Prominent amyloid plaque pathology and cerebral amyloid angiopathy in APP V717I (London) carrier – phenotypic variability in autosomal dominant Alzheimer’s disease

The discovery of mutations associated with familial forms of Alzheimer’s disease (AD), has brought imperative insights into basic mechanisms of disease pathogenesis and progression and has allowed researchers to create animal models that assist in the elucidation of the molecular pathways and development of therapeutic interventions. Position 717 in the amyloid precursor protein (APP) is a hotspot for mutations associated with autosomal dominant AD (ADAD) and the valine to isoleucine amino acid substitution (V717I) at this position was among the first ADAD mutations identified, spearheading the formulation of the amyloid cascade hypothesis of AD pathogenesis. While this mutation is well described in multiple kindreds and has served as the basis for the generation of widely used animal models of disease, neuropathologic data on patients carrying this mutation are scarce. Here we present the detailed clinical and neuropathologic characterization of an APP V717I carrier, which reveals important novel insights into the phenotypic variability of ADAD cases. While age at onset, clinical presentation and widespread parenchymal beta-amyloid (Aβ) deposition are in line with previous reports, our case also shows widespread and severe cerebral amyloid angiopathy (CAA). This patient also presented with TDP-43 pathology in the hippocampus and amygdala, consistent with limbic predominant age-related TDP-43 proteinopathy (LATE). The APOE ε2/ε3 genotype may have been a major driver of the prominent vascular pathology seen in our case. These findings highlight the importance of neuropathologic examinations of genetically determined AD cases and demonstrate striking phenotypic variability in ADAD cases.


Introduction
Alzheimer's disease (AD) is the most common form of dementia, currently affecting more than 5 million people in the United States [6]. Neuropathological hallmarks of AD include extracellular deposits of beta-Amyloid (Aβ), intracellular deposits of neurofibrillary tangles (NFT) and neuron loss [35]. The majority of cases occurs as sporadic disease (sporadic AD, SAD), modified by genetic, behavioral and environmental risk factors, while a subset of cases is caused by autosomal-dominant mutations [14,21,35]. These mutations in autosomal dominant forms of AD (ADAD) are clustered in genes associated with the metabolism of Aβ-peptides, which are generated from Amyloid Precursor Protein (APP) in sequential cleavage events mediated by βand γ-secretase [9]. ADAD associated mutations in APP mainly cluster around these secretase cleavage sites, while codon 717 is a mutational hotspot at the γ-secretase cleavage site [27]. To date, four different pathogenic amino acid changes for APP on codon 717 have been described: Valine to Phenylalanine (V717F, Indiana [37]), Valine to Glycine (V717G [10]), Valine to Isoleucine (V717I, London [18,63]) and Valine to Leucine (V717L [38]). All of these mutations shift the ratio of Aβ 1-42 /Aβ 1-40 towards increased production of Aβ   [27], which is more aggregation prone and can drive pathological protein accumulation. The V717I (London) mutation, was among the first mutations described to cause ADAD and this discovery has put Aβ center stage in AD pathogenesis. Animal models overexpressing mutant human APP are a staple of AD research [17,24,46,47,49,52,54] and the APP V717I mutation was used to generate some widely used models [34,43,54]. Despite the numerous and detailed descriptions of pathological findings in these animal models, neuropathological characterization of patients carrying the APP V717I mutation is scarce. The brain of the original case from England was reported to show AD neuropathological changes with mild cerebral amyloid angiopathy (CAA) as well as Lewy body (LB) pathology in cortical and brainstem regions, while findings from an American family with this mutation showed AD pathology but no CAA or LB [8,22,36].

Case presentation
The patient was a sixty-six (66) year-old right-handed Caucasian female with a past medical history of thyroid disease. Her family history was notable for extensive AD, involving her mother (deceased from disease at age 62), two aunts (deceased from disease at ages 66 and 68), and a grandfather (also deceased from disease). Additionally, she had a brother diagnosed with AD, still living in a nursing facility.
She was first noted to develop neurologic symptoms in her mid-fifties, manifesting primarily as progressive memory loss. She presented for formal neurologic evaluation at age 60, at which time her husband described significant memory difficulty, confusion, and occasional difficulty in finishing sentences. While she had discontinued working five years prior to evaluation due to difficulty with completing occupational tasks, she maintained her ability to finish routine housework. On initial evaluation, she was oriented to person, place, and time but not year, with a Mini Mental Status Exam (MMSE) score of 17. Physical exam findings included the presence of a tremor of her head and bilateral hands, with a negative Romberg's test. These findings were assessed to be consistent with AD of moderate intensity, and she was started on donepezil and memantine therapy. Concurrent computed tomography (CT) imaging of her brain demonstrated subtle areas of low-attenuation in the periventricular white matter of the parietal lobe, suggestive of microvascular ischemic change.
Two years after initial diagnosis, she was noted to demonstrate significant clinical deterioration during a followup clinical visit. Her husband described a loss of her ability to maintain independent activities of daily living, becoming dependent on him for bathing and dressing on a daily basis, and that she had additionally developed urinary incontinence. Her recent medical history was notable for a hospital admission due to dehydration and severe hypothyroidism secondary to thyroid medication noncompliance. On evaluation, she exhibited depression, anxiety, confusion, and an MMSE score of 8. These findings were assessed to be consistent with a progression to severe AD. CT imaging at this time revealed mild global brain parenchymal loss, with no evidence of focal lesions or asymmetric atrophy.
Her symptoms continued to progress, whereupon at a follow-up visit four years after initial diagnosis, her husband described the development of intermittent jerking movements by the patient, occurring a few times per week and lasting approximately 15 min in duration. At this visit, she was oriented only to person, and her MMSE score was 0, because she was unable to follow commands. A subsequent electroencephalogram revealed no focal abnormalities or epileptiform activity, noting only the presence of diffuse slowing activity, consistent with moderate encephalopathy.
By the time of her final follow-up visit approximately, six years after initial diagnosis, she had developed a wide-based gait with frequent falls, aphasia, personality changes, poor insight, and complete lack of orientation to time, place, and person. She ultimately passed away within six months of her last visit, at age 66. Her family consented to neuropathologic evaluation of her brain by the Center for Translational Research in Neurodegenerative Disease (CTRND) at the University of Florida.

Molecular studies
A directed Sanger Sequencing screening panel for autosomal dominant mutations of APP, PSEN1, and PSEN2 identified a guanine-to-adenine single nucleotide substitution at codon 717, resulting in a Valine to Isoleucine amino acid change (APP NM_000484.3 c2149G > A pVa-l717Ile, Fig. 1a). A PCR-based molecular assay for the APOE gene revealed the patient's genotype to be ε2/ ε3 (for details see Additional file 1).

Neuropathological evaluation
The post-mortem interval prior to brain procurement was eight hours, with a fresh brain weight of 1080 g (for details see Additional file 1). Diffuse cerebral atrophy, with relative preservation of the cerebellum (Fig. 1b, c) was noted. Minimal to no atherosclerotic changes associated with the basal vasculature were identified. Serial coronal sections of cerebral hemispheres confirmed a mild to moderate degree of atrophy, with blunting of the lateral angles of the ventricles and sulcal widening most appreciable along the Sylvian fissure. No focal lesions were otherwise observed in the remainder of the cerebrum, brain stem, or cerebellum.
Microscopic examination demonstrated extensive neuronal loss and associated gliosis in the hippocampus and neocortical areas, with numerous pyramidal neurons notable for flame-shaped neurofibrillary tangles. Multiple areas demonstrated prominent neuritic plaques (Fig. 1d, insert) with associated areas of neuronal loss, gliosis, and variable vacuolization and spongiosis of superficial cortical layers (Fig. 1e). In addition, extensive cerebral amyloid angiopathy (CAA) was apparent throughout parts of cerebrum and cerebellum, concentrating on superficial cortical and leptomeningeal blood vessels (Fig. 1f). In contrast, only mild small vessel hyalinization of the basal ganglia and white matter were identified, with focal calcification of globus pallidus blood vessels. A remote microhemorrhage was identified in the primary sensory cortex. The brainstem and cerebellum demonstrated no major neuropathologic changes, with no significant neuronal loss, gliosis, or Lewy bodies identified in the substantia nigra or locus coeruleus.
Immunohistochemistry (for details see Additional files 1 and 2) with a pan-Aβ antibody (4G8) demonstrated a very high Aβ plaque burden throughout the cerebral neocortex ( Fig. 2a-d), the amygdala (Fig. 2e), basal ganglia, and tegmentum of the midbrain and pontine brainstem. Aβ deposition was also identified in the cerebellum, presenting as scattered fleecy diffuse plaques and neuritic plaques in the molecular layer of the cerebellar cortex (Fig. 2f). These findings translated to Thal phase 5 of Aβ deposition, corresponding to an "A3" plaque score according to the 2012 NIA-AA criteria [35]. The majority of Aβ plaques were surrounded by dystrophic neurites (Fig. 3d), with the frequency of neuritic plaques throughout the neuroaxis corresponding to a CERAD semiquantitative score of "frequent" (C3) [35].
Immunostaining for tau demonstrated a concordant pattern of severe, widespread inclusion pathology, manifesting as a heavy burden of intraneuronal neurofibrillary tangles (NFT) and dystrophic neurites associated with amyloid plaques (Fig. 3a-d). Disease topography extended from the entorhinal cortex and adjacent mesial temporal lobe cortex, deep cerebral gray matter structures, to several areas of neocortex including primary visual cortex (Fig. 3a-c). These findings correspond to an advanced Braak stage (VI), translating to a "B3" NFT score [35].
In addition, intracytoplasmic neuronal inclusions were highlighted in the hippocampal subiculum and amygdala by immunohistochemistry for TDP-43 ( Fig. 3e-f), while neocortical areas were devoid of TDP-43 pathology, corresponding to stage 2 of the recently defined limbicpredominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC [41]). No α-synuclein immunoreactive pathology was identified in the examined sections of cerebrum, brainstem, or cerebellum (data not shown).

Discussion and conclusions
The identification of missense mutations in APP underlying familial forms of AD has paved the way for the formulation of the "amyloid cascade hypothesis" [16,22] by placing the generation of Aβ peptides as central to disease pathophysiology. To date, more than 50 mutations in APP associated with early onset AD have been described [9,16,29,50,55]; rare APP variants associated with protective properties have also been reported [28]. Several different substitutions of the intramembranous Valine residue at position 717 of APP have been described to be associated with familial forms of AD [10,18,37,38]. This position is near the γ-secretase cleavage site of APP, such that amino acid substitutions at this functional locus lead to an increased ratio of Aβ 1-42 /Aβ 1-40 with a trend towards increased production of Aβ 1-42 [15,27,49].
The patient described here carrying the APP V717I mutation presented with extensive AD neuropathological changes, including abundant and widespread Aβ-pathology in cerebrum, subcortical nuclei, and cerebellum. Multiple different types of plaques were noted, including coreplaques, diffuse plaques and subpial band-like Aβ deposits. All of the deposits showed a uniformly strong staining pattern with Aβ 1-42 specific antibodies and relative scarcity of Aβ 1-40 positivity compared to SAD cases, in line with the reported increase in Aβ 1-42 with this mutation in cell culture studies [27]. The majority of Aβ-deposits were associated with dystrophic neurites containing phosphorylated tau species. In addition, widespread neuronal tau pathology in the form of NFT, as well as neuropil thread pathology were noted. While no concomitant α-synuclein pathology was detected, TDP-43 positive inclusions were observed in the amygdala and hippocampus. Co-occurring TDP-43 pathology in carriers of APP mutations is not as common as in sporadic (late-onset) AD but has been reported [11]. This is in line with a contribution of age to the preponderance of TDP-43 positive pathology and ties in with the recently proposed entity of limbic-predominant TDP-43 neuropathological changes (LATE-NC) [41]. In addition,  1.1, c). Vascular amyloid in two SAD cases with different APOE genotype (ɛ3/ɛ3 (d-f) and ɛ4/ɛ4 (g-i)), showed a similar staining pattern with strong positivity for pan-Aβ antibodies (4G8, d, g), as well as Aβ 1-42 (e, h) and Aβ 1− severe and widespread CAA was noted, affecting leptomeningeal and cortical blood vessels, but sparing fine capillaries. Systematic studies on CAA in ADAD cases are scarce, but a recent report from the National Alzheimer Coordinating Center (NACC) showed an increased CAA score in ADAD compared to SAD [48]. The number of APP mutation carriers in this study were limited, but phenotypic variability with respect to CAA severity was observed. Vascular amyloid in our ADAD case was strongly labelled with pan-Aβ, as well as Aβ 1-42 and Aβ 1-40 specific antibodies in a similar pattern as observed in two SAD cases with different APOE genotype. The relative abundance of Aβ species in parenchymal and vascular deposits has been a matter of intense debate. Initial reports suggested that the majority of vascular Aβ is Aβ 1-40 [1,19,25,26,45], but subsequently a substantial contribution of Aβ 1-42 to vascular Aβ-deposition was acknowledged [1,19,40,51,60], with some reports suggesting Aβ 1-42 deposition driving more severe CAA [4,20]. Mechanistic studies in murine models indicate that initial deposition of Aβ 1-42 may be necessary to drive subsequent Aβ 1-40 deposition in blood vessels [33,42], but the impact of different APOE genotypes has not been analyzed systematically in this context. The relative sparing of capillary vessels by pathologic Aβ deposits, referred to as type 2 CAA [58] was previously reported to be associated with the presence of at least one APOE ε2 allele [2,32,59]. APOE is currently the strongest known genetic risk factor of AD, with the ε4 isoform correlating to an increased incidence of AD in people of European descent [56]. The ε3 allele is associated with preservation of synaptic integrity in old human APP (hAPP) mice [7], and mediation of amyloid clearance in comparison to ε4 [5], but it was also correlated to an earlier age of onset than ε4 in this model [7,30]. The ε2 genotype was studied in several Italian families with the APP V717I mutation. It was discovered that this allele was associated with a delayed age of onset compared to individuals with the same APP mutation but APOE ε3 homozygotes or ε4 carriers [23,39,53]. Furthermore, a recent report demonstrated protective effects of the Christchurch APOE variant in a carrier of the Presenilin (PSEN) E280A mutation [3]. These diseasemodifying effects of the APOE genotype may provide one possible explanation for the divergent phenotypes seen in the clinical and neuropathological presentation of ADAD.
The case presented herein underscores the importance of neuropathological characterization of genetically determined cases of AD. Such examinations serve to identify phenotypic diversity within the disease, clarify potential modifiers of disease progression (such as, but no limited to, APOE genotype), explore the complex interrelations between disease mechanisms, and ultimately aid in elucidating potential therapeutic targets.