Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology

Transgenic mice on a C57BL/6j background were generated with an expanded CGG99 trinucleotide repeat segment (Gfa2-CGG99-eGFP) or a typical mouse-sized CGG11 (Gfa2-CGG11-eGFP) repeat sequence. For simplicity, the Gfa2-CGG99-eGFP mice are referred to as Gfa2-CGG99 and the Gfa2-CGG11-eGFP mice as Gfa2-CGG11 control mice, respectively. Wildtype (WT) non-transgenic littermates were generated from breeding the Gfa2-CGG99 transgenic mice with WT C57BL/6j mice. These WT littermates mice were used as controls for behavioral studies to avoid litter effects and because sufficient numbers of the Gfa2-CGG11 transgenic mice were not available. Genotype was verified in all mice by PCR from tail-snips. A diagram of the DNA constructs and the nucleotide sequences used for pronuclear injection are shown in Fig. 1a and b, respectively. Expression vector maps are included in Additional file 1: Figure S1. Expression was restricted to astrocytes and Bergmann glia using the astrocyte-specific Gfa2 promoter, with the enhanced green fluorescent protein (eGFP) reporter used to identify cells expressing the Gfa2-CGG99-eGFP or the normal length Gfa2-CGG11 transgene. The eGFP sequence was derived from the pBR-eGFP vector. Expression of the CGG99 and CGG11 trinucleotide repeat expansions and eGFP reporter are driven by ~ 2-kb of the human Gfa2 promoter, which drives astrocyte-specific expression in transgenic mice [5]. cDNA derived from patient and control peripheral blood lymphocytes was used to isolate ~ 226 bp of FMR1 5′-UTR sequence as well as the CGG repeats, which were cloned into Blp I and Pst I restriction sites. The construct also contains a chimeric intron upstream of eGFP to enhance expression levels. Gfa2-CGG11-eGFP and Gfa2-CGG99-eGFP clones (i.e., clones 3 and 11, respectively) were digested with ApaLI and SnaBI (10 μg each) and the respective 4.8 and 5.1 kb restriction fragments were purified away from the 0.9 kb vector backbone on an agarose gel. The purified Gfa2-CGG11-eGFP (86 ng/ul & 1.8260/280 ratio) and Gfa2-CGG99-eGFP (62 ng/ul & 1.8260/280 ratio) DNA fragments were then microinjected into pronuclei of oocytes from C57BL/6J x C3H/HeJ F1 hybrids at the University of Washington Microinjection Service Laboratory. Six of the 26 Gfa2-CGG11-eGFP mice screened (LS-2997, LS-3006, LS-3009, LS-3011, LS-3025, LS-3030 were found to be positive for the transgene by PCR. Six of the 27 Gfa2-CGG99-EGFP mice screened (LS-3046, LS-3049, LS-3060, LS-3061, LS-3065, LS-3072) were also found to be positive for the transgene by PCR. The Gfa2-CGG11 and Gfa2-CGG99 mouse lines used in these experiments were selected based on matched, high expression levels by TaqMan real time PCR using primers and probes to the eGFP gene as follows: forward, 5′- GTC CGC CCT GAG CAA AGA -3′; reverse, 5′- TCC AGC AGG ACC ATG TGA TC -3′; Fam-probe, 5′- CCC AAC GAG AAG CG -3′.

Eight male Gfa2-CGG99 mice and five male Gfa2-CGG11 control mice between 4 and 16 months old were used for histological and molecular studies. In addition, brains of two male CGG_n knock-in (CGG128 and CGG159) and 2 WT mice 16 months of age were immunostained for ubiquitin-positive intranuclear inclusions in neurons and astroglia for comparison with Gfa2-CGG99 mice and Gfa2-CGG11 mice, as previously described [56]. Transgenic and WT mice were housed under conditions of constant temperature and a 12 / 12 h light-dark cycle, and with food and water ad libitum. These experiments followed the “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985), and were approved by the UC Davis Institutional Animal Care and Use Committee.

DNA was extracted from mouse tails by incubating with 10 mg/ml Proteinase K (Roche Diagnostics) in 300 μl lysis buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% SDS overnight at 55 °C. One hundred μl saturated NaCl was then added and the suspension was centrifuged. One volume of 100% ethanol was added, gently mixed, and the DNA was pelleted by centrifugation and the supernatant discarded. The DNA was washed and centrifuged in 500 μl 70% ethanol. The DNA was then dissolved in 100 μl milliQ-H₂0. CGG repeat lengths were determined by PCR using the Expanded High Fidelity Plus PCR System (Roche Diagnostics). Briefly, approximately 500–700 ng of DNA was added to 50 μl of PCR mixture containing 2.0 μM of each primer, 250 μM of each dNTP (Invitrogen), 2% DMSO (Sigma), 2.5 M Betaine (Sigma), 5 U Expand HF buffer with Mg (7.5 μM). For the CGG KI mice the forward primer was 5′-GCTCAGCTCCGTTTCGGTTTCACTTCCGGT-3′ and the reverse primer was 5′-AGCCCCGCACTTCCACCACCAGCTCCTCCA-3′. For the Gfa2-CGG99 mice the forward primer was 5′- GTC CGC CCT GAG CAA AGA -3′; and the reverse primer was 5′- TCC AGC AGG ACC ATG TGA TC -3.’ PCR steps were 10 min denaturation at 95 °C, followed by 34 cycles of 1 min denaturation at 95 °C, annealing for 1 min at 65 °C, and elongation for 5 min at 75 °C to end each cycle. PCR ended with a final elongation step of 10 min at 75 °C. DNA CGG band sizes were determined by running DNA samples on a 2.5% agarose gel and staining DNA with ethidium bromide.

The behavioral tests used in the present study for GFAP-CGG99-EGFP mice were selected to examine similar neurobehavioral deficits in FXTAS, including anxiety in the elevated plus-maze, ataxia and motor deficits in the ladder rung test, rotarod and TredScan apparatus, and cognitive loss using contextual fear conditioning [20].

Immunocytochemical and immunofluorescence techniques were used to visualize the occurrence and distribution of intranuclear inclusions, specifically in brain astroglia and cerebellar Bergmann glia of Gfa2-CGG99, of Gfa2-CGG11 control and WT mice. Subsets of alternate sections were processed for immunocytochemistry using a modification of the avidin-biotin complex (ABC)-peroxidase technique [29] as previously described [56]. Briefly, free-floating sections were rinsed in PB (pH 7.4) and pretreated with 0.1% sodium borohydride for antigen retrieval. Endogenous peroxidases were inactivated by treatment with 0.5–2% H₂O₂. Sections were then treated with 3% goat, horse and/or swine serum (Sigma, St. Louis, MO; DAKO, Inc., Carpinteria, CA) and 0.3% Triton X (TX) in 0.01 M PB, 0.15 M NaCl, pH 7.4 (PBS) for 1 h to reduce nonspecific staining. Following rinses in PBS, sections were incubated for 48–72 h at 4^o C in the primary antibodies: mouse monoclonal anti-glial fibrillary acidic protein (GFAP), (DAKO, Inc.), 1:2000 (1:750 for immunofluorescence (IF); rabbit polyclonal anti-S100β (Abcam, Inc., Cambridge, MA), 1:1000; mouse monoclonal anti-myelin basic protein (MBP), (Chemicon International, Inc., Temecula, CA), 1:500; mouse monoclonal anti-MAP 2 (Sigma), 1:2000 (1:1000 for IF); rabbit polyclonal anti-Iba1 (ionized calcium binding adaptor molecule 1; Wako Chemicals USA, Inc., Richmond, VA), 1:2000 (1:1000 for IF); mouse-monoclonal anti-Kv2.1 (kindly provided by Dr. J.S. Trimmer; UC Davis), 1:500 for IF; rabbit polyclonal anti-eGFP (Invitrogen, A11122: 1:1000) and rabbit polyclonal antibody against ubiquitin (DAKO, Inc.), 1:2000, (1:1000 for IF) in PBS containing 1% goat, horse or swine serum, 2% BSA and 0.3% TX. Following rinses in PBS, sections were incubated in biotinylated goat or swine anti-rabbit IgG (DAKO, Inc.; Vector Laboratories, Burlingame, CA), diluted 1:500 for 24 h at 4 °C. After rinses in PBS, sections were incubated in ABC (Elite ABC Kit, Vector Laboratories), diluted 1:500 in 2% BSA, 0.3% TX and PBS for 24 h at 4 °C. After rinses in PB followed by Tris-HCl buffers (pH 7.4; 7.6), sections were incubated in 0.025% 3,3′-diaminobenzidine (DAB, Sigma) with 0.003% H₂O₂ in TB (pH 7.6). The incubation was stopped by rinses in TB and PB. For double-immunostaining (e.g., GFAP/ubiquitin-colocalization) differently-colored chromogens were used (DAB; Vector SG Substrate Kits, Vector Laboratories). Specificity of the immunostaining was evaluated by omitting primary antibodies from the regular staining. Sections were mounted on slides, dehydrated, cleared, and cover-slipped with Permount.

For single and multiplex immunofluorescent labeling of ubiquitin and neuronal/glial cell markers, frozen sections were transferred into buffered 30% and/or 10% sucrose, then rinsed in 0.1 M PB and treated with 0.1% sodium borohydride for 15 min. Thereafter, sections were rinsed again with 0.1 M PB and then permeabilized with 0.5% H₂O₂ in 0.1 M PB for 15 min followed by rinses in 0.1 M PB and 0.01 M PBS. Free-floating sections were treated with 10% goat serum in 0.01 M PBS containing 0.3% TX-100 (vehicle) for 1 h and then incubated overnight at 4 °C in vehicle containing different combinations of mouse monoclonal/rabbit polyclonal antibodies of different IgG isotypes (see above). After rinses in 0.01 M PBS and 10% goat serum (vehicle), sections were incubated in isotype-specific Alexa-conjugated secondary antibodies (1:2000): Alexa 568- and/or 488-labeled goat anti-rabbit IgG and/or Alexa 488 and/or 568-labeled goat anti-mouse IgG (Invitrogen, Carlsbad, CA) for 1–2 h as described previously [57]. Following rinses in vehicle, sections were mounted on gelatin-coated slides and cover-slipped with mounting medium containing DAPI (4′, 6-diamidino-2-phenyindole di-lactate) for nuclear staining (Vectashield “Hard Set”, Vector Laboratories).

Repeat-associated non-ATG (i.e., RAN translation) translation of a novel potentially toxic peptide, FMRpolyG, was recently described in the brains of CGG KI mice [47]. Therefore we carried out immunostaining for FMRpolyG in order to determine whether this peptide was also present in the inclusions in the Gfa2-CGG99 mice. Whole Gfa2-CGG99 and WT mouse brains were sectioned sagitally and the hemispheres were fixed overnight in 4% paraformaldehyde and embedded in paraffin according to standard protocols. Sections (6 μm) were cut on a rotary microtome and deparaffinized, followed by antigen retrieval using microwave treatment in 0.01 M sodium citrate. Endogenous peroxidase activity was blocked and immunostaining was performed overnight at 4 °C using mouse anti-GFP (Roche 181446011000), rabbit anti-ubiquitin (Dako Z0458; 1:250), or mouse-anti FMRpolyG (8FM) [9]; 1:10) antibodies. Antigen-antibody complexes were visualized by incubation with DAB substrate (Dako) after incubation with Brightvision poly-HRP-linker (Immunologic). Slides were counterstained with haematoxylin and mounted with Entellan.

Multiplex immunofluorescence staining for FMRpolyG, ubiquitin, GFAP and NeuN were carried out in separate brain sections. Sections were blocked for autofluorescence with Sudan Black in 70% ethanol. Primary antibodies included rabbit-anti ubiquitin (DAKO Z0458; 1:50), mouse-anti ubiquitin (Cytoskeleton AUB01-S; 1:200), mouse-anti FMRpolyG (1:10) [9], rabbit anti-GFAP (Sigma G9269; 1:200), and mouse anti-Map 2 (Roche, 1:400). Secondary antibodies included anti-rabbit Fab Alexa 488 (Life technologies A11070; 1:100) and anti-mouse Cy3 (Jackson Immuno research 715–165-150; 1:100). Nuclei were visualized with Hoechst. Analysis was done with a Leica confocal microscope and Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems, Buffalo Grove, IL).

Frozen 30 μm brain sections were collected from CGG KI and Gfa2-CGG99 transgenic mice perfused with 4% PB-buffered PFA, post-fixed for 1 h and stored in 30% sucrose at 80 °C. Sets of sections were thawed and postfixed with Karnovsky fixation solution for 1 h. The sections were then postfixed with 1% buffered osmium tetroxide for 1 h, then thoroughly rinsed with PB and dehydrated in a series of ethanol solutions and flat embedded in a mixture of Epon and Araldite between two aclar sheets for 24 h at 70 °C as previously described [55]. Serial ultrathin sections were collected on TEM grids, and stained with uranyl acetate and lead citrate. Sections were then examined in a Philips 120 electron microscope. Electron microscopic images were acquired digitally using a 2 k × 2 k high resolution CCD camera (Gatan, Pleasanton, CA), and post-processed using Photoshop software.

The sections were analyzed using a Nikon ECLIPSE E600 microscope with epifluorescence attachment and digital camera. Images were converted to a file format for processing as an Adobe Photoshop document. Images were analyzed to verify the presence of ubiquitin-positive intranuclear inclusions in different cell types identified with various neuronal and glial cell markers in brains of Gfa2-CGG99, Gfa2-CGG11 and WT mice. Cresyl violet and/or H&E-stained sets of brain sections from transgenic and WT mice at different ages were used for comparison and evaluation of gross anatomical differences. The different cell types in the brain were determined based on standard morphological criteria using Nissl cell staining and neuronal and glial cell markers as described previously [23, 56].

Neurons were identified by their size, large round nuclei, single or multiple nucleoli, and their abundant cytoplasm, as well as by using molecular neuronal markers (e.g., NeuN, Kv2.1 channel protein). These descriptions were confirmed using electron microscopy.

Astroglia were identified by their round/ovoid nuclei with light euchromatin, and absence of nucleoli and cytoplasm. In addition, GFAP- and S100β-immunocytochemistry and/or immunofluorescence were used to identify subpopulations of astroglia based on their differing immunoreactivities (i.e, protoplasmic and/or fibrous astroglia) in different brain regions. These descriptions were confirmed using electron microscopy.

Oligodendrocytes (MBP-immunopositive cells) were identified based on their typical appearance with small, round, hyperchromatic nuclei surrounded by thin somatic cytoplasm, and their localization to white and grey matter.

Microglia were identified primarily on cellular morphology obtained from Iba1 immunostaining which displayed small cell bodies with a round nucleus and fine, ramified processes that are characteristic of resting microglia, and were clearly distinguishable from activated (but non-phagocytic) microglia and phagocytotic cells (i.e., brain macrophages). For all cell types, immunocytochemical staining for ubiquitin was used to specifically label intranuclear inclusions in combination with cell-specific markers to identify the cell type (i.e., neuronal and/or non-neuronal cells). Finally, all cell and inclusion types were determined and confirmed based on their ultrastructural appearance and characteristic features using electron microscopy.

To evaluate the cellular specificity of Gfa2-CGG99-eGFP expression, single cell laser capture microdissection (LCM) was performed on ubiquitin-immunolabeled cells. Gfa2-(CGG99)-eGFP, CGG knock-in (KI) and WT mice were euthanized by lethal overdose with sodium pentobarbital after which their brains were rapidly removed and immediately frozen in OCT compound (Ted Pella Inc., Redding, CA). Coronal sections were cut at 12 μm on a cryostat (Leica Microsystems Inc., Buffalo Grove, Il). Sections were direct-mounted onto MMI Membrane Slides (Molecular Machines & Industries AG, Switzerland) and dried for 10 min at room temperature. Sections were then rinsed briefly in water, fixed in 70% ethanol for 1 min, and incubated in rabbit polyclonal antibody against ubiquitin (DAKO, Inc.), 1:100 in 0.1 M PB with 5% goat serum for 1 h at room temperature. After 3 brief washes in 0.1 M PB, sections were incubated in Alexa 488-labeled goat anti-rabbit secondary IgG containing 5% goat serum for 1 h at room temperature. Finally, sections were rinsed 3x briefly in 0.1 M PB, counterstained with DAPI in 0.1 M PB for 1 min and dehydrated through a descending series of alcohols. After complete drying, slides were either directly processed for LCM or stored at -80 °C.

LCM was performed on coronal sections using a MMI CellCut Laser Capture Microscope (Molecular Machines & Industries AG, Switzerland). Individual neurons were captured onto isolation caps of specifically designed centrifuge tubes (MMI) and maintained frozen at -80 °C until RNA purification. Approximately 100–200 individual neurons containing ubiquitin-positive inclusions, but negative for eGFP histofluorescence, were captured from 18 tissue sections from the ventromedial hypothalamus and combined into a single PCR tube. In addition, astrocytes expressing eGFP fluorescence and bearing ubiquitin-positive inclusions were laser-captured from 10 neocortical tissue samples from Gfa2-CGG99-eGFP mice, combined and used as a eGFP-positive control. For comparison 100–200 cells from the amygdala of a CGG KI mouse with a CGG168 repeat expansion but no eGFP were isolated and combined as a negative control.

Total RNA was isolated from LCM samples using a Qiagen RNeasy micro kit (Qiagen, Germantown, MD) according to manufacturer’s recommendations. RNA quantity and quality was estimated using a NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA) and Agilent Bioanalyzer (Agilent Techologies Inc., Santa Clara, CA). RNA from each sample was subjected to linear amplification using Nugen Inc. SPIA technology (Nugen Tehnologies Inc., San Carlos, CA). The quantity and quality of resulting amplified cDNA was assessed using a NanoDrop and Bioanalyzer.

Real-time PCR was performed using an iCycler (Bio-Rad) to measure incorporation of the fluorescent dye SYBR Green I. For each reaction, a master mix of the following was made: 1× PCR buffer (QIAGEN), 400 mM dNTP, 0.5 mM forward (5′-AGTGGAGAGGGTGAAGGTGA) and reverse (5′-GGTAAAAGGACAGGGCCATC) eGFP primers (Operon), 0.01× SYBR Green I (Invitrogen), 1.5 mM MgCl2, 10 nM FITC (Bio-Rad), and 1 U of TaqDNA polymerase (QIAGEN). All PCRs were optimal for the following cycle conditions, 94 °C (15 s), 60 °C (30 s), and 72 °C (30 s), and were run for approximately 40 cycles. After the PCR, a melting-curve analysis was performed to confirm the specificity of the PCR. In addition, samples of the PCRs were subjected to electrophoresis to verify product size and specificity. The relative quantification of RNA targets was performed as follows: The threshold cycle (Ct) at which a gene of interest first rose above background was determined and subtracted from that of the housekeeping gene, β-actin, the PCR for which was performed in a separate reaction tube. This was termed ΔCt. The ΔCt for each reaction was plotted as 2 − ΔCt. Therefore, all values are for RNA expression normalized to β-actin mRNA.

Behavioral data were analyzed using R 2.14.0 language and environment. Data for each variable were examined for normality using the Shapiro-Wilk test and Kolmogorovo-Smirnov test. Normally distributed data were analyzed by Analysis of Covariance (ANCOVA) with body weight as a covariate. If the assumption of normality of distribution was violated, then group comparisons were carried out using nonparametric rank-based ANCOVA with body weight as a covariate. The minimum levels for statistical significance set at p < 0.05 for all statistical analyses. Data in figures are means ± standard error of the mean (SEM). Detailed statistical results for behavioral experiments are provided in Additional file 1: Figure S2.

Ubiquitin-positive intranuclear inclusions are the hallmark neuropathology in FXTAS patients [26, 27], and similar appearing inclusions are found in a CGG knock-in (KI) mouse model of the fragile X premutation [61, 64]. Figure 5 shows representative red immunofluorescent staining for ubiquitin-positive intranuclear inclusions in neurons (arrowheads) and in a astrocytes (arrow). Astrocyte was labeled immunofluorescent green for GFAP. Brain section is from layer I of the parietal cortex of a 16 month old CGG KI mouse with a 128 CGG trinucleotide repeat expansion. Ubiquitin-positive inclusions were never observed in neurons or astroglia of WT mice used in this study, or in our previous studies in any brain region at any age [46].

As expected, astrocytes showed green eGFP histofluorescence through the brain in Gfa2-CGG99-eGFP (Fig. 6a) and Gfa2-CGG11-eGFP (Fig. 6b) mice. This is shown for the rostral neocortex where eGFP expression was higher in Gfa2-CGG99 (Fig. 6a) compared to Gfa2-CGG11 mice (Fig. 6b). Gfa2-CGG99 mice showed eGFP histofluorescence in the majority of astroglia across all brain regions (e.g., neocortex, hippocampus, cerebellum, brain stem nuclei). High levels of eGFP histofluorescence were seen in somata, as well as the larger processes and majority of fine processes which appeared as a “green-fluorescent cloud” around the cell body (i.e., see Fig. 6f and g). Immuno-labeling astrocytes with GFAP, microglia with Iba1 and neurons with NeuN or MAP 2 was carried out to determine specificity of expression of eGFP in the different cell types. The results revealed that eGFP histofluorescence was only observed in astrocytes and Bergmann glia, and not in microglia, oligodendroglia or neurons.

Although greater than 50% of astroglia in the Gfa2-CGG99 mice expressed eGFP, ubiquitin-positive inclusions were only observed in 0.1–0.5% of predominantly protoplasmic astroglia, depending on age and brain region examined. Astrocytes with inclusions, although low in number, were widely distributed throughout the brain including neocortex, cerebellum and occasionally in subcortical brain regions (e.g., hypothalamus and some brain stem nuclei). Figs. 6c-d show astroglia visualized by eGFP histofluorescence (6C) or co-labeled for the glial marker GFAP (6D) with ubiquitin positive intranuclear inclusions (arrows). The inclusions were spherical bodies averaging 1.82 ± 0.29 μm in diameter located within DAPI-stained nuclei of astroglia (Figs. 6c-d). No ubiquitin-positive intranuclear inclusions were found in astrocytes from WT mice (e.g., Fig. 6e) identified by anti-GFAP immunofluorescence, or in the Gfa2-CGG11 transgenic control mice (e.g., Fig. 6f). Examples of a Gfa2-CGG99 neocortical astrocyte lacking or harboring a ubiquitin-positive intranuclear inclusion are shown in Fig. 6g and h, respectively. Interestingly, eGFP expression appeared to be lower in astrocytes from Gfa2-CGG99 mice that contained ubiquitin-positive intranuclear inclusions. For example, compare eGFP histofluorescence in an astrocyte without an inclusion in 6G to an astrocyte with an inclusion in 6H. Developmentally, intranuclear ubiquitin-positive inclusions in astroglia were first observed in neocortex at 4 months of age, but the number was low and only increased slightly at older age (greater than 10 months). We did not find evidence of increased astrocyte proliferation (i.e., gliosis) in the Gfa2-CGG99 or Gfa2-CGG11 mice.

Glia with characteristic Bergmann cell morphology [7, 32, 42, 56, 59] were observed by eGFP histofluorescence throughout the cerebellum in Gfa2-CGG99 and Gfa2-CGG11 mice. Figure 6i shows a representative example of a Bergmann glia cell from a Gfa2-CGG11 transgenic mouse which has the characteristic morphology of Bergman glia (small arrows point to radial processes). No ubiquitin-positive inclusions were found in Bergmann glia from Gfa2-CGG11 mice. In contrast, ubiquitin-positive intranuclear inclusions averaging 1.88 ± 0.36 μm in diameter were observed in somata of Bergmann glia from Gfa2-CGG99 mice (Figs. 6j, k, l, arrows). It is notable that ubiquitin-positive inclusions were more frequently found in Bergmann glia of the cerebellar lobuli 1, 2 and 10, and rarely in the other lobules. All of the ubiquitin-positive inclusions associated with Bergmann glia had defined spherical structures and were located within DAPI-positive nuclei. In addition to Bergmann glia, ubiquitin-positive inclusions were also seen in the velate protoplasmic astroglia of the granule layer, and in astroglia located within the molecular layer and white matter of the cerebellum (not shown). Ubiquitin-positive inclusions were not found in nuclei of Purkinje cells or other cerebellar neurons in Gfa2-CGG99 mice or from Gfa2-CGG11 control mice. We also did not observe a pattern of Purkinje cell loss in Gfa2-CGG99 mice resembling that reported earlier in transgenic mice expressing a similar, but not identical construct [47]. Further, there were no ubiquitin-positive inclusions identified in selectively stained oligodendroglia (not shown) or in microglia (an Iba1-labeled red fluorescent microglia without an inclusion is shown in the bottom right of panel 6 L, small arrow; larger arrow points to an inclusion in an adjacent Bergmann glia).

Neuronal intranuclear inclusions are key pathological features of CGG KI mice that appear as early as 3 months of age [7, 32, 56, 59]. We did not see and did not expect to see intranuclear inclusions in Gfa2-CGG11 control mice because we were using the Gfa2-specific promoter to limit expression to glia (e.g., Fig. 7c). Unexpectedly, all of the Gfa2-CGG99 mice studied exhibited some ubiquitin-positive inclusions in neuronal nuclei in distinct brain regions, particularly in the hypothalamus, including the paraventricular (Fig. 7a) and ventromedial (Fig. 7b) nuclei. In contrast to these regions, neuronal inclusions in Gfa2-CGG99 mice, although present, occurred less frequently in neocortex and cerebellum. Intranuclear inclusions were particularly large in periventricular nuclei (Fig. 7a). Neuronal intranuclear inclusions were also found in brainstem nuclei (e.g., substantia nigra, reticular formation; data not shown). The appearance of neuronal inclusions in Gfa2-CGG99 mice was age-dependent, with 4–8 month old mice exhibiting only a few neuronal intranuclear inclusions, while older mice displayed higher numbers of inclusions in the brain regions described above. The presence of intranuclear ubiquitin-positive inclusions in neurons was further established by showing co-localization of the inclusions in cells that were identified as neurons by immunofluorescence for well-accepted neuronal markers, including NeuN (Figs. 7d & e, arrowheads), and showing localization within nuclei by DAPI staining. The inclusions showed a similar appearance to those previously described in neurons in the CGG KI mouse (Fig. 7f) [56].

In order to determine whether inclusion formation could be related to “leaky” expression in neurons of the astrocyte-specific Gfa2 promoter in Gfa2-CGG99 mice, brain sections were immunoreacted using multiple antisera for NeuN, GFAP, and/or eGFP. These experiments revealed that (i) ubiquitin-positive inclusions were present in neurons, in addition to astroglia, in various brain regions; (ii) NeuN-positive cells (i.e., neurons) did not show any detectable eGFP histofluorescence and/or GFAP immunoreactivity in the nucleus or cytoplasm; and (iii) only astroglia expressing eGFP (not neurons) were immuno-positive when reacted with antiserum against eGFP and GFAP.

The unexpected finding that neurons without eGFP histofluorescence had inclusions suggested the possibility of cell-to-cell transfer of pathology from astrocytes to neurons in Gfa2-CGG99-eGFP mice. However, it is also possible that eGFP expression in neurons was below the level of detection by histofluorescence. To test this possibility, PCR analysis of laser-capture microdissected cells (LCM) was used to detect possible eGFP mRNA expression in inclusion-bearing neurons in the ventromedial hypothalamus (VMH) of Gfa2-CGG99 mice, a region with numerous neuronal inclusions (e.g., see Fig. 7b). Specifically, neurons in the VMH that lacked eGFP histofluorescence but contained ubiquitin-positive intranuclear inclusions were identified by immunofluoresent staining (Figs. 8a1 & a2), isolated by LCM (Fig. 8a3) and combined for PCR analysis (Fig. 8b & c). Total RNA was isolated, quantified and linearly amplified using NuGEN SPIA technology (www.​nugen.​com). The resulting cDNA was processed for semi-quantitative real-time PCR with primers specific for EGFP and β-actin. As a positive control, RNA was also isolated from regions of neocortex that included astroglia that expressed eGFP (i.e., GFA2 cortex). In addition, we isolated single cells with inclusions from the amygdala of a CGG168 KI mouse (i.e., CGG KI Amygdala) that should not express eGFP. These neurons express an expanded CGG repeat under the control of an endogenous Fmr1 promoter, develop neuronal intranuclear inclusions but do not express eGFP [3, 18, 56]. We did not observe expression of eGFP mRNA in VMH neurons with ubiquitin-positive intranuclear inclusions (GFA2 LCM Neurons; Fig. 8b). Similarly, neurons isolated from the amygdala of a CGG168 KI mouse did not express eGFP (Fig. 8b). In contrast, larger cortical samples from Gfa2-CGG99 mice expressed high levels of eGFP mRNA (Fig. 8b) reflecting the presence of astroglia expressing the eGFP protein. The expected size and relative purity of PCR reactions were confirmed by gel electrophoresis (Fig. 8c).

Ubiquitin-positive inclusion bodies in Gfa2-CGG99 mice did not appear to be restricted to the nuclear compartment (Fig. 9). Initial observations in brain sections from Gfa2-CGG99 mice stained with neutral red and immunostained for ubiquitin revealed inclusion bodies that did not appear to be closely associated with somata of astrocytes or neurons. These ectopic inclusion bodies shown in Fig. 9a were prevalent across grey and white matter of all brain regions examined, but particularly frequent in upper neocortical layers, hippocampus, olfactory bulb and certain brain stem nuclei. These bodies (aggregates) were spherical and averaged 3.62 ± 1.26 μm in diameter. In some regions (e.g., ventromedial hypothalamus) they appeared to form larger clusters of several bodies. They were immunopositive for ubiquitin (dark-blue reaction product) using DAB peroxidase reaction (inset 9a), and/or exhibited ubiquitin immunofluorescence (arrowheads, Fig. 9b-f). Some appeared to be engulfed or surrounded by fine astroglial processes suggesting the inclusion bodies were intracytoplasmic in astroglia (Figs. 9b-c, inset b; arrowheads). Co-localization of inclusions with eGFP (Fig. 9d & e) and GFAP (Fig. 9f) confirmed the association of these inclusions with astrocytes. This observation was confirmed by electron microscopy (EM) as described below (Figs. 11i & k).

Figure 10a shows an FMRpolyG-positive (red fluorescent) intranuclear inclusion in a GFAP-positive (green) neocortical astroglia from Gfa2-CGG99 mouse (arrow). This observation provides the first evidence for RAN translation in astroglia in a mouse model of the FXTAS. In addition, FMRpolyG immunostaining was also seen in an inclusion body in MAP 2-positive (green) neuron in the hypothalamus (arrow, Fig. 10b). Arrowheads in 10A and 10B, show FMRpolyG-positive inclusions that are likely in an unlabeled neuron and an unlabeled astrocyte, respectively. Combined with the evidence against eGFP transgene expression in neurons (i.e., Fig. 8; laser-microdissection single-cell PCR for eGFP), the finding of FMRpolyG in neurons suggests that some form of cell-to-cell transfer of pathology, possibly involving FMRpolyG, may occur in Gfa2-CGG99 mouse brains. This could be similar to a recent report of cell-to-cell transfer of RAN translation peptides in other models of trinucleotide (or hexanucleotide) repeat disorders [58].