Tau pathology in frontotemporal lobar degeneration with C9ORF72 hexanucleotide repeat expansion

Abstract

An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration associated with TDP-43 pathology (FTLD-TDP). In addition to TDP-43-positive neuronal and glial inclusions, C9ORF72-linked FTLD-TDP has characteristic TDP-43-negative neuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites in the hippocampus and cerebellum. These lesions are immunopositive for ubiquitin and ubiquitin-binding proteins, such as sequestosome-1/p62 and ubiquilin-2. Studies examining the frequency of the C9ORF72 mutation in clinically probable Alzheimer's disease (AD) have found a small proportion of AD cases with the mutation. This prompted us to systematically explore the frequency of Alzheimer-type pathology in a series of 17 FTLD-TDP cases with mutations in C9ORF72 (FTLD-C9ORF72). We identified four cases with sufficient Alzheimer-type pathology to meet criteria for intermediate-to-high-likelihood AD. We compared AD pathology in the 17 FTLD-C9ORF72 to 13 cases of FTLD-TDP linked to mutations in the gene for progranulin (FTLD-GRN) and 36 cases of sporadic FTLD (sFTLD). FTLD-C9ORF72 cases had higher Braak neurofibrillary tangle stage than FTLD-GRN. Increased tau pathology in FTLD-C9ORF72 was assessed with thioflavin-S fluorescent microscopy-based neurofibrillary tangle counts and with image analysis of tau burden in temporal cortex and hippocampus. FTLD-C9ORF72 had significantly more neurofibrillary tangles and higher tau burden compared with FTLD-GRN. The differences were most marked in limbic regions. On the other hand, sFTLD and FTLD-C9ORF72 had a similar burden of tau pathology. These results suggest FTLD-C9ORF72 has increased propensity for tau pathology compared to FTLD-GRN, but not sFTLD. The accumulation of tau as well as lesions immunoreactive for ubiquitin and ubiquitin-binding proteins (p62 and ubiquilin-2) suggests that mutations in C9ORF72 may involve disrupted protein degradation that favors accumulation of multiple different proteins.

Fig. 1

Macroscopic comparison of two C9ORF72 mutation carriers presenting with pathological AD (Case 24: a and b; Case 10: c and d). (a) Left hemisphere of AD/FTLD-U case with frontal, motor, and parietal cortical atrophy. (b) Coronal section at the level of the subthalamic nucleus shows widening of sulcal spaces, enlargement of the temporal horn of the lateral ventricle, and slight enlargement of the frontal horn of the lateral ventricle. (c) Right hemisphere of FTLD-TDP/AD case with frontal (predominantly frontal pole) “knife-edge” cortical atrophy as well as opercular and superior parietal atrophy. (d) Coronal section at the level of the subthalamic nucleus shows cortical atrophy and widening of sulcal space, thinning of the corpus callosum, and severe ventricular enlargement of the lateral ventricle.

Fig. 2

Microscopic comparison of AD pathology in two C9ORF72 mutation carriers presenting with pathological AD (Case 24: a and c; Case 10: b and d). Cases stained with a tau antibody (PHF-1; a, c) and an Aβ antibody (33.1.1; b, d). Red inserts show x5 magnification of the hippocampus endplate, dentate fascia, and molecular layer. Blue inserts show x5 magnification of the inferior temporal cortex. Case 24 has abundant tau and Aβ pathology in the hippocampus and temporal cortex consistent with Braak Stage V (a, b). While Case 10 has less overall tau and Aβ pathology in comparison to Case 24, the distribution of tangles is consistent with a similar Braak Stage V–VI. (insert bar = 100 μm)

Fig. 3

Microscopic C9ORF72 pathology in a pathological AD case. Ubiquitin (Ubi-1), sequestosome-1 (p62), ubiquilin-2 (Ubqln2), and phospho-TDP-43 (pS409/410) immunohistochemistry in the cerebellum (a, c, e, and g) and hippocampus (b, d, f, and h) of Case 24. Ubi-1 and p62 antibodies stain hallmark cerebellar NCIs in the Purkinje and granule cell layer, and hippocampal NCIs in the dentate fascia (a-d). Ubi-1 additionally labels normal aging pathology in the cerebellar white matter (unseen with p62), and senile plaques (SP) and neurofibrillary tangles (NFT) in the endplate and molecular layer of the hippocampus (a, b). p62 labels SP in the hippocampus (d). Ubqln2 marks cerebellar NCIs in the granule cell layer, but not in the Purkinje layer (e). In the hippocampus, the dentate contains Ubqln2-positive NCIs. The molecular layer has many immunopositive neurites and no Ubqln2-positive AD pathology (f; Fig. 4). There is no TDP-43 staining in the cerebellum and occasion TDP-43 stained NCIs in the dentate fascia (g, h). (bar = 20 μm for a, c, e, and g; 50 μm for b, d, f, and h)

Fig. 4

Immunofluorescence in the hippocampus of a pathological AD case. (a) Double stain with Ubqln2 and phospho-tau (PHF-1) reveals comorbidity of C9ORF72 pathology (neurites and NCIs) and AD pathology (plaques and tangles) with minimal colocalization. (b) Double stain with ubiquilin 1 (Ubqln1), a known marker of tangles, and Ubqln2 reveals co-labeled NCIs which could reflect sequence homology between the ubiquilins and poor antibody epitope specificity. While there are co-labeled puncta swellings in the molecular layer neurites, Ubqln1 does not label the entire process. Additionally, Ubqln1 labels a potential tangle in the dentate fascia; however, this neuron is also marked by Ubqln2 which is not known to stain tangles. (Case 24, ×10 magnification)