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Clinical and neuropathological features of ALS/FTD with TIA1 mutations Hirsch-Reinshagen, Veronica; Pottier, Cyril; Nicholson, Alexandra M; Baker, Matt; Hsiung, Ging-Yuek R; Krieger, Charles; Sengdy, Pheth; Boylan, Kevin B; Dickson, Dennis W; Mesulam, Marsel; Weintraub, Sandra; Bigio, Eileen; Zinman, Lorne; Keith, Julia; Rogaeva, Ekaterina; Zivkovic, Sasha A; Lacomis, David; Taylor, J. P; Rademakers, Rosa; Mackenzie, Ian R A Dec 7, 2017

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RESEARCH Open AccessClinical and neuropathological featuresof ALS/FTD with TIA1 mutationsVeronica Hirsch-Reinshagen1, Cyril Pottier2, Alexandra M. Nicholson2, Matt Baker2, Ging-Yuek R. Hsiung3,Charles Krieger4, Pheth Sengdy3, Kevin B. Boylan2, Dennis W. Dickson2, Marsel Mesulam5, Sandra Weintraub5,Eileen Bigio6, Lorne Zinman7, Julia Keith8, Ekaterina Rogaeva9, Sasha A. Zivkovic10, David Lacomis10,11,J. Paul Taylor12,13, Rosa Rademakers2 and Ian R. A. Mackenzie1,14*AbstractMutations in the stress granule protein T-cell restricted intracellular antigen 1 (TIA1) were recently shown to causeamyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD). Here, we provide detailedclinical and neuropathological descriptions of nine cases with TIA1 mutations, together with comparisons tosporadic ALS (sALS) and ALS due to repeat expansions in C9orf72 (C9orf72+). All nine patients with confirmedmutations in TIA1 were female. The clinical phenotype was heterogeneous with a range in the age at onset fromlate twenties to the eighth decade (mean = 60 years) and disease duration from one to 6 years (mean = 3 years).Initial presentation was either focal weakness or language impairment. All affected individuals received a finaldiagnosis of ALS with or without FTD. No psychosis or parkinsonism was described. Neuropathological examinationon five patients found typical features of ALS and frontotemporal lobar degeneration (FTLD-TDP, type B) withanatomically widespread TDP-43 proteinopathy. In contrast to C9orf72+ cases, caudate atrophy and hippocampalsclerosis were not prominent. Detailed evaluation of the pyramidal motor system found a similar degree ofneurodegeneration and TDP-43 pathology as in sALS and C9orf72+ cases; however, cases with TIA1 mutations hadincreased numbers of lower motor neurons containing round eosinophilic and Lewy body-like inclusions on HEstain and round compact cytoplasmic inclusions with TDP-43 immunohistochemistry. Immunohistochemistry andimmunofluorescence failed to demonstrate any labeling of inclusions with antibodies against TIA1. In summary, ourTIA1 mutation carriers developed ALS with or without FTD, with a wide range in age at onset, but without otherneurological or psychiatric features. The neuropathology was characterized by widespread TDP-43 pathology, but amore restricted pattern of neurodegeneration than C9orf72+ cases. Increased numbers of round eosinophilic andLewy-body like inclusions in lower motor neurons may be a distinctive feature of ALS caused by TIA1 mutations.Keywords: Amyotrophic lateral sclerosis, Frontotemporal dementia, Frontotemporal lobar degeneration,T-cell restricted intracellular antigen-1, TDP-43IntroductionWe recently reported the identification of mutations inthe T-cell restricted intracellular antigen-1 gene (TIA1)as a cause of amyotrophic lateral sclerosis (ALS) andfrontotemporal dementia (FTD) [19]. Similar to severalother ALS/FTD related proteins (e.g. transactiveresponse DNA-binding protein 43 (TDP-43), fused insarcoma (FUS) and heterogeneous nuclear ribonucleo-protein A1 (hnRNPA1)), TIA1 is an RNA binding pro-tein that contains a C-terminal, prion-like, lowcomplexity domain (LCD) which promotes its self-assembly and the formation of membrane-less organellesthrough the process of liquid-liquid phase separation(LLPS) [16, 22, 31]. Specifically, TIA1 plays a central rolein the formation of stress granules (SG) that form in re-sponse to environmental stress to temporarily store andprotect mRNA [1, 9, 14, 25]. SG dysfunction has beenimplicated in the pathogenesis of a number of* Correspondence: ian.mackenzie@vch.ca1Department of Pathology and Laboratory Medicine, University of BritishColombia, British Columbia, Canada14Department of Pathology, Vancouver General Hospital and VancouverCoastal Health, 855 West 12th Avenue, Vancouver, BC V5Z 1M9, CanadaFull list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 DOI 10.1186/s40478-017-0493-xneurodegenerative conditions including ALS [1, 30] andTIA1 was previously identified as a candidate ALS gene ina yeast functional screen [5]. Moreover, a founder muta-tion affecting the TIA1 LCD (E384K) has been reported inSwedish/Finnish patients to cause Welander distal myop-athy (WDM) [10, 15], a type of vacuolar myopathy withclinical and histopathological similarity to the myopathiescaused by mutations a number of other genes that canalso cause ALS/FTD (e.g. valosin containing protein andsequestosome-1) [8, 12].In the previous study, we identified a different hetero-zygous missense TIA1 mutation (P362L) in affectedmembers of a family with autosomal dominant ALS andFTD [19]. This variant affects a highly conserved residuein the LCD and is predicted to be deleterious. Subse-quent analysis of a large cohort of patients with ALS,with and without FTD, identified TIA1 mutations in ap-proximately 2% of familial ALS (fALS), and 0.4% of spor-adic ALS (sALS), but not in neurologically normalcontrols [19]. Autopsy material from five TIA1 mutationcarriers showed widespread TDP-43 immunoreactive(TDP-ir) pathology as a consistent feature. Biophysicaland cell culture studies demonstrated that the diseaseassociated mutations altered phase transition of TIA1and resulted in SG that failed to normally disassemblefollowing the removal of stress. It is known that TDP-43is recruited into SG under a variety of stress conditions[1] and we showed that prolonged localization of TDP-43 within persistent SG promotes TDP-43 aggregationand reduces its solubility. Based on these findings, weproposed that TIA1 mutations are a cause of ALS andFTD; thus, reinforcing the central role of RNA metabol-ism and SG dynamics in the pathogenesis of thisspectrum of disease [19].Whereas the original study focused on the geneticanalysis and functional effects of TIA1 mutations, in thisreport we provide a more detailed description of theassociated clinical features and neuropathology. In par-ticular, we highlight phenotypic and pathological charac-teristics that distinguish cases with TIA1 mutation fromother types of familial and sporadic ALS and FTD.Materials and methodsCase identificationDetails of the genetic analysis are provided in the originalreport [19]. Briefly, whole exome sequencing was per-formed on two affected second-degree relatives who weremembers of a family with autosomal dominant ALS andFTD, negative for mutations in known ALS- and FTD-causing genes (UBCU2, Fig. 1). Variants that were presentin a heterozygous state in both patients were filtered basedon standard criteria of frequency, brain expression and pre-dicted functional effect. The P362L missense variant inTIA1 was determined to be the most likely candidatecausal mutation, based on the protein’s normal functionand structure and its association with another neurologicaldisorder (WDM) (see above). Sanger sequencing confirmedthe P362L mutation in the two affected family membersand in a clinically asymptomatic family member who wasan obligate carrier (UBCU2-2) (Fig. 1, Table 1). We thenanalyzed the TIA1 LCD (encoded by exons 11-13) in a co-hort of 1039 ALS (± FTD) patients and identified five add-itional TIA1 mutations in six unrelated patients; whereas,none was identified in 3036 neurologically normal controls(p = 8.7 × 10−6). In total, nine TIA1 mutation carriers wereidentified (three members of UBCU2 and six unrelated pa-tients), representing 2.2% of fALS and 0.4% of sALS casesin our study population.Clinical evaluationClinical information was obtained through retrospectivereview of the patients’ clinical records. All subjects hadbeen evaluated by neurologists with expertise inneuromuscular disorders, behavioral neurology and/orlanguage disorders. Clinical diagnosis of ALS was basedon El Escorial criteria [3]. All patients signed informedconsent and this study was approved by the ethicscommittee of all respective institutions.Fig. 1 Pedigree of family UBCU2. Family of European ancestry showingan autosomal dominant pattern of inheritance of ALS ± dementia.Black symbols represent clinically affected individuals and diagonal linesindicate those who are deceased. Genetic analysis was performed onthe proband (1), her affected niece (14) and her early affected sister(2); all of whom carried the P362L mutation in TIA1Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 2 of 13NeuropathologyPost mortem examination restricted to the central nervoussystem had been performed on five of the affected TIA1mutation carriers (Tables 1 and 2). In four of the fivecases, the entire spinal cord was available for examination;whereas, in case NWU-1, only the upper segments of cer-vical spinal cord were available. Microscopic evaluationwas performed on 5 μm-thick sections of formalin fixed,paraffin-embedded material representing a wide range ofanatomical regions (Table 2). Histochemical stains in-cluded hematoxylin and eosin (HE), HE combined withLuxol fast blue (HE/LFB), modified Bielschowsky silverstain, Gallyas silver stain, Masson trichrome, Periodic AcidSchiff with and without diastase, Alcian Blue (pH 2.5) andCongo red. Standard immunohistochemistry (IHC) wasperformed using the Ventana BenchMark XT automatedstaining system with primary antibodies against alpha-synuclein (Thermo Scientific; 1:10,000 following micro-wave antigen retrieval), beta amyloid (DAKO; 1:100 withinitial incubation for 3 h at room temperature), hyperpho-sphorylated tau (clone AT-8; Innogenetics, Ghent,Belgium; 1:2000 following microwave antigen retrieval),phosphorylation-independent TDP-43 (ProteinTech;1:1000 following microwave antigen retrieval), ubiquitin(DAKO; 1:500 following microwave antigen retrieval),FUS (Sigma-Aldrich; 1:1000 following microwave antigenretrieval), p62 (BD Biosciences; 1:500 following microwaveantigen retrieval), poly-(A) binding protein (PABP; SantaCruz; 1:200 following microwave antigen retrieval),hnRNP A1 (Santa Cruz; 1:100 following microwave anti-gen retrieval), hnRNP A3 (Sigma-Aldrich; 1:100 followingmicrowave antigen retrieval) and hnRNP A2/B1 (SantaCruz; 1:500 following microwave antigen retrieval). Inaddition, we tested a number of commercial monoclonaland polyclonal antibodies against different epitopes ofhuman TIA1, raised in different species (Table 3).Double-labelling immunofluorescence (IF) experi-ments were performed on paraffin-embedded tissuesections that were heated to 60°C for 20 min, thenimmediately deparaffinized and rehydrated. Antigen re-trieval was performed in citrate buffer (10 mM, pH 6.0,10 min at 95°C in a water bath). The sections wereblocked for 1 h with 5% donkey serum in 0.1% triton X-100 in TBS. Incubation with various combinations ofprimary antibodies was performed in the same blockingsolution overnight at 4C. The combinations included arat anti-phosphorylated TDP-43 (from M. Neumann,1:1000) [24] with one of three anti-TIA1 antibodies:Santa Cruz goat anti-TIA1 (1:300), Santa Cruz rabbitanti-TIA1 (1:300) or Proteintech rabbit anti-TIA1(1:100) (Table 3).The sections were then washed, and in-cubated with appropriate Alexa Fluor- or biotin-conjugated secondary antibodies at 1:1000 dilution for1 h at room temperature. When needed, a third stepwith Alexa Fluor-conjugated streptavidin (1:1000) wasadded for 40 min. Background fluorescence was thenquenched by staining with 0.1% Sudan Black in 70%ethanol for 15 min. Slides were mounted after a 15-minincubation in DAPI with Prolong-Gold anti-fade reagent(Invitrogen). Microscopy was performed using a NikonEclipse i-80 epifluorescent microscope and NIS-Elements software. Images were further processed andmerged using Image J.The severity of chronic degenerative changes and theburden of TDP-ir pathology in different brain regionswas evaluated using a semi-quantitative scoring system,Table 1 Clinical and demographic characteristics of TIA1 mutation carriersCase Sex Onset (yrs) Death or[Current Age] (yrs)PresentingSymptomPark Psych Final/CurrentClinical DiagnosesFamily History TIA1 Mutation[19]Post MortemExamaUBCU2-1 F 51 55 limb weakness no no ALS, early bvFTD ALS, FTD,dementiap.P362L yesaUBCU2-2 F 55 [55] abnormalmemory testsno no CSND ALS, FTD,dementiap.P362L n/aaUBCU2-14 F 28 30 aphasia,behavioralchangesno no FTD (PPA), ALS ALS, FTD,dementia,dyslexiap.P362L yesNWU-1 F 65 68 aphasia no no FTD (PPA), ALS dementia p.M334I yesTOR-1 F 73 79 aphasia no no FTD (PNFA), ALS cardiovasculardiseasep.A381T yesALS701-1 F 74 76 bulbar weakness no no ALS, aphasia none p.G355R noALS458-1 F 63 [64] bulbar weakness no no ALS ALS, Parkinson’s p.V294 M n/aALS752-1 F 58 59 bulbar weakness no no ALS none p.V360 M yesPITT-87 F 64 66 limb weakness no no ALS none p.A381T noALS amyotrophic lateral sclerosis, bvFTD behavioral variant FTD, CSND clinically symptomatic not demented, F female, FTD frontotemporal dementia,n/a not applicable, Park parkinsonism, PNFA progressive non-fluent aphasia, PPA primary progressive aphasia, not otherwise specified, Psych psychosis, yrs. yearsadenotes members of the same familyHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 3 of 13as follows: −, absent; +, mild/few (easy to find but notpresent in every medium power field); ++ moderate (atleast a few in most fields); +++, severe/numerous (manyin virtually every field). In addition, the number of lowermotor neurons (LMN) (defined as medium to large cellswith prominent peripheral Nissl substance) and the num-ber of LMN containing various types of neuronalcytoplasmic inclusions (NCI), including Bunina bodies,round inclusions and cored Lewy body-like inclusions(LBLI) seen with HE stain, as well as TDP-ir NCI withgranular, filamentous or compact morphology werecounted in sections of cervical and lumbosacral spinal cordin the cases with TIA1 mutations (N = 5) and in sections ofsALS and C9orf72+ ALS (N = 10 each). In each case, oneslide from each of the available tissue blocks representingdifferent levels of cervical or lumbar spinal cord enlarge-ment was evaluated and the counts averaged (mean num-ber per tissue section). Similar evaluation was performedon sections of medulla; however, this data was not in-cluded in the statistical analysis because the variation inthe anatomical level resulted in significant differences inthe representation of the hypoglossal nucleus among cases.Statistical analysisStatistical analysis and data graphing were performedusing GraphPad Prism 6.0 software. Non-parametricKruskal-Wallis test with Dunn’s multiple comparisontest were used to analyze the differences among thegroups in round eosinophilic and cored LBLI; whereas,ANOVA with multiple comparisons was used to analyzethe differences in TDP-ir NCI in LMN.Table 2 Semiquantitative analysis of neurodegeneration and TDP-ir pathology in TIA1 mutation carriersNeurodegeneration TDP-43 ImmunohistochemistryNWU-1 TOR-1 UBCU2-14 UBCU2-1 ALS752-1 NWU-1 TOR-1 UBCU2-14 UBCU2-1 ALS752-1FTD,ALSFTD, prob.ALSFTD, prob.ALSALS, earlyFTDALS FTD,ALSFTD, prob.ALSFTD, prob.ALSALS, earlyFTDALSpyramidal motor system motor cortex + + + ++ + + ++ + + +++CN XII +++ ++ +++ ++ +++ ++ +++ ++ ++ ++CST ++ + +++ ++ ++ n/a n/a n/a n/a n/aant. horn ++ ++ +++ +++ +++ ++ ++ +++ +++ +++neocortex prefrontal ++ + +++ ++ – +++ +++ +++ +++ ++temporal + + ++ + – ++ ++ ++ ++ ++parietal – – ++ + – +++ ++ ++ ++ +striatonigralsystemcaudate + – ++ + – + ++ +++ +++ +++putamen – – – – – + + ++ +++ +GP – – – – – + + + + +SN +++ + +++ ++ ++ ++ ++ +++ +++ ++limbicsystemhip. CA – – – – – + + + + +hip. dentate ++ + – – – ++ +++ ++ ++ ++thalamus + – – – – + – + – +midbrain PAG + – ++ + – + + ++ + +cerebello-pontinesystemcb cortex – – – – – – – – – –cb dentate + – – – – – – – – –basis pontis – – – – – + – + – +The patients are ordered according to their earliest and/or predominant clinical features from predominant FTD (left) to pure ALS (right). ALS amyotrophic lateralsclerosis, ant. anterior, cb cerebellar, CA cornu ammonis, CN XII twelfth cranial nerve (hypoglossal) nucleus, CST corticospinal tract, deg. non-specific changes ofchronic degeneration, FTD frontotemporal dementia, GP globus pallidus, hip. hippocampal, n/a not applicable, PAG periaqueductal grey matter, prob. probable,SN substantia nigra. Semiquantitative grading of pathology; −, none; +, mild; ++, moderate; +++, severeTable 3 TIA1 antibodies used for immunohistochemistry anddouble label immunofluorescenceAntibody Species Epitope DilutionSanta Cruz#sc-1751Goat polyclonal(clone C-20)near C-terminus 1:300Santa Cruz#sc-48371Mouse monoclonal(clone D-9)aa 21-140(near N-terminus)1:200Santa Cruz#sc-28237Rabbit polyclonal(clone H-120)aa 21-140(near N-terminus)1:300Abcam#ab140595Rabbit monoclonal aa 350 to theC-terminus1:100Abcam#ab40693Rabbit polyclonal aa 350 to theC-terminus1:500ProteinTech#12133-2-APRabbit polyclonal human TIA1-GSTfusion protein1:100#, catalogue number; GST glutathione S-transferaseHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 4 of 13ResultsClinical featuresBrief summaries of the demographic and clinical infor-mation are provided in Table 1. Below are detaileddescriptions of each case.UBCU2-1This previously healthy woman came from a family ofEuropean ancestry (Fig. 1) [19]. Her mother had ALSonset at age 51 and died at age 56 and her maternaluncle died in his 60s of ALS with dementia. At age 51,UBCU2-1 developed progressive, asymmetric upper limbweakness and occasional tripping. Initial examinationfound mild-to-moderate atrophy and weakness of upperand lower limb muscles, without fasciculations. Deeptendon reflexes were symmetrically brisk. Bulbar muscu-lature was intact. Nerve conduction studies of medianand ulnar nerves showed low amplitude but normal con-duction velocities; whereas, sensory studies were normal.EMG showed denervation of several muscles in all fourextremities and of paraspinal muscles. She was felt to becognitively normal at that time but was not formallytested. A clinical diagnosis of definite ALS was made.Over the subsequent 4 years, her extremity weaknessprogressed to the point of requiring full assistance fordaily activities. She became increasingly dysarthric andhad difficulty breathing, but was able to swallow. Achange in personality was first noted 6 months beforeher death when she became disinhibited, mildly disor-iented, inappropriately emotional and repetitive. Shedied at age 55 with a clinical diagnosis of ALS and earlybehavioral variant FTD (bvFTD). An autopsy limited tobrain and spinal cord was performed. Neuropathologicalexamination showed ALS-TDP and FTLD-TDP (subtypeB) and tau-ir neurofibrillary tangles restricted to theentorhinal and transentorhinal cortex (Braak stage I).UBCU2-2The sister of the proband was assessed at age 55. Neithershe nor her family reported any motor or cognitivesymptoms. Clinical neurological assessment was normal;however, detailed neuropsychological assessment founddeficits on measures of verbal and non-verbal memory.She was considered symptomatic (early memory abnor-malities), but not demented.UBCU2-14The niece of the proband developed difficulty withword-finding at age 28. Around the same time, herfamily noted that she was becoming emotionally flat,withdrawn, apathetic and displaying little empathy. Afew months later she developed dysarthria, difficultychewing and swallowing and she became clumsy andprone to minor injury. Language comprehension beganto decline, she was increasingly forgetful and had diffi-culty planning and organizing simple household tasks.She had suffered from dyslexia since childhood, as hadseveral of her paternal relatives. When initially evalu-ated, 6–9 months following her disease onset, her Mini-Mental Status Examination (MMSE) score was 21/30,the Montreal Cognitive Assessment test (MoCA)showed prominent visuospatial dysfunction and theFrontal Assessment Battery (FAB) was 3/15. There wasmild emotional incontinence. She had a slight spasticdysphonia and mild Gegenhalten in the right arm andleg. MRI demonstrated moderate symmetric frontotem-poral atrophy.When next evaluated, at age 29, she was found to havemore severe cognitive impairment (MMSE score of 11/30and MoCA score of 8/30). She was friendly but apatheticand emotionally blunt. Her speech was anomic, dysarthricand perseverative, but grammatically intact. She could fol-low simple commands and repeat sentences. Althoughher aphasia was difficult to classify, it was felt to have fea-tures of apraxia of speech and progressive non-fluentaphasia (PNFA). Physical examination was limited by herinability to fully cooperate. Strength of her facial muscleswas normal, but tongue movement was impaired. Jaw jerkwas brisk and she was hyperreflexic and mildly spastic inall extremities. Muscle bulk and strength were normal inall extremities and no fasciculations were noted. EMGshowed active denervation, fibrillation potentials, irritabil-ity and some positive sharp waves in several muscles ofboth legs; however, due to patient noncompliance, theEMG study was discontinued before the upper extremitiescould be tested. The clinical diagnosis was that of primaryprogressive aphasia (PPA, difficult to classify) and“probable” ALS (due to the incomplete EMG study).She progressed rapidly and became almost mute witha very limited degree of language comprehension withinthe next 6 months. Her bulbar dysfunction worsenedand the right lower extremity became weak. She died atage 30 with a clinical diagnosis of FTD with both behav-ioral symptoms and progressive aphasia and probableALS. Autopsy limited to brain and spinal cord demon-strated ALS-TDP and FTLD-TDP (subtype B).NWU-1This woman presented at age 65 with intermittent confu-sion and aphasia characterized by laconic speech, wordfinding difficulties and paraphasic errors in writing, butwith intact language comprehension. No motor featureswere identified at that time and she was given a prelimin-ary clinical diagnosis of primary progressive aphasia (PPA).More detailed evaluation at age 67 found apraxia ofspeech, dysarthria, telegraphic phrases, anomia, problemswith sentence comprehension and agrammatic writing.There were also impairments in executive function,Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 5 of 13motivation and insight. Motor examination demonstratedbulbar weakness, but normal limb strength and reflexeswithout fasciculations. MRI showed extensive cerebralwhite matter hyperintensities, attributed to chronic ische-mia, and SPECT scan showed mild hypoperfusion of theleft anterior temporal lobe.Her disease progressed rapidly and by age 68 she hadglobal aphasia, swallowing difficulties and fasciculationsin the tongue and all limbs. EMG revealed findings ofmotor neuropathy and spontaneous motor activity, andswallowing studies were abnormal. She died later thatyear. Her family history was positive for late onset de-mentia, but not for ALS.An autopsy was performed but was limited to thebrain. Neuropathological examination showed FTLD-TDP (type B) and ALS-TDP pathology in the brainstemand high cervical spinal cord. There was very mildAlzheimer-type pathology with rare neuritic senile pla-ques and neurofibrillary tangles (Braak stage II).TOR-1This woman presented at age 73 with speech abnormal-ities characterized by frequent errors in grammar andsyntax. Her speech progressively deteriorated and shealso developed swallowing difficulties with frequentchoking. Neuroimaging studies were unremarkable andshe was diagnosed with PNFA and probable ALS. Shedeveloped depressive symptoms, but no other behavioralabnormalities. Her family history was negative for neuro-logical disorders. Neuropathological examination showedALS-TDP and FTLD-TDP (type B).ALS701-01This 75-year old woman presented with approxi-mately 7 months of bulbar weakness and pseudobul-bar affect and was diagnosed with clinically definiteALS. Her ALS Functional Rating Scale (ALSFRS)score was 25/48 and the ALS Cognitive BehavioralScreen (ALS-CBS) was compatible with probablefrontotemporal cognitive impairment with expressiveaphasia. She died at age 76. There was no family his-tory of ALS, dementia or Parkinson disease. Post-mortemexamination was not performed.ALS458-01This woman presented at age 63 with progressive bulbarweakness. By age 64 she met clinical criteria for definiteALS and required ventilatory support. Her ALSFRSscore was 29/48. ALS-CBS was normal and she did nothave any cognitive or behavioral symptoms. She wassubsequently lost to follow up. Family history waspositive for ALS and Parkinson disease.ALS752-01This woman presented at age 58 with 4 months of bul-bar symptoms and was diagnosed with clinically definiteALS. Her ALSFRS score was 43/48 and ALS-CBS scoreswere normal. She died 21 months after disease onset.There was no family history of ALS, dementia or Parkin-son disease. Autopsy limited to brain and spinal cordshowed ALS-TDP and mild TDP-43 pathology in theextramotor cerebral cortex.PITT-87This woman presented at age 64 with sudden onset ofbilateral leg weakness and back pain. EMG performed 4months later showed widespread denervation in the legsand thoracic paraspinal muscles; however, weakness wasonly demonstrated in the distal legs. Over the followingyear, her weakness became more severe and spread toinvolve proximal legs, arms and face with a hyperactivejaw jerk and increased tone in the legs. Her respiratoryfunction declined to a forced vital capacity of 33%. Cog-nitive testing performed at age 65 was normal withMMSE 30/30 and ALSCBS 19/20. She died at age 66.There was no history of neurological disorders in thefamily. Post-mortem examination was not performed.NeuropathologyGross pathologyThe fresh brain weight ranged from 1132 to 1450 g(mean 1300 g) with two cases showing bifrontal lobar at-rophy (UBCU2-1 and UBCU2-14) and one with left sidepredominant frontotemporal atrophy (TOR-1). Thehippocampi were normal in size and only one caseshowed mild atrophy of the head of the caudate nucleus.Mild or moderate reduction in the pigmentation of thesubstantia nigra was noted in four cases.General histologyThe pyramidal motor system showed chronic degenera-tive changes in all cases (Table 2). The primary motorcortex tended to show mild neuronal loss and reactivechanges, there was variable axonal and myelin loss inthe corticospinal tracts (Fig. 2a) and moderate or severeloss of LMN in the brainstem and spinal cord. In allcases, small, brightly eosinophilic Bunina bodies werepresent in some of the remaining LMN (Fig. 2b). Inaddition, all cases were found to have some LMN con-taining sharply demarcated, round cytoplasmic inclu-sions that were often larger than the cell nucleus(Fig. 2c-e). These were pale pink or amphophilic withHE stain and approximately half had a compact centralcore, surrounded by a paler halo, similar in appearanceto a Lewy body (Lewy body-like inclusion, LBLI). Theseround inclusions were distinct from the more irregularlyshaped and more brightly eosinophilic hyaline inclusionsHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 6 of 13that are frequently found in LMN in both ALS and nor-mal aging (not shown). They were also easily identifiedon HE/LFB stained sections but did not stain with otherhistochemical stains such as Masson trichrome, PeriodicAcid Schiff, Congo red or silver stains. Round inclusionswere present in LMN of both the spinal cord ventralgrey matter and the hypoglossal nucleus but were notseen in other neuronal populations. They averagedapproximately two per tissue section, with a maximumfrequency of 5 in one section (see quantitation below).The four cases with clinical features of FTD alsoshowed degeneration of the extramotor neocortex, withthe prefrontal regions being most consistently and se-verely affected (Table 2, Fig. 2f ). Apart from the pyram-idal system motor nuclei, the only other subcorticalregions that commonly showed degeneration were thecaudate nucleus, periaqueductal grey matter and sub-stantia nigra. The hippocampus was usually spared andno case showed selective loss of CA1 pyramidal neurons(hippocampal sclerosis).TDP-43 immunoreactive pathologyTDP-ir pathology tended to be more severe and anatomic-ally widespread than the degenerative changes (Table 2).There was moderate to severe involvement of the extra-motor cerebral neocortex with the prefrontal cortex beingmost severely affected. In all cases, the pattern of path-ology was most consistent with FTLD-TDP subtype B [18]with NCI in all cortical layers that were more often granu-lar than compact (Fig. 3a). There were relatively few shortthick dystrophic neurites (DN), but wispy threads and dotlike structures were often concentrated in layer II. Therewere no neuronal intranuclear inclusions. Similar TDP-irpathology was present in the primary motor cortex(Fig. 3b), but was milder in all cases with the exception ofthe patient with clinically pure ALS (ALS752-1) (Table 2).The hippocampus showed moderate numbers of TDP-irNCI in the dentate granule cells (Fig. 3c) but few in thepyramidal layer and no TDP-ir wispy threads in the CA1/subiculum, characteristic of hippocampal sclerosis. Vary-ing degrees of TDP-ir granular NCI and mild DN path-ology was a consistent finding throughout the basalganglia and substantia nigra and periaqueductal grey mat-ter (Table 3, Fig. 3d); whereas, the thalamus and ponswere only mildly and inconsistently involved and the cere-bellum was spared.Many of the remaining LMN in the spinal cord andhypoglossal nucleus contained TDP-ir NCI of varyingmorphology (Fig. 3e-i). Most common were small gran-ules, diffusely distributed throughout the perikaryal cyto-plasm (Figs. 3e, and 4). Filamentous NCI were alsopresent (Fig. 3f ), but were less common than large com-pact NCI that were often round and of similar size tothe round/LBL inclusions seen on HE (Figs. 3g-i and 4).It was not uncommon to find a LMN that containedmore than one inclusion type (Fig. 3i).Fig. 2 Histological changes in TIA1 mutation carriers. Cross section of spinal cord showing severe loss of myelin stain in the corticospinaltracts (CST) (a). Lower motor neurons containing Bunina bodies (arrow) (b) and large, round cored Lewy body-like inclusions (LBLI) (c, d) orround eosinophilic inclusions without distinct cores (e) were present in the medulla and spinal cord. Extra-motor pathology included chronicdegeneration with superficial, laminar microvacuolation of the prefrontal cortex (f). a and e, HE/LFB stain; b - d and f, HE stain. Scale bar:a, 1200 μm; b, 7 μm, c, 23 μm; d and e, 15 μm; f, 205 μmHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 7 of 13TDP-ir glial inclusions were not quantified separatelybut were a common feature in affected grey matter re-gions as well as the subcortical white matter. They wererelatively uncommon in the corticospinal tracts and rarein other spinal cord funiculi.Other pathological findingsMost cases did not show any other significant neuro-degenerative pathology. Mild Alzheimer-type path-ology was present in two cases with tau-irneurofibrillary tangles limited to the entorhinal andtransentorhinal cortex (Braak stage I or II, UBCU2-1and NWU-1) and infrequent neuritic senile plaques(CERAD rare, NWU-1). No alpha-synuclein-ir Lewybodies or neurites were present.Comparison of pyramidal motor system pathology in ALSpatients with and without TIA1 mutationsNo differences were seen in the degree of chronic neuro-degeneration in the primary motor cortex, corticospinaltracts, hypoglossal nucleus or ventral grey matter of thecervical or lumbar spinal cord when comparing ALS pa-tients with TIA1 mutations, C9orf72 mutations and sALSwithout either mutation (LMN counts shown in Fig. 4a).There were also no differences in the burden of TDP-irpathology in the primary motor cortex or the numbersof LMN containing Bunina bodies (data not shown).However, cases with TIA1 mutations had significantlymore round inclusions and LBLI seen on HE stainedsections of spinal cord than either the sALS or C9orf72+group (Fig. 4b and c, p < 0.005 and p < 0.05, respectively).This difference was all the more striking given that theFig. 3 TDP-43 immunoreactive pathology in TIA1 mutation carriers. Numerous predominantly granular TDP-43 immunoreactive (TDP-ir) neuronalcytoplasmic inclusions (NCI, arrows) were present in the prefrontal cortex (a) and primary motor cortex (b). Hippocampal dentate granule cells (c)and dopaminergic neurons in the substantia nigra (d) were consistently affected in all cases. Lower motor neurons (LMN) of the medulla andspinal cord (e – i) contained NCI that were granular (e), filamentous (f) or round and compact (g and h). Single LMN containing combinations ofNCI types were not uncommon (i, arrow points to filamentous inclusions in close proximity to a compact, round NCI). Phosphorylation-independent TDP-43 immunohistochemistry. Scale bar: a and i, 25 μm; b, c and e, 18 μm; d and g, 36 μm; f and h, 9 μmHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 8 of 13analysis included the TIA1 mutation case with only asingle section of high cervical spinal cord available forexamination (NWU-1). Although that case had only oneround inclusion and no LBLI in the single cervical sec-tion (Fig. 4c, red circle), multiple round and LBLI werepresent in the hypoglossal nucleus. On average, one totwo round inclusions were present in each section ofspinal cord and medulla from the TIA1 mutation car-riers; whereas, in the non-TIA1 cases, most sections didnot have any round inclusions and cored LBLI wereexceptionally rare.With TDP-43 IHC, granular LMN NCI were the mostfrequent type in all three patient groups, representing60-75% of the total. No differences were found amongthe groups in the frequency of granular or filamentousNCI (Fig. 4d and e); however, the TIA1 mutation caseshad significantly more compact NCI (p < 0.05) thanC9orf72+ or sALS cases (Fig. 4f ).Immunostaining for TIA1, other SG components and otherRNA-binding proteins (RBP)IHC using a number of anti-TIA1 primary antibodiesthat recognize different TIA1 epitopes (Table 3) failed todemonstrate any abnormality. Most of the antibodiesshowed moderately intense diffuse staining of neuronalcytoplasm and some also stained the nucleus, althoughnone showed preferential nuclear staining (Fig. 5a). Thestaining patterns were similar in ALS cases with andwithout TIA1 mutations and in sections from normalcontrols. Specifically, no TIA1-ir pathological inclusionswere demonstrated in cases with TIA1 mutations. Fordouble label IF an antibody that recognizes phosphory-lated pathological TDP-43 (pTDP-43) was used (to avoidthe normal nuclear positivity) in combination with eachof the three TIA1 antibodies that gave the best resultswith IHC. The same types of NCI were labelled forpTDP-43 as had been seen on light microscopy withTDP-43 IHC (Fig. 5b-d). The TIA1 antibodies againshowed diffuse cytoplasmic +/− nuclear reactivity; how-ever, there was no specific co-localization of TIA1 withcompact, filamentous or granular pTDP-43-ir NCI(Fig. 5b-d). IHC using antibodies against another SGmarker (PABP) and against a number of other RBP(FUS, hnRNPA1, hnRNPA3 and hnRNPA2B1) also failedto show any distinctive staining pattern in the TIA1mutation cases (data not shown).DiscussionThe purpose of this report is to provide a detailed de-scription of the clinical and neuropathological featuresFig. 4 Quantitative comparison of spinal cord lower motor neurons (LMN) and different types of neuronal cytoplasmic inclusions (NCI) in LMN inALS cases with TIA1 mutations, the C9orf72 mutation (C9) and sporadic ALS (sALS). Quantitation in a-c was performed on HE stained sections;whereas d–f was based on TDP-43 immunohistochemistry. There was no difference in the number of spinal cord LMN among groups (a). RoundNCI (b) and Lewy body like inclusions (c) were significantly more frequent in TIA1 mutation carriers than in either C9 or sALS cases. In c, the redcircle denotes the data point corresponding to the case with only a single section of upper cervical cord available for evaluation. No differenceswere seen among groups in the number of granular (d) or filamentous (e) TDP-43 NCI; however, round compact NCI were more frequent in TIA1mutation carriers (f). (** = p < 0.005, * = p < 0.05 compared to C9 and sALS groups, respectively)Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 9 of 13associated with the recently identified TIA1 mutationsthat cause ALS ± FTD. Although the number of casescurrently available is not extensive, it is sufficient toidentify some features that may be helpful in distinguish-ing these cases from other ALS subtypes.In the initial discovery series, TIA1 mutations wereidentified in 2.2% of familial ALS and 0.4% of sporadicALS cases [19]; far less frequent than the most commongenetic cause of ALS, which is the C9orf72 repeat expan-sion (responsible for 37% of familial and 6% sporadicALS [26]), but similar to the frequencies reported forVCP and SQSTM1 mutations and more common thanmany other genetic variants that have been associatedwith ALS [2]. The mean age of disease onset in our caseswas 60 years, with an average disease duration of 3 years;similar to what has been reported for ALS caused by theC9orf72 mutation and for cases of ALS with no identi-fied mutation [28]. Only three of our seven probands(43%) had a family history of neurological disease, whichis significantly less than for C9orf72 and SOD1 muta-tions [28] and suggests that TIA1 mutations may bevariably penetrant. Interestingly, all of our TIA1 muta-tions carriers were female, despite there being no sexbias in the case series in which they were identified. Thisfinding is all the more striking, given that ALS, in gen-eral, is reported to be more common in men with a ratioof males to females of 1.7:1 [21] and could indicate thatsome factor associated with biological sex affects thepenetrance of TIA1 mutations, similar to what has beenreported for some other genetic subtypes of ALS/FTD[6]. This association will need to be investigated furtherin additional case series and we note that one affectedmember of UBCU2 was male, although no source ofDNA was available to confirm his genetic status.The initial clinical features of our TIA1 mutation car-riers varied, with five presenting with symptoms of ALS,three with early changes of FTD and one who wasasymptomatic but found to have abnormal memoryfunction of neuropsychological testing (Table 1). Of theeight cases that developed ALS symptoms, bulbar weak-ness was a prominent feature in six (75%), which is morecommon than in cases of sALS (26%) or those with theC9orf72 mutation (44%) [28]. Of the five patients whopresented with, or later developed FTD, the initial andmost prominent problem was expressive aphasia in four,whereas only one patient showed mainly behavioral ab-normalities. The high frequency of primary progressiveaphasia (PPA) in our TIA1 mutation carriers is differentfrom those with the C9orf72 mutation whose FTDphenotype is more often bvFTD [11]. It is important tonote that the case series used to identify mutation car-riers had a primary ALS diagnosis with only a smallFig. 5 TIA1 immunohistochemistry (IHC) and double-label immunofluorescence (IF) of lower motor neurons from cases with TIA1 mutations.Representative image of TIA1 IHC with the rabbit polyclonal (Santa Cruz #sc-28,237, clone H-120) antibody showing delicate granular cytoplasmicstaining (a). Double label IF with pTDP (green) and TIA1 (red) antibodies failed to show co-localization of TIA1 in compact (b), filamentous (c) orgranular pTDP-43-ir NCI (d). Scale bar: a and d, 24 μm; b and c, 16 μmHirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 10 of 13proportion (< 4%) also having FTD, which may under-estimate the incidence of TIA1 mutations in patientswith mainly FTD. Interestingly, none of our TIA1 muta-tion carriers developed significant parkinsonism orpsychotic features, both of which are commonly re-ported in series with the C9orf72 mutation [11, 27].It is intriguing that another founder mutation affect-ing the LCD of TIA1 (E384K) was previously identifiedin Swedish and Finnish populations to cause WDM,which is characterized by late-onset slowly progressiveweakness of hand and distal leg muscles and is associ-ated with a rimmed vacuolar myopathy which is TDP-ir[10, 15]. To our knowledge, no patients with WDMhave been reported to also develop ALS or FTD andnone of our ALS patients with other TIA1 mutationshad a personal or family history of muscle disease. It ispossible that something about the specific amino acidsubstitution caused by the E384K mutation leads to adistinct and selective phenotype, or that the expressionis influenced by other genetic factors common to thespecific ethnic population in which the WDM TIA1mutation occurs [15]. However, given the fact that theE384K mutation is reported to show similar effects tothe ALS associated TIA1 mutations in biophysical andcell culture studies [19], it is also possible that greateroverlap in the clinical features exist but has not yetbeen recognized due to referral or reporting biases.This might not be unexpected given the number ofother genes (including VCP, SQSTM1, HNRNPA1,HNRNPA2B1 and MATR3), in which mutations causeclinical syndromes, referred to as multisystem proteino-pathies, with ALS, FTD, inclusion body myopathy andbone disease each showing variable penetrance [29].Overall, the neuropathology of our patients with TIA1mutations was characterized by chronic degenerativechanges, primarily involving the pyramidal motor sys-tem, prefrontal neocortex and substantia nigra, withmore anatomically widespread TDP-ir pathology(Table 2, Figs. 2 and 3). In all four cases with clinicalFTD, the pattern of neocortical TDP-ir pathology fit bestwith FTLD-TDP type B and was similar to what is foundin most patients with FTD combined with ALS, includ-ing those with the C9orf72 mutation [18]. None of theTIA1 mutation cases showed severe degeneration of thecaudate nucleus or hippocampal sclerosis which are bothcommon in other genetic causes of FTLD-TDP (e.g.C9orf72 and GRN mutations) [11, 20].The most striking aspect of the pyramidal systempathology in the TIA1 mutation carriers was the num-ber of round, sometimes LBL inclusions seen in LMNof the spinal cord and medulla with HE stain (Fig. 2).Although similar round inclusions were also found insome cases of sALS and C9orf72+ cases, they never av-eraged more than one per tissue section and werecompletely absent in most cases. In contrast, round andLBL inclusions were a consistent feature in the TIA1mutation carriers, with at least one, and often multipleexamples, present in each section of spinal cord andmedulla. Consistent with this was the finding of signifi-cantly more compact round TDP-ir NCI in LMN in theTIA1 mutation cases, which were often of a similar sizeand shape as the round/LBL inclusions seen on HEstain. This finding suggests that frequent round/LBLIin LMN may represent a pathological signature of ALS-TDP caused by TIA1 mutations and that the formationof these particular inclusion bodies may somehow berelated to the altered SG dynamics that has been shownto be associated with expression of the mutant TIA1protein [19]. Although this is somewhat speculative, itis interesting that cases of ALS caused by mutations inthe RNA-binding protein FUS are also characterized bylarge round cytoplasmic inclusions in LMN that arevisible with HE stain (although generally more baso-philic) and that these have been proposed to form inpersistent SG [7]. Round, hyaline, LBLI have also beendescribed in some (but not all) cases of ALS caused bySOD1 mutations [13, 23], where they are composed ofmisfolded SOD1, rather than TDP-43, and may be in-duced by ER stress [33].The pathomechanism that has been proposed for ALSassociated with TIA1 mutations is that the amino acidchange in the LCD enhances its intermolecular inter-action, which promotes LLPS and results in SG that aremore persistent, thus creating an environment where thecontents are more likely to begin to aggregate and be-come insoluble [19]. Although TDP-43 is one of themost abundant and most aggregate prone constituentsof SG [1], this model raises the possibility that theresulting pathological inclusions might also containTIA1, other SG markers, and other RBP that are typic-ally stored in SG. However, our IHC and IF studies failedto demonstrate any co-localization of TIA1 with TDP-43in the inclusions, and also failed to show any abnormalaccumulation of other RBPs in cases with TIA1 muta-tions. Although some other studies have suggested thatTIA1 may co-localize with TDP-43 in ALS [17, 32],others have refuted this finding [4]. Consistent with ourimmunostaining results are previous biochemical studiesthat failed to show any enrichment of TIA1 in the insol-uble protein fraction extracted from post mortem braintissue of TIA1 mutation carriers [19]. This suggests thateven if TDP-43 begins to form insoluble aggregates inSG, further protein aggregation may occur independentof the presence and function of TIA1. None-the-less, itis possible that the commercial TIA1 antibodies we usedare not sufficiently sensitive for use in post mortem tis-sue that has undergone prolonged fixation and that fur-ther TIA1/TDP-43 co-localization studies are warranted.Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 11 of 13ConclusionsIn summary, this analysis of a small series of cases pro-vides an initial characterization of the clinical and patho-logical features in patients with ALS ± FTD caused bymutations in TIA1. The clinical presentation is distin-guished by frequent bulbar onset weakness and a pre-dominance of expressive aphasia, and by the absence ofassociated parkinsonian or psychotic features. Althoughthe disease appears to be inherited in an autosomaldominant fashion, the lack of family history in manycases suggests that penetrance may be variable. Thestriking predominance of females in our series needs tobe confirmed, but suggests that expression of the muta-tion may be influenced by some sex-related factor. Theanatomical pattern of neurodegeneration correlates withthe main clinical features and these cases tend not toshow the caudate atrophy or hippocampal sclerosis thatis common with the C9orf72 mutation. The molecularneuropathology is a combination of FTLD-TDP type Band ALS with TDP-ir inclusions. However, the moststriking pathological feature is the consistent presence ofround, often LBL inclusions that are visible on HE stainand correlate with an increased frequency of compactTDP-ir NCI in LMN. There is currently no evidence forany abnormal distribution or accumulation of TIA1 pro-tein in postmortem brain tissue of TIA1 mutation car-riers; however, this warrants more detailed investigation.Additional studies are clearly needed to confirm and ex-pand upon this initial characterization. In particular, thepresence of TIA1 mutations needs to be investigated inpatients with clinically pure FTD and families with TIA1mutations should be reviewed for evidence of multisyste-mic features such as muscle and bone disease. Eventhough this initial case series is relatively small, the de-tailed clinical and pathological characterization will behelpful in identifying additional cases that might carry aTIA1 mutation and provides a baseline upon whichfuture studies can build.AcknowledgementsWe would like to thank Margaret Luk and Simon Cheung for their excellenttechnical assistance. This work was supported by the Canadian Institutes ofHealth Research (74580), the Canadian Consortium on Neurodegeneration inAging (137794) and the ALS Canada-Brain Canada Hudson Grant (IM, RH);NIH/NINDS grants R35 NS097261 and P01 NS084974 (RR); and EHB, MMM,and SW are supported by NIA P30 AG13854 (EB, MM, SW).Authors’ contributionsVHR performed the immunofluorescence studies, the quantification andstatistical analysis of the pathological data and drafted the manuscript, CPparticipated in the genetic analysis, AMN participated in the genetic analysis,MB participated in the genetic analysis, GYRH contributed clinical data, CKcontributed clinical data, PS contributed clinical data, KBB contributed clinicaldata, DWD contributed pathological material and data, MM contributedclinical data, SW contributed clinical data, EB contributed pathologicalmaterial and data, LZ contributed clinical data, JK contributed pathologicalmaterial and data, ER contributed material for genetic analysis, SAZcontributed clinical data, DLJ contributed clinical data, JPT performed thefunctional studies, RR supervised all the genetic analysis, IRAM conceived,designed and coordinated the study and participated in the assessment ofpathological data. All authors read and approved the final manuscript.Competing interestsThe authors declare they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Department of Pathology and Laboratory Medicine, University of BritishColombia, British Columbia, Canada. 2Department of Neuroscience, MayoClinic Jacksonville, Jacksonville, FL, USA. 3Division of Neurology, University ofBritish Columbia, Vancouver, BC, Canada. 4Department of BiomedicalPhysiology & Kinesiology, Simon Fraser University, Burnaby, BC V5A 1S6,Canada. 5Department of Neurology, Northwestern University FeinbergSchool of Medicine, Chicago, IL, USA. 6Department of Pathology,Northwestern University Feinberg School of Medicine, Chicago, IL, USA.7Division of Neurology, Sunnybrook Health Sciences Centre, Toronto, ON,Canada. 8Department of Anatomic Pathology, Sunnybrook Health SciencesCentre, Toronto, ON, Canada. 9Tanz Centre for Research inNeurodegenerative Diseases, University of Toronto, Toronto, ON, Canada.10Department of Neurology, University of Pittsburgh School of Medicine,Pittsburgh, PA, USA. 11Department of Pathology, University of PittsburghSchool of Medicine, Pittsburgh, PA, USA. 12Department of Cell and MolecularBiology, St. Jude Children’s Research Hospital, Memphis, USA. 13Tennesseeand Howard Hughes Medical Institute, Chevy Chase, MD, USA. 14Departmentof Pathology, Vancouver General Hospital and Vancouver Coastal Health, 855West 12th Avenue, Vancouver, BC V5Z 1M9, Canada.Received: 9 November 2017 Accepted: 9 November 2017References1. 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PLoS One 2:e1030. 10.1371/journal.pone.0001030•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Hirsch-Reinshagen et al. Acta Neuropathologica Communications  (2017) 5:96 Page 13 of 13


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