Neuropathologic analysis of Tyr69His TTR variant meningovascular amyloidosis with dementia
© Ziskin et al. 2015
Received: 26 May 2015
Accepted: 29 May 2015
Published: 10 July 2015
Transthyretin/TTR gene mutations usually cause systemic amyloidotic diseases. Few TTR variants preferentially affect the central nervous system, manifesting as oculoleptomeningeal amyloidosis. Patients with TTR meningovascular amyloidosis often show dementia, however the neuropathologic features of dementia in these cases have not been elucidated. We report the neuropathologic findings from a brain autopsy of a 72-year-old man with the rare Tyr69His (Y69H) TTR gene variant, dementia and ataxia. Severe amyloid deposits were observed in the leptomeninges and in a subpial and subependymal distribution. Mass spectrometry analysis demonstrated that the amyloid deposits were comprised of over 80 % of the variant TTR. TTR was undetectable by mass spectrometry in the neocortex subjacent to the subpial amyloid deposits. Subpial TTR amyloid deposits were associated with brisk superficial reactive gliosis and siderosis in the neocortex and cerebellar cortex. Subependymal TTR amyloid deposits were associated with subjacent myelin pallor in the hippocampal outflow tract structures including the alveus, fimbria and fornix. Phospho-tau immunostains demonstrated transentorhinal-stage neurofibrillary degeneration (Braak stage II) which, in the absence of neocortical amyloid-beta and neuritic plaques, was indicative of primary age-related tauopathy (PART). However, distinctive phospho-tau aggregates were observed subjacent to the subpial TTR amyloid deposits in all regions of the neocortex, including the primary motor and striate cortices, suggesting a potential link between TTR amyloid and neocortical tauopathy. Our report reveals novel insights into the potential neuropathologic substrates of dementia in variant TTR amyloidosis that need to be investigated in larger autopsy series.
KeywordsTransthyretin Amyloid Tau/MAPT Dementia
Transthyretin (TTR) is a soluble protein tetramer that carries thyroxine and retinol binding protein in the circulation . Insoluble TTR amyloid deposition is most commonly seen as a systemic disease in elderly individuals . Patients with TTR mutations also most often present with systemic amyloidotic diseases including familial amyloidotic polyneuropathy (FAP)  and familial amyloid cardiomyopathy . Mild cerebral TTR amyloid angiopathy and choroid plexus amyloidosis can be observed in systemic amyloidotic diseases like FAP [4, 5]. In contrast, severe meningovascular amyloidosis is associated with certain TTR gene mutations, including Leu12Pro , Asp18Gly [7, 8], Ala25Thr [9, 10], Val30Gly [11–13], Val30Met [14, 15], Thr49Pro , Leu58Arg , Phe64Ser , Tyr69His [19–21] and Tyr114Cys , and can lead to dementia and ataxia. To gain potential insights into the pathogenesis of these deficits in meningovascular amyloidosis, we report our postmortem neuropathologic findings from a patient with dementia, ataxia and the rare Tyr69His (Y69H) TTR substitution.
Patient and methods
Detailed methods can be found in Additional file 1: Supplemental Methods.
Ethics, consent and permissions
Informed consent to publish the results of this autopsy study was obtained from the patient’s next of kin.
A 72-year-old Italian-American male demonstrated progressive cognitive decline over 13 years punctuated by multiple encephalopathic episodes that included headache, confusion, ataxia and short-term memory loss. Six years prior to death, radiographic workup revealed superficial siderosis and an arteriovenous malformation involving the thoracolumbar spinal cord (T11), findings which were previously reported . Resection of the arteriovenous malformation alleviated the patient's encephalopathic episodes but did not ameliorate his moderate ataxia nor halt his progressive cognitive decline. A follow-up visit three months after his resection was notable for severe cognitive impairment. He scored a 9 on the 30-point mini-mental state examination with deficits in language, memory, executive function, and visuospatial skills. A complete autopsy demonstrated that the patient died of aspiration pneumonia, sepsis and multiple organ system failure. Mild to moderate amyloidosis was also noted in the systemic organs examined histologically (Additional file 1: Table S1). A standard dementia neuropathologic workup  was performed.
Tissue sections (6 μm thickness) were stained with hematoxylin and eosin. Immunoperoxidase reactions with the following antibodies were performed with standard methods: α-synuclein (Cell Signaling #2642, 1:1000); amyloid-beta (clone 6F/3D, Dako, M0872, 1:400; clone 4G8, BioLegend, SIG-39220, 1:500); Fused in sarcoma (FUS; Sigma, HPA008784, 1:3000); glial fibrillary acidic protein (GFAP; Dako, Z0334, 1:2000); myelin basic protein (Dako A0623, 1:400); phospho-MAPT (clone AT8; Thermo Scientific, MN1020, 1:2000); 3-repeat isoform MAPT (3R MAPT, RD3; clone 8E6/C11, Millipore, 05–803, 1:250); 4-repeat isoform MAPT (4R MAPT, RD4; clone 1E1/A6, Millipore, 05–804, 1:250); TDP-43 (Proteintech 10782-2-AP, 1:10,000); TTR (Dako A0002, 1:4000). Special stains, including the modified Bielschowsky stains, Gallyas silver stains, Luxol fast blue-periodic acid Schiff stains, were also performed. Formalin-fixed, paraffin-embedded (FFPE) tissues were sampled and reprocessed for transmission electron microscopy (TEM) using standard techniques.
APOE genotyping was performed on genomic DNA extracted from FFPE tissue sections via restriction fragment analysis according to the method of Kamboh and colleagues . All 4 exons of the patient’s TTR gene were sequenced from the same genomic DNA. Tissue cores (3 mm diameter; 0.8 to 1.7 mg) punched from the formalin-fixed, paraffin embedded tissue blocks were analyzed by mass spectrometry (MS) .
We employed the immunostains at our disposal, Amyloid-beta and TTR, to attempt to identify the amyloid protein in FFPE tissue block sections with negative results (Additional file 1: Figure S1a, b). However, TEM images demonstrated leptomeningeal amyloid fibrils that resemble previously reported TTR amyloid fibrils (Additional file 1: Figure S1c) . We subsequently employed MS-based proteomic analysis on core samples of the amyloid-laden leptomeninges harvested from the paraffin blocks. The most abundant protein identified by MS in the meningovascular amyloid samples was TTR (Additional file 1: Figure S2a). Extracted ion chromatograms (EICs) demonstrated that mutant Y69H TTR comprised over 80 % of the of TTR peptides T49-K70. TTR was not detected in the control brain parenchyma samples from the basis pontis and subcortical white matter. Furthermore, TTR was not detected in a sample of the insular cortex, demonstrating that amyloidogenic TTR does not penetrate past the subpial space into the cortical parenchyma. We sequenced the patient’s TTR gene exons and detected a heterozygous T to C point mutation in the first nucleotide position of codon 69, exon 3, which encodes for the amino acid substitution of tyrosine for histidine (Additional file 1: Figure S2b). Exons 1, 2 and 4 demonstrated wild type sequences. The patient’s APOE genotype was determined to be E2/E3 (Additional file 1: Figure S2c).
Immunostains for amyloid-beta (clones 6F3D and 4G8) demonstrated a complete absence of amyloid-beta plaques in the neocortex (Additional file 1: Figure S5a). Bielschowsky stains likewise revealed an absence of neocortical neuritic plaques. We incidentally observed focal sparse neuritic plaques, immunoreactive for amyloid-beta and AT8, in the stratum oriens of the hippocampal CA1 sector (not shown). These few focal neuritic plaques were distinct from the TTR amyloid deposits in the alveus. Grade I cerebral amyloid-beta angiopathy was seen in only a few leptomeningeal vessels with the 4G8 antibody (Additional file 1: Figure S5b) . Immunostains for α-synuclein (Additional file 1: Figure S5c), TDP-43 (Additional file 1: Figure S5d) and FUS (Additional file 1: Figure S5e) revealed no abnormal neuronal inclusions.
Several TTR substitutions are associated with biopsy- or autopsy-proven meningovascular amyloidosis [6–22]. Many patients with these mutations suffer from dementia and/or ataxia. Previously, Blevins and colleagues  reported only sparse neocortical neuritic plaques and amyloid-beta plaques and no evidence of hemorrhages or siderosis in a non-demented patient from a Swedish kindred with the Tyr69His TTR substitution. Post-mortem neuropathologic dementia workups in cases of meningovascular TTR amyloidosis with dementia have not been reported in the literature. The neuropathologic underpinnings of meningovascular amyloidosis-associated dementia are therefore unknown.
To provide novel insights into the potential neuropathologic substrates of dementia and ataxia in this disorder, we present the postmortem neuropathologic findings from a patient with dementia, ataxia and meningovascular amyloidosis associated with the rare Tyr69His (Y69H) substitution in TTR. To our knowledge, our patient, whose maternal and paternal ancestors originated from northwest Italy, is not related to the prior 2 reported kindreds with the Tyr69His TTR substitution from Sweden and Saskatchewan [19–21]. Our histologic and MS data demonstrate that TTR amyloid does not penetrate the cortex and subcortical white matter. Rather, TTR amyloid is deposited in leptomeningeal vessels and in subpial and subependymal deposits. This observation strongly argues that injuries to periventricular structures and superficial cortex are likely relevant for the neurologic deficits. We observed subependymal TTR amyloid and myelin loss in hippocampal efferent tracts including the alveus, fimbria and fornix that may underlie our patient’s memory deficit. We also observed brisk reactive gliosis and dystrophic astrocytic processes, which were previously noted by Herrick and colleagues , in the superficial neocortex. Our findings may implicate neuronal toxicity subjacent to subpial and subependymal TTR amyloid deposits in the pathogenesis of the dementia and ataxia.
A novel finding in this report is the association of neocortical tauopathy with meningovascular TTR amyloidosis. AT8 immunostains revealed Braak stage II , or transentorhinal B1 stage , of neurofibrillary degeneration based on the presence of neurofibrillary tangles in the Pre α neurons of the entorhinal cortex and only a few neurofibrillary tangles in the CA1 sector of the hippocampus. These neurofibrillary tangles were immunoreactive for both 3R and 4R MAPT and appeared to have paired-helical filament ultrastructure. In our patient, whose APOE genotype was E2/E3, there were no neocortical neuritic plaques, no neocortical Aβ deposits and no hippocampal granulovacuolar degeneration. We identified only focal sparse neuritic plaques of uncertain significance in the stratum oriens of the hippocampal CA1 sector. In the absence of neocortical amyloid-beta and neuritic plaques in our patient, the transentorhinal stage neurofibrillary degeneration is indicative of early primary age-related tauopathy (PART) .
Interestingly, we found AT8ir threads and neurons in nearly all of the neocortical regions examined, including the primary motor cortex and striate cortex, which are classically considered the last to be affected by tauopathy in PART and Alzheimer disease (AD). The density of threads and neurons appeared increased over the levels typically seen with Braak stage II PART. Furthermore, in all sections of the neocortex, we observed distinctive AT8ir granules, globules and threads in the molecular layer subjacent to the subpial TTR amyloid deposits. Thus, while we cannot be certain that all of the tau pathology in our case is not related to PART, these observations suggest the possibility of a link between the subpial amyloid and the neocortical tau pathology. Only rare neocortical neurons showing staining consistent with neurofibrillary tangles were seen, and overall, the tauopathy was much less severe than the neocortical tauopathy seen in patients with high AD neuropathology . However, given the association of isocortical tauopathy/tangles with cognitive impairment in the elderly  and evidence supporting a role for pMAPT in the functional impairment of synapses , it is reasonable to hypothesize that the neocortical tauopathy observed in this case of meningovascular TTR amyloidosis contributed to the cognitive impairment. Further studies are needed to elucidate the prevalence and contribution of neocortical tauopathy to dementia in meningovascular amyloidosis, its potential relationship to PART and early AD neuropathologic changes, and a possible causal or contributory role for TTR amyloidosis in the neocortical tauopathy.
We evaluated for co-morbid dementia neuropathologies and found no evidence of Lewy bodies, TDP-43 proteinopathy or FUS proteinopathy. Ischemic lesions, which were reported in a kindred with an unspecified TTR mutation , were not prominent in our case. Our sections demonstrated only a solitary minute cortical microvascular ischemic lesion in the postcentral gyrus  and no evidence of ischemic white matter degeneration, suggesting that a significant burden of ischemic lesions is not necessary for dementia in meningovascular TTR amyloidosis. There was, however, evidence of vascular damage and chronic hemorrhage from the meningovascular amyloid leading to toxic superficial siderosis , which likely contributed to his cognitive impairment and ataxia.
In summary, we have described the neuropathologic autopsy findings from a 72 year old male with variant Y69H TTR meningovascular amyloidosis. Our findings suggest that neocortical injury secondary to subpial TTR amyloid, injury to hippocampal efferent tracts secondary to subependymal TTR amyloid, and superficial siderosis may play important roles in the cognitive impairment and ataxia associated with variant TTR meningovascular amyloidosis. Our case, which demonstrated early PART, also showed neocortical tauopathy that was unusual for its subpial distribution. Further autopsy studies on patients with TTR meningovascular amyloidosis are necessary to elucidate the significance of tauopathy in the cognitive impairment and to further delineate the neuropathologic changes that underlie dementia. A better understanding of the pathogenic events leading to dementia in variant TTR meningovascular amyloidosis may lead to novel treatment strategies for this debilitating and fatal disease.
EDP is supported by a K08 grant from the National Institute of Neurologic Disorders and Stroke (NS085324). The authors thank the NIH for Award Number S10RR027425 from the National Center for Research Resources supporting the mass spectrometry. The authors report no conflicts of interest.
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