Received: 3 March 2020 | Accepted: 13 January 2021 DOI: 10.1111/bpa.12942 R E SEA RCH A RTICLE Cognitive decline in amyotrophic lateral sclerosis: Neuropathological substrate and genetic determinants Sergi Borrego-­Écija1 | Janina Turon-­Sans2,3 | Teresa Ximelis4 | Iban Aldecoa4,5 | Laura Molina-­Porcel1,4 | Mónica Povedano6 | Miguel Angel Rubio7 | Josep Gámez8,9 | Antonio Cano10 | Martí Paré-­Curell11 | Lorena Bajo12 | Javier Sotoca13 | Jordi Clarimón2,3 | Mircea Balasa1 | Anna Antonell1 | Albert Lladó1 | Raquel Sánchez-­ Valle1,4 | Ricard Rojas-­García2,3 | Ellen Gelpi4,14 1 Alzheimer’s Disease and Other Cognitive Disorders Unit, Neurology Department, Hospital Clínic, Institut d’Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Barcelona, Spain 2 Neurology department, Research Institute, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 3 Center for Networked Biomedical Research into Neurodegenerative Diseases (CIBERNED), Madrid, Spain 4 Neurological Tissue Bank, Biobanc-­Hospital Clínic-­IDIBAPS, Barcelona, Spain 5 Pathology Department, CDB, Hospital Clinic Barcelona, Barcelona, Spain 6 Service of Neurology, Motor Neuron Unit, IDIBELL, Bellvitge University Hospital, Hospitalet de Llobregat, Spain 7 Neuromuscular Unit, Department of Neurology, Hospital del Mar, Barcelona, Spain 8 ALS Unit, Neurology Department, Vall d’Hebrón University Hospital, Vall d’Hebrón Research Institute (VHIR) 9 European Reference Network on Rare Neuromuscular Diseases (ERN EURO-­NMD), Department of Medicine, UAB, Barcelona, Spain 10 Neurology Department, Hospital de Mataró, Mataró, Spain 11 Neurology Department, Hospital Germans Trias i Pujol, Badalona, Spain 12 Servei de Geriatria, Fundació Hospital de la Santa Creu, Hospital Universitari de la Santa Creu de Vic, Vic, Spain 13 Neurology Department, Hospital Mutua de Terrassa, Terrassa, Spain 14 Division of Neuropathology and Neurochemistry, Department of Neurology, Medical University of Vienna, Vienna, Austria Correspondence Ellen Gelpi, Division of Neuropathology Abstract and Neurochemistry, Department of Neurolgy, Medical University of Vienna Cognitive impairment and behavioral changes in amyotrophic lateral sclerosis Medizinischer Universitaetscampus Wien, (ALS) are now recognized as part of the disease. Whether it is solely related to Allgemeines Krankenhaus der Stadt Wien, Leitstelle 4J, Waehringer Guertel 18-­20, the extent of TDP-­43 pathology is currently unclear. We aim to evaluate the in- 1090 Vienna, Austria. Email: ellen.gelpi@idibaps.org, fluence of age, genetics, neuropathological features, and concomitant patholo- ellen.gelpi@meduniwien.ac.at gies on cognitive impairment in ALS patients. We analyzed a postmortem series Ricard Rojas García, Neurology of 104 ALS patients and retrospectively reviewed clinical and neuropathologi- Department, Institut d’Investigacions Biomèdiques-­Hospital de la Santa Creu cal data. We assessed the burden and extent of concomitant pathologies, the i Sant Pau, Carrer de Sant Quintí, 89, Barcelona 08041, Spain. role of APOE ε4 and mutations, and correlated these findings with cognitive Email: rrojas@stpau.cat status. We performed a logistic regression model to identify which pathologies Funding information are related to cognitive impairment. Cognitive decline was recorded in 38.5% of SBE is a recipient of the Rio-­Hortega post-­residency grant from the Instituto de the subjects. Neuropathological features of frontotemporal lobar degeneration Ricard Rojas-­García and Ellen Gelpi are contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­c ommercial and no modifications or adaptations are made. © 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology. Brain Pathology. 2021;31:e12942.  wileyonlinelibrary.com/journal/bpa | 1 of 14 https://doi.org/10.1111/bpa.12942 2 of 14 |    BORREGO-­ÉCIJA et al. Salud Carlos III, Spain. This study was partially funded by Fundació Marató de (FTLD) were found in 32.7%, explaining most, but not all, cases with cogni- TV3 (grant no. 20141610 to EG and no. 20143710 to RRG) and Fondo Europeo de tive impairment. Extent of TDP-­43 pathology and the presence of hippocam- Desarrollo Regional (FEDER) (PI15/01618 pal sclerosis were associated with cognitive impairment. Mutation carriers to RRG). AA is funded by Departament de Salut de la Generalitat de Catalunya, Pla presented a higher burden of TDP-­43 pathology and FTLD more frequently estratègic de recerca i innovació en salut (PERIS) 2016–­2020 (SLT002/16/00329). than sporadic cases. Most cases (89.4%) presented some degree of concomi- JG is recipient of the Instituto de Salud tant pathologies. The presence of concomitant pathologies was associated with Carlos III-­FEDER grants (PI16/01673 and PI19/00593) older age at death. FTLD, but also Alzheimer’s disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non-­FTLD related findings can influence the cognitive status, particularly in older age groups. K EY WOR DS Alzheimer’s disease, amyotrophic lateral sclerosis, ALS-­FTD, frontotemporal dementia, neuropathology, TDP-­43 protein 1 | I N T RODUC T ION or driven by common physiopathological mechanisms leading to global protein misfolding and aggregation. Amyotrophic lateral sclerosis (ALS) is a neurodegen- Genetic factors, especially the presence of apolipopro- erative disorder characterized by progressive weakness tein E (APOE) ε 4 allele, an extensively studied potential and atrophy because of loss of motor neurons. Cognitive genetic risk in Alzheimer’s disease (AD), has been asso- decline and behavioral impairment have been recently ciated with the presence of AD-­related co-­pathologies recognized as part of the disease (1,2). Approximately (9–­ 11). Previous work suggests that APOE haplotype half of the patients with ALS have cognitive or behav- also influences the risk of developing hippocampal scle- ioral impairment and around 10–­20% fulfill clinical rosis (HS) with TDP-­43 proteinopathy in aging (12,13), as diagnostic criteria of any of the clinical variants of well as FTD in patients with ALS (14,15). Whether cog- frontotemporal dementia (FTD), mainly behavioral nitive impairment in ALS patients is related to underly- variant (bvFTD), but also of primary progressive apha- ing FTLD or because of another concomitant pathology sia (PPA) (2–­5). The majority of ALS cases and almost remains unclear. half of FTD patients harbor neuronal, and a propor- The purpose of this study was to evaluate the influ- tion also glial, inclusions of TAR DNA-­binding pro- ence of demographic, genetic, and pathologic features, tein 43 (TDP-­43) at neuropathological examination. In including the presence of concomitant pathologies, on addition, both diseases share genetic alterations such cognitive impairment in a neuropathologically defined as the hexanucleotide expansion in chromosome 9 open cohort of ALS-­TDP patients. reading frame 72 (C9orf72) gene, as well as mutations in the tank-­binding kinase 1 (TBK1), sequestosome-­1 (SQSTM1), TAR DNA-­binding protein (TARDBP), or 2 | M ET HOD S valosin-­containing protein (VCP). For these reasons, ALS and FTD are now recognized as a disease con- 2.1 | Standard protocol approvals, tinuum (6). FTLD is the pathological substrate under- registrations, and patient consents lying most FTD cases. The term frontotemporal lobar degeneration (FTLD) is used as an umbrella term for All cases meeting criteria for a primary clinicopatho- the different clinical variants (bvFTD, semantic, and logical diagnosis of ALS associated with TDP-­43 pro- non-­fluent PPA) as well as a neuropathological term teinopathy were selected from the Neurological Tissue that refers to neuronal loss and gliosis of the frontal Bank (NTB), Hospital Clínic -­IDIBAPS Biobank in and temporal lobes. As a matter of clarity, we refer to Barcelona from the period 1994 to 2018. In order to the neuropathological concept when using the term get a homogeneous sample, patients without TDP-­43 FTLD throughout the text. pathology and mimics were excluded. The study was On the other hand, concomitant neurodegenera- approved by the Ethics Committee of Hospital Clínic tive disease-­related proteinopathies are frequent and de Barcelona. All individuals or relatives had given have been primarily associated with age at death (7–­9). their informed consent for the use of brain tissue for It remains unclear whether this overlap is coincidental research. ALS COGNITIVE DETERMINANTS    | 3 of 14 2.2 | Clinical classification following the criteria proposed by Brettschneider for ALS (19). Presence of FTLD was established when neu- We retrospectively reviewed the medical records avail- ronal loss and gliosis were observed in temporal and/or able at the NTB and contacted the neurologists who frontal regions (20). FTLD-­TDP subtype classification attended the patients during life. As we included ALS do- was performed based on TDP-­43 or pTDP-­43 immuno- nors since 1994, some cases had not been systematically histochemistry following current recommendations (21). screened for cognitive impairment. In those subjects, the HS was considered on HE-­stained sections when neuro- presence and the grade of cognitive impairment were as- nal loss and gliosis at least in the subiculum and/or CA1 sessed by the global impression of the treating neurolo- sector of the hippocampus were observed (22). Cases that gist. They were asked to fill in a form that included the showed only mild gliosis in subiculum were considered following clinical information: age at onset, age at death, incipient HS. In all cases, we evaluated the presence of disease duration, site of onset (bulbar/spinal), presence fine TDP-­43 immunoreactive neurites in the CA1 sector. or absence of cognitive or behavioral impairment, and As previous reports stated that the presence of TDP-­43 diagnosis of bvFTD or PPA. The severity of the cogni- inclusions in the anterior cingulate may identify patients tive/behavioral impairment was retrospectively assessed with clinical bvFTD, we specifically assessed this area in according the CDR® Dementia Staging Instrument plus cases with available tissue (23). National Alzheimer’s Coordinating Center Behaviour For the analysis of concomitant pathologies, we and Language Domains (CDR plus NACC-­FTLD) (16). considered the following findings: neurofibrillary This rating scale classifies the severity of the dementia pathology, staged according to Braak criteria (24), according eight domains in: no cognitive impairment β -­amyloid phases, evaluated according to Thal criteria (score of 0), mild cognitive impairment (score of 0.5) or (25), and neuritic plaque score, assessed according to mild, moderate or severe dementia (scores of 1, 2, and the Consortium to Establish a Registry for Alzheimer 3, respectively). The cognitive domains affected were as- Disease criteria (26). The National Institute on Aging-­ signed according to the expert opinion of the attending Alzheimer’s Association (NIAA) Guidelines for neuro- neurologist or the neuropsychological evaluation. For pathologic assessment of AD were applied and a final every patient, we considered the last information avail- ABC score was assigned (27). Cerebral amyloid angiop- able before death. Cases with insufficient clinical infor- athy was recorded as present or absent. Additionally, mation to be classified were excluded from the study. possible and definite PART pathology was defined fol- With this information, patients were classified into lowing the current pathological criteria (28). In order to three clinical subtypes: i) ALS cognitively not impaired assess whether progressive spinal cord degeneration is (ALSni) when cognitive decline was not present or re- associated to astroglial tau pathology, we also evaluated ported, ii) ALS with cognitive impairment (ALSci) when the presence of age-­related tau astrogliopathy (ARTAG) cognitive or behavioral decline were reported but with- at the cervical, dorsal, and lumbosacral spinal level when out fulfilling bvFTD or PPA criteria and iii) ALS with available and defined thorn-­shaped astrocytes and gran- frontotemporal dementia (ALS-­FTD) when patients ful- ular fuzzy astrocytes as present/absent, and recorded filled criteria for bvFTD or PPA (2,17,18). their location (subependymal, subpial, and perivascular) and the affected level (cervical, dorsal, and/or lumbo- sacral) (29). Assignments of Lewy body pathology were 2.3 | Neuropathological work-­up performed following McKeith criteria (30). Argyrophilic grain disease (AGD) was staged according to the Saito Neuropathological examination was performed accord- criteria (31). Limbic-­ predominant age-­ related TDP-­ 43 ing to standardized protocols at the NTB. Half brain encephalopathy (LATE) was evaluated according to the was dissected in the fresh state, frozen and stored at recently described criteria, but only in those subjects −80°C and the other half was fixed in formaldehyde so- without FTLD, as both are TDP-­43 proteinopathies and lution for 3 weeks. At least 25 representative brain areas may overlap in temporomedial regions (32). In addition, were embedded in paraffin, cut at 5 µm and stained with the presence of granulovacuolar degeneration (GVD) hematoxylin & eosin and luxol fast blue in selected brain was assessed on HE-­and ubiquitin-­stained sections in areas. Immunohistochemistry was performed using var- the hippocampus and, if present, was extended to fur- ious antibodies including anti-­βA4, anti-­pTau, anti-­RD3 ther limbic areas and staged according to Thal et al. (33). and anti-­ RD4 Tau, anti-­ α-­synuclein, anti-­ubiquitin, Vascular pathology including microvascular lesions and anti-­p62, anti α-­internexin, anti-­FUS, and anti-­TDP-­43, territorial infarcts were recorded as present or absent. and phTDP-­ 43. Immunoreaction was visualized by the EnVision + system peroxidase procedure (DAKO). Details on tissue section pretreatment, antibody dilu- 2.4 | Genetic analysis tion, and incubation time are shown in Table S1. Disease assessment was performed according to in- DNA was extracted from fresh-­frozen cerebellum using ternational consensus criteria. All cases were staged the QIAamp DNA Mini kit for DNA purification from 4 of 14 |    BORREGO-­ÉCIJA et al. tissues (QIAGEN Co.) following the manufacturer’s in- Cognitive decline was recorded in 40 subjects (38.5%). structions. For APOE genotyping two single nucleotide Thirty-­one of them (29.8%) fulfilled diagnostic cri- polymorphisms (SNPs) in APOE gene were determined teria for bvFTD or PPA (29 and 2 cases, respectively), (rs429358 and rs7412) using TaqMan genotyping technol- while nine were classified as ALSci. Table 1 shows de- ogies (Life technologies, Carlsbad, USA). mographic, neuropathological, and genetic features of Systematic screening for potential C9orf72 expansion ALSni, ALSci, and ALS-­FTD patients. No demographic mutation carriers was performed searching for ubiqui- differences in age at onset, age at death or disease du- tin/p62-­positive inclusions in the cerebellum and hip- ration were found between ALS, ALSci, and ALS-­FTD pocampus as surrogate and as reported previously (34). patients. Bulbar onset was more frequent in subjects with The C9orf72 repeat was confirmed in suspected cases by ALS-­FTD (p = 0.03). repeat-­primed PCR and fragment-­length analysis (35). Most patients (67.6%) were homozygous for APOE The identification of other mutations was not performed ε3/ε3, 5.1% had ε2/ε3, 22.2% ε3/ε4, and 5.1% ε4/ε4. None by systematic screening but by previous studies or usual of them were homozygous for APOE ε2. No significant clinical practice according to the criteria of the treating differences in APOE genotypes were found between neurologist (36). Information on other affected family groups. Mutations in genes reported as causative of or members was not available. at risk for ALS were found in 21 subjects: 14 C9orf72, 2 TARDBP (p.A90V and p.I383V), 2 SQSTM1 (both c.1157C > T), 1 TBK1 (p.T79del), 1 VCP (p.I27V), and 1 2.5 | Statistical analysis TAF15 (p.G462S). Most of these mutation carriers have been previously reported (34,36,37). More than half of We compared clinical, genetic, and pathology vari- ALS-­FTD subjects presented an ALS/FTD related mu- ables between ALS, ALSci, and ALS-­FTD groups with tation (61.3%), this percentage was higher than in ALSci χ 2 or Fisher tests for categorical data and Wilcoxon or (0%) and ALSni (3.1%) subjects (p < 0.001). Kruskal–­Wallis test for ordinal and continuous data. The association of the clinical, demographic, and genetic data with the neuropathological findings was assessed 3.2 | Neuropathological by Kruskal–­Wallis test and Fisher test. Finally, we per- features and their relationship with cognitive formed a logistic multivariate regression to test the asso- impairment and genetics ciation between cognitive status and neuropathological variables. In this logistic multivariate regression, we Figure 1 summarizes the main neuropathological fea- included the presence of cognitive decline as dependent tures of ALS patients. variable and the presence of the following neuropatho- logical items as independent variables: frontotemporal lobar degeneration, hippocampal sclerosis, Alzheimer’s 3.2.1 | Brettschneider staging (Figure 2A) disease, Lewy body disease, CAA, PART, ARTAG, AGD, GVD, LATE, and vascular pathology. As some The distribution of the TDP-­43 pathology according to of these covariates may suffer from relevant collinearity, the Brettschneider score for ALS was as follows: 9 subjects other reduced models including less covariates were also were scored as stage 1, 17 as stage 2, 26 as stage 3, and 51 assessed. Statistical significance was set at p < 0.05 for as stage 4. In one subject the Brettschneider staging was all analyses. not at all applicable because of the presence of TDP-­43 pathology in motor cortex and entorhinal region but not in inferior olive, medullary reticular formation, prefrontal 3 | R E SU LT S cortex or striatum. The Brettschneider stage was strongly associated with the CDR plus NACC FTLD stage of de- 3.1 | Demographic and genetic features of the mentia (p < 0.01, Figure 2A). ALS-­FTD patients showed ALS cohort significant higher Brettschneider stages than ALSni pa- tients (p < 0.001; see Table 1). The presence of a genetic mu- Of the 112 donors with a neuropathological diagnosis tation was associated with a higher Brettschneider stage of ALS, four were excluded because of the absence of (p < 0.01, Figure 2A), while APOE was not. No significant TDP-­43 pathology (three ALS-­FUS cases and one sub- differences concerning disease duration were found be- ject without pathological inclusions). Another four cases tween the different Brettschneider stages. were excluded because of incomplete neuropathological exam or insufficient clinical information. Finally, a total of 104 cases (50 male and 54 female) were included in the 3.2.2 | Hippocampal sclerosis (Figure 2B) study. The onset was bulbar in 30.9% of cases. Mean age at onset of motor symptoms was 63.8 years (range 29–­87) HS was detected in 10 subjects (9.6%). Other eleven cases and the mean age at death was 67.7 (range 34–­92). showed signs of incipient HS. Subjects with ALS-­FTD or ALS COGNITIVE DETERMINANTS    | 5 of 14 TA BL E 1 Demographic, neuropathologic, and genetic features in patients ALS, ALSci, and ALS-­FTD ALSni ALSci ALS-­F TD n = 64 (61.5%) n = 9 (8.7%) n = 31 (29.8%) p value Demographic features Male/female 30/34 4/5 16/15 ns Age at onset 63.0 (14.3) 67.1 (10.9) 64.3 (11.6) ns Mean (SD) Age at death 66.6 (13.6) 71.2 (10.3) 68.7 (10.7) ns Mean (SD) Bulbar onset (%) 23.0% 22.2% 54.2% <0.05* CDR plus NACC FTLD 64/0/0/0/0 0/9/0/0/0 0/0/6/9/16 <0.001 0/0.5/1/2/3 Neuropathologic features Brettschneider stage (stage 1/2/3/4) 8/15/21/19 0/1/3/5 1/1/2/27 <0.001* FTLD (%) 4.7% 44.4% 87.1% <0.001*,**,*** HS (%) 6.2% 33.3% 41.9% <0.001*, <0.05** Genetics APOE genotype (%) ε2/ε3 6.3% 0% 3.7% ns ε3/ε3 69.8% 55.6% 66.7% ε3/ε4 20.6% 22.2% 25.9% ε4/ε4 3.2% 22.2% 3.7% Mutations (%) C9orf72 2 (3.1%) 0 (0%) 12 (38.7%) <0.001*,*** TARDBP 0 (0%) 0 (0%) 2 (6.5%) VCP 0 (0%) 0 (0%) 2 (6.5%) TBK1 0 (0%) 0 (0%) 1 (3.2%) SQSTM1 0 (0%) 0 (0%) 1 (3.2%) TAF15 0 (0%) 0 (0%) 1 (3.2%) Abbreviations: ALS, amyotrophic lateral sclerosis; ALSci, amyotrophic lateral sclerosis with cognitive impairment; ALS-­FTD, amyotrophic lateral sclerosis with frontotemporal dementia; CDR plus NACC FTLD, Clinical Dementia Rating Dementia Staging Instrument PLUS National Alzheimer’s Disease Coordinating Center frontotemporal lobar degeneration Behaviour and Language Domains; FTLD, frontotemporal lobar degeneration; HS, Hippocampal Sclerosis; ns, statistically not significant. *Significant differences between ALSni and ALS-­FTD. **Significant differences between ALSni and ALSci. ***Significant difference between ALSci and ALS-­FTD. ALSci presented HS more frequently than ALSni cases 3.2.3 | Frontotemporal lobar degeneration (p < 0.01 and p < 0.05, respectively). The presence of HS (Figure 2C) was also significantly associated with higher stages in the CDR plus NACC FTLD (p < 0.01). HS was strongly Neuropathological features consistent with FTLD were correlated with Brettschneider staging and presence found in 34 subjects (32.7%). Three cases were classi- of FTLD: all subjects with HS presented a stage 4 of fied as type A, 25 as type B, 5 showed mixed features Brettschneider, and 88.2% presented also with FTLD. of type A and B, and 1 subject had FTLD type C. Most TDP-­ 43-­ positive neurites in CA1 sector were not de- cases with neuropathological FTLD fulfilled clinical tected in any of the cases with HS or incipient HS in the criteria of ALS-­FTD (77.1%) or ALSci (14.3%). FTLD ALSni group. In contrast, two cases with additional AD-­ was strongly associated with higher stages in the CDR type pathology showed frequent fine TDP-­43-­positive plus NACC FTLD (p < 0.001). It was present in 87.1% neurites in CA1 sector. The presence of a genetic muta- of the ALS-­FTD cases, in 44.0% of ALSci, and only in tion or the APOE ε4 were not associated with a higher 4.7% of ALSni cases. Moreover, FTLD was present in prevalence of HS. 63.0% cases with a Brettschneider ALS stage 4 and in 6 of 14 |    BORREGO-­ÉCIJA et al. F I G U R E 1 Characteristic neuropathological features of ALS. (A) Cross section through the thoracic spinal cord shows prominent degeneration of the lateral and anterior corticospinal tract and atrophy of the anterior horns (higher magnification in E). There is also myelin pallor in anterior roots compared to posterior roots. The degeneration of the lateral corticospinal tract is characterized by loss of myelin sheaths (B), reduction of axonal profiles (C), and increased macrophagic activity (D), as compared with posterior horns (b, c, d). (F) some residual motor neurons (here from the n. XII) may contain granular eosinophilic cytoplasmic inclusions or Bunina bodies (arrow and inset). Others may appear as pale spherical inclusions (G). Axonal damage with swellings (H) in anterior horns is well depicted by anti-­neurofilament immunohistochemistry (large brown structures). I–­L: Immunohistochemistry for pTDP-­43 reveals a spectrum of inclusions including a fine granular pattern in large motor neurons (I), fibrillar and skein like inclusions (J), compact spherical neuronal inclusions (K), and coiled-­body like inclusions in oligodendrocytes (L). Scale bars: 20 µm in Bb, Cc, Dd, F; 10 µm in G–­L, 100 µm in E 4.3% of cases in stage 3, while it was absent in stages 1 3.3 | Concomitant pathology and 2. ALS subjects with any genetic mutation presented sig- Figure 3 shows some of the representative concomitant nificantly more risk to develop FTLD than ALS without pathologies found in ALS patients. Figure 4 shows the known mutations (80.9% vs. 20.4%, OR 15.92, IC 95% proportion of these concomitant pathologies found in 4.45–­73.66, p < 0.001). By contrast, APOE ε4 was not as- our series. sociated with a higher risk of FTLD or a specific FTLD subtype. Cases carrying the C9orf72 expansion were neu- ropathologically classified as subtype B or A/B. We did 3.3.1 | Presence of concomitant not find statistically significant differences in the neuro- neurodegenerative pathologies (Figure 4A) pathological subtypes according the APOE genotype. Considering all concomitant pathologies together, most (89.4%) ALS patients presented at least one 3.2.4 | Cingulate involvement (Table 2) co-­pathology at neuropathological examination. Concomitant AD neuropathologic changes, accord- Cingulate involvement was present in 70.0% of cases ing to “ABC” score, were found in 52 subjects (50.0%) (sparse 16.7%, mild 18.9%, moderate 25.5%, and severe at low, in 8 (7.7%) at intermediate, and in 7 patients grade in 8.9%). All subjects with FTLD showed, at least, (6.7%) at high level. CAA was found in 17 cases (16.3%). a mild grade of TDP-­43 pathology in the anterior cin- Nearly half (48.1%) of the subjects fulfilled criteria for gulate. In contrast, 70% of subjects without FTLD pre- PART pathology (25.0% possible, 23.1% definite). In sented none or only sparse pathology in this area. addition, 38.5% presented ARTAG (18.3% granular ALS COGNITIVE DETERMINANTS    | 7 of 14 F I G U R E 2 Barplots representing the proportion of ALS cases with (A) the different Brettschneider stage, (B) the presence of hippocampal sclerosis and (C) Frontotemporal Lobar degeneration according to their clinical diagnosis, CDR plus NACC FTLD stage, presence of APOE ε4, or presence of mutations in genes reported as causative of or at risk for ALS/FTLD T A B L E 2 Prevalence of TDP-­43 pathology in anterior cingulate (p < 0.05). AGD was detected in 14 subjects (13.5%; 11 cortex in ALS spectrum subjects at Saito stage 1, 1 subject at stage 2, and 2 sub- ALSni ALSci ALS-­FTD jects at stage 3), while Lewy body pathology was iden- Presence of TDP-­43 in tified in 9.6% (seven subjects restricted to brainstem anterior cingulate n (%) n = 58 n=6 n = 26 nuclei, three with limbic involvement, and only one No 25 (43.1%) 1 (16.7%) 1 (3.8%) with neocortical involvement). Vascular lesions were Sparse 14 (24.1%) 1 (16.7%) 0 (0%) identified in 14 subjects (13.5%); 11 of them with small Mild 13 (22.4%) 0 (0%) 4 (15.4%) vessel pathology and only 3 of them presenting large vessel infarcts. We observed LATE in 19 (27.1%) of the Moderate 6 (10.3%) 4 (66.7%) 13 (50%) 70 ALS cases without FTLD. Frequent 0 (0%) 0 (0%) 8 (30.8%) Note: n = 90 cases out of 102. Abbreviations: ALSci, amyotrophic lateral sclerosis with cognitive 3.3.2 | Relationship of concomitant impairment; ALS-­FTD, amyotrophic lateral sclerosis with frontotemporal dementia; ALSni, amyotrophic lateral sclerosis without cognitive impairment. pathologies and age at death (Figure 4B) Concomitant pathologies were more frequent in subjects fuzzy astrocytes, 13.5% thorn-­shaped astrocytes, 6.7% who died at older age. ABC score for AD was signifi- both), with 8.3% presenting astroglial pathology also cantly higher in subjects with older age at death (p < 0.01). in the spinal cord. ARTAG pathology was found more Particularly, Braak and Braak stages for neurofibrillary frequently, but not exclusively, in subjects with AD pa- pathology and amyloid Thal phases were significantly thology (44.8% vs. 27.0%, p < 0.05). GVD was found increased in older subjects (p < 0.01). The presence of in 42.3% of the subjects and was mostly very mild and LATE, ARTAG, Lewy body pathology, GVD, and vas- restricted to the hippocampal subfields CA2/CA1 with cular lesions also was significantly higher in older age only mild entorhinal involvement. GVD was not only groups (p < 0.05). CAA, PART, and AGD concomitant strongly related to AD pathology (p < 0.001), but also pathologies did not show significant association with age to the presence of FTLD and the C9orf72 expansion at death. 8 of 14 |    BORREGO-­ÉCIJA et al. F I G U R E 3 Frequent co-­pathologies in ALS. (A–­C) Segmental hippocampal sclerosis is sometimes better identified in more anterior segments of the hippocampus (A, arrowheads). It is characterized by neuronal loss and prominent fibrillar gliosis (B) in the CA1 sector and subiculum. In most cases granule cells of the dentate gyrus harbor TDP-­43 protein inclusions in the cytoplasm (C, upper row) and in some cases there are abundant fine threads in the CA1 sector within and adjacent to the sclerotic area (C, lower row). (D and E) Argyrophilic grain pathology is well depicted by the AT8 anti-­t au antibody (D) and consists of grain-­l ike structures along neuronal processes, abundant threads, and a diffuse cytoplasmic neuronal staining (pretangle type) instead of compact tangles. They are frequently associated with ballooned neurons in the amygdala (E, upper row) and oligodendroglial coiled bodies in temporomedial white matter (E, lower row). Astrocytic tau pathology in form of granular fuzzy astrocytes (F, upper row) and thorn-­shaped astrocytes in the glia limitans (F, lower row, here as subependymal and perivascular thorn-­shaped astrocyte) are features of age-­related tau astrogliopathy (ARTAG). (G and H) Alzheimer’s disease neuropathologic changes have been also found as co-­pathology in a fraction of cases. It is characterized by dense cored amyloid plaques with or without amyloid angiopathy (G) and tau positive neurofibrillary pathology with tangles, neuropil threads, and dystrophic neurites around amyloid deposits (H). (I) Lewy body pathology has been detected less frequently and can be encountered in brainstem neurons (HE, upper row, medulla oblongata) and/or limbic system and is well identified with anti-­alpha-­synuclein antibodies (lower row), where Lewy-­neurites are also frequently seen (here in the locus coeruleus). Scale bars: 50 µm in B, F, G, H; 20 µm in C (upper and lower panel), D, E, and I (upper and lower panel) 3.3.3 | Relationship of concomitant of amyloid, CAA, PART, ARTAG, AGD, Lewy body, pathologies with CDR-­NACC FTLD (Figure 4C) LATE, or vascular co-­pathologies. Concomitant pathologies were found more frequently in subjects with reported cognitive impairment (97.5% 3.3.4 | APOE status and concomitant vs. 84.0%, p < 0.05). The CDR-­NACC-­FTLD score was pathologies (Figure 4D) significantly related to the Braak and Braak stage for neurofibrillary pathology (p < 0.01). Subjects with severe CAA was found more frequently in subjects with an stages of dementia also presented more GVD pathol- APOE ε4 allele (34.6%, vs. 9.6%; p < 0.05). For the rest ogy (p < 0.05) No significant associations were found of the concomitant pathologies, no statistical differences between the CDR-­NACC-­FTLD score and Thal phase were according to the APOE status. ALS COGNITIVE DETERMINANTS    | 9 of 14 F I G U R E 4 Presence of total concomitant pathologies in ALS cases (A), distribution of pathology according to age at death (B), distribution of pathology according to the CDR NACC plus FTLD staging (C), and distribution of pathology according to the presence of APOE ε4 allele (D). Neurofibrillary tangles were staged following Braak stages (24). Amyloid represents Aβ Thal phases (25). Globally, Alzheimer’s disease pathology was staged according the NIA/AA guidelines and an ABC score was assigned (27). Cerebral Amyloid angiopathy (CAA) has been reported as absent or present. Vascular lesions were classified according to small or large vessel pathology. Assignments of Lewy Body pathology were performed following McKeith criteria (30). Possible or definite primary age related tauopathy (PART) was assessed according the current neuropathological criteria (28). Age-­ related tau astrogliopathy (ARTAG) was evaluated according the current criteria (29). Argyrophilic grain disease (AGD) was staged according to the Saito criteria (31). Granulovacuolar degeneration (GVD) was staged according to Thal et al (33) 3.4 | Determinants of cognitive impairment of AD concomitant pathology independently influ- in ALS enced the development of cognitive impairment in ALS patients, especially in those with higher burden Finally, to assess whether neuropathologic and concomi- of pathology (OR 36.5, 95% CI 3.4–­4 44.2, p < 0.05). tant pathologies influence the presence of cognitive im- Other concomitant pathologies did not influence cog- pairment in ALS patients a logistic regression analysis nitive symptoms in ALS subjects with these analyses. was performed (Table 3). Reduced models considering less covariates in order Regarding the neuropathological substrate, the to avoid collinearity problems showed similar results presence of FTLD was the strongest determinant for (Table S2). cognitive decline in our model (OR 359.5, 95% CI 20.4–­ Five subjects with reported cognitive impairment 1940.2, p < 0.001). We also found that the presence (three ALSci and two ALS-­FTD) did not present an 10 of 14 |    BORREGO-­ÉCIJA et al. TA BL E 3 Logistic multivariate regression FTLD were associated with sex, age at death or disease Neuropathologic features Odds ratio (95% CI) p value* duration. Spencer et al. recently reported that patients Age at death 0.9 (0.8–­1.0) 0.177 with long-­duration ALS (>10 years) showed less frequent TDP-­43 pathology and less severe lower motor neuron Sex (female) 2.7 (0.6–­14.5) 0.219 involvement. In our series, only six patients had a dis- Frontotemporal lobar 359.5 (20.4–­1940.2) <0.001 ease duration of 10 years or more and two of them had degeneration no TDP-­ 43 but FUS pathology (39). However, those Hippocampal sclerosis 0.3 (0.1–­5.5) 0.459 harboring TDP-­43 pathology had no different TDP-­43 Alzheimer’s disease 36.5 (3.4–­4 44.2) <0.05 severity stages (as assessed by Brettschneider staging) neuropathological change compared to the standard duration group in their study Cerebral amyloid angiopathy 2.9 (0.4–­20.9) 0.279 (40). This was also observed in our series. Also in accor- α-­synuclein 2.3 (0.1–­13.7) 0.559 dance with previous studies, the presence of a mutation PART 2.3 (0.4–­7.1) 0.325 was the strongest determinant for comorbid ALS and ARTAG 1.9 (0.4–­6.5) 0.461 FTLD (14), even in the absence of family history within Argyrophilic grain disease 1.0 (0.1–­5.5) 0.995 the ALS/FTD spectrum (36). Bulbar onset was more fre- quent in subjects with ALS-­FTD, a finding that also has LATE 3.4 (0.3–­11.3) 0.315 been found in other studies (37,41). Granulovacuolar degeneration 1.7 (0.3–­8.0) 0.542 We found that an FTLD neuropathological pattern Vascular lesions 1.5 (0.1–­7.7) 0.748 and/or HS are the most frequent neuropathologic sub- Note: The dependent (outcome) variable of the model was the presence of strate of cognitive impairment in ALS subjects. Neither cognitive impairment (ALSci and ALS-­FTD were considered together). the Brettschneider stage nor the presence of FTLD or Frontotemporal lobar degeneration was established according to the presence of neuronal loss and gliosis in temporal and/or frontal cortices. Alzheimer’s HS were associated with disease duration (40). At the disease neuropathological change was considered as a moderate or high same time, the presence of cognitive impairment was burden of tau and amyloid pathology according the NIA-­A A. associated with a Brettschneider ALS stage 4 and the Abbreviations: ARTAG, age-­related tau astrogliopathy; LATE, limbic-­ predominant age-­related TDP-­43 encephalopathy. PART, primary age-­related presence of TDP-­43 pathology in anterior cingulate. In tauopathy. such cases, showing extensive ALS and FTLD-­related *p values were obtained from Wald’s test, bold indicates statistical TDP-­43 proteinopathy it might be useful either to apply significance. both proposed staging systems in parallel, that is, that for ALS-­TDP and that bvFTD with TDP-­43 inclusions, unequivocal neuropathological substrate explaining their or even to combine both and propose a new staging fus- symptoms. This situation was found more frequently in ing both particularly when one or the other system fails the ALSci group than in the ALS-­FTD group (33.3% vs. to appropriately classify the pathology. One of the ca- 6.5%; p < 0.05). The two subjects fulfilling criteria for veats of Brettschneider ALS stages is that the topogra- ALS-­ FTD but lacking an obvious neuropathological phy alone may not explain cognitive symptoms. Previous substrate presented genes reported as pathogenic of or at studies have suggested that the burden of TDP-­43 pa- risk for ALS (1 TARDBP and 1 VCP). The TARDBP car- thology in the anterior cingulate cortex discriminates rier presented frequent TDP-­43 inclusions in amygdala cases with clinical bvFTD (23). In our work, almost all with focal gliosis and neuronal loss. The VCP carrier, subjects diagnosed as ALS-­FTD showed at least mild by contrast, had very few TDP-­43 inclusions restricted TDP-­43 pathology in the anterior cingulate, and this to motor neurons (Brettschneider stage I), but presented was also present in 32.7% of subjects in the ALSni group. concomitant AGD pathology (Saito I). This finding suggests that the cingulate burden of TDP-­ 43 could be more sensitive than specific to discriminate cognitive involvement in ALS patients. However, be- 4 | DI SC US SION cause of the retrospective methodology of our study, it is difficult to elucidate if subtle cognitive alterations might In this work, we have analyzed the potential effects of have been underdiagnosed in these subjects, especially in the extent of TDP-­43 pathology, concomitant patholo- the last stages of the disease. gies, and genetic variables on the cognitive-­behavioral Moreover, in HS the presence of TDP-­43 proteinopa- status in a large series of neuropathologically confirmed thy is a frequent finding, independently of motor neuron ALS patients. involvement. In ALS cases with HS, the application of Though the growing evidence of ALS and FTLD TDP-­43-­stages according to Brettschneider’s proposal overlap, only few neuropathological series have been might result in stage 4 although hippocampal involve- reported so far (9,38). In our series of neuropathologi- ment by TDP-­43 would be more likely related to HS and cally confirmed ALS patients, cognitive impairment was not necessarily to a sequential progression of pathology reported in more than one-­third (38.5%) of cases. Most from motor regions. Therefore, the staging systems for of them showed a pattern of FTLD at neuropathology. ALS, FTLD, and HS, when appearing concomitantly, Neither the Brettschneider stage nor the presence of should be critically rethought. This also applies to the ALS COGNITIVE DETERMINANTS    | 11 of 14 recently described “LATE,” which by itself may also in the hippocampus and has been linked to a cell death contribute to cognitive decline. It should be considered mechanism (44,45). GVD could, therefore, represent an a separated entity from FTLD, as it manifests with a additional, TDP-­43-­related mechanism leading to cog- different phenotype, it occurs particularly in older age nitive impairment in ALS besides its relationship with groups (the “oldest old,” >85 years) and has a relatively Alzheimer’s disease neuropathological change. restricted neuroanatomical distribution (32). In the con- In our series the strongest determinant for the pres- text of ALS-­TDP, whether the presence of TDP-­43 pa- ence of concomitant pathologies was the age at death: thology in the limbic system represents LATE pathology, while subjects aged 60 or less rarely had co-­pathologies, or whether it is a restricted form of FTLD, remains a those >60 years had at least 1, and those >70 years had matter of discussion. 2 or more co-­pathologies. Although it is not possible to Our study also reveals a high frequency of concom- further elucidate this observation given the absence of itant pathologies in ALS patients, however, at different a matched control group in our study, this frequency of severity grades. It is currently well known that the accu- co-­pathologies seems to appear at relatively early ages in mulation of brain pathologies is common and that these ALS cases. Moreover, subjects with reported cognitive may influence the cognitive state or lower the threshold impairment presented with more severe AD pathology. for the development of cognitive decline (7,8). The fre- The presence of the APOE ε4 allele is a well-­k nown quencies of concomitant pathologies found in our series risk factor to develop AD. For other dementias, this rela- are in accordance with those reported in previous works tionship is not well established. Chio et al. found that the (9,38). Most ALS patients presented any grade of tau presence of the allele ε2 significantly increased the risk pathology in the form of neurofibrillary tangles: PART of cognitive impairment in ALS patients (14). Other stud- pathology was found in approximately half of the cases; ies, including two meta-­analyses, found a relationship around one-­third to one-­half also presented amyloid-­β between FTLD and the presence of the ε4 allele (15,46). pathology; and about a 10th presented any grade of α-­ These studies were limited, as they were not performed synuclein pathology. Our study also evaluated the pres- in a pathologically confirmed cohort or in ALS patients. ence of ARTAG pathology in the brain and spinal cord Recently, Yang et al. found an age-­dependent effect of of ALS subjects, as it represents the most frequent as- APOE ε4 on TDP-­43 and HS, (13) and Wennberg et al. on trocytic tau pathology found in aging brains. We found TDP-­43 deposition independently of Aß in AD pathol- that more than a third cases (38.5%) presented ARTAG ogy in a large postmortem series (12). In our postmortem pathology. In a few subjects, ARTAG pathology was also study, we also analyzed the effect of APOE on ALS and found in the spinal cord, particularly in older age groups. cognition, but we did not find statistically significant Whether the spinal astroglial pathology may be elicited associations between APOE ε4 allele and the presence by the chronic neurodegenerative process or not remains of FTLD, FTLD subtypes or HS. Except for CAA, we a matter of further studies. neither found differences in the presence of concomitant In addition, we found a quite considerable preva- pathologies in APOE ε4 carriers. Because of the low fre- lence of AGD co-­pathology in our series (15.5%). AGD quency of the APOE ε2 allele observed in our series, an is a common sporadic neurodegenerative disease of old association with the presence or absence of FTLD can- age but is rarely seen in young subjects. Of note, some of not be established. the subjects with AGD pathology were relatively young When evaluating the clinical impact of neuropatho- (mean age at death 70.9 years, range 58–­84). Previous logical findings, we found that the presence of FTLD reports point that AGD and TDP-­43 are occasionally is, by large, the predominant underlying pathology that concurrent in ALS/FTLD cases (42,43). These findings better explained the cognitive impairment in ALS pa- may strengthen the hypothesis of the existence of a link tients. However, our analysis reveals that AD also can between TDP-­ 43 pathology and argyrophilic grains. influence the development of cognitive symptoms in However, the pathogenesis of this potential interaction these patients. In addition, we found a few cases with re- remains unclear. ported cognitive decline but without FTLD or enough GVD, a feature observed in cellular stress conditions AD pathology to justify cognitive impairment. The two and some neurodegenerative pathologies including AD subjects with ALS-­FTD diagnosis lacking an obvious was not a frequent feature and, if present, was mostly neuropathological substrate for cognitive impairment very mild and restricted to the hippocampal subfields were carriers of mutations in genes reported as patho- CA2/CA1 with only mild entorhinal involvement (33). genic of or at risk for ALS (1 TARDBP and 1 VCP). The GVD was not only more frequently related to AD pa- TARDBP mutation carrier showed TDP-­43 inclusions thology, including both, neurofibrillary tangles and am- in amygdala associated with focal gliosis and neuronal yloid plaques, but was also observed more frequently in loss, while the VCP mutation carrier showed mild argy- individuals with FTLD pathology or in those carrying rophilic grain pathology with ballooned neurons. The the C9orf72 expansion. An association between GVD involvement of the amygdala in behavioral disturbances and TDP-­43 pathologies has been recently reported. It is well known (47). Therefore, despite not having a clas- has been suggested to be potentially triggered by TDP-­43 sical FTLD-­ pattern, the presence of focal amygdala 12 of 14 |    BORREGO-­ÉCIJA et al. degeneration could have been sufficient explanation for AC K NOW L E D G M E N T S the cognitive impairment. So finally, in three ALS cases SBE is a recipient of the Rio-­Hortega post-­residency grant in which cognitive impairment was reported, we found from the Instituto de Salud Carlos III, Spain. This study no adequate neuropathological explanation. Given the was partially funded by Fundació Marató de TV3 (grant retrospective nature of the study, we cannot draw any no. 20141610 to EG, grant no. 20143810 to RSV and no. conclusion concerning this finding. However, it may be 20143710 to RRG) and Fondo Europeo de Desarrollo an interesting focus of interest in future studies with pre- Regional (FEDER) (PI15/01618 to RRG). JG is recipi- cisely phenotyped series. ent of the Instituto de Salud Carlos III-­FEDER grants Our study has several limitations. First, due the ret- (PI16/01673 and PI19/00593). We are indebted to the rospective nature of the study, which included cases Neurological Tissue Bank of the Biobanc-­Hospital Clinic-­ that had been evaluated years before the acceptance of IDIBAPS, Barcelona, Spain, for data and sample procure- the concept of the ALS-­FTLD continuum, two-­thirds ment. We thank Sara Charif, Veronica Santiago, Leire of the subjects had not been systematically screened Etxarri, and Abel Muñoz for technical assistance. The with a formal neuropsychological evaluation, cognition authors wish to thank the invaluable and generous col- was assessed by the global impression of their neurol- laboration of all brain donors and their families. Without ogist. While this might underestimate the presence of them, the work of Brain Banks would not be possible. cognitive impairment particularly when it was subtle or in the last stages of the motor neuron disease, we still C ON F L IC T OF I N T E R E ST found a good association between the cognitive infor- The authors declare that they have no conflict of interest. mation reported by the neurologist and the neuropatho- logical examination. In that sense, further prospective AU T HOR C ON T R I BU T ION S studies with accurate screening tools for cognitive JTS, MB, RSV, AL, MP, MAR, JG, AC, MPC, LB, JS, decline are needed to validate the conclusions of this and RRG collected clinical data. SBE, IA, LMP, and EG work. Second, as a single Brain Bank series, it is pos- collected neuropathological data. TX, AA, and JC per- sible that our results do not represent the whole ALS formed genetic analysis. SBE drafted and prepared the population. Therefore, some clinical or genetic features manuscript. SBE and MB performed statistical analysis. may be overrepresented because of a selection bias for RRG and EG conceived the study and its design. All au- brain donation. Finally, while a morphological screen- thors read, revised, and approved the final manuscript. ing for C9orf72 was performed in all cases, genetic tests for other mutations have not been performed systemat- DATA AVA I L A B I L I T Y STAT E M E N T ically (34). The data that support the findings of this study are avail- able from the corresponding author upon reasonable request. 5 | CONC LUSION S ORC I D Cognitive impairment is a frequent finding in patients Sergi Borrego-­Écija https://orcid. with ALS. Most, but not all cases, present with FTLD org/0000-0003-2557-0010 with or without hippocampal sclerosis as the underly- Martí Paré-­C urell https://orcid. ing pathology, have higher ALS-­Brettschneider stages org/0000-0002-0627-5350 and higher burden of TDP-­43 pathology in the anterior Ellen Gelpi https://orcid.org/0000-0003-2948-4187 cingulate. AD can also influence the development of cognitive symptoms in ALS patients. The presence of a R EF ER ENCE S mutation, but not the APOE genotype, is a strong deter- 1. Goldstein LH, Abrahams S. Changes in cognition and behaviour minant for the presence of cognitive decline. Moreover, in amyotrophic lateral sclerosis: nature of impairment and impli- cations for assessment. Lancet Neurol. 2013;12:368–­80. concomitant neurodegenerative pathologies, particu- 2. Strong MJ, Abrahams S, Goldstein LH, Woolley S, Mclaughlin larly AD, are more frequent in older ALS subjects with P, Snowden J, et al. Amyotrophic lateral sclerosis -­frontotempo- cognitive decline, while AGD may appear earlier than in ral spectrum disorder (ALS-­FTSD): revised diagnostic criteria. non-­ALS patients. Amyotroph Lateral Scler Front Degener. 2017;18:153–­74. These findings contribute to broaden the knowledge 3. Prudlo J, König J, Schuster C, Kasper E, Büttner A, Teipel S, on the overlap between ALS and FTD and emphasize et al. TDP-­ 43 pathology and cognition in ALS. Neurology. 2016;87:1019–­23. the importance of an accurate assessment of non-­motor 4. Saxon JA, Harris JM, Thompson JC, Jones M, Richardson features in ALS patients including clinical, neuropsycho- AMT, Langheinrich T, et al. Semantic dementia, progressive logical, neuroimaging, and biofluid biomarkers through non-­f luent aphasia and their association with amyotrophic lat- an integrative approach. Well-­ characterized patients eral sclerosis. J Neurol Neurosurg Psychiatry. 2017;88:711–­12. will undoubtedly profit from better directed therapeutic 5. Strong MJ, Grace GM, Freedman M, Lomen-­ Hoerth C, Woolley S, Goldstein LH, et al. Consensus criteria for the di- interventions and care. agnosis of frontotemporal cognitive and behavioural syndromes ALS COGNITIVE DETERMINANTS    | 13 of 14 in amyotrophic lateral sclerosis. Amyotroph Lateral Scler. 25. Thal DR, Rüb U, Orantes M, Braak H. Phases of A beta-­ 2009;10:131– ­46. deposition in the human brain and its relevance for the develop- 6. Burrell JR, Halliday GM, Kril JJ, Ittner LM, Götz J, Kiernan ment of AD. Neurology. 2002;58:1791–­800. MC, et al. The frontotemporal dementia-­motor neuron disease 26. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee continuum. Lancet. 2016;388:919–­31. LM, Vogel FS, Hughes JP, van Belle G, Berg L. The Consortium 7. Kovacs GG, Alafuzoff I, Al-­Sarraj S, Arzberger T, Bogdanovic to Establish a Registry for Alzheimer's Disease (CERAD): N, Capellari S, et al. Mixed brain pathologies in dementia: The Part II. Standardization of the neuropathologic assessment of BrainNet Europe consortium experience. Dement Geriatr Cogn Alzheimer's disease. Neurology. 1991;41(4):479. Disord. 2008;26:343–­50. 27. Montine TJ, Phelps CH, Beach TG, Bigio EH, Cairns NJ, 8. Rahimi J, Kovacs GG. Prevalence of mixed pathologies in the Dickson DW, et al. National institute on aging-­ A lzheimer’s aging brain. Alzheimer’s Res Ther. 2014;6:1–­11. association guidelines for the neuropathologic assessment of 9. Robinson JL, Lee EB, Xie SX, Rennert L, Suh E, Bredenberg Alzheimer’s disease: a practical approach. Acta Neuropathol. C, et al. Neurodegenerative disease concomitant proteinopa- 2012;123:1–­11. thies are prevalent, age-­related and APOE4-­associated. Brain. 28. Crary JF, Trojanowski JQ, Schneider JA, Abisambra JF, Abner 2018;141:2181–­193. EL, Alafuzoff I, et al. Primary age-­related tauopathy (PART): 10. Hauser PS, Ryan RO. Impact of apolipoprotein E on Alzheimer’s a common pathology associated with human aging. Acta disease. Curr Alzheimer Res. 2013;10:809–­17. Neuropathol. 2014;128:755– ­66. 11. Yu J, Tan L, Hardy J. Apolipoprotein E in Alzheimer’s disease: 29. Kovacs GG, Ferrer I, Grinberg LT, Alafuzoff I, Attems J, Budka an update. Annu Rev Neurosci. 2014;37:79–­100. H, et al. Aging-­related tau astrogliopathy (ARTAG): harmo- 12. Wennberg AM, Tosakulwong N, Lesnick TG, Murray ME, nized evaluation strategy. Acta Neuropathol. 2016;131:87–­102. Whitwell JL, Liesinger AM, et al. Association of apolipoprotein 30. McKeith IG, Boeve BF, Dickson DW, Halliday G, Taylor JP, E ϵ4 with transactive response DNA-­binding protein 43. JAMA Weintraub D, et al. Diagnosis and management of dementia with Neurol. 2018;75:1347–­54. Lewy bodies. Neurology. 2017;89(1):88–­100. 13. Yang HS, Yu L, White CC, Chibnik LB, Chhatwal JP, Sperling 31. Saito Y, Ruberu NN, Sawabe M, Arai T, Tanaka N, Kakuta Y, RA, et al. Evaluation of TDP-­43 proteinopathy and hippocampal et al. Staging of argyrophilic grains: an age-­associated tauopa- sclerosis in relation to APOE ε4 haplotype status: a community-­ thy. J Neuropathol Exp Neurol. 2004;63:911–­8. based cohort study. Lancet Neurol. 2018;17:773–­81. 32. Nelson PT, Dickson DW, Trojanowski JQ, Jack CR, Boyle PA, 14. Chiò A, Brunetti M, Barberis M, Iazzolino B, Montuschi A, Arfanakis K, et al. Limbic-­predominant age-­related TDP-­43 en- Ilardi A, et al. The role of APOE in the occurrence of frontotem- cephalopathy (LATE): consensus working group report. Brain. poral dementia in amyotrophic lateral sclerosis. JAMA Neurol. 2019;142:1503–­27. 2016;73:425–­30. 33. Thal DR, Del Tredici K, Ludolph AC, Hoozemans JJM, 15. Rubino E, Vacca A, Govone F, De Martino P, Pinessi L, Rainero Rozemuller AJ, Braak H, et al. Stages of granulovacuolar degen- I. Apolipoprotein E polymorphisms in frontotemporal lobar de- eration: their relation to Alzheimer’s disease and chronic stress generation: A meta-­analysis. Alzheimer’s Dement. 2013;9:706–­13. response. Acta Neuropathol. 2011;122:577–­89. 16. Miyagawa T, Brushaber D, Syrjanen J, Kremers W, Fields J, 34. Ramos-­ c ampoy O, Avila-­ polo R, Rojas-­ garc R, Charif S, Forsberg LK, et al. Utility of the global CDR® plus NACC Santiago-­valera V, Hernandez I, et al. Systematic screening of FTLD rating and development of scoring rules: Data from Ubiquitin/p62 aggregates in cerebellar cortex expands the neu- the ARTFL/LEFFTDS Consortium. Alzheimers Dement. ropathological phenotype of the C9orf72 expansion mutation. J 2020;16:106–­17. Neuropathol Exp Neurol. 2018;77:703–­9. 17. Gorno-­Tempini ML, Hillis AE, Weintraub S, Kertesz A, Mendez 35. Renton AE, Majounie E, Waite A, Simón-­Sánchez J, Rollinson S, M, Cappa SF, et al. Classification of primary progressive apha- Gibbs JR, et al. A hexanucleotide repeat expansion in C9ORF72 sia and its variants. Neurology. 2011;76:1006–­14. is the cause of chromosome 9p21-­ l inked ALS-­ FTD. Neuron. 18. Rascovsky K, Hodges JR, Knopman D, Mendez MF, Kramer 2011;72:257–­268. JH, Neuhaus J, et al. Sensitivity of revised diagnostic criteria 36. Dols-­Icardo O, García-­Redondo A, Rojas-­García R, Borrego-­ for the behavioural variant of frontotemporal dementia. Brain. Hernández D, Illán-­Gala I, Muñoz-­Blanco JL, et al. Analysis 2011;134(9):2456–­77. of known amyotrophic lateral sclerosis and frontotemporal 19. Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin dementia genes reveals a substantial genetic burden in patients DJ, Grossman M, et al. Stages of pTDP-­43 pathology in amyo- manifesting both diseases not carrying the C9orf72 expansion trophic lateral sclerosis. Ann Neurol. 2013;74:20–­38. mutation. J Neurol Neurosurg Psychiatry. 2018;89:162–­8. 20. Cairns NJ, Bigio EH, Mackenzie IRA, Schneider JA, Paulo 37. Cortés-­Vicente E, Turon-­Sans J, Gelpi E, Clarimón J, Borrego-­ UDS, Paulo S, et al. Neuropathological diagnostic and nosologic Écija S, Dols-­Icardo O, et al. Distinct clinical features and out- criteria for FTLD : consensus of the Consortium for FLTD. Acta comes in motor neuron disease associated with behavioural Neuropathol. 2010;114:5–­22. variant frontotemporal dementia. Dement Geriatr Cogn Disord. 21. Mackenzie IR, Neumann M. Reappraisal of TDP-­43 pathology 2018;45:220–­31. in FTLD-­U subtypes. Acta Neuropathol. 2017;134:79–­96. 38. Coan G, Mitchell CS. An assessment of possible neuropathol- 22. Rauramaa T, Pikkarainen M, Englund E, Ince PG, Jellinger ogy and clinical relationships in 46 sporadic amyotrophic lateral K, Paetau A, et al. Consensus recommendations on pathologic sclerosis patient autopsies. Neurodegener Dis. 2015;15:301–­12. changes in the hippocampus: a postmortem multicenter inter-­ 39. Borrego-­É cija S, Cortés-­Vicente E, Cervera-­Carles L, Clarimón rater study. J Neuropathol Exp Neurol. 2013;72:452–­61. J, Gámez J, Batlle J, et al. Does ALS-­FUS without FUS mutation 23. Tan RH, Kril JJ, Fatima M, McGeachie A, McCann H, represent ALS-­FET? Report of three cases. Neuropathol Appl Shepherd C, et al. TDP-­43 proteinopathies: Pathological iden- Neurobiol. 2019;45:421– ­6. tification of brain regions differentiating clinical phenotypes. 40. Spencer KR, Foster ZW, Rauf NA, Guilderson L, Collins D, Brain. 2015;138:3110–­22. Averill JG, et al. Neuropathological profile of long-­ duration 24. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici amyotrophic lateral sclerosis in military veterans. Brain Pathol. K. Staging of Alzheimer disease-­associated neurofibrillary pa- 2020. https://doi.org/10.1111/bpa.12876. thology using paraffin sections and immunocytochemistry. Acta 41. Burrell JR, Kiernan MC, Vucic S, Hodges JR. Motor neuron dys- Neuropathol. 2006;112:389– ­404. function in frontotemporal dementia. Brain. 2011;134:2582–­94. 14 of 14 |    BORREGO-­ÉCIJA et al. 42. Arnold SJ, Dugger BN, Beach TG. TDP-­43 deposition in pro- spectively followed, cognitively normal elderly individuals: S U PP ORT I NG I N F OR M AT ION Correlation with argyrophilic grains but not other concomitant Additional Supporting Information may be found online pathologies. Acta Neuropathol. 2013;126:51–­7. in the Supporting Information section. 43. Soma K, Fu Y-­ J, Wakabayashi K, Onodera O, Kakita A, Supplementary Material Takahashi H. Co-­ occurrence of argyrophilic grain disease in sporadic amyotrophic lateral sclerosis. Neuropathol Appl Neurobiol. 2012;38:54–­60. TABLE S1 Antibodies used for immunohistochemistry 44. Riku Y, Duyckaerts C, Boluda S, Plu I, Le Ber I, Millecamps S, and their pretreatments et al. Increased prevalence of granulovacuolar degeneration in TABLE S2 Reduced model of logistic regression: The C9orf72 mutation. Acta Neuropathol. 2019;138:783–­93. dependent (outcome) variable of the model was the pres- 45. Van Schoor E, Koper MJ, Ospitalieri S, Dedeene L, Tomé SO, ence of cognitive impairment (ALSci and ALS-­ FTD Vandenberghe R, et al. Necrosome-­ positive granulovacuolar degeneration is associated with TDP-­ 43 pathological lesions were considered together). The reduced model avoids co- in the hippocampus of ALS/FTLD cases. Neuropathol Appl variates with collinearity problems Neurobiol. 2020. https://pubmed.ncbi.nlm.nih.gov/32949​ 047/. Accessed 9 Dec 2020. 46. Su WH, Shi ZH, Liu SL, Wang XD, Liu S, Ji Y. Updated meta-­ How to cite this article: Borrego-­Écija S, Turon-­Sans analysis of the role of APOE ε2/ε3/ε4 alleles in frontotemporal J, Ximelis T, et al. Cognitive decline in amyotrophic lobar degeneration. Oncotarget. 2017;8:43721–­32. lateral sclerosis: Neuropathological substrate and 47. Takeda T, Seilhean D, Le Ber I, Millecamps S, Sazdovitch V, Kitagawa K, et al. Amygdala TDP-­43 pathology in frontotempo- genetic determinants. Brain Pathology. 2021;31: ral lobar degeneration and motor neuron disease. J Neuropathol e12942. https://doi.org/10.1111/bpa.12942 Exp Neurol. 2017;76:800–­12.