CNS demyelination in fibrodysplasia ossificans progressiva

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder of progressive heterotopic ossification (HO) caused by a recurrent activating mutation of ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. FOP is characterized by progressive HO, which is associated with inflammation in the setting of dysregulated BMP signaling, however, a variety of atypical neurologic symptoms are also reported by FOP patients. The main objective of this study is to investigate the potential underlying mechanism that is responsible for the observed atypical neurologic symptoms. We evaluated two mouse models of dysregulated BMP signaling for potential CNS pathology through non-invasive magnetic resonance imaging (MRI) studies and histological and immunohistochemical approaches. In one model, BMP4 is over-expressed under the control of the neuron-specific enolase promoter; the second model is a knock-in of a recurrent FOP mutation of ACVR1/ALK2. We also retrospectively examined MRI scans of four FOP patients. We consistently observed demyelinated lesions and focal inflammatory changes of the CNS in both mouse models but not in wild-type controls, and also found CNS white matter lesions in each of the four FOP patients examined. These findings suggest that dysregulated BMP signaling disturbs normal homeostasis of target tissues, including CNS where focal demyelination may manifest as the neurologic symptoms frequently observed in FOP.

Fig. 1

T2-weighted MRI detects hyperintense lesions in the CNS of Nse-BMP4 mice. a T2-weighted imaging (continuous sections) revealed large irregular hyperintense lesions in left anterior olfactory nucleus (A1), piriform area (A2 and A3), and substantia innominata and nucleus accumbens (A4). b Images show a large hyperintense area in the left piriform and amygdalar nucleus (B1) and a relatively small area of hyperintensity in the adjacent region (B2). c A sagittal section shows multiple hyperintense lesions in the lumbar spinal cord. d Serial cross sections of thoracic spinal cord show asymmetrical signal intensity in the white matter of the top 3 sections but not the bottom panel. White arrows in all panels point to the hyperintense regions

Fig. 2

Histologic evaluation identifies multiple demyelinated areas which co-localize with activated microglia in the CNS of Nse-BMP4 mice, a, b Multiple areas of demyelination in the cerebellum (a, sagittal section) and spinal cord (b, longitudinal section) detected by luxol fast blue staining in adult Nse-BMP4 mice. Black arrows in (a and b) point to areas of demyelination. c Typical low power image of myelin basic protein (MBP) staining (green) confirmed the multiple areas of demyelination in the cerebellum of adult Nse-BMP4 mice. White arrow points to areas of demyelination. d MBP (green)/GFAP (red) double staining showed that GFAP, an astrocyte marker, is detected inside and in adjacent areas to the lesion (absence of MBP). White arrow points to area of demyelination. (e, f) IBA1 (red) staining shows local accumulation of IBA1⁺ activated microglia in young (1 month old) transgenic mice (e), but not in WT (f) controls. Note that the accumulated activated microglia have short and wide processes in (e), compared to control mice (f). White arrows in e point to areas of lesions, where clusters of multiple activated microglia are located, g–i MBP (Green)/MHC (activated microglial marker, red) double staining shows that MBP and MHC are almost always mutually exclusive, and the lesion sizes and numbers increase with age. g MHC (green)/MBP (red) double staining showed that MHC⁺ cells with multiple processes surrounding an area of demyelination in cerebellum of a 1-month-old (1 M) Nse-BMP4 mouse. h MHC (green)/MBP (red) double staining showed that at least three small lesion sites with MHC⁺ cells are present in a 3-month old (3 M) Nse-BMP4 mouse, i MHC (green)/MBP (red) double staining showed that a larger lesion with profound accumulation of MHC⁺ cells and severe demyelination in a 4-month old (4 M) Nse-BMP4 mouse. White arrows in g–i point to areas of lesions, where clusters of multiple activated microglia are located. DAPI counterstaining (blue) was performed in c–i, Bar = 200 µm in c, bar = 40 µm in all other panels

Fig. 3

Focal demyelination is consistently found in the cerebellum and spinal cord of FOP knock-in (ACVR1/ALK2 (R206H)) chimeric mice. Luxol fast blue staining of tissues from ACVR1 R206H mice (a, c, d, and e) and control tissues (b and f) identified demyelination only in chimeric mice (a, d and e). a, b Luxol fast blue staining identified demyelination in white matter and molecular layer of cerebellum. Black arrows in (b) point to myelinated fibers in the molecular layer in control tissue; these myelinated fibers are absent in ACVR1 R206H mice (a). Note also that the white matter is under-myelinated in chimeric mice. c–e Demyelination in the spinal cord of an ACVR1 R206H mouse. c Typical image of normal Luxol fast blue staining of white matter in a region of spinal cord from an ACVR1 R206H mouse. d Typical image of local demyelination of white matter in spinal cord of an ACVR1 R206H mouse. Broken lines in c and d indicate the border between white matter and gray matter. e Typical image of local demyelination of gray matter in the spinal cord of an ACVR1 R206H mouse. f Typical image of normal Luxol fast blue staining of gray matter in the spinal cord of control mouse. Black arrows in f point to myelinated fibers in gray matter; these myelinated fibers are absent in chimeric mice (e). CC central canal, M molecular layer, W white matter, G gray matter

Fig. 4

Inflammation markers (IBA1 and F4/80) are up-regulated in areas of demyelination in chimeric ACVR1 R206H FOP knock-in mice. a–d In cerebellum (a and b) and spinal cord (c and d), IBA1 is up-regulated in ACVR1 R206H mice (a and c) compared to controls (b and d). e–h In spinal cord (g and h) and cerebellum (e and f), F4/80 is up-regulated in ACVR1 R206H mice (e and g) compared to controls (f and h). In both spinal cord and cerebellum, F4/80 staining is closely associated with small blood vessels. Bar = 40 µm in all panels

Fig. 5

MRI detects progression of hyperintense lesions in the CNS of a FOP patient. a–c T2-weighted brain images of patient 1 in 2006 showed isolated hyperintense lesions in the left (a), and right (b) frontal lobe, and cerebellar peduncle (c) (axial views). (a′–c′) showed the lesions in the similar locations 5 years later (in 2011), however the sizes and numbers of the detected lesions increased. At the later time point, some of the lesions were merging with each other. d1–d6 show additional hyperintense lesions in additional brain regions (at 2011, axial views). White arrows in all panels indicate hyperintense regions

Fig. 6

MRI detects hyperintense lesions in the CNS of FOP patients. (a–a″) T2-weighted brain images of patient 2. An extensive hyperintense lesion in the left frontal periventricular white matter is shown from sagittal (a), coronal (a′), and axial (a″) views. b T2-weighted image of the spinal cord of patient 2. Hyperintense lesions are shown from a sagittal view. Insert in b shows a higher-power image surrounding the lesion. c T2-weighted image shows hyperintensity of the dorsal pons and the dentate nuclei bilaterally in patient 3 from an axial view. d T2-weighted image shows hyperintense lesions in the dentate nuclei and surrounding the fourth ventricle in patient 4 from an axial view. White arrows in all panels indicate hyperintense regions