The genetics and neuropathology of frontotemporal lobar degeneration

The first significant genetic linkage found in FTLD families was with markers at chromosome 17q21 [69, 120, 154]. Linkage was described in 13 families in which a consensus clinical syndrome of autosomal dominant disinhibition, dementia, parkinsonism, and amyotrophy was found, termed FTDP-17 [47]. As tau was implicated in FTLD pathology and the chromosomal location of MAPT coincided with the linkage at chromosome 17, MAPT was the most obvious candidate gene and mutations were identified in FTDP-17 families [69]. Today, 44 different MAPT mutations are reported in 134 FTLD families [30] (Table 2). The mutations are mainly clustered in the five most 3′ exons from 9 to 13, encoding the four microtubule-binding domains of tau. In normal brain, the tau protein occurs as six isoforms of which three contain three microtubule-binding domains (3R tau) and three contain four microtubule-binding domains (4R tau) [41]. A substantial number of mutations were located in the intron 10 splice donor site or intronic splice regulatory elements resulting in aberrant splicing of exon 10. Interestingly, MAPT exon 10 encodes one of the microtubule-binding domains and the mutations affecting the splicing of this exon result in aberrant ratios of 3R and 4R tau [38]. Synonymous and nonsynonymous mutations in exon 10 were shown to locate in exonic splice regulatory elements resulting in similar aberrant splicing effects [39]. In addition, missense mutations are identified that affect the amino acid sequence of the microtubule-binding domains. Mutations affect the binding of tau to tubulin either due to an increased expression of 4R tau relative to 3R tau isoforms, or due to the altered binding properties of mutant tau protein [123]. The mutations thus disturb the subtle equilibrium between cytoskeletal assembly and disassembly affecting neuronal plasticity and axonal transport across the microtubules. In addition, coding MAPT mutations increase the tendency of tau to form neurotoxic aggregates. Mutations in exon 10 lead to 4R tau in both neurons and glia, while mutations outside exon 10 lead to neuronal accumulation of 3R tau and 4R tau [133]. Except for p.P301L and IVS10+16C>T that occur in 32 and 27 families, respectively, MAPT mutations are rare and seen in single families.

FTLD due to MAPT mutations is invariantly of the pathological type FTLD-tau, including PiD. In PiD pathology, the cardinal microscopic features are neuronal intracytoplasmatic spheroid tau-containing Pick bodies in the granular neurons of the dentate gyrus and in pyramidal neurons of the hippocampus and affected neocortical regions (Fig. 2a–c). Pick bodies, mainly localized in cortical layers II and III, and to a lesser extent also in deeper layers [41], specifically accumulate insoluble 3R tau [35, 41, 48, 73]. In addition to FTLD-tau, MAPT mutations have also been associated with other tauopathies including PSP, CBS, FTD with parkinsonism and the rarely seen AGD [73, 79]. The typical tau lesions in PSP are globose neurofibrillary tangles (NFT) found in neurons of the subcortical nuclei [35, 41, 73]. In CBS patients, heterogeneous tau-immunoreactive inclusions are also found in cortical neurons and neurons in substantia nigra and locus coeruleus. Other characteristic hallmarks are astrocytic plaques containing hyperphosphorylated tau and oligodendroglial inclusions [35, 41]. The neuropathological hallmark of FTD with parkinsonism is the combination of tau-immunoreactive aggregates in neurons, astrocytes and oligodendroglial cells, throughout cortex, deep gray and subcortical white matter [35, 41]. Often, these lesions are already extensive in early and intermediate stages of the disease. In AGD, on external macroscopic examination, mild diffuse cortical atrophy is seen, with a predilection of the ambient gyrus [35, 44]. The histopathological hallmark is the presence of argyrophilic grains (ArG). These ArG are present throughout the neuropil of cortical and subcortical structures. Lesions are initially present in the amygdala and temporal allocortex, spreading toward temporal neocortex as disease progresses.

FTLD-tau is mainly associated with bvFTD, however, forms of PPA are also reported [105]. Symptoms of MND are rare. On average, FTLD-tau is characterized by the earliest onset age in the FTLD syndromes, although the range of onset ages is large and strongly overlapping with other types of FTLD. A literature survey of 228 FTLD patients carrying a MAPT mutation demonstrated that the average onset age was 48 ± 10 years with a disease duration of 9 ± 5.5 years [27].

In some families linked to chromosome 17q21, extensive mutation analyses could not identify a MAPT mutation. It became apparent that, in FTLD families negative for MAPT mutations, the characteristic pathological inclusions were of the tau-negative, ubiquitin-positive types suggesting that mutations in another gene at the same genetic locus were causing this FTLD-U type of disease. Extensive mutation analyses of other positional candidate genes identified mutation in the nearby GRN gene [7, 28]. To date, 69 different GRN mutations have been reported in 231 families [30] (Table 2). GRN mutations are distributed across the complete coding region and splice sites of the gene. They are loss-of-function mutations leading to reduced functional protein and resulting in haplo-insufficiency [52]. Most mutations produce null alleles as a result of mRNA decay mediated by a nonsense or frameshift mutation [7, 28, 50], but other mechanisms including gene deletion [53] and defective protein sorting [143] are described as well. GRN encodes progranulin, a ubiquitously expressed growth factor precursor consisting of 7.5 granulin peptides [1]. Both full-length progranulin and the granulin peptides are implicated in a wide range of biological processes such as inflammation and wound repair, as well as in pathological conditions including tumorigenesis [62]. Although their roles in the CNS are not well established, in vitro and in vivo studies suggest a neurotrophic function involved in neuronal survival and neurite outgrowth [2, 138, 164, 178].

The effect of GRN missense mutations on reduced function and resulting phenotype is not straightforward: some compromise protein stability or cellular sorting, which might result in complete loss of function and lead to haplo-insufficiency, thereby behaving as highly penetrant FTLD mutations. Clear examples of loss-of-function (LOF) missense mutations are those disrupting the signal peptide [104, 143] or affecting the disulfide bonds controlling the characteristic granulin fold [166]. Other missense mutations might only partially compromise function and behave as risk alleles [29]. GRN missense mutations have been implicated in AD risk [15, 150] and survival in ALS [148]. A useful measure of the reduced GRN function, discriminating between pathogenic and neutral missense mutations, is the granulin protein level in cerebrospinal fluid [22, 164], plasma [21, 45] and serum [149], although the correlation between protein expression level and associated disease risk is poorly studied.

All GRN-associated FTLD patients have a FTLD-TDP proteinopathy [4, 117]. The presence of TDP-43 positive inclusions is invariably associated with a decreased physiologically normal nuclear staining [36, 95]. Recent neuropathological studies elicited four FTLD-TDP subtypes A to D based on the cortical distribution, intracellular location and morphology of the inclusions [99] (Table 1; Fig. 3). Based on the large clinicopathological studies, the most common FTLD-TDP subtype is type A accounting for 41–49 % of the cases, followed by type B with 28–34 % and type C with 17–25 %, while type D is rare [19, 73]. All GRN mutations are associated with FTLD-TDP type A (Fig. 3a–c), and in turn mutations in GRN explain about 40 % of these cases [133]. In FTLD-TDP type A, many neuronal cytoplasmic inclusions (NCI), short dystrophic neurites (DN) and some lentiform neuronal intranuclear inclusions (NII) are observed in layer II of the affected neocortex. NCI in the dentate granular gyrus of the hippocampus are variable in number [99].

Despite the fact that haplo-insufficiency is the common disease mechanism in all patients carrying a GRN mutation, the associated clinical phenotype is variable, including bvFTD and PNFA [88, 125]. Parkinsonian symptoms are often observed, but motor neuron symptoms are rare [23]. Sporadically, clinical diagnoses of related neurodegenerative diseases including AD and parkinsonian disorders have been associated with GRN mutations [14, 21, 22, 88]. A literature survey of 183 patients with GRN-associated FTLD demonstrated that the onset age was on average 61 ± 9 years and 13 years later than in MAPT mutation carriers although as in FTLD-tau, onset ages are highly variable, ranging from 35 to 87 years [29, 30]. A considerable age-dependent penetrance was described with 50–60 % of the mutation carriers being affected by the age of 60 years and 90–95 % by the age of 70 years [50, 125]. Consequently, family history is not always apparent and GRN mutations are found in a significant number of FTLD patients classified as being sporadic [87].

Genetic linkage [12, 54, 89, 93, 102, 162, 163] and association [83, 144, 170] studies in FTLD-TDP, FTLD-ALS and ALS suggested a common genetic defect located at chromosome 9p21 in the complete spectrum of diseases in the FTLD–MND complex [55]. Extensive mutation analyses of positional candidate genes identified an expanded noncoding G₄C₂ hexanucleotide repeat in C9orf72 explaining linkage and association [33, 55, 128]. In the normal population, the size of the G₄C₂ repeat ranges from 3 to 25 units, which is expanded to at least 60 units in patients [33, 55, 128]. Accurate size estimations of the expanded repeat in a substantial number of carriers are lacking, as the commonly used detection method based on the repeat-primed PCR does not differentiate repeat sizes >60 units. In one study, Southern blot analyses of a limited number of repeat expansion carriers estimated repeat sizes between 700 and 1,600 units [33]. In an extended study, pathological C9orf72 repeat expansions were observed in 11.4 % of the 1,381 FTLD patients of European origin, rising to 24.8 % in familial patients [101]. The same study reported a C9orf72 repeat expansion frequency of 6.0 % in sporadic FTLD. Whether this high mutation frequency in patients without family history of disease is explained by a high de novo repeat expansion rate remains to be determined, but a preliminary study suggests that this is more likely explained by incomplete penetrance [116].

C9orf72 encodes a ubiquitously expressed protein of unknown function. It is expressed as three major transcripts [33] and the expanded G₄C₂ repeat is located in the proximal regulatory region of C9orf72 [55], upstream of one and in the first intron of the two other transcripts. Repeat expansion results in near complete loss of expression of the major gene transcripts [33, 55, 128]. In addition, accumulation of transcripts harboring the expanded G₄C₂ repeat in nuclear RNA foci was described [33] although this was not replicated in another study [146]. Whether haplo-insufficiency due to the loss of transcription, RNA toxicity due to sequestration of RNA-binding proteins in RNA foci, and/or yet unidentified mechanisms are contributing to disease, needs further investigation.

Before the identification of the C9orf72 repeat expansions, FTLD linked to chromosome 9p21 was neuropathologically characterized as being of the TDP-43 proteinopathy type B. The pathological characteristics of FTLD-TDP type B (Fig. 3d–f) include NCI throughout the entire cortical thickness [99]. NCI are seen in the granular cells of the dentate gyrus of the hippocampus. In some patients, NCI can also be found in the motor neurons of the hypoglossal nuclei and ventral horn of the spinal cord. Next to these TDP-43 inclusions, granular “pre”-inclusions are seen in affected cortical regions [36]. Ubiquitin staining of these pre-inclusions is negative. Furthermore, GCI are often seen in brain stem and spinal cord [36]. When the pathological C9orf72 repeat expansion was identified, a larger number patients with neuropathological documentation could be attributed to this mutation, revealing a wider pathological diversity. In a substantial number of patients, the number and distribution of TDP-43 inclusions were more consistent with a TDP-43 type A proteinopathy [107] or intermediate to type B and type A proteinopathy [152]. Alternatively, the TDP-43 lesion load could be relatively limited or even absent resulting in a pathological diagnosis of FTLD-UPS in rare patients [55, 107]. Abundant p62 positive, TDP-43 negative NCI and rare NII were seen in the pyramidal cell layer of the hippocampus and cerebellar granular layer of most patients [3, 13]. Remarkably, similar brain inclusions were also observed in ALS patients without associated cognitive decline [158]. Interestingly, the sequestosome 1 gene (SQSTM1) encoding the p62 protein harbors mutations leading to ALS [42] and Paget disease of bone (PDB) [58]. Nevertheless, p62 immunoreactivity of the TDP-43-negative NCI might indicate general staining of proteins of the ubiquitin–proteasome system (UPS) for protein degradation, and the hallmark protein of these aggregations remains to be identified. The presence of DN and aggregates that were immunoreactive to ubiquilin (UBQLN) in the molecular layer of the hippocampus and throughout the neocortex was shown to be highly specific for the C9orf72 mutation. Further, UBQLN and p62 colocalized in NCI in the dentate gyrus of the hippocampus and the granular layer of the cerebellum [13]. Staining of C9orf72 itself has until today remained inconclusive. Polyclonal antibodies against both protein isoforms showed a punctiform staining of synaptic terminals in the CA4 region of the hippocampus in patients with the C9orf72 mutation, but also in patients with AD and controls [13, 33, 146].

C9orf72 repeat expansions clinically present with a widely variable phenotype including FTLD, ALS or FTLD-ALS [33, 55, 128]. With a mutation frequency of 30 % in FTLD-ALS [55], it is the only known common disease gene in this condition. Independent of concomitant ALS, FTLD is mostly of the behavioral type but patients with PPA have been described [33, 55, 146, 152]. Reported mean onset ages ranged between 55.3 and 58.3 years [33, 55, 146, 152] and are intermediate to onset ages in FTLD associated with MAPT and GRN. Also here, a high variability in onset age was noted, even among patients of the same family [33, 55]. Typical of repeat expansion diseases, genetic anticipation has been suggested with decreasing onset ages in younger generations [5, 24, 43, 156], and a decrease in onset age of 7 years in the younger of two subsequent generations has been suggested for ALS [24]. However, reports correlating sizes of the expanded repeat allele and onset age are lacking.

VCP mutations were identified by linkage analysis studies in autosomal dominant families with disabling muscle weakness due to inclusion body myopathy (IBM), osteolytic bone lesions consistent with PDB and FTLD (IBMPFD) [174]. Today, 17 different mutations have been identified in 41 independent families [30]. Incomplete penetrance was noted for all three clinical characteristic features and patients may present with classical FTLD.

VCP encodes a ubiquitously expressed member of a family of ATPases associated with a wide range of cellular functions through interactions with different adaptor proteins [175]. All IBMPFD mutations reside at the interface between the D1 ATPase and the N-domain of the CDC48-like protein [175]. The best supported hypotheses of the disease mechanism of VCP mutations are disturbed ubiquitin–proteasome mediated protein degradation [31], autophagy [74], or both [75]. FTLD patients with a VCP mutation are associated with TDP-43 proteinopathy type D [176] characterized by large numbers of NCI, NII and DN in affected neocortical regions [99] (Fig. 3g–i). Some inclusions also stain for VCP protein p97 [140].

Symptoms of FTLD due to a VCP mutation become apparent in the mid-50s in 25–30 % of the IBMPFD patients [76, 78, 174]. The incomplete penetrance of the three clinical characteristics IBM, PDB and FTLD is independent of the underlying mutation [76, 78, 108, 174]. However, in FTLD, VCP mutations are rare and represent less than 1 % of the familial FTLD (Table 2). The most frequently reported FTLD subtypes are bvFTD and SD [176]. Interestingly, a single study reported VCP mutations in 1–2 % of the ALS patients in the absence of dementia symptoms [72, 77].

Linkage analyses in a large Danish FTLD family identified a mutation in CHMP2B at chromosome 3p11.2 [147]. CHMP2B encodes a component of the heteromeric ESCRT-III complex with functions in the endosomal–lysosomal and the autophagic protein degradation pathway. The gene is expressed in neurons of all major brain regions. Mutations affect the C-terminal end of the protein due to aberrant splicing.

CHMP2B mutations were associated with enlarged vacuoles in cortical neurons in the frontal, temporal, parietal and occipital cortices, due to impaired endosome–lysosome fusion [160], and impairment of autophagy [25]. Ubiquitin-immunoreactive NCI do not stain for tau, TDP-43 or FUS antibodies [65, 66], consistent with a pathological classification of FTLD-UPS [100].

The general clinical diagnoses in patients of the large Danish family corresponded to bvFTD, with early personality changes being the most common feature [60]. In other patients, progressive aphasia involvement was described, although no diagnosis of PNFA, SD or LPA could be made. The aphasia is characterized by a reduction in spontaneous speech sometimes leading to mutism and preserved reading and repetition, most consistent with a dynamic aphasia [70, 167]. The average onset age is 58 years, ranging between 46 and 65 years [60, 70]. The CHMP2B p.Gln206His mutation was reported in two ALS patients [25, 117]. Other missense mutations have been described in patients with FTLD and/or MND; however, their pathogenic nature remains unclear [30].

Mutations in the genes encoding the TDP-43 (TARDBP) and fused-in-sarcoma (FUS) proteins are typically associated with ALS. TARDBP mutations were initially identified [155] as a direct consequence of the identification of TDP-43-derived protein species as the major constituent of the aggregates found in upper and lower motor neurons of ALS patients without SOD1 mutations and in FTLD-U [4, 117]. Whereas 5 % of the familial ALS patients have a TARDBP mutation, mutations are rarely found in FTLD and FTD-MND [8, 10].

TDP-43 is an RNA-binding protein that forms heterogeneous nuclear ribonucleoprotein complexes (hnRNP) which function in RNA processing activities of several cellular functions, including transcription, RNA splicing and microRNA processing [16, 17, 118]. Missense mutations were found in the C-terminal glycine-rich region involved in protein–protein interactions [118]. Similar to TARDBP, FUS is a member of the hnRNP family. Its location at chromosome 16p11.2, made it an excellent candidate gene to explain previously established genetic linkage to the same chromosomal region in multiple ALS families [137, 139] and mutation analyses of patients in these and other families identified FUS mutations [80]. Although FUS represents an ALS gene, a mutational analysis of the FUS gene in 122 patients with FTLD revealed one novel p.Met254Val mutation in a patient with pure bvFTD. The silico analysis of this missense mutation predicted a pathogenic affect; however, the biologic relevance of this mutation remains elusive [157, 171].

Interestingly, an accumulation of FUS protein in inclusion bodies in neuronal cytoplasm and nucleus was associated with three clinicopathological subtypes of FTLD, defined by specific characteristics and location of NCI and NII [92, 97, 98, 114]. The first type is pathologically characterized by severe atrophy of the caudate nucleus and the frontotemporal cortex. Compact, round to oval kidney-shaped NCI and vermiform NII are localized in neocortex, granule cells of the dentate gyrus, striatum and to a lesser extent in globus pallidus, thalamus and periaqueductal gray matter (Fig. 4a–c). The cerebellar cortex is never affected. NCI and NII are not detected upon hematoxylin–eosin staining and were not immunoreactive to intermediate neurofilament on IHC. This type of FUS pathology is associated with a severe clinical syndrome resembling bvFTD and is referred to as atypical FTLD-U (aFTLD-U) [94]. Onset of disease is early, in 30s or 40s, and disease duration is approximately 7 years [85]. aFTLD-U is the most frequent FTLD-FUS subtype, accounting for 7–20 % of FTLD-U cases [94, 131, 142, 161]. The second FTLD-FUS type is characterized by an asymmetric atrophy of frontotemporal cortex and neostriatum, which is less severe than in aFTLD-U [85]. NCI and NII stain with FUS antibodies and to a lesser extent with antibodies against type IV interfilaments, alpha-internexin and neurofilaments. Therefore, this FTLD-FUS type is referred to as neuronal intermediate filament inclusion disease (NIFID) [94, 113]. Clinically NIFID patients usually develop a rapidly evolving FTD syndrome, mostly bvFTD, in combination with motor disorders such as parkinsonian or motor neuron symptoms. Patients usually develop the disease between 40 and 60 years of age, but earlier onset has been described and disease duration is on average approximately 3 years [85]. Considerable neuropathological similarities exist between aFTLD-U and NIFID; however, distinctive differences are observed. In NIFID, FUS-immunoreactive NCI are more extensive and widespread and are particularly numerous in CA1 and subiculum, but not in the granular layer of the dentate gyrus. The NCI morphology in NIFID patients is very heterogeneous, varying from small round to oval or tangle-like and annular shapes [73, 98, 100]. While less FUS-immunoreactive inclusions are seen in aFTLD-U, microscopic evaluation of aFTLD-U patients often shows sclerosis of the hippocampus and subcortical structures, for example, caudate nucleus, putamen and substantia nigra [85]. Another difference could be the presence of the NII, which are variably found in the hippocampus, but rare in other brain regions of NIFID patients, and are far more numerous in aFTLD-U patients [98, 100]. In the third FTLD-FUS type, referred to as basophilic inclusion body disease (BIBD), basophilic NCI staining with hematoxylin and eosin are seen in the pontine nuclei and to a lesser extent also in cerebral cortex [73, 98, 100, 106]. The basophilic NCI show FUS immunoreactivity in frontal cortex, basal ganglia and brain stem [98, 100]. BIBD presents with a clinical syndrome of early-onset ALS, occasionally accompanied with bvFTD.