neuronal cell types, including other glial types, might be involved in the disease process.

In sum, although impaired neurons ultimately underlie nervous system failure in RTT, restoring MeCP2 in glia can ameliorate four consistent and robust features of mouse models of RTT: premature lethality, aberrant respiration, hypoactivity, and decreased dendritic complexity. Future studies identifying the key molecules that are restored after glial MeCP2 re-expression may provide further clues into the mechanism of recovery, thereby providing new potential targets for therapeutic intervention.

Methods summary

Male mice harboring an hGFAPcreERT2 transgene were crossed to female Mecp2+/Stop or Mecp2+/Jaenisch.Flox mice and the F1 progeny were injected with 100mg/kg tamoxifen or oil when appropriate. Mice used in astrocyte-rescue experiments were backcrossed for at least seven generations to a C57BL6 background. Mice used in astrocyte-knockout experiments were of a FVB/N/C57BL6 background. Histology was performed on transcardially perfused, frozen sections. Behavior was analyzed using CleverSystems StereoScan software. Body plethysmography was performed on unanesthetized restrained mice. Statistics were performed with Graphpad PRISM V5.0C software. Mouse maintenance, breeding, and genotyping, tamoxifen treatments, phenotype scoring, tissue preparation and immunohistochemistry, fluorescence intensity measurements, FACS, plethysmography, motor activity and anxiety assessments, western blotting, and statistics were performed as described in Supplementary Methods.

Supplementary Material

Supplementary

2

Supplementary Methods

All animal studies were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee.

Mice - maintenance, breeding, and genotyping

Tamoxifen (TAM) treatments

Phenotype scoring

Mice were removed from their home cage and placed onto a metal laminar flow hood for observation.

4. Tremor: 0 = no tremor. 1 = intermittent mild tremor. 2 = continuous tremor or intermittent violent tremor.

5. General Condition: 0 = Shiny coat, clear and opened eyes, normal body stance. 1 = dull or squinty eyes, dull or ungroomed coat, somewhat hunched stance. 2 = piloerection, hunched stance.

Tissue preparation, immunohistochemistry, cell counts, and neuronal soma measurements

Fluorescence intensity measurements

Fluorescence-activated cell sorting (FACS) and semi-quantitative PCR

Plethysmography

Motor activity and anxiety assessment

Western blot

AAV9 production and injections

Statistics

Acknowledgements

We thank P. Brehm, R. H. Goodman, C. Bond, M. McGinley, and C. Mandel-Brehm for helpful discussions, P. Micha, J. Eng, S. Knopp, and T. Shaffer for technical support, and M. Murtha for generating the CBA/CMV-MeCP2 construct. ViraPur, LLC generated the AAV9 virus. The work was supported by grants from the National Institutes of Health (G. M and N. B.), International Rett Syndrome Foundation (N. B. and J. M. B.), Rett Syndrome Research Trust (G. M. and B. K. K.), Oregon Brain Institute (D. T. L.), and OHSU Cell and Developmental Biology Training Program (D. T. L.). Gail Mandel is an Investigator of the Howard Hughes Medical Institute.

Footnotes

Author contributions D. T. L., S. K. G., J. R., J. M. B., N. B., and G. M. designed the astrocyte knockout and rescue experiments. B. K. K. and K. D. F. helped design the AAV9 experiments. D. T. L., S. K. G., C. E. M., and J. M. B. performed the experiments. P. G. H. and F. K. provided the hGFAPcreT2 transgenic mice. D. T. L., S. K. G., N. B., and G. M. wrote the manuscript with input from the other co-authors.

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