A postmortem MRI study of cerebrovascular disease and iron content at end-stage of fragile X-associated tremor/ataxia syndrome

Brain changes at end-stage of fragile X-associated tremor/ataxia syndrome (FXTAS) are largely unknown due to mobility impairment. We conducted a postmortem MRI study of FXTAS to quantify cerebrovascular disease, brain atrophy, and iron content and examined their relationships using principal component analysis (PCA). Intracranial hemorrhage (ICH) was observed in 4/17 FXTAS cases among which one was confirmed by histologic staining. Compared with seven control brains, FXTAS cases showed higher ratings of T2-hyperintensities (indicating cerebral small vessel disease) in the cerebellum, globus pallidus, and frontoparietal white matter and significant atrophy in cerebellar white matter, red nucleus, and dentate nucleus. PCA of FXTAS cases revealed negative associations of T2-hyperintensity ratings with anatomic volumes and iron content in the white matter, hippocampus, and amygdala, that were independent from highly correlated number of regions with ICH and iron content in subcortical nuclei. Post hoc analysis confirmed PCA findings and further revealed increased iron content in the white matter, hippocampus, and amygdala in FXTAS cases than controls after adjusting for T2-hyperintensity ratings. These findings indicate that both ischemic and hemorrhagic brain damage may occur in FXTAS, with the former marked by demyelination/iron depletion and atrophy and the latter, ICH and iron accumulation in basal ganglia.


Introduction
At least 6.77% of the human genome is highly polymorphic short tandem repeats that are prone to mutate and become a source of genetic variation in human populations 1 . Large expansions of these repeat tracts, however, can cause neurological or developmental disorders presenting shared clinical phenotypes including cerebellar ataxia, tremor, cognitive impairment, and peripheral neuropathy 2 .
Currently, over fty repeat expansion disorders have been identi ed 3 . Fragile X-associated tremor/ataxia syndrome (FXTAS) is one of these disorders due to the expansion of CGG repeat element at a noncoding region of the fragile X messenger ribonucleoprotein 1 (FMR1) gene in the premutation range  repeats) 4,5 .
FXTAS is an age-related neurodegenerative disorder affecting premutation carriers with a prevalence in males increasing from 17% in those in their 50's to 75% in those aged 80 and above 6 . Clinically, FXTAS has a variable presentation with core features comprising cerebellar ataxia, intention tremor, parkinsonism, autonomic dysfunction, cognitive decline, and psychological disorders 7 . Primary radiological markers are hyperintensities on T2-weighted MRI in the middle cerebellar peduncle ("MCP sign"), pons, corpus callosum, and cerebral white matter as well as generalized brain atrophy 8- 10 . We recently expanded T2 ndings in FXTAS to include abnormal signals in the globus pallidus ("pallidal sign") displaying hyperintensities in the center surrounded by hypointensive T2 signals. We explored clinical signi cance of both MCP and pallidal signs 11 . The prominence of the MCP sign in FXTAS pathophysiology was demonstrated by its independent associations with cerebellar ataxia, intention tremor, and executive function de cits. Although the pallidal sign was not associated with motor or cognitive de cits independently, having both MCP and pallidal signs was associated with greater impairment in executive function and iron content variability in the globus pallidus 11 .
Pathophysiologic mechanisms underlying the occurrence of T2 hyperintensities in FXTAS have not been explored. In elderly individuals with or without dementia, white matter hyperintensities (WMHs) on T2weighted MRI are prevalent and thought to be associated with cognitive and motor de cits 12,13 . WMHs are commonly regarded as MRI features of cerebral small vessel disease (CSVD). Other MRI features of CSVD encompass small infarct, cerebral microbleed, enlarged perivascular spaces, and brain atrophy 14,15 . Consistently, our recent neuropathological examination of microangiopathy 16 provided support for cerebrovascular dysfunction in FXTAS. Increased number of microbleeds in cerebral cortical white matter and cerebellum was discovered in FXTAS cases compared to age-and sex-matched control cases. Ubiquitin + intranuclear inclusions, the pathological hallmarks of FXTAS, in the endothelial cells of capillaries were revealed as well, expanding the list of cell types that are compromised with the inclusions including neurons, astrocytes, ependymal cells, subependymal cells, epithelial lining cells of the choroids plexus, and Purkinje cells [17][18][19][20] . Neuropathologic examination of a male carrier with FXTAS revealed moderate CSVD in the arteries of deep white matter that exhibited wall thickening, perivascular gliosis, and enlarged perivascular spaces and severe CSVD in the globus pallidus showing calci cation of the walls of perforating arteries 21 ; Other prominent pathological features of FXTAS include elevated FMR1 mRNA levels 22 , iron accumulation in the putamen and choroid plexus 23,24 , and mitochondrial dysfunction 25,26 . Cerebrovascular disease and its relationship with the appearance of T2-hyperintensities in FXTAS have not yet been investigated. Postmortem MRI has the advantages over in vivo MRI for examining brain changes at end stage of FXTAS when patients are often bedridden. It also allows longer scanning time to provide improved image resolution bene cial for detecting microbleeds with submillimeter-diameters. The goal of this study is to conduct a postmortem MRI study in FXTAS to examine (1) severity of cerebrovascular disease characterized by intracranial hemorrhage, microbleeds, and WMHs; (2) iron content in the white matter and 10 deep nuclei; (3) regional brain atrophy; and (4) relationship between MRI measures and subgrouping among patients via principal component analysis (PCA).

T2-Hyperintensities
Brain specimens were collected from 17 premutation carriers (males/females: 14/3) diagnosed of FXTAS during life and 7 non-carrier controls (males/females: 5/2) between 2009-2020 ( Table 1). The diagnoses were con rmed in xed brain tissue by the presence of intranuclear inclusions. The average age of death for the premutation carriers (75.3 ± 8.0 years, range 66-93 years) was higher than that of non-carrier controls (70.1 ± 8.4 years, range 60-83 years).  Intracranial Hemorrhage and Microbleeds R2* transverse relaxation rate are affected by the degree of myelination and variations in iron concentration and thus can be used to detect demyelination and iron-containing blood degradation products due to intracranial hemorrhage or pathologic changes associated with small vessel disease such as microbleeds 27,28 . Three FXTAS cases (P4M, P5M, and P7M) exhibited increased R2* consistent with intracranial hemorrhages affecting 2-3 brain regions ( Fig. 1a-c). One case, P17M, showed absence of the temporal white matter (Fig. 1d), which could be caused by hemorrhages. H&E staining of the residual temporal cortex con rmed presence of numerous small microbleeds with diameters in the order of micrometers (Fig. 1e, f) and a large intracranial hemorrhage. None of the control cases showed intracranial hemorrhages. However, as a group, the FXTAS cases did not show signi cantly increased number of brain regions with intracranial hemorrhages compared with the controls (β = 0.66 ± 0.43, p = 0.14) ( Table 2). Ratings of microbleeds were not signi cantly different between the two groups in the cerebral cortex, deep white matter, or cerebellum (β = -0.78 to 0.27, SE = 0.16 to 0.67, p = 0.06 to 0.62) ( Table 2).
Since the PCA revealed negative correlation between R2* in the cerebral and cerebellar white matter, hippocampus, and amygdala (having positive values of component 1) and T2-hyperintensity ratings in the frontoparietal white matter (having negative values of component 1), we further explored their relationships by conducting multiple linear regression using age of death, group membership, and individual T2-hyperintensity rating as the explanatory variables and R2* as the outcome variables. The results con rmed the negative correlations between R2* in these regions and frontoparietal T2hyperintensity ratings. The analysis further indicated increased R2* in the cerebral white matter in FXTAS cases than controls after adjusting for frontal T2-hyperintensity ratings (β = 5.   Fig. 2 revealed clustering of the two female carriers who died at old age (93 years old for P2F and 89 years old for P3F) and contained low R2* in the subcortical nuclei (i.e., positioned in the opposite directions of the d.~ variables) while the third female that died at 79 (P14F) was positioned close to the center of gravity of the male FXTAS cases, indicating similar MRI changes as the male cases (Fig. 2a). The three male cases with intracranial hemorrhages (P4M, P5M, P7M) exhibited high coordinates of component 1 and showed relatively high R2* in the cerebral and cerebellar white matter and subcortical nuclei (Fig. 2a). Males cases displaying relatively high volumes of the white matter and subcortical nuclei (top middle) also positioned away from those with relatively high ratings of WMHs in the frontoparietal regions (lower left) (Fig. 2a).

Discussion
We performed the rst postmortem MRI study in FXTAS to quantify features of cerebrovascular disease and changes in R2* transverse relaxation rate in the white matter and subcortical nuclei that can be caused by demyelination, the presence of blood degradation products, and iron accumulation in the subcortical nuclei. Correlations among different types of MRI measures and heterogeneity in patients were examined using the dimension reduction multivariate analysis technique, PCA.
All brains with FXTAS displayed MRI changes consistent with cerebrovascular disease. T2hyperintensities in the white matter are commonly regarded as indications of CSVD 14,15 . All FXTAS cases exhibited multiple regions with con uent WMHs except for two cases that showed smooth "halo" (rating of 2) in the anterior periventricular white matter as the highest rating (Table 6). FXTAS cases showed signi cantly higher ratings of T2-hyperintensities in the MCP, globus pallidus, corpus callosum, and frontoparietal white matter than controls after the adjustment for age of death (Table 2). PCA revealed that high ratings of the frontoparietal WMHs were associated with low R2* decay in the cerebral white matter, implicating loss of oligodendrocytes, the predominant iron-containing cells in the brain 29 , as well as ischemic as oppose to hemorrhagic injury that may underlie the occurrences of WMHs. The cerebral white matter is more vulnerable to ischemia than the cortex due to the much lower artery and capillary density (2-3 times lower) 30 . Among the cerebral white matter regions, the frontal white matter is particularly susceptible to ischemia, where the blood is supplied by long and thin medullary arteries. This is in contrast with the subcortical U-ber region where shorter arteries provide the perfusion 15,30 . However, 4/17 FXTAS cases also showed increased R2* transverse relaxation rate in 1-3 cortical regions in concord with intracranial hemorrhages. Although ratings of microbleeds were not signi cantly higher in FXTAS cases than control cases, they were associated with number of regions with intracranial hemorrhages (Fig. 2b). These ndings indicate that all brains with FXTAS show MRI changes consistent with ischemia although hemorrhages could occur concurrently or even were the predominant injury in about 24% of the brains. Consistent with our ndings, a recently published study 31 reported p62-positive intranuclear inclusions in the pericytes and endothelial cells of brain vasculature as well as vascular infarcts such as lacunae and strokes throughout the brain and iron deposits resulting from disrupted vasculature throughout the cerebral cortex and hippocampus in two male premutation carriers. These two men displayed mild motor impairments, no MCP sign or con uent WMHs, but prominent clinical symptoms of fragile X-associated neuropsychiatric disorders (FXAND) including apathy, aggression, and depression 32 . In addition, the toxic polyglycine-containing protein, FMRpolyG protein 33 , was also detected throughout the brain and brain vasculature 31  symptomology. However, we were not able to adjust for individual differences in skull size in the statistical analysis because skull size was unavailable.
Unexpectedly, of the ten subcortical nuclei investigated in this study, only the hippocampus, subthalamic nucleus, and substantia nigra suggested higher R2* in FXTAS cases than controls that were not signi cant after the correction for multiple testing. However, after adjusting for frontoparietal WMH ratings, the cerebral and cerebellar white matter, hippocampus, and amygdala revealed higher R2* in FXTAS cases than controls (Table 5). R2* in the human brain is affected by diamagnetic myelin, paramagnetic iron and blood degradation products, and orientation of myelinated axons relative to external magnetic eld 28,39 . Hence, the relatively higher R2* in the white matter in FXTAS may be due to demyelination and/or increased iron content carried by surviving oligodendrocytes 29 . This is consistent with neuropathologic ndings of white matter spongiosis with corresponding axonal loss and myelin pallor in FXTAS 18 . In contrast, the relatively higher R2* in subcortical nuclei (with low myelin content) can be caused by hemosiderin depositions that have been demonstrated histologically in parenchyma and capillaries of the putamen 24 . We were not able to replicate the nding of high iron content in the dentate nucleus in premutation carriers from our recent in vivo MRI study 11 . This may be due to changes in iron content at different stages of FXTAS since we showed that iron content decreased as dentate nucleus atrophied 11 . Further MRI-pathologic association studies are needed to clarify the source of increased R2* in the white matter and deep nuclei.
One strength of this study was the characterization of heterogeneity in FXTAS via PCA, which revealed subgroups that varied by sex, age of death, iron content in the subcortical nuclei, severity of frontoparietal WMHs, and degree of atrophy in the cerebral and cerebellar white matter and subcortical nuclei. This can be helpful for recognizing the range of MRI changes associated with FXTAS and for developing effective personalized therapeutic treatments to alleviate or reverse these changes. However, we were not able to explore clinical signi cance of the subgrouping since clinical data were unavailable from many cases with FXTAS. In addition, quantitative susceptibility mapping was conducted following our published method 11 but was not usable because of substantial artifacts from residual air and/or water bubbles.
In conclusion, we revealed MRI changes in the brain consistent with cerebrovascular disease in all 17 cases of FXTAS. All FXTAS cases exhibited WMHs that were associated with reduced R2*, indicating loss of iron-containing oligodendrocytes and ischemic damage in the white matter. Four FXTAS cases (23.5%) also showed increased R2* in 1-3 brain regions consistent with intracranial hemorrhages. Those with intracranial hemorrhages tended to show increased R2* in the basal ganglia supporting hemorrhagic nature of both types of changes.

Postmortem MRI Acquisition
To remove background eld effects and to keep the brain moist, formalin-xed brains were placed in a plastic container lled with 3M uorinert electronic liquid (FC-770, Parallax Technology, Inc.), which had the similar susceptibility as the brain 40 . The brains were rocked gently in room temperature for 12-24 h to allow air bubbles to escape [40][41][42] . MRI scans were acquired at a 3T Siemens Trio MRI scanner with a 32-channel head coil (Siemens Medical Solutions) using 3D multi-echo gradient recalled echo (GRE) and 3D T2-weighted turbo spin echo sequences. For the initial 5 brains, multi-echo GRE scans were acquired

Postmortem MRI Processing
Anterior-and posterior-commissures were aligned using DTI Studio 43

MRI Quanti cations
Ratings of microbleeds and intracranial hemorrhages and estimation of R2* were performed for speci c brain regions. Microbleeds were classi ed as well-de ned, circular hyperintensities with diameters ranging from 2-10 mm on R2* map 28 . Only de nite microbleeds in seven anatomic regions (i.e., frontal, parietal, temporal, occipital, insula, deep white matter, and cerebellar regions) were counted and converted to the scale of 0-4, using cut-points (< 1, 1-4, 5-9, 10-19, ≥ 20) 28, 49 . Microbleeds rating of the cerebral cortex was then computed as the average rating of the frontal, parietal, temporal, occipital, and insular regions. We also counted total brain regions showing intracranial hemorrhages 50,51 according to the following anatomical division: frontal, parietal, temporal, occipital, and cerebellar regions. Rating of hyperintensities on T2-weighted scans followed Fazekas method 52 in the following regions: anterior, posterior, and inferior periventricular white matter; frontal, parietal, temporal, and occipital deep white matter; MCP/cerebellar white matter; globus pallidus; brainstem; and the genu and splenium of the corpus callosum. Periventricular WMHs were rated as 0 (absence), 1 ("caps" or pencil-thin lining), 2 (smooth "halo"), and 3 (irregular hyperintensities extending into the deep white matter) whereas hyperintensities in the remaining brain regions were rated as 0 (absence), 1 (punctate foci), 2 (beginning con uence of foci), and 3 (large con uent areas). For the ve whole brain specimens, number of microbleeds and anatomic volume were estimated in both hemispheres and then divided by two. For hyperintensities, the higher ratings among the two hemispheres were utilized. Missing/incomplete brain regions were excluded from the analysis ( Table 1). All ratings were performed after intra-rater reliability assessed using Cohen's kappa reached 0.80 or above (almost perfect).

Histology
A sample of temporal cortex from P17M was dissected, rehydrated in 30% sucrose and embedded in optimal cutting temperature compound (Fisher HealthCare). Blocks were cut using a cryostat at 14 µm thickness. Tissue samples were processed according to standard procedures. Slides were rehydrated and stained with hematoxylin-eosin (H&E) dyes (Sigma-Aldrich SLCC6883/SLCJ2543), followed by dehydration in ethanol 50%-100%, cleared in 3 changes of xylene, mounted and coverslip. Stained slides were imaged at 10x and 40x using a bright-eld microscope (Olympus DP71).

Statistical Analysis
All statistical analyses were conducted using R 4.1.1. The comparisons of MRI data between brains with FXTAS and control brains were conducted using multiple linear regression using age of death as a covariate. Multiple comparisons were corrected using false discovery rate (FDR) 53 . Correlation among MRI measures and FXTAS subgroups were examined via PCA using the R package 'factoextra'.
Declarations nancial and instrumental support, and supervised the study. All authors critically reviewed the manuscript.

Data Availability
All data included in this study will be shared by the corresponding authors as anonymized data through request from any quali ed investigator. Disclosure R.J. H. has received funding from Zynerba and the Azrieli Foundation to carry out treatment studies in fragile X syndrome, and has also consulted with Zynerba regarding treatment studies in fragile X syndrome. The remaining authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

Role of the Funder/Sponsors
The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. The four brains with intracranial hemorrhages. The T2 scan for rating hyperintensities, T2* scan for anatomic segmentation, and R2* mapping for rating cerebral microbleeds and estimating R2* transverse relaxation rate are shown for each brain. (a) The R2* mapping of P4M shows an intracranial hemorrhage affecting the occipital lobe and cerebellum (arrow). The T2 scan shows con uent T2-white matter hyperintensities in the adjacent regions while other white matter regions appear to be normal. (b) The R2* mapping of P5M shows intracranial hemorrhages affecting the occipital lobe, anterior temporal lobe, and cerebellum (arrows). The frontoparietal white matter shows con uent white matter hyperintensities on the T2 scans and reduced R2* transverse relaxation rate on the R2* mapping (*). (c) The R2* mapping of P7M shows a large intracranial hemorrhage affecting the frontal, parietal, and occipital lobes. (d) The scans of P17M show con uent frontal white matter hyperintensities on the T2 scan and loss of iron content on R2* mapping in the same region (*). P17M also shows T2-hyperintensities in the globus pallidus (arrow) and corresponding signal variations on the R2* mapping. All T2, T2*, and R2* mapping show the absence of anterior temporal white matter (arrow). (e, f) The hematoxylin and eosin (H&E) staining of the residual temporal cortex of P17M shows numerous free erythrocytes, indications of microbleeds. Scale bar: 100 µm in E and 20 µm in F. On T2* scans, the cerebral cortex is labeled as lilac; cerebral white matter, dark yellow; corpus callosum, blue; caudate nucleus, pink; tail of caudate nucleus, purple; putamen, green; globus pallidus, red; amygdala, light blue; hippocampus, yellow; unnamed subcortical gray matter, vanilla; pons, dark blue; cerebellar cortex, salmon; cerebellar white matter, dark green; cerebellar dentate nucleus, sand.