Sequential distribution of pTDP-43 pathology in behavioral variant frontotemporal dementia (bvFTD)

Tissue preparation, staining, and immunohistochemistry

Pathology was examined in the following regions of the central nervous system (CNS): middle frontal gyrus, orbital gyri, gyrus rectus, agranular motor cortex (Brodmann areas 4 and 6), sensory cortex, superior or middle temporal gyrus, visual cortex (Brodmann areas 17 and 18), anterior cingulate gyrus, hypothalamus, amygdala, hippocampal formation, striatum and pallidum, thalamus, midbrain (including the substantia nigra and red nucleus), pontine tegmentum at the level of the locus coeruleus, lower pons (including the motor nucleus of cranial nerve VII), medulla oblongata at level of the hypoglossal nucleus (XII), cerebellum (including both a portion of the cerebellar cortex and dentate nucleus), and cervical spinal cord (Table 2). For nine cases, olfactory bulbs were also available.

After fixation, tissue samples were embedded in paraffin using standardized cassettes, sectioned at 6–7 μm, and stained with hematoxylin and eosin (HE) and for immunohistochemistry (IHC) as described elsewhere [14, 15, 32, 33]. Briefly, IHC was performed with antibodies to hyper-phosphorylated tau (monoclonal antibody PHF1; 1:1,000, gift from Dr. Peter Davies), α-synuclein (monoclonal antibody Syn303; 1:4,000, generated in CNDR) [24], pTDP-43 (rat antibody p409/410, 1:1,000, gift from Dr. Manuela Neumann) [75], and amyloid-β (monoclonal antibody NAB228; 1:15,000; generated in CNDr) [58]. Neurofibrillary (NF) tangle stages and CERAD neuritic plaque (NP) scores are shown in Table 2. Double-labeling IHC was also performed on selected 70 μm sections using the pTDP-43 antibody above together with the Vector Sg (SK-4700, Vector) blue chromogen and either the SMI-311 monoclonal antibody (1:1,000, Covance, Princeton, NJ, USA), the rabbit polyclonal MAP2 antibody (1:500, Millipore) or the NFH 200 monoclonal antibody (1:300, Sigma-Aldrich, St. Louis, MO, USA) with the brown chromogen 3,30-diamin-obenzidine tetrahydrochloride (DAB).

To study each of the CNS regions in all 60 cases in greater neuroanatomical detail, two additional sets of 70 μm sections were prepared as described previously [26] from the same paraffin blocks used immediately above as done earlier for ALS staging [14]. This thick section technique is performed on free-floating sections. It permits recognition of specific cell types and cytoarchitectonic units (subcortical nuclei and cortical layers) owing to the visualization of large numbers of biological structures and it allows accurate assessment of pathological changes, e.g., those associated with synucleinopathies, tauopathies, and pTDP-43 proteinopathies [26]. The first set of 70 μm sections was stained with aldehyde fuchsin and Darrow red for topographical orientation and evaluation of neuronal loss (lipofuscin pigment deposits, basophilic Nissl material). pTDP-43 pathology was analyzed in the second set of 70 μm sections using a commercially available 409/410 rabbit polyclonal antibody (1:10,000, Cosmo Bio, Carlsbad, CA, USA) and assessed according to a semiquantitative rating scale (0, none/not detectable or ≤2 aggregates per region; +, mild; ++, moderate; 3, +++severe/numerous in Table 2). In assigning cases a semiquantitative score, the extent (topographic distribution pattern) was assigned more weight than the degree of pTDP-43 pathology.

Genetic analysis

Genomic DNA was extracted from peripheral blood or brain tissue following the manufacturer's protocols (Flexi-gene Qiagen, Valencia, CA, USA) or Quick Gene DNA whole blood kit L (Autogen, Holliston, MA, USA) for blood, and QIA symphony DNA Mini Kit (Qiagen) for brain for all available cases (58/60). DNA sequencing of the entire coding region of GRN (n = 58) was performed in selected cases by Beckman Coulter Genomics, as described [105, 111]. Data were analyzed with Mutation Surveyor software (Soft Genetics, State College, PA, USA). C9orf72 hexanucleotide repeat expansions (defined as >30 repeats) were tested (n = 57) with repeat-primed PCR and fragment analysis, as described [16].

Four regional distribution patterns of pathology in bvFTLD-TDP

We analyzed the regional distribution patterns of pTDP-43 in individuals who presented as bvFTD (Table 2). These cases presented mainly as cFTLD (n = 32, 82.1 %, Suppl. Fig. 2), whereas nFTLD was much less frequent (n = 7, 17.9 %).

Pattern I (Fig. 5: yellow): cases with the lowest burden of pTDP pathology (Table 2) were characterized by the presence of pTDP-43 immunoreactive inclusions in projection neurons of basal and anterior portions of the prefrontal neocortex (orbital gyri, gyrus rectus, inferior frontal gyrus, Figs. 1, 5a, b, d). Within the prefrontal cortex, pTDP-43 pathology occurred most extensively in projection neurons of neocortical layers II and III and was observed to a lesser extent in small projection neurons of layers V, and VI. pTDP-43 inclusions were also observed in the basolateral subnucleus of the amygdala (Fig. 5e). Neuronal pTDP-43 immunoreactive inclusions were accompanied by TDP-43 positive aggregates in oligodendrocytes.

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Fig. 5

Summary diagram of four regional distribution patterns of pathology in bvFTLD illustrating the hypothesis that pTDP-43 pathology may spread sequentially, possibly propagating (white arrows) along axonal pathways. Preferentially vulnerable cortical and subcortical sites are involved. Pattern I cases with the lowest burden of pTDP pathology display the presence of pTDP-43 immunoreactive inclusions in projection neurons and oligodendrocytes of basal and anterior portions of the prefrontal neocortex (orbital gyri, gyrus rectus, inferior frontal gyrus). Pattern II cases show the same lesion distribution as pattern I, but with pTDP pathology also in prefrontal neocortical areas located further caudal to the frontal pole, including the middle frontal gyrus, insular cortex, and anterior cingulate gyrus, and also in anteromedial areas (transentorhinal region, adjoining allocortical entorhinal region and hippocampal formation), the superior and middle temporal gyri, striatum (accumbens nucleus, putamen, claustrum), parvocellular portion of the red nucleus, medial and lateral portions of the thalamus, and in precerebellar nuclei of the pons and medulla oblongata. Cases with additional pathology beyond that seen in the first two patterns are characterized by the presence of pTDP-43 immunoreactive inclusions in the agranular motor neocortex (Brodmann fields 4 and 6), in parietal neocortical areas (sensory cortex, angular gyrus), in bulbar somatomotor neurons, and in α-motoneruons of the cervical spinal cord anterior horns which is pattern III. Cases with the highest burden of pathology (pattern IV) show the presence of pTDP-43 lesions in the occipital neocortex (visual cortex, Brodmann 17, 18) in addition to involvement of all of the areas involved in patterns I–III. a pc precerebellar nuclei; c XII hypoglossal nucleus, io inferior olive; e ca caudate nucleus, pu putamen, pi internal pallidum, pe external pallidum, fo fornix, cl claustrum, mc mediocentral subnuclei of the bl basolateral subnuclei of amygdala, en entorhinal cortex (en); f pu putamen, md mediodorsal thalamus, rn red nucleus, sn substantia nigra (sn), ca1 Ammon's horn region 1, en entorhinal cortex

Pattern II (Fig. 5: light orange): with increasing burden of pathology, pTDP-43 immunoreactive lesions were visible, in addition to the regions described above, in prefrontal neocortical areas located further caudal to the frontal pole, including the middle frontal gyrus, insular cortex, and anterior cingulate gyrus, and involving the same cortical layers as described above. Furthermore, pTDP-43 pathology was seen in anteromedial areas (transentorhinal region and adjoining allocortical entorhinal region and hippocampal formation, Figs. 2, ​,5f),5f), the superior and middle temporal gyri, striatum, parvocellular portion of the red nucleus of the midbrain, in medial and lateral portions of the thalamus (Figs. 4a, ​,5e),5e), and in precerebellar nuclei of the pons and medulla oblongata (Fig. 5c). Mild pTDP-43 pathology was observed in the anterior olfactory nucleus and olfactory tract of 9/9 cases (Fig. 3a) where the bulb was available.

Within the hippocampal formation, it was particularly the granular cells of the dentate fascia that showed extensive pTDP-43 pathology (Fig. 3b–d), accompanied by inclusions in pyramidal neurons of Ammon's horn (CA), including sectors CA1–CA2 (Figs. 3e, ​,5f).5f). In the entorhinal cortex, pTDP-43 inclusions were mainly observed in layers pre-α, pre-β, and, to a lesser extent, in pri-α (Fig. 3f).

Striatal lesions were seen mainly in the ventral striatum (accumbens nucleus, Fig. 3f, g) but also in the putamen within medium-sized projection neurons. The projection neurons of the claustrum also contained pTDP-43 pathology, whereas inclusions were not observed in the pallidum (Fig. 5e). Representative of the involvement of the precerebellar nuclei [13] was the inferior olivary complex. Within this complex, pTDP-43 inclusions occurred chiefly in circumscribed groups of neurons within ventral portions of the principal olivary nucleus (Fig. 4b), within the dorsal accessory olivary nucleus, and, more infrequently, in the medial accessory olive. Similarly to the inferior olivary complex, pTDP-43 aggregates in the pontine gray matter were seen in isolated groups of neurons, forming an insular pattern. Inasmuch as the neurons in the parvocellular red nucleus also contained pTDP-43 pathology and are known to project to the inferior olivary complex, we assessed the pathology there within the context of the precerebellar nuclei.

Pattern III (Fig. 5: dark orange): cases with additional pathology beyond what was described above were characterized by the presence of pTDP-43 immunoreactive inclusions in the agranular motor neocortex (Brodmann fields 4 and 6), in parietal neocortical areas (sensory cortex, angular gyrus) (Fig. 5b), and in bulbar somatomotor neurons as well as the cervical spinal cord anterior horns. Within the motor cortex, pTDP-43 pathology predominated in projection neurons of layers II and III, but also in the primary motor field in Betz pyramidal cells of layer Vb and in projection neurons of layers V–VI. In the anterior horn of the spinal cord, pTDP-43 pathology was present in α-motoneurons of lamina 9 (Fig. 4c). Notably, oligodendrocytes of the anterior and lateral portions of the corticospinal tract were also pTDP-43-immunoreactive (Fig. 4c). In the lower brainstem, pTDP-43 pathology was present bilaterally in the hypoglossal nucleus (XII) (Figs. 4d–f, ​,5c)—and,5c)—and, occasionally, to a mild extent in intramedullary axons arising from the hypoglossal motor nucleus (Fig. 4f)—in the ambiguus nucleus of the vagal nerve (X), and in motor nuclei of cranial nerves XI, VII, and V. In contrast, the motor neurons of cranial nerves III, IV, and VI did not display pTDP-43 aggregates (see above). Similarly, brainstem nuclei that remained free of pTDP-43 pathology included the serotoninergic nuclei of the raphe system and noradrenergic locus coeruleus.

Pattern IV (Fig. 5: red): cases with the highest burden of pathology showed the presence of pTDP-43 lesions in the occipital neocortex (visual cortex, Brodmann 17, 18, Figs. 4g, 5a, b, d) in addition to involvement of all of the areas described above. Finally, a minority of stage 4 cases (n 4) also displayed mild involvement of the dentate nucleus in the cerebellum, whereas the cerebellar cortical gray matter generally remained unaffected by pTDP-43 pathology.

Types of neocortical pTDP-43 pathology

In 70 μm sections from the cases studied here, we observed two major types of cortical pTDP-43 pathology: cFTLD was characterized by predominantly cytoplasmic neuronal inclusions (with variable presence of short dystrophic neurites), and nFTLD by long dystrophic neurites, which could be identified as dendrites using double-labeling IHC, and by less abundant cytoplasmic inclusions (Fig. 2). These subdivisions constitute a simplification of the previously described four morphological types of cortical pTDP-43 pathology based on an analysis of 7–10 μm sections [68]. In 70 μm sections, cFTLD approximated Mackenzie types A and B, whereas nFTLD approximated Mackenzie type C (Table 2). The Mackenzie types of cortical pTDP-43 pathology were not readily ascertained in 70 μm sections [26], where we observed that the pTDP-43 pathology was much more widely and severely distributed across all neo-cortical layers (Fig. 1). Furthermore, we think that thick slices disambiguate the two types of neuritic inclusions reported in thin sections that may have cross-cut the neurites. In addition, we observed essentially different regional distribution patterns for the two types we propose here, and these patterns, in turn, make it possible to speculate that two different pathways of pTDP-43 progression exist in FTLD.

Whereas cases with nFTLD pathology showed widespread and severe neocortical involvement from orbito-frontal areas along the entire convexity of the brain that frequently reached the occipital cortex in the most advance cases (Fig. 4g), pTDP-43 lesions were comparatively scarce in brainstem somatomotor nuclei, the precerebellar nuclei, and the anterior horn of the cervical spinal cord (Suppl. Fig. 1). This observation is consonant with prior reports that Mackenzie type C cases display only mild involvement of the diencephalon, brainstem, and spinal cord anterior horn [45, 46]. In contrast, cFTLD cases showed less severe neocortical involvement, whereas bulbar somatomotor neurons and the spinal cord anterior horns were more involved. While neocortical layers II and III were extensively involved in both types (Fig. 1), pTDP-43 lesions in Betz pyramidal cells of layer Vb in the primary motor cortex were more frequent in cFTLD. Inasmuch as these cells are the origin of major corticofugal projections in the corticospinal and corticobulbar tracts, it is plausible that they contribute to the more frequent involvement of bulbar somatomotor neurons and spinal cord α-motoneurons encountered in cFTLD.

Consequently, within the continuum of pTDP-43 proteinopathies [51, 83], cFTLD type pathology appears to be more closely related to AlS than nFTLD both with regard to neuronal pTDP-43 lesion morphology and the anatomical/regional distribution pattern of the lesions [32, 33, 76]. Notably, these differing distribution patterns were of significant clinical relevance: cFTLD cases more frequently displayed clinical signs of MND (p < 0.001) and showed a tendency towards a shorter survival period as compared to nFTLD cases (Table 2).

The two morphological types of neocortical pTDP-43 distribution observed here could point to the existence of different neuronal mechanisms for responding to toxic pTDP-43 protein assemblies [57]. Whereas some neurons (e.g., in nFTLD) can localize pTDP-43 aggregates to the dendritic compartment, this might not be possible for other neurons (e.g., in cFTLD). The cellular mechanisms underlying these different morphological distributions of pTDP-43 lesions are unknown. There is increasing recognition of possible prion-like phenomena of cell-to-cell transmission in neurodegenerative diseases [84, 85], and the C-terminal domain of TDP-43 shows structural similarities to prions [78]. given the acknowledged relevance of various strains of the pathogenic prion protein for disease heterogeneity in prion diseases [20, 30, 96], it can be speculated that pTDP-43 may also form diverse strains of pathogenic protein that may be associated with a differential morphology of pTDP-43 inclusions as well as with different spreading patterns—and, hence, varying clinical phenotypes in FTLD. While this has not yet been described for TDP-43, at least two different strains of pathological α-synuclein have been described by guo et al. [39] and it is likely that other non-prion neurodegenerative disease proteins will be shown to exist as different strains of the same pathological protein.

Spreading of pathology in FTLD-TDP presenting as bvFTD

We analyzed the possible spreading of pTDP-43 pathology in cases that had presented with bvFTD (Table 2). given the clinical, pathological, and genetic heterogeneity of bvFTD [83, 97], the cohort size (n = 39) was too small to permit our proposing a neuropathological staging procedure for bvFTLD, as done previously for ALS [14]. Nonetheless, we observed the existence of four characteristic regional distribution patterns indicative of sequential pTDP-43 dissemination. Additional studies are required to determine whether the four patterns we describe here are disparate or, alternatively, when taken together, form a continuum ranging from pattern I to pattern IV. In contrast to the reports of previous authors, our findings indicate that (1) the degree of protein deposition (here, the severity of TDP-43-associated pathology) tends to be related (i.e., increase) in successive patterns [53, 54], and (2) the primary visual cortex and lower brainstem will be useful for future immunohistochemically-based staging protocols [17, 50]. On the other hand, our data also enable us to note that some of the earliest TDP-43-associated lesions occur in the orbitofrontal cortex [17, 50]. The data for pattern I, however, show that the amygdala is also among the earliest preferentially vulnerable sites in FTLD-TDP presenting as bvFTD (see below).

In cases with the lowest burden of pTDP-43 pathology (pattern I), widespread pTDP-43 lesions were observed in basal parts of the prefrontal neocortex (orbital gyri, gyrus rectus) (Fig. 5a, b, d). The orbitofrontal cortex is thought to play a pivotal role in executive function and emotional enhancement of memory [56, 100] and it is characterized by widespread connections with primary olfactory areas, high-order sensory association and other prefrontal cortical areas, with the amygdala, and with memory-related temporal cortices [3].

Our observations of early pTDP-43 pathology in the prefrontal cortex confirm earlier descriptions of severe pTDP-43 lesions in prefrontal neocortical areas of cases with FTLD [18, 33, 45, 68, 76, 98] and corroborate neuroimaging findings of prefrontal brain atrophy in FTD [36, 41, 55, 62, 81, 94, 99, 106].

pTDP-43 lesions were abundant in layers II and III of the prefrontal neocortex (Fig. 1), and both layers are closely linked to adjacent neocortical areas via association fibers, thereby providing a potential pathway for pTDP-43 dissemination within the neocortex [25, 66, 108]. It has been suggested that cortico-cortical connections directed from less differentiated cortices (e.g., the phylogenetically young prefrontal cortex) towards primary areas with a higher degree of laminar differentiation originate primarily from deeper cortical layers (e.g., layers 5, 6) and terminate in upper cortical layers (layers I, II, III) [6, 93]. Spreading of pTDP-43 pathology via such connections within the pre-frontal cortex could contribute to the pronounced involvement of the upper cortical layers in FTLD.

Pattern I was also characterized by an involvement of basolateral portions of the amygdala, which is in line with previous reports of pTDP-43 inclusions and neuronal loss [33, 52] as well as with neuropathology and neuroimaging findings of amygdalar atrophy in patients with FTD [7, 52, 64, 87, 107]. Axons from the amygdala terminate in layers I–II of many prefrontal fields [86], and the prefrontal limbic areas in the posterior orbitofrontal and anterior cingulate cortex send major projections to the amygdala [34]. Therefore, close anatomical interconnectivities exist between the orbitofrontal cortex and the amygdala, and these, in turn, could also facilitate early dissemination of pTDP-43 pathology in FTLD. Inasmuch as the number of cases with a low burden of pTDP-43 pathology in this study was limited, additional efforts are needed to confirm the earliest distribution of the lesions in FTLD presenting as bvFTD.

With increasing burden of pTDP-43 pathology, the lesions occurred in additional prefrontal areas adjacent to the orbital gyri and/or gyrus rectus (e.g., the middle frontal gyrus) and also appeared in the anterior cingulate gyrus, mediotemporal cortex, and hippocampal formation (pattern II, Fig. 3). early involvement of entorhinal areas in FTLD could be facilitated by cortico-cortical projections from the orbitofrontal gyrus to the entorhinal cortex, which projects to the hippocampus [91].

Hippocampal neuronal damage is usually linked to episodic memory deficits [21, 43], and clinical observations indicate that patients with bvFTD experience episodic memory deficits, which may reach a magnitude similar to AD in some individuals [42, 83]. Indeed, neuroimaging studies show hippocampal atrophy in bvFTD patients that can be as severe as that seen in AD [74, 103].

The anterior cingulate gyrus is part of the prefrontal limbic system [110] and receives cortico-cortical afferences from the posterior orbitofrontal cortex [3], thereby providing a possible pathway for early propagation of pTDP-43 pathology. Similar to the orbitofrontal cortex, the anterior cingulate gyrus is known to have dense connections with the basolateral nucleus of the amygdala [4].

As in previous studies, we encountered pTDP-43 lesions in medium spiny projection neurons of the striatum [2, 33], which were most severe in the accumbens nucleus (Fig. 3f, g). Such striatal involvement is reflected by neuroimaging findings of striatal atrophy in cases with bvFTD [31]. Striatal pTDP-43 pathology in the putamen could be anticipated to cause extrapyramidal motor symptoms (e.g., bradykinesia or rigidity). Indeed, all of the bvFTD cases with clinically detectable signs of extrapyramidal motor deficits showed extensive pTDP-43 lesions in the putamen (Table 2). However, accumulating pTDP-43 aggregates are likely to only gradually impair neuronal function; therefore appearance of clinical symptoms could considerably trail behind the occurrence of pTDP-43 lesions [14]. The accumbens nucleus has been associated with emotional/ motivational functions of the striatum [90] and is closely connected to limbic structures (amygdala, hippocampus) as well as the prefrontal neocortex [40]. Moreover, lesions in ventral striatal regions have been associated with various forms of behavioral dysregulation, e.g., impulsivity [8]. As such, early involvement of the accumbens nucleus by pTDP-43 pathology could contribute to some of the neuropsychological deficits reported in individuals with bvFTD [79]. Cases corresponding to patterns I and II furthermore displayed TDP-43 pathology in portions of the thalamus (Fig. 4a), which is closely connected with the orbitofrontal gyrus and other prefrontal cortical areas [5]. Intriguingly, all regions that we observed to be involved in early pTDP-43 dissemination in bvFTD (patterns I and II) are characterized by close anatomical connections with the orbitofrontal cortex. Although the small number of early stage cases (patterns I, II, Table 2) preclude any definitive statements regarding the earliest onset of FTLD that presents as bvFTD, our data strongly point to an important role of the orbital gyri/gyrus rectus in the early dissemination of pTDP-43 pathology. In cases with the third pattern (III), involvement of the primary motor cortex, bulbar somato-motor neurons, and the cervical spinal cord anterior horn (Fig. 4) is consonant with previous neuropathological findings [2, 18, 33, 76] and with clinical reports that up to 30 % of FTD patients develop MND signs [61, 65]. While all of our bvFTD cases with clinical signs of MND displayed a pronounced involvement of the agranular neocortex, the pTDP-43 lesions in the hypoglossal nucleus and the spinal cord anterior horn were less prominent in comparison to those seen in ALS [14].

Cases with the highest burden of pTDP-43 pathology (pattern IV) were characterized by the additional presence of pTDP-43 inclusions in occipital neocortical areas (Brodmann areas 17, 18, Fig. 4g). This involvement of the occipital neocortex was more frequent than in ALS [14] and it was predominantly seen in cases with nFTLD (Suppl. Fig. 1). Visual deficits have not been described as a leading or characteristic symptom of bvFTD. However, for the most part, pTDP-43 lesions in the occipital cortex were mild, and it is plausible that this pathology might only gradually impair neuronal function before crossing a threshold that leads to neuronal death [57].

Whereas genetic subtypes of FTLD-TDP presenting as bvFTD (e.g., cases with C9orf72 repeat expansion or GRN mutation carriers) displayed some differences regarding the regional severity of pTDP-43 pathology, the overall patterns were similar and could indicate a common underlying spreading scheme. However, the number of C9orf72 expansion carriers and cases with GRN mutation included here were comparatively small, underlining the need for further studies on possible differences in TDP-43 propagation among these genetic subtypes.

We thank the many patients who contributed to this study. We are also grateful to Kevin Raible, Terry Schuck, Sigrid Baumann, Gabriele Ehmke, Simone Feldengut, Julia Straub, and Thi Phuong Thu Brettschneider for technical support, together with David Ewert (University of Ulm) for assistance with the graphics. This study was supported by the NIH (AG033101, AG017586, AG010124, AG032953, AG039510, NS044266), the Wyncote Foundation, and the Koller Family Foundation. VM-YL is the John H. Ware, 3^rd, Professor of Alzheimer's Disease research. JQT is the William Maul Measey-Truman G. Schnabel, Jr. Professor of Geriatric Medicine and Gerontology. DJI is supported by T32-Ag000255. This study was supported by the German BMBF FTLD Consortium.