Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies

Abstract

Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) is a common neuropathological subtype of frontotemporal dementia. Although this subtype of frontotemporal dementia is defined by the presence of ubiquitin-positive but tau- and alpha-synuclein-negative inclusions, it is unclear whether all cases of FTLD-U have the same underlying pathogenesis. Examination of tissue sections from FTLD-U brains stained with anti-ubiquitin antibodies revealed heterogeneity in the morphological characteristics of pathological inclusions among subsets of cases. Three types of FTLD-U were delineated based on morphology and distribution of ubiquitin-positive inclusions. To address the hypothesis that FTLD-U is pathologically heterogeneous, novel monoclonal antibodies (mAbs) were generated by immunization of mice with high molecular mass (Mr > 250 kd) insoluble material prepared by biochemical fractionation of FTLD-U brains. Novel mAbs were identified that immunolabeled all of the ubiquitin-positive inclusions in one subset of FTLD-U cases, whereas other mAbs stained the ubiquitin-positive inclusions in a second subset of cases. These novel mAbs did not stain inclusions in other neurodegenerative disorders, including tauopathies and alpha-synucleinopathies. Therefore, ubiquitin immunohistochemistry and the immunostaining properties of the novel mAbs generated here suggest that FTLD-U is pathologically heterogeneous. Identification of the disease proteins recognized by these mAbs will further advance understanding of molecular substrates of FTLD-U neurodegenerative pathways.

FIGURE 1

Patterns of FTLD-U pathology based on ubiquitin immunohistochemistry. Frontal cortex in FTLD-U cases. A and B: Type 1 is characterized by abundance of long neuritic profiles predominantly in the superficial cortical layers (case 11). C and D: Type 2 is characterized by numerous cytoplasmic inclusions in superficial and deep cortical layers as well as infrequent neuritic profiles (case 17). E and F: Type 3 is characterized by pathology predominantly in the superficial cortical layers with numerous, often ring-shaped cytoplasmic inclusions, and frequent small neuritic profiles (case 18). Scale bars: 100 μm (A, C, E); 50 μm (B, D, F).

FIGURE 2

Immunoreactivity profile for newly generated antibodies in FTLD-U cases with different patterns of inclusion pathology. A–E: Type 1: mAbs 164 and 132 label numerous cytoplasmic and neuritic inclusions in dentate gyrus (A; case 4, mAb 164), frontal cortex (C; case 4, mAb 164), and striatum (D; case 11, mAb 132) as well as intranuclear inclusions in striatal neurons (E; case 11, mAb 132). Inclusions in the dentate gyrus of a type 1 case are not labeled with mAb 137 (B; case 4, mAb 137). F–J: Type 2: Cytoplasmic inclusions are labeled with mAbs 137 and 471 in dentate gyrus (G; case 13, mAb 137), frontal cortex (H; case 13, mAb 137), and striatum (I; case 14, mAb 471). Intranuclear neuronal inclusion in temporal cortex labeled with mAb 471 (J, case 14). Inclusions in the dentate gyrus of a case with type 2 pathology are negative with mAb 164 (F, case 13). K–M: Type 3: Ubiquitin-positive inclusions in the dentate gyrus (K) of case 35, type 3, are negative for mAbs 164 (L) and 471 (M). Scale bars: 50 μm (A–D, F–I, K–M); 10 μm (E, J).

FIGURE 3

Specificity of newly generated antibodies for FTLD-U inclusions. A–C: FTLD-U type 1: ubiquitin-positive inclusions in the dentate gyrus (A) stain positive using mAb 132 (B). D–F: FTLD-U type 2: ubiquitin-positive inclusions in the dentate gyrus (D) stain positive using mAb 137. G–J: α-Synuclein-positive Lewy bodies (G, I) in DLB are not recognized by mAb 132 (H) or mAb 137 (J). K–R: Tau-positive neurofibrillary tangles, neuropil threads, and dystrophic neurites in the hippocampus in AD (K, M) as well as Pick bodies in the dentate gyrus in PiD (O, Q) are not labeled by mAb 132 (L, P) or mAb 137 (N, R). Scale bars = 50 μm.