Concurrent tau pathologies in frontotemporal lobar degeneration with TDP‐43 pathology

Abstract Aims Accumulating evidence suggests that patients with frontotemporal lobar degeneration (FTLD) can have pathologic accumulation of multiple proteins, including tau and TDP‐43. This study aimed to determine the frequency and characteristics of concurrent tau pathology in FTLD with TDP‐43 pathology (FTLD‐TDP). Methods The study included 146 autopsy‐confirmed cases of FTLD‐TDP and 55 cases of FTLD‐TDP with motor neuron disease (FTLD‐MND). Sections from the basal forebrain were screened for tau pathology with phosphorylated‐tau immunohistochemistry. For cases with tau pathology on the screening section, additional brain sections were studied to establish a diagnosis. Genetic analysis of C9orf72, GRN and MAPT was performed on select cases. Results We found 72 cases (36%) with primary age‐related tauopathy (PART), 85 (42%) with ageing‐related tau astrogliopathy (ARTAG), 45 (22%) with argyrophilic grain disease (AGD) and 2 cases (1%) with corticobasal degeneration (CBD). Patients with ARTAG or AGD were significantly older than those without these comorbidities. One of the patients with FTLD‐TDP and CBD had C9orf72 mutation and relatively mild tau pathology, consistent with incidental CBD. Conclusion The coexistence of TDP‐43 and tau pathologies was relatively common, particularly PART and ARTAG. Although rare, patients with FTLD can have multiple neurodegenerative proteinopathies. The absence of TDP‐43‐positive astrocytic plaques may suggest that CBD and FTLD‐TDP were independent disease processes in the two patients with both tau and TDP‐43 pathologies. It remains to be determined if mixed cases represent a unique disease process or two concurrent disease processes in an individual.

FTLD-tau includes progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and Pick's disease [5]. Intracellular aggregates of phosphorylated tau protein in neurons and glia associated with neurodegeneration are pathologic hallmarks of FTLD-tau [5]. Increased age is a common risk factor for neurodegenerative disorders. Age-related changes, such as cellular senescence, mitochondrial dysfunction and epigenetic alterations, have been reported in neurodegenerative processes [6][7][8]; thus, elderly individuals may often develop more than one neurodegenerative disease, with the accumulation of multiple types of pathological protein aggregates [9][10][11][12]. Indeed, concurrent TDP-43 pathology has been reported in a range of tauopathies, including Alzheimer's disease [12,13], PSP [14][15][16] and CBD, [16][17][18][19] as well as synucleinopathies [20][21][22]. Our previous study found that 45% of CBD patients had TDP-43 pathology [19]. Some cases had extensive TDP-43 pathology in the neocortex, which can be considered a mixed FTLD-tau and FTLD-TDP. More recently, Kim et al [23]. reported nine cases of mixed FTLD-TDP and FTLD-tau, in which three unclassifiable FTLD-tau and two PSP cases had a primary diagnosis of FTLD-TDP, while FTLD-TDP was found in four cases with CBD. These studies suggest that FTLD can be caused by coexisting TDP-43 and tau pathologies; however, the frequency and characteristics of tau and TDP-43 copathology have not been investigated.
In the present study, we aimed to determine the frequency and characteristics of tau pathology in a series of cases of FTLD-TDP and FTLD-MND that were considered 'primary' TDP-43 proteinopathies as their original neuropathologic diagnosis. To do this, we screened tau pathology in 146 patients with FTLD-TDP and 55 patients with FTLD-MND. All cases were from the Mayo Clinic brain bank for neurodegenerative disorders.

Case selection
This study included 201 cases with FTLD-TDP with MND (N = 55) or without MND (N = 146) from 1998 to 2020. All brain autopsies were performed after consent of the legal next of kin or individual with power of attorney to grant permission. Studies of autopsy samples are considered exempt from human subjects research by Mayo Clinic Institutional Review Board.

General neuropathologic assessment
Formalin-fixed hemibrains underwent systematic and standardised sampling with neuropathologic evaluation by a single, experienced neuropathologist (D.W.D.). The whole brain weight was estimated by multiplying by two the weight of the available hemibrain. Regions sampled in all cases included six regions of the neocortex, two levels of the hippocampus, a basal forebrain section (including the amygdala, lentiform nucleus and hypothalamus), corpus striatum at the level of the nucleus accumbens, thalamus at the level of the subthalamic nucleus, midbrain, pons, medulla and two sections of the cerebellum, one including the deep nuclei. Paraffin-embedded 5-μm thick sections mounted on glass slides were stained with haematoxylin and eosin (H&E) and thioflavin S (Sigma-Aldrich, St. Louis, MO). Braak neurofibrillary tangle stage (NFT), Thal amyloid phase and severity of cerebral amyloid angiopathy were assigned using thioflavin S fluorescent microscopy according to previously described methods [24][25][26][27][28].
• Tau pathology consistent with corticobasal degeneration (CBD) was detected in two FTLD-TDP patients.
• Both TDP-43 and tau should be included in neuropathologic assessment of FTLD.
FTLD-TDP or FTLD-MND. The neuropathologic diagnosis of FTLD-MND required motor neuron loss with Bunina bodies and a variable degree of corticospinal tract degeneration, demonstrated with myelin stains (Luxol fast blue-periodic-Schiff) and immunohistochemistry for activated microglia (IBA-1, rabbit IG, 1:3,000, Wako Chemicals, USA [29]). Hippocampal sclerosis was diagnosed when neuronal loss and gliosis were selective in the CA1 sector and/or subiculum of the hippocampus without other pathologic findings that could account for neuronal loss in this region.

Screening of tau pathologies
We immunostained 5-μm-thick sections of the basal forebrain section using a phosphorylated-tau antibody (phosphorylated-tau Ser202, CP13; mouse monoclonal; 1:1,000; a gift from the late Dr A diagnosis of CBD was made based on the presence of astrocytic plaques and numerous tau-positive threads in the grey and white matter in cortical and subcortical regions [30]. A diagnosis of argyrophilic grain disease (AGD) required tau-positive (argyrophilic) grains in medial temporal lobe structures (i.e., amygdala and hippocampus), accompanied by pretangles, coiled bodies, balloon neurons and granular/fuzzy astrocytes or bush-like astrocytes [31].
Silver stains (Gallyas) were used to confirm the diagnosis of AGD. For cases with AGD in the amygdala, additional sections from the hippocampus, entorhinal cortex, inferior temporal gyrus and cingulate gyrus were stained with tau immunohistochemistry and assessed to assign an AGD stage according to Saito et al [32]. The diagnosis of ageing-related tau astrogliopathy (ARTAG) was associated with variable thorn-shaped astrocytes or granular/fuzzy astrocytes in subependymal, subpial, perivascular, grey matter and white matter [33].

Clinical assessment
Clinical information was abstracted by two investigators (SK and AM) from the available medical records and brain bank questionnaires filled out by a close family member. The information included the age at symptom onset, disease duration, age at death, clinical diagnosis, clinical symptoms, neurological signs and family history of dementia or parkinsonism.

Genetic analysis
We performed genetic analyses in two patients with FTLD-TDP and CBD. For genotyping, genomic DNA was extracted from frozen cerebellum tissue using standard procedures. MAPT H1/H2 haplotype (SNP rs1052553 A/G, A = H1, G = H2) was assessed with TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). Genotype calls were obtained with QuantStudio™ Real-Time PCR Software (Applied Biosystems). MAPT sequencing was performed in exons 7 and 9-13, as well as known pathogenic intronic mutations located at 50 bp on either side of each exon (e.g., IVS10 + 16 C > T). GRN sequencing and screening for C9orf72 hexanucleotide repeat expansion were performed as previously described [34,35].

Statistical analysis
All statistical analyses were performed using R 4.1.1. Fisher's exact test was performed for group comparisons of categorical data, as appropriate. Mann-Whitney rank-sum test, analysis of variance (ANOVA) on ranks, followed by Steel-Dwass post hoc test, or oneway ANOVA, followed by post hoc Tukey test, was used for analyses of continuous variables as appropriate. P values <0.05 were considered statistically significant.

Summary of the cohort
The study set included 146 patients (84 men and 62 women) of FTLD-TDP and 55 patients (30 men and 25 women) of FTLD-MND (Table 1). The average age at death was significantly older in FTLD-TDP than in FTLD-MND (74 AE 10 vs. 67 AE 9 years; p < 0.001). FTLD-TDP also had a significantly longer disease duration than FTLD-MND (9 AE 5 vs. 4 AE 2 years; p < 0.001). The average formalin-fixed brain weight was significantly lower in FTLD-TDP than in FTLD-MND (970 AE 160 g vs. 1120 AE 170 g; p < 0.001). Alzheimer-type pathologies, measured by the Braak NFT stage and Thal amyloid phase, were not significantly different between the two groups.
Alzheimer's-type pathology assessed by thioflavin S microscopy

Frequency of tau pathologies in TDP-43 proteinopathies
The second most common concurrent tau pathology was ARTAG, which was detected in 85 cases (42%) ( Table 1). Subpial/ subependymal type (36%) was the most frequent, followed by perivascular type (27%). On tau immunohistochemistry, thorn-shaped astrocytes were detected in subpial ( Figure 1A) and perivascular spaces ( Figure 1B) in the mediobasal forebrain and variably in the amygdala ( Figure 1C). Thorn-shaped astrocytes were also observed in the peri-amygdaloid white matter ( Figure 1D). The age at death was significantly older in cases with ARTAG than in those without ARTAG (76 AE 9 vs. 69 AE 10 years; p = 5.7 Â 10 À7 ), regardless of the disease group (i.e., FTLD-TDP, FTLD-MND; Figure 1E) AGD was detected in 45 cases (22%). Argyrophilic grains in the amygdala were accompanied by pretangles, coiled bodies, balloon neurons and granular/fuzzy astrocytes ( Figure 1F-I). Screening of additional regions revealed that 23 cases had AGD restricted to the amygdala (stage 1), 15 also had AGD in the entorhinal cortex or subiculum (stage 2) and in 7 cases had pathology in the cingulate gyrus (stage 3). As with ARTAG, the age at death was significantly older in cases with AGD compared to cases without AGD (77 AE 10 vs. 71 AE 10 years; p = 9.1 Â 10 À4 ). As shown in Figure 1J, the median age at death was highest in stage 3, followed by stages 2 and 1. A multivariable logistic regression model adjusting for age, sex, Braak NFT stage and Thal amyloid phase revealed that older age (OR 1.05; CI 1.01-1.09; p = 0.017) and higher Braak NFT stage (OR 1.33; CI 1.03-1.71; p = 0.026) were independent risk factors for AGD.
We found two patients with tau pathology consistent with CBD (Table 2). Immunohistochemistry for tau revealed astrocytic plaques in the superior frontal gyrus and premotor cortex, tau-positive threads and coiled bodies in the adjacent white matter, and pretangles and threads in the subthalamic nucleus, pontine base, inferior olivary nucleus and cerebellar dentate nucleus in both patients (Table 3)

CASE 2
The fixed left hemibrain weighed 540 g. Macroscopic evaluation of the fixed brain revealed severe cortical atrophy over the dorsolateral and medial frontal lobe, including the frontal pole and the orbital frontal lobe ( Figure 3A,B). The medial temporal lobe had moderate atrophy and the parietal lobe had mild atrophy in the superior lobule. The anterior corpus callosum was markedly thinned ( Figure 3C). The hippocampal formation and amygdala were both atrophic, especially in the subiculum. Basal ganglia showed severe atrophy of the caudate nucleus and attenuation of the anterior limb of the internal capsule ( Figure 3C). The globus pallidus was markedly atrophic and had brown discolouration. The anterior thalamus was atrophic. The substantia nigra had marked loss of pigment ( Figure 3D).

DISCUSSION
In this series of 201 autopsy cases of TDP-43 proteinopathies, many patients had concurrent tau pathologies. Not unexpectedly, ARTAG (42%) and PART (36%) were the most frequent tau pathologies, followed by AGD (22%). In addition, we found two patients with CBD, which is a four-repeat tauopathy form of FTLD-tau. TDP-43 and tau are the most common molecular subtypes of FTLD, but the coexistence of FTLD-TDP and FTLD-tau is uncommon [23]. These cases raise the issue of which should be considered the 'primary' pathologic process and which should be considered the 'secondary' process.
Case 1 was clinically diagnosed with familial AD based upon his cognitive impairment, initially characterised by amnestic type dementia, as well as dementia in multiple family members. Neuropathologic assessment revealed mild Alzheimer's-type pathology (Braak NFT stage III and Thal amyloid phase 3) insufficient to account for dementia. Although the cortical atrophy of the frontal and temporal lobes was relatively mild, immunohistochemistry for TDP-43 and presence of C9RANT inclusions were diagnostic of FTLD-TDP [38]. A hexanucleotide repeat expansion in the C9orf72 gene was confirmed with repeat-primed polymerase chain reaction assay [35]. The presence of a pathogenic mutation in a gene for FTLD makes a strong case for the primary diagnosis in the case to be FTLD-TDP. Of note, amnestic Alzheimer's dementia is a common clinical diagnosis of genetically confirmed FTLD-TDP in elderly individuals [39,40], and in an autopsy series from the State of Florida brain bank, late onset patients with C9orf72 mutations often present with Alzheimer type dementia or Lewy body dementia [41].
Tau pathology in this patient was consistent with CBD based upon morphology and neuroanatomical distribution; however, tau pathology was mild and subcortical nuclei vulnerable to neuronal loss in CBD, such as the globus pallidus and substantia nigra, were well preserved. Moreover, the severity of tau pathology was mild in subcortical regions. These findings are like those reported as 'preclinical' CBD [42]. In the current situation, the term 'preclinical' is not appropriate since the patient presented with cognitive impairment, behavioural changes and parkinsonism. Taken together, genetically confirmed FTLD-TDP with 'incidental' CBD seems to be the best neuropathologic diagnosis.
Interestingly, this patient is like a patient in the study of Kim and co-workers. Their patient had FTLD-TDP type A and unclassifiable FTLD-tau with C9orf72 mutation (case 1) [23]. Given that C9orf72 mutation is the most common risk factor for familial amyotrophic lateral sclerosis and FTLD, its strong association with TDP-43 pathology has been established. In contrast, it is still unknown whether C9orf72 mutation is also associated with FTLDtau. Bieniek et al [43]. investigated Alzheimer's-type tau pathology in the temporal cortex and hippocampus in patients with FTLD who carried C9orf72 mutation (c9FTLD) and found that tau pathology burden was not different between c9FTLD and sporadic FTLD.
Snowden et al [44]. screened for C9orf72 mutations in 398 patients with clinical presentations of frontotemporal dementia and found one patient who had CBD pathology without TDP-43 pathology.  The present study needs to consider the fact that TDP-43 pathology can be found in a subset of CBD cases. Our previous study identified astrocytic plaque-like TDP-43 lesions in the motor cortex and superior frontal gyrus in CBD patients with TDP-43 pathology. Therefore, we assumed that TDP-43 pathology was 'secondary' to CBD pathology [19]. In contrast, the two patients in the present study did not have astrocytic plaques with TDP-43 immunoreactive processes.
This finding supports the conclusion that TDP-43 pathology occurs independently from tau pathology in CBD, not 'secondary' to tau pathology.
A limitation of this study is that the clinical information of two patients with FTLD-TDP and CBD was limited due to the retrospective nature of the autopsy cohort. Systematic, longitudinal data with neurological examinations and neuropsychological testing were not available. Motor symptoms suggestive of corticobasal syndrome may have been overlooked or not well documented. Another limitation is that we did not assess clinical features in patients with PART, ARTAG or AGD because the primary focus was FTLD-tau, rather than these 'age-related' tau pathologies.
In conclusion, by screening for tau pathology in TDP-43 proteinopathies, we identified two patients with mixed FTLD-TDP and CBD that are likely independent ('co-primary') disease processes.
Many of elderly individuals with neurodegenerative disorders have multiple coexisting pathologies [11,12]; therefore, both TDP-43 and tau should be part of the screening process for the neuropathologic assessment of FTLD. We are not able to determine the relative contribution of each pathology to the clinical presentations in these patients. This is not too dissimilar to the issue of assigning relative contributions of mixed pathology in other settings, for example, cases with both Alzheimer's disease and diffuse Lewy body disease. Implementation of molecular imaging modalities for each protein may help determine the timing and relative contributions of the proteins to clinical presentations.