Distribution and frequency of intranuclear inclusions in female CGG KI mice modeling the fragile X premutation

Abstract

The fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by CGG trinucleotide repeat expansions in the fragile X mental retardation 1 (FMR1) gene. The neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and in astroglia. Intranuclear inclusions have also been reported in the neurons of male CGG KI mice carrying an expanded CGG trinucleotide repeat and used to model FXTAS, but no study has been carried out quantifying inclusions in female CGG KI mice heterozygous for the fragile X premutation. We used histologic and immunocytochemical methods to determine the pathological features of intranuclear inclusions in astroglia and neurons. In female CGG KI mice, ubiquitin-positive intranuclear inclusions were found in neurons and astroglia throughout the brain in cortical and subcortical regions. These inclusions increased in number and became larger with advanced age and increasing CGG repeat length, supporting hypotheses that these pathologic features are progressive across the lifespan. The number of inclusions in neurons was reduced by ∼25% in female CGG KI mice compared to male CGG KI mice, but not so low as the 50% predicted. These data emphasize the need to evaluate the neurocognitive and pathological features in female carriers of the fragile X premutation with and without FXTAS symptomatology is warranted, as this population shows similar neuropathological features present in male FXTAS patients.

Fig. 1

Intranuclear inclusions in the dentate gyrus of a human female carrier of the fragile X premutation. Inclusions are present in the granule cell layer (arrow) and an astroglial cell in the hilus (arrowhead) of a 76 year old female premutation carrier that did not show FXTAS symptoms (Case 1 from Tassone et al., 2012). Magnification 1000 ×. Scale bar = 50 μm. H&E stain. Inset: inclusions in a pyramidal neuron (lower) and astroglial cell (upper) in the hilus. Original magnification 1000 ×. Scale bar = 10 μm. H&E stain.

Fig. 2

Intranuclear inclusions in dentate gyrus of female CGG KI mice. Hematoxylin and eosin (H&E) stained section of the dentate gyrus showing inclusions in granule cells (arrow; inset) and putative GABAergic cells in the inner molecular layer (arrowhead). Original magnification 1000 ×. Scale bar = 50 μm. Inset: numerous brown intranuclear inclusions by immunoperoxidase staining in hematoxylin stained cells of the posterior nucleus of the amygdala of a female CGG KI mouse. Original magnification 1000 ×. Scale bar = 10 μm. Main figure H&E stain, inset imunoperoxidase stain for ubiquitin. These images are from a female CGG KI mouse, 48 weeks of age with 12 repeats in the Fmr1 gene one X chromosome and 128 on the second Fmr1 gene. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Example correlations between CGG repeat length and intranuclear inclusion number in female CGG KI mice. Top row: the slopes of the correlations are similar between young and old mice (p>1), suggesting in these brain regions age may be a major factor in the progression of inclusion formation. Bottom row: the slope of the correlations are dissimilar (p<0.05) between young and old mice, suggesting a combination of CGG repeat length and age may accelerate pathological features in these brain regions. Circles = female CGG KI mice older than 6 months of age; diamonds = female CGG KI mice younger than 6 months of age. These correlations are from the data presented in Table 1.

Fig. 4

Intranuclear inclusion in astroglia and Bergmann glia. Protoplasmic (open arrowhead) and velate astroglial cells (filled arrowhead) stained for GFAP (green) and ubiquitin (red). Bergmann glial cell (arrow) stained for GFAP (green) and ubiquitin (red). Neurons negative for GFAP with intranuclear inclusions are shown with boxes. Cell nuclei stained with DAPI (blue). Arrowheads point to an astroglial cell in the inner molecular layer with an intranuclear inclusion as well as a velate astroglial cell in the granule cell layer with an intranuclear inclusion. Single plane confocal image. Original magnification 600 ×. Scale bar = 50 μm. This image came from a female CGG KI mouse, 55 weeks of age with 8 CGG repeats in the Fmr1 gene one X chromosome and 164 on the second Fmr1 gene. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Intracellular masses in microglia. (A) Intracellular masses in CGG KI mice and (B) wildtype mice at 72 weeks of age. In microglia there appear to be intracellular, cytoplasmic masses (arrowheads) that stain positive for ubiquitin (red) as well as iba1 (green) in microglia. Nuclei stained blue with DAPI. These masses appear larger and less organized in wildtype mice (B) compared to female CGG KI mice (A). Original magnification 1000 ×. Scale bar = 10 μm. The image in A came from a female CGG KI mouse with 11 CGG repeats in the Fmr1 gene one X chromosome and 128 on the second Fmr1 gene and the image in B came from a wildtype littermate with both Fmr1 genes containing 10 CGG repeats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)