Distinct molecular patterns of TDP-43 pathology in Alzheimer’s disease: relationship with clinical phenotypes

The co-existence of multiple pathologies and proteins is a common feature in the brains of cognitively impaired elderly individuals. Transactive response DNA-binding protein (TDP-43) has been discovered to accumulate in limbic brain regions of a portion of late-onset Alzheimer’s disease (AD) patients, in addition to amyloid-β and τ protein. However, it is not yet known whether the TDP-43 species in the AD brain differ in their composition, when compared among different AD cases and to frontotemporal lobar degeneration cases with TDP-43 inclusions (FTLD-TDP). Furthermore, it is not known whether TDP-43 pathology in AD is related to symptoms of the frontotemporal dementia (FTD) spectrum. In this study, we investigated the molecular pattern of TDP-43 lesions with five different antibodies against different phosphorylated (pTDP-43) and non-phosphorylated TDP-43 epitopes. We analyzed a cohort of 97 autopsy cases, including brains from 20 non-demented individuals, 16 cognitively normal pathologically-defined preclinical AD (p-preAD), 51 neuropathologically-confirmed AD cases and 10 FTLD-TDP cases as positive controls. We observed distinct neuropathological patterns of TDP-43 among AD cases. In 11 neuropathologically-confirmed AD cases we found dystrophic neurites (DNs), neuronal cytoplasmic inclusions (NCIs) and/or neurofibrillary tangle (NFT)-like lesions not only positive for pTDP-43409/410, but also for pTDP-43 phosphorylated at serines 403/404 (pTDP-43403/404) and non-phosphorylated, full-length TDP-43, as seen with antibodies against C-terminal TDP-43 and N-terminal TDP-43. These cases were referred to as ADTDP + FL because full-length TDP-43 was presumably present in the aggregates. FTLD-TDP cases showed a similar molecular TDP-43 pattern. A second pattern, which was not seen in FTLD-TDP, was observed in most of p-preAD, as well as 30 neuropathologically-confirmed AD cases, which mainly exhibited NFTs and NCIs stained with antibodies against TDP-43 phosphorylated at serines 409/410 (pTDP-43409, pTDP-43409/410). Because only phosphorylated C-terminal species of TDP-43 could be detected in the lesions we designated these AD cases as ADTDP + CTF. Ten AD cases did not contain any TDP-43 pathology and were referred to as ADTDP-. The different TDP-43 patterns were associated with clinically typical AD symptoms in 80% of ADTDP + CTF cases, 63,6% of ADTDP + FL and 100% of the ADTDP- cases. On the other hand, clinical symptoms characteristic for FTD were observed in 36,4% of ADTDP + FL, in 16,6% of the ADTDP + CTF, and in none of the ADTDP- cases. Our findings provide evidence that TDP-43 aggregates occurring in AD cases vary in their composition, suggesting the distinction of different molecular patterns of TDP-43 pathology ranging from ADTDP- to ADTDP + CTF and ADTDP + FL with possible impact on their clinical picture, i.e. a higher chance for FTD-like symptoms in ADTDP + FL cases.


Introduction
Alzheimer's Disease (AD) is a progressive, neurodegenerative disorder and constitutes the most common form of dementia in people over 65 years of age [41]. AD is characterized by two main neuropathological hallmarks: extracellular amyloid-β (Aβ) deposition in senile plaques and intracellular generation of neurofibrillary tangles (NFTs), neuropil threads, and neuritic plaques containing abnormally phosphorylated τ protein (p-τ) [7]. Transactive response DNA-binding protein (TDP-43)positive cytoplasmic inclusions in limbic areas occur in AD brains as well. They are present in up to 74% of AD cases [1,2,33,40].
TDP-43 pathology has been described to expand in the AD brain in a stereotypical manner, starting in the amygdala and progressing into the medial temporal lobe and later into further regions such as temporal and frontal cortices [27,47]. Furthermore, TDP-43 pathology in AD has been associated with a later onset of the disease, memory loss and hippocampal atrophy, playing a role in the clinical manifestation of the disease [28,29]. Similar lesions have also been described in non-AD cases [40,62].
Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a neurodegenerative disorder typically manifesting with behavioral changes or signs of aphasia [59], i.e., symptoms of the frontotemporal dementia (FTD) spectrum. Brettschneider et al. suggested that TDP-43 pathology spreads in a stereotypical manner in the behavioral variant of FTLD-TDP, with the prefrontal neocortex, middle-frontal gyrus, superior and middle-temporal gyri being heavily involved and with the amygdala being involved early in the disease [9]. FTLD-TDP has been classified into different subtypes, according to the morphology and topographic distribution of TDP-43 lesions in the cortex [39].
TDP-43-positive inclusions in limbic brain regions have been recently considered as limbic-predominant ageassociated TDP-43 encephalopathy-related neuropathological changes (LATE-NC) [47]. This means that lateonset AD cases that have TDP-43 pathology may present concomitant LATE-NC, even without the clinical manifestation of LATE, according to the recent consensus working group report [47]. LATE-NC has recently been established as TDP-43 neuropathology in individuals without amyotrophic lateral sclerosis (ALS) or FTLD, which can be found in elderly individuals (mostly 80 years of age or older) with and without AD [47].
A controversial point raised by recent studies is whether TDP-43 deposition in AD cases represents the co-existence of AD and FTLD-TDP, or whether TDP-43 proteinopathy in AD is substantially different from that of FTLD-TDP [25][26][27]49]. Given the recently developed, controversial concept of LATE-NC encompassing TDP-43 lesions in AD, non-AD and non-demented patients, the question whether TDP-43 pathology in these cases displays distinct molecular signatures is even more relevant. Furthermore, it was not yet addressed whether such patterns are different from that observed in FTLD-TDP [25].
TDP-43 is a nuclear protein that under pathological conditions can be cleaved and phosphorylated [10]. Due to the loss of the nuclear localization signal after cleavage, N-terminal truncated fragments of TDP-43 mislocalize and aggregate in the cytoplasm. In turn, the nucleus is depleted from normal TDP-43. Hence, a gain of toxic function in the cytoplasm as well as a loss of nuclear function seem to constitute TDP-43 disease mechanisms [50,51]. Phosphorylation at serines 403/404 and 409/410 of TDP-43 constitutes pathological features of ALS and FTLD-TDP [2,5,18,23,48,50]. However, the pattern of TDP-43 phosphorylation and species distribution present in AD cases is not yet understood.
Hence, we studied 97 autopsy cases including 20 non-AD/non-FTLD-TDP cases as controls, 16 preclinical AD cases, 51 neuropathologically-confirmed AD cases and 10 FTLD-TDP cases (used as positive controls). We screened our cohort with five different antibodies against several phosphorylated and nonphosphorylated TDP-43 epitopes. We report distinct molecular patterns of TDP-43 pathology among the cases with AD neuropathology based upon the detection of full-length TDP-43 or phosphorylated TDP-43 C-terminal fragments. These molecular differences were associated with a clinical presentation of AD or FTD symptoms.

Material and methods
Neuropathology A total of 97 autopsy cases between 36 and 98 years of age (mean age: 72 years old, 45 females and 52 males) were investigated: 20 non-diseased controls, 16 preclinical AD, 51 neuropathologically-confirmed AD cases and 10 FTLD-TDP cases as positive controls for TDP-43 pathology ( Table 1, Additional file 1- Table A1). Cases with hippocampal sclerosis were not included, since this pathology is considered as separate entity or separate type of LATE [1,47,54]. All autopsy brains were received from university or municipal hospitals in Leuven (Belgium), Bonn, Offenbach am Main and Ulm (Germany), and collected in accordance with local ethical committee guidelines and the federal laws governing the use of human tissue for research in Belgium and Germany. Dementia was diagnosed according to the DSM-IV criteria. The neuropathological diagnosis of AD was made when dementia was observed and when at least an intermediate degree of AD-related neuropathology was determined according to current criteria for the neuropathological diagnosis of AD as published by the National Institute of Aging and Alzheimer Association working group (NIA-AA criteria) [21]. The degree of dementia at the time of death was determined retrospectively using the Clinical Dementia Rating (CDR) score [19,43]. For this purpose, the information from the clinical files was used to provide a CDR score. The CDR score was applied in controls and AD cases with AD clinical symptoms [43], whereas CDR with FTLD modules was used when scoring cases with a clinical picture of FTD [34]. The diagnosis of FTD behavioral variant and of primary progressive aphasia variants FTLD was made using consensus criteria [16,53].
The left hemispheres were fixed in formalin for 2 to 4 weeks and dissected. Blocks from frontal, parietal, temporal, occipital, and entorhinal cortex, the hippocampal formation at the level of the lateral geniculate body, basal ganglia, hypothalamus, thalamus, amygdala, basal nucleus of Meynert (NBM), midbrain, pons, medulla oblongata and cerebellum were embedded in paraffin. Five μm sections were cut using a microtome. The sections were stained with hematoxylin and eosin (H&E) for identification of pathologies different from AD and FTLD-related lesions.

Immunohistochemistry
Sections of the hippocampus, entorhinal, frontal, temporal and occipital cortex, amygdala, and NBM were stained with antibodies against TDP-43. Briefly, after epitope retrieval in heated citrate buffer (pH 6) and deparaffinization (using a Dako autostainer Link 48, Dako, Glostrup, Denmark), the sections were treated with peroxidase blocking reagent (Envision flex Peroxidase-Blocking Reagent, Dako) for 5 min. Primary antibodies (Additional file 1- Table A2) were applied for 30 min or overnight. The antibodies against TDP-43 provide the ability to detect specific sites of TDP-43 protein as well as the phosphorylation status at serines 403/ 404 and 409/410 (Fig. 1). Afterwards, the slides were incubated with an appropriate secondary antibody (Envision Dual flex, Dako). 3.3′-diaminobenzidine (Liquid DAB+ Substrate Chromogen System, Dako) was used as a chromogen to yield brown reaction products. Counterstaining with hematoxylin was performed. In sections of the hippocampus, entorhinal, temporal and occipital cortex, Aβ and p-τ immunostaining was performed using a similar protocol as described above, but with formic acid pre-treatment for antigen retrieval prior to the immunostaining procedure.
For immunofluorescence procedures, paraffin sections were incubated overnight with a cocktail of antibodies of different species (mouse TDP-43 C-terminus, rabbit TDP-43 N-terminus) (Additional file 1- Table A2) after the respective pre-treatment and deparaffinization. The sections were then incubated with a goat Cy2-labelled anti-mouse and goat Cy3-labelled anti-rabbit antibody cocktail (Jackson ImmunoResearch Ltd., West Grove, PA, USA) and mounted with ProLong™ Gold with DAPI (ThermoFisher Scientific, Rockford, IL, USA). The sections were microscopically analyzed with a Leica DM2000 LED microscope and images were taken with a Leica DFC7000 T camera, at 200 or 400x magnification. The extent of p-τ, Aβ and TDP-43 pathology was assessed with antibodies against p-τ, Aβ and TDP-43 (Additional file 1- Table A2).

TDP-43 pathology analysis
A case was considered as TDP-43-positive in a given brain region, if there was TDP-43 immunoreactivity for one or more of the following lesions: neuronal cytoplasmic inclusions (NCIs), dystrophic neurites (DNs) or neuronal intranuclear inclusions (NIIs) (Fig. 2). Glial inclusions were not separately assessed, since none of our cases showed glial inclusions without associated neuronal TDP-43 pathology. Of note, a case was only considered to be TDP-43 negative when no TDP-43-positive lesion of any type, including glial inclusions, was detected. The identification of TDP-43 pathology was based on stainings with antibodies raised against pTDP-43 409/410 . pTDP-43 409/410 staining in granulovacuolar degeneration (GVD) was not considered as a relevant TDP-43 lesion in this study, because it contains other phosphorylated proteins, such as compounds of the necrosome [35,65] and it is not associated with nuclear clearance of TDP-43 [47]. NCIs, DNs and NIIs positive for TDP-43 have been previously associated with ALS and FTLD-TDP, as well as with AD [1,18,29,48,58,66]. pTDP-43 pathology was analyzed for the relevant phosphorylation sites with antibodies against pTDP-43 409/410 , pTDP-43 409 and pTDP-43 403/404 (Additional file 1- Table A2). For confirmation, a monoclonal rat (clone 1D3) antibody against pTDP-43 409/410 was used. The non-phosphorylated TDP-43 C-terminus (C-t TDP-43) was stained with a rabbit polyclonal antibody raised against the non-phosphorylated amino acids 260-414 of TDP-43 and confirmed with a rabbit polyclonal antibody against the non-phosphorylated amino acids 405-414 (Additional file 1- Table A2). The N-terminus of TDP-43 (N-t TDP-43) was detected with a rabbit polyclonal antibody raised against the amino acids 1-50 ( Fig. 1).
TDP-43 pathology types α and β as defined by Josephs et al. were determined in non-FTLD-TDP/non-ALS cases, as proposed [26], by using sections stained with anti-pTDP-43 409/410 antibodies. The presence of DNs and NCIs in the amygdala, hippocampal formation, and the frontotemporal cortex were classified as type α whereas cases with NFT-like pathology restricted to limbic regions (amygdala and hippocampus) were classified as type β. In the event that cases exhibiting a type α distribution pattern also presented NFT-like TDP-43 pathology, we considered them as type α + β (Additional file 1- Table A1). This classification was applied by two independent observers (ST and EVS) with an 85,3% agreement rate. Adjustment for discrepancies was reduced in a consensus diagnosis after discussing the results together at the microscope. AD cases with widespread lesions in the temporal or frontal cortex and FTLD-TDP cases were subtyped into the known FTLD-TDP subtypes (A-D), as defined by Mackenzie et al. [38], by analyzing the morphology and topography of DNs and NCIs in temporal and frontal cortices (see Additional file 1- Table A1). If a case did not fit into any of these subtypes, as it presented large NCIs, scattered among all layers of the temporal cortex, little DNs and occasionally NFT-like lesions, it was not further classified (indicated by * in Additional file 1- Table A1). AD cases without TDP-43 pathology in cortical layers were also not further classified into an FTLD-TDP subtype A-D, as this classification requires TDP-43 pathology in cortical layers. For the assessment of LATE-NC pathology, cases with more than 80 years of age and TDP-43 pathology in limbic regions (amygdala and hippocampal formation) without exclusive FTD-related symptoms were considered to be positive [47]. Cases younger than 80 years of age but older than 60 years with severe TDP-43 pathology restricted to limbic regions were also considered to represent LATE-NC. The amygdala, hippocampal formation and frontal cortex were used to stage for LATE-NC as proposed: Stage 0 = no LATE-NC; stage 1 = TDP-43 pathology restricted to the amygdala; stage 2 = TDP-43 pathology in the amygdala and hippocampal formation; stage 3 = TDP-43 pathology extending to the frontal cortex, in addition to stage 2 regions. LATE-NC staging was based on sections stained with a polyclonal antibody against pTDP-43 (pS409/pS410, Cosmobio). These criteria were not applied to the FTLD-TDP cases (positive controls). Of note, none of our cases fulfilled the criteria for hippocampal sclerosis according to the consensus criteria [54]. Genetic mutations (i.e. C9ORF72) in some cases are also referred in Additional file 1- Table A1.

Aβ and τ pathology assessment
To determine the anatomical distribution of Aβ plaques, phases of Aβ deposition in the medial temporal lobe (AβMTL phases) were assessed as follows: Aβ plaques in the temporal neocortex (layers III, V and VI) characterize AβMTL phase 1. In AβMTL phase 2, the plaque deposition spreads into the layers pre-βpri-γ of the entorhinal cortex, CA1, and the subiculum.
AβMTL phase 3 is characterized by Aβ deposition in all six layers of the temporal neocortex including subpial band-like Aβ accumulation. In addition, Aβ plaques also occur in the outer molecular layer of the dentate gyrus and the parvopyramidal cell layer of the presubicular region. Finally, in AβMTL phase 4 there is fully developed β-amyloidosis in the medial temporal lobe with additional Aβ plaques in the CA4 region of the hippocampus and in the pre-α layer of the entorhinal cortex [64].
NFT distribution was assessed using the Braak NFTstaging method: Stage I is characterized by p-τ-positive neurons and threads, limited to the transentorhinal region, stage II by p-τ pathology in the entorhinal region, extending to CA1 and CA2, stage III by affection of the neocortex of the fusiform and lingual gyri, stage IV by progression into superior temporal neocortex areas and the dentate gyrus, stage V by the involvement of frontal and occipital cortex, reaching the peristriate region (layer V also begins to be affected) and finally, stage VI is identified by p-τ pathology in secondary and primary neocortical areas and extends into striate area of the occipital lobe [6,7]. The consortium to establish a registry for AD (CERAD) scores for neuritic plaque density were assessed based on sections stained with an antibody against p-τ (AT8, Additional file 1-Tab. A1) [42]. The National Institute of Aging -Alzheimer Association (NIA-AA) degree of AD-pathology was determined according to Hyman et al. [21], based on the AβMTL phase, Braak NFT-stage, and the CERAD score for neuritic plaque pathology. CAA was diagnosed whenever Aβ deposits were found in the wall of cerebral and leptomeningeal blood vessels [14]. NFT pathology in the absence of Aβ plaque pathology was classified as definite primary age-related tauopathy (PART) [11].

Statistical analysis
Non-parametric, Kruskal-Wallis H-test was used for independent samples, to perform comparisons between disease groups (Braak NFT-stage, AβMTL phase, NIA-AA, Age at death, CDR score). Friedman test for related samples was used to compare different antibodies within the same disease group. Bonferroni correction for multiple testing was applied. Post-hoc power analysis was calculated based on mean values with the help of Gpower software (University of Düsseldorf, Germany).
Binary logistic regression was used to assess associations between the molecular TDP-43 patterns found in this study with clinical symptoms, AD-related neuropathological changes (Braak NFT-stages and AβMTL phases) and to associate these patterns with the morphological subtypes defined by Josephs et al. [26]. These analyses were controlled for age at death and sex, with a 95% confidence interval (CI).
Multinomial regression controlled for age and sex was used to further confirm the results obtained by Kruskal-Wallis H-test. For these regressions we used the symptomatic AD group as reference group.
IBM SPSS software (IBM, USA) was used in all instances.

Results
Here, we analyzed the biological patterns of TDP-43 proteinopathy in demented cases with moderate-high degrees of AD pathology, pathologically defined preclinical AD (p-preAD) cases [61], FTLD-TDP cases and control cases (non-AD), by screening the whole cohort with five different TDP-43 antibodies. We distinguished distinct molecular patterns of TDP-43 pathology based on the different TDP-43 species and on the pattern of TDP-43 phosphorylation sites. Ten AD cases showed no TDP-43 pathology and were referred to as AD TDPcases. Thirty AD cases were positive for NCIs and NFT-like lesions stained with antibodies raised against pTDP-43 409/410 , as well as pTDP-43 409 , but neither with anti-pTDP-43 403/404 nor with antibodies against C-or N-terminus epitopes of TDP-43. This subgroup of AD cases was designated as AD TDP + CTF . Of note, there was one exceptional AD case with predominant pTDP-43 409/410 epitope expression that also had very sparse N-t TDP-43 pathology in amygdala and temporal cortex, but no pTDP 403/404 -positive lesions. For this reason, we classified this case also as an AD TDP+CTF case. The remaining 11 neuropathologically-confirmed AD cases were positive not only for anti-pTDP43 409/410 or anti-pTDP43 409 , but also for anti-pTDP-43 403/404 and antibodies raised against non-phosphorylated TDP-43 species, such as C-t TDP-43 and N-t TDP-43. FTLD-TDP cases showed a similar expression pattern of these TDP-43 epitopes. Given the similarity of the TDP-43 staining pattern with the predominant expression of full-length TDP-43, the AD cases exhibiting all types of TDP-43 species were referred to as AD TDP + FL . Furthermore, 7 out of these 11 AD TDP + FL cases presented a morphological pattern of TDP-43 pathology compatible with one of the FTLD-TDP subtypes A-C as described before [39], whereas the remaining four cases did not fit into any of these subtypes (Additional file 1- Table A1).

Distinct patterns of TDP-43 and its modified forms in cases fulfilling the neuropathological criteria for AD
To clarify the biological differences between TDP-43 patterns in AD cases, we investigated the prevalence of pTDP-43 and non-phosphorylated TDP-43 species. For that, we used phospho-dependent and phosphoindependent antibodies against the N-and C-t epitopes of TDP-43 ( Fig. 1). To test whether the TDP-43 staining patterns are consistent among different brain regions we analyzed hippocampal sub-regions (dentate gyrus, CA4, CA3/2, CA1, subiculum), amygdala, basal nucleus of Meynert (NBM), entorhinal, temporal, frontal and occipital cortices.
The number of positive cases for pTDP 409 and pTDP 409/410 in most regions analyzed in this study were similar among AD TDP + CTF and AD TDP + FL , with the exception of dentate gyrus, CA4, frontal and occipital cortex for pTDP 409/410 and dentate gyrus, occipital cortex for pTDP 409 . This was due to the low abundance of TDP-43 pathology in AD TDP + CTF cases in these regions (Fig. 3 a-c, 4 Additional file 1- Fig. A1-A3, Tab. A3, 4). In turn, the predominance of TDP-43 403/404 pathology in AD TDP + FL cases compared to AD TDP + CTF cases was higher in all regions analyzed except NBM and occipital cortex, which exhibited low pTDP-43 403/404 pathology in both groups ( Fig. 4, Additional file 1- Fig. A1-A3, A5). Furthermore, AD TDP + FL cases displayed a higher percentage of cases positive for non-phosphorylated TDP-43 in all regions except NBM compared to AD TDP + CTF cases, as seen with antibodies raised against the C-t TDP-43 ( Fig. 4 a-c, Additional file 1- Fig. A3, A6). This was also true for Table 2 Summary of the neuropathological groups used in this study. The number of cases with each observed symptom(s) and respective percentages are indicated (see also Additional file 1- Table A1) Neuropathological Diagnosis
AD TDP + FL and FTLD-TDP groups displayed a very similar neuropathological profile regarding all TDP-43 species in most regions ( Fig. 3 a-c; Additional file 1- Fig.  A1-A3). FTLD-TDP cases still displayed a higher abundance of pTDP 403/404 , C-and N-t TDP-43 lesions in most regions when compared to AD TDP + FL cases (Additional file 1- Fig. A3).
In addition, nuclear clearance of C-t TDP-43 was present in AD TDP + CTF cases, and even more pronounced in AD TDP + FL and FTLD-TDP cases. However, this was purely observational and it was not quantified.
To further investigate the TDP-43 aggregate composition, we performed fluorescence double-labelling experiments with antibodies raised against N-t and C-t-TDP-43. We confirmed that the inclusions in FTLD-TDP as well as in AD TDP + FL cases prominently exhibited the full-length protein at least in the dentate gyrus (   Tables A3-A4). This was also true for C-t TDP-43, where 45,5-100% cases of these two groups were positive for this antibody in the investigated regions except for NBM (Additional file 1- Table A6). Furthermore, all regions except for NBM and occipital cortex were positive for pTDP 403/404 in 45.5-100% of cases AD TDP + FL cases, whereas 50-100% of FTLD-TDP cases were positive for this antibody in the investigated regions, including NBM and occipital cortex (Additional file 1- Table  A5). The number of severely affected regions was lower with an antibody against N-t-TDP-43. Nevertheless, in AD TDP + FL cases at least seven regions were heavily impacted by N-t-TDP-43 pathology, and all investigated regions except for NBM showed lesions detectable with this antibody (Additional file 1- Table A7). Similarly, in FTLD-TDP cases, all regions except for NBM and occipital cortex were considerably affected by N-t TDP-43 positive lesions (Additional file 1- Table A7). This means that despite AD TDP + FL and FTLD-TDP cases were molecularly similar in terms of TDP-43 species, FTLD-TDP cases showed a more widespread distribution of TDP-43 pathology.
On the other hand, in p-preAD cases, as well as AD TDP + CTF cases, the pTDP-43 409/410 and pTDP-43 409 pathology seemed to be mainly restricted to the medial temporal lobe incl. Amygdala, with the exception of the dentate gyrus of the hippocampus, which was generally spared (Additional file 1-Tables A3). Therefore, the most affected regions were amygdala, CA1, subiculum and entorhinal cortex (56.7-90% of AD TDP + CTF cases) whereas the frontal and occipital cortices were less affected (up to 44.8 and 14.3% of positive cases, respectively). Similarly, in the non-AD cases with pTDP-43 409/410 pathology, these lesions were also restricted to the medial temporal lobe, incl. Amygdala.

LATE-NC distribution in AD TDP + CTF and AD TDP + FL
We classified our cases that were not typical FTLD-TDP with the recently proposed staging scheme for TDP-43 pathology by Nelson and colleagues [47]. Therefore, we considered LATE-NC positive whenever cases were older than 60 years and had at least pTDP-43 pathology ( Table 2, Additional file 1- Table A1). We observed that a minority of non-AD cases displayed LATE-NC stage 1 or 2 (5 or 10%, respectively), meaning that the TDP-43 pathology was limited to the amygdala and hippocampal formation, whereas the remaining control cases did not display LATE-NC (Fig. 6). This was either because they were too young to be considered part of the LATE-NC spectrum and/or because there was no TDP-43 pathology. Around half of p-preAD and AD TDP + CTF cases showed TDP-43 pathology in LATE-NC stage 2, extending into the hippocampus (50% and 63,3%, respectively), whereas 31,3% of p-pre AD cases did not show LATE-NC. Additionally, 30% of AD TDP + CTF cases displayed TDP-43 pathology extending to the frontal cortex (LATE-NC stage 3, Fig. 6).
As for the AD TDP + FL cases, more than half of the cases (54,5%) presented LATE-NC stage 3, whereas the remaining cases were classified as stage 2 LATE-NC (Fig. 6). These findings corroborate our previous results and further demonstrate that in AD TDP + CTF cases, TDP-43 pathology was mostly restricted to the limbic system, whereas the TDP-43 pathology in AD TDP + FL cases was more frequently widespread in the brain.

Relationship of molecular TDP-43 patterns with other ADrelated neuropathological lesions
Notably, AD TDP + FL cases were not distinguishable from AD TDP + CTF cases and AD TDPregarding Aβ, neuritic plaque and NFT pathology (Braak NFT stages III-VI, AβMTL phases 3-5; CERAD score: 1-3; NIA-AA stage 2-3) (p = 1, Fig. 4 a3-a4,b3-b4, Fig. 7 a-d). Kruskall-Wallis with Bonferroni correction for multiple testing was used. For these neuropathological parameters, a post-hoc power analysis revealed a power of 100% for the differences observed among all six groups of cases. The majority of non-AD, control cases (75%) presented PART (Fig. 7 a, Additional file 1- Table A1). Moreover, 2 out of the 3 non-AD cases with TDP-43 pathology mentioned earlier were contemplated among the PART group.
We then tested by binary logistic regression (while controlling for age at death and sex) if there was an association between the molecular patterns identified here with the morphological types of TDP-43 lesions as previously described by Josephs et al. [26]. In this study, the authors defined two morphological types of TDP-43 pathology in non-FTLD brains: type α -DNs, NIIs or NCIs widespread in the brain, and type β -NFTassociated material, restricted to the medial temporal lobe. We classified our cases according to this system (see Additional file 1- Table A1). We further classified all our cases with the molecular patterns found in this study: 'TDP + CTF' pattern, positive for pTDP 409 , and pTDP 409/410 ; or 'TDP + FL+' pattern, positive for all TDP-43 markers. The 'TDP + CTF' pattern (present in some controls, most p-preAD and all AD TDP + CTF cases) was strongly associated with type β (p < 0.0001, Additional file 1- Table A12) characterized by NFT-like lesions. The 'TDP + FL' pattern (observed in AD TDP + FL (See figure on previous page.) Fig. 5 TDP-43 aggregates in AD TDP + FL and FTLD-TDP mostly comprise the full-length protein or just CTFs. Double-labeling with N-t TDP-43 and C-t TDP-43 antibodies in a (a-i) FTLD-TDP, and (j-r) AD TDP + FL . The majority of NCIs and DNs positive for the full-length protein are detected in FTLD-TDP in the DG, temporal and frontal cortices (c, f, i, respectively, arrows) and in AD TDP + FL cases in the DG, temporal and frontal cortices (l, o, r, respectively, arrows). C-t exclusive inclusions were detected in FTLD-TDP cases in the DG (c, inset, arrowhead) and in the frontal cortex (g, h, i, arrowheads and i, inset, arrowhead). C-t exclusive inclusions were also detected in AD TDP + FL cases in the temporal cortex (o, inset, arrowhead). Scale bars = 50 μm and FTLD-TDP cases) did neither show an association with type α nor with type β (Additional file 1- Table  A13). We analyzed each molecular pattern individually due to collinearity effects. In our cases, we did not observe pure type α pattern. This pattern was always associated with at least few NFT-like TDP-43 inclusions and, therefore, considered as type α + β.

Association of the TDP-43 molecular patterns with clinical phenotypes
To further investigate these molecular patterns statistically, we re-grouped our cohort according to the clinical diagnosis: non-demented (including the neuropathological non-AD and p-preAD cases, n = 36), symptomatic AD (sympAD = all cases with a typical AD phenotype, n = 40), AD/FTD (cases with signs of both AD and FTD symptoms such as behavioral and/or language problems, n = 5) and FTD cases (cases with a clinical FTD phenotype, n = 15). We found that even when grouping our cases based on clinical phenotype, we observed significant differences in the prevalence of pTDP 409/410 , pTDP 409 , pTDP 403/404 , C-and N-t-TDP-43 reactivity, particularly in sympAD cases (Fig. 3 d-f, Additional file 1- Fig. A4) whereas no differences were observed in non-demented individuals (Additional file 1- Table A14, A15). Specifically, in sympAD we observed significant differences between the positivity for both pTDP 409/410 and pTDP 409 when compared to pTDP 403/404 , C-and N-t TDP43 in CA1 region and temporal cortex (p ≤ 0.001 and p < 0.047, Fig. 3 e-f). Moreover, significant differences were observed in the staining pattern between these antibodies in the remaining sub-regions of the hippocampus such as CA4, CA3/2, subiculum, as well as entorhinal, amygdala and NBM in sympAD (p < 0.01, Additional file 1- Fig. A4a-f, Additional file 1- Table A15). SympAD cases displayed a lower prevalence of all antibodies in frontal and occipital cortices, therefore there were no significant differences in these regions (Additional file 1- Fig. A4g-h Additional file 1- Table A15).
Moreover, there were no significant differences in positivity of the TDP-43 lesions between the anti-TDP-43 antibodies in the clinical AD/FTD group nor in the FTD group (Additional file 1-Tables A16-A17).
To further confirm the differences in the molecular profile among the clinical groups, we used multinomial logistic regression controlled for age and sex in the most severely affected region (CA1). For this, we used the sympAD group as a reference category. Consistent with our previous results, we observed significant differences Fig. 7 AD TDP + FL cases have high amounts of p-τ, Aβ, and a high dementia score, comparable to AD TDP + CTF cases. a Braak NFT-stage, b AβMTL phase, c NIA-AA degree of AD pathology, d CERAD score, e CDR score and (f) age at death among neuropathological groups. No significant differences were found between these AD groups except for age. p-preAD, AD TDP-, AD TDP + CTF and AD TDP + FL cases were significantly older when compared to non-AD controls (p = 0.002, p < 0.0001, p < 0.0001 and p = 0.003, respectively). Non-parametric, Kruskal-Wallis test for independent samples with Bonferroni correction for multiple testing was used to compare the same independent variable among neuropathological groups. When limiting the sample to the AD groups and comparing AD TDP-, AD TDP + CTF , and AD TDP + FL cases, two by two with binary logistic regression analysis controlled for age and sex and adjusted for multiple testing by the Bonferroni procedure, AD TDP + CTF cases showed higher Braak NFT stages, AβMTL phases, CERAD scores, and NIA-AA degrees of AD pathology compared to AD TDPcases (Additional file 1- Tables A8-A11) between the sympAD and the FTD group regarding the prevalence of pTDP 409/410 (p = 0.023), pTDP-43 403/404 (p = 0.001), C-and N-t TDP-43 (p = 0.001), but not pTDP-43 409 (Additional file 1-Tables A18-A22). This strengthens the hypothesis that the patterns of TDP-43 species differ among these dementias. Furthermore, the positivity for pTDP-43 409/410 and pTDP-43 409 was lower in non-demented cases, when compared to sympAD cases (Additional file 1-Tables A18-A19). Of note, age at death of FTD cases was lower than that of the sympAD cases. This was also true for non-demented cases (Additional file 1-Tables A18-A22). When using age as the only independent variable through multinomial logistic regression, we confirmed that the sympAD cases were significantly older than the FTD cases (p = 0.002, odds ratio = 1.1).
Clinically, all AD TDPcases had a clinical AD phenotype (Table 2). Twenty-four out of 30 AD TDP + CTF cases (80%) presented typical AD symptoms in life, such as amnestic deficits and executive dysfunction. Four AD TDP + CTF cases (13,3%) exhibited prominent language problems, with an initial diagnosis of semantic variantprimary progressive aphasia (svPPA). Interestingly, one AD TDP + CTF case (3,3%) showed an AD clinical phenotype and motor speech deficits later on in the disease. Another AD TDP + CTF case (3,3%) presented both clinical AD and behavioral problems, such as impulsivity and aggression ( Table 2).
The AD TDP + FL cases presented a larger variety of symptoms ranging from AD to FTD features. Specifically, 7 out of 11 AD TDP + FL cases (63,6%) exhibited a classical AD clinical phenotypememory deficits and executive dysfunction -whereas 2 AD TDP + FL cases (18, 2%) had a diagnosis of the behavioral variant of FTD (bvFTD), with pronounced behavioral problems. Moreover, 1 AD TDP + FL case (9,1%) with the C9ORF72 mutation displayed behavioral FTD-like deficits as well as AD symptoms such as memory deficits. Finally, one AD TDP + FL case (9,1%) displayed svPPA during life, with additional AD signs later on (Table 2, Additional file 1- Table A1).
Interestingly, we observed that the AD TDP + FL cases with a Josephs' morphological pattern type β in the absence of type α features (see additional file 1- Table A1) were clinically typical AD whereas the presence of type α features was observed in 57.1% of the AD TDP + FL cases with FTD symptomatology. To address this, we performed a binary logistic regression using Josephs' type α as a dependent variable and FTD symptoms, age at death and sex as independent variables. We observed an association between Josephs' type α and FTD symptoms (p = 0.039), but not between type β and FTD symptoms (p = 0.999), as expected (Additional File 1-Tables A23-24).
Finally, we addressed whether the different TDP-43 molecular patterns identified in this study are statistically associated with different clinical manifestation of the disease. For this, we only considered the demented cases with TDP-43 pathology and performed binary logistic regressions, while controlling for age at death and sex. We found that the 'TDP + CTF' molecular pattern was statistically associated with typical AD symptomsamnestic syndrome executive dysfunctionand that age at death was also associated with clinical AD (Table 3) but inversely associated with FTD symptoms (Additional file 1- Table A25). In turn, the 'TDP + FL' pattern, observed in AD TDP + FL as well as FTLD-TDP cases, was significantly associated with a clinical presentation of the FTD spectrumcases that presented behavioral problems or language deficits (Table 4) -and inversely associated with clinical AD (Additional file 1- Table A26). Post-hoc power analysis of these comparisons among the clinical phenotypes revealed a power 48-87% when considering the typical AD symptoms (87%) and FTD symptoms with or without AD-type cognitive impairment (48%) in each neuropathological group.

Discussion
In our study, we aimed to clarify the molecular characteristics of TDP-43 aggregates in AD cases with TDP-43 pathology.
We identified distinct molecular patterns of TDP-43 species in neuropathologically-confirmed AD cases. One pattern exhibiting pTDP-43 409/410 , pTDP-43 409 , pTDP-43 403/404 , and non-phosphorylated N-and C-terminal epitopes of TDP-43 indicated the presence of full-length TDP-43 aggregates with a complex phosphorylation pattern including multiple phosphorylation sites. AD cases showing this pattern were referred to as AD TDP + FL . This TDP-43 epitope exhibition pattern was also found in FTLD-TDP cases. The other pattern -seen in most p-preAD cases and in AD TDP + CTF cases was restricted to material stained with the anti-pTDP-43 409 and pTDP-43 409/410 antibodies, lacking pTDP-43 403/404 and rarely positive for non-phosphorylated epitopes of TDP-43. Additionally, 19,6% of all AD cases lacked pTDP-43 inclusions and were considered as AD TDPcases. Accordingly, TDP-43 pathology in AD showed a spectrum ranging from the complete absence of TDP-43 lesions in AD TDPcases to AD TDP + CTF , and finally to AD TDP + FL cases with a molecular pattern similar to FTLD-TDP cases (Fig. 8).
Serines 403/404 and 409/410 have been previously described as TDP-43 sites that are abnormally phosphorylated in the brains of ALS and FTLD-TDP and AD patients [2,18,30]. However, it is not yet clear whether pathomechanistic variations are responsible for these different phosphorylation patterns among TDP-43 proteinopathies. Our findings support the hypothesis that phosphorylation at distinct sites of TDP-43 has impact on the molecular type of AD-related TDP-43 pathology and its relation to FTD symptoms. Clinically, 80% of the AD TDP + CTF cases were characterized by typical AD symptoms with leading amnestic and cognitive decline and only 20% of the cases by language problems indicative for PPA. The AD TDP + FL cases presented typical AD symptoms in 63,6% and FTD-like symptoms, such as behavior or language problems in 36,4%.
Morphologically, the majority of TDP-43 lesions in AD TDP + CTF cases consisted of large NCIs, few DNs and NFT-like structures, consistent with previous studies [26,39]. On the other hand, AD TDP + FL cases displayed abundant DNs and NCIs, as well as NFT-like structures in some cases, which were particularly predominant in temporal and frontal cortices. Of note, we observed no strict association to a specific FTLD-TDP subtype (A-D) as defined by Mackenzie et al [39], considering that there was morphological variability in the AD TDP + FL cases as in FTLD-TDP. Moreover, 4 out of the 11 AD TDP + FL cases did not fit into any FTLD-TDP subtype. These cases displayed large, TDP43-positive NCIs and few DNs scattered among all layers of the temporal cortex and were clinically AD cases. Nevertheless, all AD TDP + FL cases presented a common molecular pattern, which was the focus of our study. Importantly, this pattern was also observed in the typical FTLD-TDP cases. Moreover, Josephs et al. [26] recently studied the morphology of TDP-43 lesions in non-FTLD cases. They identified two morphological signatures for their cases: one related to NCIs and DNs distributed widespread over the brain including frontal cortex (type α), and another related to NFT-associated TDP-43 inclusions, restricted to the medial temporal lobe (type β). Our results corroborate these data in the sense that all AD TDP + CTF and 45,4% of AD TDP + FL cases display type β, whereas the remaining 54,5% of the AD TDP + FL cases present features of both types, α and β, considering that they exhibited not only dystrophic neurites and NCIs in a widespread distribution, but also NFT-associated neuronal inclusions. Thus, we extend this knowledge by distinguishing distinct molecular patterns of TDP-43 species, with the 'TDP + CTF' pattern being significantly associated to type β whereas type α features were restricted to a subset of AD TDP + FL cases. Interestingly, these AD TDP + FL cases with a type α TDP-43 pattern were those to show FTD symptoms in 57% of the cases whereas none of our AD TDP + FL cases without type α features, i.e. without frontal or temporal TDP-43 pathology, exhibited signs of FTD. Thus, we can conclude that TDP-43 pathology in AD can cause FTD symptoms when TDP-43 lesions extent into the cortex. This argues strongly in favor of similar underlying processes in FTLD-TDP and AD TDP + FL with cortical TDP-43 pathology, probably to coexisting AD and FTLD-TDP in AD TDP + FL cases. Interestingly, 4 AD TDP + FL cases that were considered as Josephs' type β were symptomatically AD. In turn, Josephs' type α was statistically associated with FTD symptoms, which makes it tempting to speculate that type α may play a role in the FTD symptomatology, however more investigation regarding the association of types α and β with clinical symptoms needs to be done.
A few non-AD controls, as well as cognitively normal p-preAD cases also presented TDP-43 proteinopathy, consistent with other studies that observed TDP-43 pathology in cognitively normal individuals [3,40,45,67], which was associated with PART [28,68]. The TDP-43 molecular pattern in these cases was similar to that seen in AD TDP + CTF cases, as well as the morphology of these lesions, which consisted of NFTassociated material, reflecting Josephs' type β TDP-43 pathology.
Overall, these results point to a potential difference in the mechanism of TDP-43 proteinopathy between AD TDP + CTF and AD TDP + FL , with the latter similar to that of FTLD-TDP. Our results also indicate that Cterminal fragments (CTFs) of TDP-43 are enriched in AD TDP + CTF , consistent with other studies [22]. Such mechanisms could be related to different kinases phosphorylating serines 403/404 and 409/410, considering that the pattern of phosphorylation at these sites distinguishes these groups of cases. Alternatively, genetic differences could also explain the distinct neuropathological TDP-43 patterns [26,47]. Interestingly, one case in the AD TDP + FL group had a mutation in an FTLD-TDP causing gene, i.e. in the C9ORF72 gene [13,55]. This supports our interpretation of the 'TDP + FL' pattern as possibly biologically linked to FTLD-TDP at least in some of these cases probably exhibiting coexisting AD and FTLD-TDP. On the other hand, the C9ORF72 mutation has been previously found in a very low amount of AD cases [17].
A third explanation for the different patterns of TDP-43 pathology in AD cases could be that TDP-43 plays different roles in these patients. In AD, the accumulation of presumably N-terminal truncated pTDP-43 409/410 may represent a secondary event, maybe co-seeded by τ or Aβ, as hypothesized by others [12,20,36]. An argument supporting this hypothesis is that TDP-43 pathology in our control cases occurred in the same anatomical regions, in which PART-lesions (NFTs and . Moreover, they were restricted to limbic regions, including the medial temporal lobe and amygdala. These cases comprised high amounts of pathological Aβ (plaques) and τ NFT pathology. On the other end of the spectrum, the TDP-43 proteinopathy in FTLD-TDP cases consist of a large abundance of DNs and NCIs, that were detected not only with pTDP-43 antibodies, but also C-and N-t-TDP-43 antibodies (C-t-TDP-43 + and N-t-TDP-43 + , respectively). Moreover, these lesions were widespread in the brain. In addition, in these cases TDP-43 was phosphorylated in S403/404 residues, in addition to S409/S410. Similar to AD TDP + CTF cases, AD TDP + FL cases had high amounts of Aβ and p-τ. However, they comprised an FTLD-TDP-like molecular pattern, which mostly consisted of NCIs, DNs and NFT-like lesions that comprise the full-length protein with phosphorylation of S403/404 in addition to S409/410, and that are widespread in the brain. AD TDPcases were clinically AD. AD TDP + CTF cases mostly presented a clinical AD phenotype, with some cases presenting svPPA or additional behavioral deficits. Most AD TDP + FL cases presented symptomatic AD, with some cases presenting additional behavioral or language problems and other cases presenting bvFTD. FTLD-TDP cases presented a clinical picture of FTD neuropil threads) were co-existing. The morphological appearance of the TDP-43 lesions in AD TDP + CTF cases as NFTs may also argue for a secondary phenomenon induced by the underlying τ pathology [1,60]. Nonspecific detection of NFTs by anti-TDP-43 409/410 antibodies has also been discussed [38]. However, in our study three different antibodies against pTDP-43 409/410 , including a monoclonal antibody, labelled NFTs, arguing against non-specific staining. Furthermore, all of our AD TDP + CTF cases had high amounts of τ protein pathology in the frontal cortex, but no anti-pTDP-43 409/410 or anti-pTDP-43 409 positive material, which also argues against non-specific labelling of anti-pTDP-43 antibodies in the hippocampus or the amygdala. This is strengthened by the reports of other authors that NFT-like material can be detected with non-phosphorylated anti-TDP-43 antibodies [1,26], which suggests a strong association between τ and TDP-43. On the other hand, one may speculate that TDP-43 acts as the primary pathology in AD TDP + FL cases, similarly to FTLD-TDP. In light of these arguments, it is tempting to speculate that both secondary accumulation of TDP-43 and primary TDP-43 pathology may occur in AD cases: Secondary accumulation of pTDP43 409/410 and pTDP-43 409 -positive material in AD TDP + CTF cases and primary development of TDP-43 aggregates in AD TDP + FL cases.
There has been growing evidence regarding the existence of concomitant neuropathologies, in which a neurodegenerative disease might have additional aggregated proteins besides the primary pathology, accumulating as co-pathologies [4,15,32,57,63]. AD TDP + FL cases described in this study appear to be an example of this, as they present a molecular pattern of TDP-43 pathology similar to that seen in FTLD-TDP cases, as well as histological full-blown AD pathology (as observed with antibodies against Aβ and τ). Furthermore, 7 of these 11 cases presented a morphological and topographic distribution of TDP-43 compatible with one of the FTLD-TDP subtypes (A-C). This stresses the importance of considering multiple pathologies contributing to the development of dementia, as seen in previous studies [52]. The recent consensus work regarding LATE might also be important in this context [47]. LATE-NC was described as the presence of TDP-43 pathology in the limbic areas of elderly, with or without co-existing AD pathology. As seen here, a few non-AD controls, the majority of p-preAD and all AD TDP + CTF , as well as AD TDP + FL cases fit into this new classification ( Table 2, Additional file 1- Table A1). The controls, p-preAD, and AD TDP + CTF cases that exhibited LATE-NC, mostly presented LATE-NC stage 2, which means the TDP-43 pathology is predominantly present in amygdala and hippocampus. On the other hand, 54,5% of AD TDP + FL cases had additional TDP-43 lesions in the frontal cortex, indicative for LATE-NC stage 3. Of note, 19,6% of all neuropathologically-confirmed AD cases did not present TDP-43 pathology (i.e LATE-NC) at all, consistent with other studies [44,47]. Interestingly, the p-preAD cases in our cohort showed a high prevalence of TDP-43 pathology, reflected in the 68,7% of cases with LATE-NC stage 1 or stage 2. Given the hierarchical detectability of first pTDP-43 409 and pTDP-43 409/410 in cases with LATE-NC stages 1-2 and second other TDP-43 epitopes in AD TDP + FL cases with LATE-NC stages 2 and 3, one could hypothesize that TDP-43 aggregates in AD undergo maturation changes similar to Aβ aggregates [56]. An argument against this hypothesis is that both AD-related τ and Aβ pathology showed late stages of AD pathology in AD TDP-, AD TDP + CTF and AD TDP + FL cases, and that there was no significant difference in age among these groups. Secondly, the identification of one AD TDP + FL case with a C9ORF72 gene mutation strongly suggests a specific FTLD-like influence of TDP-43 pathology in at least a subset of these cases and, thereby, also argues against a simple maturation process of TDP-43 aggregates that distinguishes AD TDP + CTF and AD TDP + FL cases. In our opinion, it is therefore likely that the different molecular patterns of TDP-43 pathology among AD cases may constitute subtypes of LATE-NC or alternatively argue in favor of spectrum of AD ranging from AD TDPwithout TDP-43 pathology to AD TDP + FL with probably coexisting FTLD pathology. However, the LATE concept is controversial and further investigation addressing the morphology of these lesions and its relationship to the molecular and clinical patterns is needed. Nevertheless, if these patterns constitute subtypes of LATE-NC, the umbrella term 'LATE-NC' could include different molecular types of TDP-43 lesions that give rise to the hypothesis that LATE-NC covers biologically different lesions/diseases: 1. TDP-43 pathology with predominance of phosphorylated C-terminal fragments of TDP-43 as in AD TDP + CTF , 2. full-length TDP-43 pathology as in AD TDP + FL similar as in FTLD-TDP, 3. hippocampal sclerosis, and maybe others that we are not yet aware of. Whether the full-length TDP-43 predominant pattern in AD TDP + FL cases distinguishes co-existing FTLD-TDP pathology from the C-terminal fragmentpredominant lesions, needs to be clarified in the future.
Importantly, the 'TDP + CTF' molecular pattern (observed in some non-AD, most p-preAD and AD TDP + CTF cases) was significantly associated with typical AD symptoms, whereas the 'TDP + FL' molecular pattern (observed in AD TDP + FL and FTLD-TDP cases) was associated with symptoms of the FTD spectrum, especially in those AD TDP + FL cases that exhibit TDP-43 lesions in the frontal and/or temporal neocortex, i.e. representing the type α TDP-43 distribution pattern (Fig. 8). This suggests that the molecular profile of TDP-43 species in these cases together with the cortical distribution of the lesions influences the clinical presentation of the disease to a certain degree. However, typical AD symptoms were seen in more than 50% of the cases in both AD subgroups. This indicates that the 'TDP + FL' molecular pattern had impact on less than 50% of the cases with various phenotypes typical of the FTD spectrum (36,4%). Interestingly, in the AD TDP + CTF group, less than 20% of the cases showed other symptoms besides cognitive deficits and executive dysfunction. These were related to language alterations in 16,6% of the cases, with only 1 case showing additional behavioral changes.
AD and LATE-NC can occur in the same individuals and both are associated with cognitive decline and amnestic symptoms, whereas FTLD-TDP is characterized by alterations in behavior and/or language [47]. It has been shown that the co-existence of LATE-NC and AD pathology is associated with clinically more severe symptoms than pure AD (in the absence of TDP-43 pathology, i.e. LATE-NC) [37]. Indeed, TDP-43 has been demonstrated to worsen cognition in aged individuals [29,31,46,53], probably due to a synergistic effect with τ protein [37,60]. The AD TDP + FL cases in our cohort presented a broad range of clinical phenotypes, from a primary amnestic deficit (typical for AD) to personality and language changes (typical for FTD). This has impact on the clinical differential diagnosis of degenerative dementing disorders. On the one hand, cases presenting a clinical phenotype of the FTLD spectrum may display significant levels of AD pathology, as seen in our results, which might be associated with positive AD biomarkers. This is also valid for those AD TDP + CTF cases that presented a svPPA phenotype. On the other hand, cases exhibiting a typical AD clinical phenotype and biomarker profile might present a widespread FTLD-TDPlike TDP-43 pathology and molecular pattern as well. Thus, AD-related treatments might possibly be less effective than expected in these cases. This may also have implications for clinical praxis, in the sense that the screening for AD biomarkers in cases with a clinical picture of FTD might be relevant. This would avoid missing AD lesions in AD TDP + FL cases. Overall, our data highlight the relevance of underlying pathologies for the diagnosis and treatment of patients.
One limitation of this study is that the post-mortem intervals of the cases in our cohort were variable (24 to 120 h). Furthermore, the fixation time was also variable (2-4 weeks) due to the fact that the sample consists of several hospital-based cohorts, in which the tissue was acquired at different time-points. However, we did not observe obvious differences in the TDP-43 staining quality among our cases, indicating that these limitations had no significant impact on our results. Another limitation is that there is not a standard TDP-43 antibody recommended to characterize TDP-43 proteinopathies. To overcome this problem, we chose several commercially available, phosphorylation dependent and independent antibodies to provide reliable results. In addition, the number of AD TDP + FL and FTLD-TDP cases was limited (eleven and ten, respectively). The difference in age distribution among the groups and the use of hospital-based cohorts may be considered as another limitation of this study. Logistic regressions controlling for age at death showed that especially controls and FTLD-TDP cases were younger compared to the AD group. This could be due to the low number of cases. However, the younger age of death of control cases free of any AD pathology can be expected, as in older individuals we often observe AD-related pathology. Therefore, only few cases at higher ages may serve as non-AD controls [8,62], leading to the selection of younger cases as controls compared to older ones with full-blown AD. To take this age difference into account in our analyses, we included age and sex in logistic regression models as additional independent variables. Finally, the low number of cases with distinct clinical phenotypes in the AD TDP + FL group is another limitation. To determine the impact of this limitation, we performed a post-hoc power analysis for our data and obtained a statistical power of ≥80% when analyzing the different TDP-43 antibody patterns among the disease groups, as well as other neuropathological parameters (i.e.: Braak NFT-stages, AβMTL phases, CERAD score, etc). For the clinical parameters, we had a statistical power of 48-87% indicating that the interpretation of our results is solid for the neuropathological groups and parameters. Conclusions about clinical phenotypes/parameters, on the other hand, must be considered with caution. Since we did not have a community-based cohort of cases, we cannot exclude influence of the hospital-based sampling on our results. However, the prevalence of TDP-43 pathology in elderly individuals of community-based samples in the literature showed a similarly high prevalence of TDP pathology in the brain [24,40].
In conclusion, we were able to identify molecular differences in TDP-43 pathological lesions, distinguishing distinct patterns of TDP-43 pathology in neuropathologically-confirmed AD cases, one of which being similar to the pattern observed in FTLD-TDP. These patterns differed in the prevalence of truncated and non-truncated TDP-43 species and of distinct phosphorylation epitopes. Furthermore, we showed that these patterns were associated with the frequency of FTD symptoms. These differences may have an impact for future diagnostic algorithms and treatment of patients with clinical signs of dementia. Therefore, underlying pathologies need to be considered when diagnosing and consequently treating demented patients. Whether these molecular patterns of TDP-43 pathology represent types of LATE-NC and, thereby, are features of one disease entity (LATE) or whether they represent a spectrum of late-life neuropathologies in AD ranging from AD TDPto AD TDP + CTF and finally AD TDP + FL with molecular similarities to FTLD-TDP needs to be explored in the future.
Additional file 1: Figure A1. -pTDP-43 409/410 species are predominant in p-preAD and AD TDP + CTF whereas AD TDP + FL and FTLD-TDP display positivity for all pTDP-43 epitopes in several regions. Immunohistochemistry of a non-AD, p-preAD, AD TDP + CTF , AD TDP + FL and FTLD-TDP case in dentate gyrus (a,d,h), temporal (b,e,i) and frontal (c,f,j) cortices with (a1-c5) pTDP-43 409/410 (clone 1D3), (d1-f5) pTDP-43 409 and (h1-j5) pTDP-43 403/404 , displaying lesions (arrowheads). P-preAD and AD TDP + CTF cases displayed mostly NFT-like inclusions with pTDP-403 409 in temporal cortex (e2-e3). AD TDP + FL and FTLD-TDP cases showed NCIs in the DG (a4-a5 respectively, arrowheads), NCIs and DNs in temporal cortex (b4-b5 respectively, arrowheads) and DNs in frontal cortex (c4-c5 respectively, arrowheads) when stained with all pTDP-43 antibodies. AD TDPcases were not included in this figure because no TDP-43 inclusions were observed. Scale bar = 50 μm. Figure A2. -AD TDP + FL and FTLD-TDP display inclusions positive for non-phosphorylated TDP-43, but not p-preAD or AD TDP + CTF cases. Immunohistochemistry of a non-AD, p-preAD, AD TDP + CTF AD TDP + FL and FTLD-TDP case in dentate gyrus (a, d), temporal (b, e) and frontal (c, f) cortices with C-and N-t TDP-43, displaying cytoplasmic lesions (arrowheads) and nuclear clearance (arrows). AD TDP + FL and FTLD-TDP cases also displayed NCIs in the DG (d4-d5 respectively, arrowheads) with clearance of normal C-t-TDP-43 from the nucleus (a4-a5, arrows), DNs in the temporal cortex (b4-b5 respectively, arrowheads) and DNs in frontal cortex (c4-c5 respectively, arrowheads) when stained with C-t-TDP-43. Finally, AD TDP + FL and FTLD-TDP cases showed NCIs in the DG (d4-d5 respectively, arrowheads) with clearance of normal N-t-TDP-43 from the nucleus (arrows), DNs in the temporal cortex (e4-e5 respectively, arrowheads) and DNs in the frontal cortex (f4-f5 respectively, arrowheads) when stained with N-t-TDP- 43 Nonparametric, Friedman test for related samples with Bonferroni correction for multiple testing was used to compare the amount of positive cases of each TDP-43 antibody. Each group was analyzed separately, *p < 0.05, **p < 0.01. Non-demented cases included the cases from the non-AD and p-preAD neuropathological groups (n = 36). SympAD cases referred to cases with exclusive AD symptoms (including AD TDP + CTF , AD TDPand some AD TDP + FL cases, n = 40), AD/FTD refer to cases with both AD and FTD signs (n = 5) and FTD group represents cases with an exclusive FTD presentation (n = 15).  . Table A6percentage of positive cases for C-t-TDP-43. Table A7-percentage of positive cases for N-t-TDP-43. Table A8 a -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDP + FL regarding Braak NFT-staging, when controlled for age and sex. b -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDPregarding Braak NFT-staging, when controlled for age and sex. c -Binary logistic regression addressing the differences between AD TDP + FL vs. AD TDPregarding Braak NFT-staging, when controlled for age and sex. * after Bonferroni correction for multiple testing. Table A9 a -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDP + FL regarding AβMTL phase, when controlled for age and sex. b -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDPregarding AβMTL phase, when controlled for age and sex. c -Binary logistic regression addressing the differences between AD TDP + FL vs. AD TDPregarding AβMTL phase, when controlled for age and sex. * after Bonferroni correction for multiple testing. Table A10 a -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDP + FL regarding CERAD score, when controlled for age and sex. b -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDPregarding CERAD score, when controlled for age and sex. c -Binary logistic regression addressing the differences between AD TDP + FL vs. AD TDPregarding CERAD score, when controlled for age and sex. * after Bonferroni correction for multiple testing. Table A11 a -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDP + FL regarding NIA-AA degree of AD pathology, when controlled for age and sex. b -Binary logistic regression addressing the differences between AD TDP + CTF vs. AD TDPregarding NIA-AA degree of AD pathology, when controlled for age and sex. c -Binary logistic regression addressing the differences between AD TDP + FL vs. AD TDPregarding NIA-AA degree of AD pathology, when controlled for age and sex. * after Bonferroni correction for multiple testing. Table A12 -Binary logistic regression addressing the association of the TDP + CTF pattern with types α and β, controlled for age at death and sex. Cases with type α + β were considered as positive for type α as well as for type β, respectively. Table A13 -Binary logistic regression addressing the association of the TDP + FL pattern with types α and β, controlled for age at death and sex. Cases with type α + β were considered as positive for type α as well as for type β, respectively. Table A14 -P values of comparisons between TDP-43 antibodies for non-demented cases, n = 36. Friedman test with Bonferroni correction for multiple testing. Abbreviations; NBM: basal nucleus of Meynert, DG: dentate gyrus. Table A15 -P values of comparisons between TDP-43 antibodies groups for symptomatic AD cases, n = 33. Friedman test with Bonferroni correction for multiple testing. Abbreviations; NBM: basal nucleus of Meynert, DG: dentate gyrus. Table A16 -P values of comparisons between TDP-43 antibodies groups for AD/FTD cases, n = 5. Friedman test with Bonferroni correction for multiple testing. Abbreviations; NBM: basal nucleus of Meynert, DG: dentate gyrus. Table A17 -P values of comparisons between TDP-43 antibodies groups for FTD cases, n = 15. Friedman test with Bonferroni correction for multiple testing. Abbreviations; NBM: basal nucleus of Meynert, DG: dentate gyrus. Table  A18 -Multinomial logistic regressions addressing the differences between disease groups, for pTDP 409/410 , in CA1 region. Symptomatic AD cases were considered as the reference category for comparison. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Table A19 -Multinomial logistic regressions addressing the differences between disease groups, for pTDP 409 , in CA1 region. Symptomatic AD cases were considered as the reference category for comparison. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Table A20 -Multinomial logistic regressions addressing the differences between disease groups, for pTDP 403/404 , in CA1 region. Symptomatic AD cases were considered as the reference category for comparison. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Table  A21 -Multinomial logistic regressions addressing the differences between disease groups, for C-t TDP-43, in CA1 region. Symptomatic AD cases were considered as the reference category for comparison. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Table A22 -Multinomial logistic regressions addressing the differences between disease groups, for N-t TDP-43, in CA1 region. Symptomatic AD cases were considered as the reference category for comparison. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Table A23 Binary logistic regression addressing the association of Josephs' TDP-43 pathology type α with FTD symptoms, controlled for age at death and sex. Cases with type α + β were considered as positive for type α as well as for type β, respectively. Table A24 -Binary logistic regression addressing the association of Josephs' TDP-43 pathology type β with FTD symptoms, controlled for age at death and sex. Fields were marked with "-" when the software could not assess the statistical value due to overflow. Cases with type α + β were considered as positive for type α as well as for type β, respectively. Table A25 Binary logistic regression addressing the association of FTD symptoms with TDP + CTF pattern, controlled for age at death and sex. Only demented cases were considered. Table A26 -Binary logistic regression addressing the association of typical AD symptoms with TDP + FL pattern, controlled for age at death and sex. Only demented cases were considered.