FUS immunogold labeling TEM analysis of the neuronal cytoplasmic inclusions of neuronal intermediate filament inclusion disease: a frontotemporal lobar degeneration with FUS proteinopathy.

Fused in sarcoma (FUS)-immunoreactive neuronal and glial inclusions define a novel molecular pathology called FUS proteinopathy. FUS has been shown to be a component of inclusions of familial amyotrophic lateral sclerosis with FUS mutation and three frontotemporal lobar degeneration entities, including neuronal intermediate filament inclusion disease (NIFID). The pathogenic role of FUS is unknown. In addition to FUS, many neuronal cytoplasmic inclusions (NCI) of NIFID contain aggregates of α-internexin and neurofilament proteins. Herein, we have shown that: (1) FUS becomes relatively insoluble in NIFID and there are no apparent posttranslational modifications, (2) there are no pathogenic abnormalities in the FUS gene in NIFID, and (3) immunoelectron microscopy demonstrates the fine structural localization of FUS in NIFID which has not previously been described. FUS localized to euchromatin, and strongly with paraspeckles, in nuclei, consistent with its RNA/DNA-binding functions. NCI of varying morphologies were observed. Most frequent were the "loosely aggregated cytoplasmic inclusions," 81% of which had moderate or high levels of FUS immunoreactivity. Much rarer "compact cytoplasmic inclusions" and "tangled twine ball inclusions" were FUS-immunoreactive at their granular peripheries, or heavily FUS-positive throughout, respectively. Thus, FUS may aggregate in the cytoplasm and then admix with neuronal intermediate filament accumulations.

Pin1 protein associates with neuronal lipofuscin: potential consequences in age-related neurodegeneration.

Pin1 protein is a peptidyl-prolyl cis-trans isomerase that modulates the activity of a range of proteins involved in cell function. We and others have demonstrated neuronal Pin1 deficits in Alzheimer's disease (AD) and have shown similar deficits in frontotemporal dementia and in aging. Pin1 may, in fact, be a susceptibility factor; others have shown that Pin1 depletion causes apoptosis in HeLa cells. Further, patterns of AD pathology correlate with regions of lower Pin1 expression in normal human brain; Pin1 knockout mice suffer neurodegeneration; and Pin1 can ameliorate p-tau pathology by isomerizing p-tau, facilitating its trans-specific dephosphorylation and restoring its ability to bind to and restabilize microtubules and thence cytoskeletal integrity. Here, we report a novel localization of high levels of Pin1 with lipofuscin in aging neurons. This association could progressively drain available Pin1 and be deleterious to neuronal function during aging. We also show that Pin1 associates with lipofuscin when lipofuscin accumulations become marked and correlate with susceptibility to neurodegenerative disease. Our data are consistent with the possibility that neuronal Pin1 deficits may be a contributory factor in neurodegeneration associated with aging.

Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease.

Abnormal neuronal cytoplasmic inclusions (NCIs) containing aggregates of alpha-internexin and the neurofilament (NF) subunits, NF-H, NF-M, and NF-L, are the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID). The disease has a clinically heterogeneous phenotype, including frontotemporal dementia, pyramidal and extrapyramidal signs presenting at a young age. NCIs are variably ubiquitinated and about half of cases also have neuronal intranuclear inclusions (NIIs), which are also ubiquitinated. NIIs have been described in polyglutamine-repeat expansion diseases, where they are strongly ubiquitin immunoreactive. The fine structure of NIIs of NIFID has not previously been described. Therefore, to determine the ultrastructure of NIIs, immunoelectron microscopy was undertaken on NIFID cases and normal aged control brains. Our results indicate that the NIIs of NIFID are strongly ubiquitin immunoreactive. However, unlike NCIs which contain ubiquitin, alpha-internexin and NF epitopes, NIIs contain neither epitopes of alpha-internexin nor NF subunits. Neither NIIs nor NCIs were recognised by antibodies to expanded polyglutamine repeats. The NII of NIFID lacks a limiting membrane and contains straight filaments of 20 nm mean width (range 11-35 nm), while NCIs contain filaments with a mean width of 10 nm (range 5-18 nm; t-test, P<0.001). Biochemistry revealed no differences in neuronal IF protein mobilities between NIFID and normal brain tissue. Therefore, NIIs of NIFID contain filaments morphologically and immunologically distinct from those of NCIs, and both types of inclusion lack expanded polyglutamine tracts of the triplet-repeat expansion diseases. These observations indicate that abnormal protein aggregation follows separate pathways in different neuronal compartments of NIFID.

Shortfalls in the peptidyl-prolyl cis-trans isomerase protein Pin1 in neurons are associated with frontotemporal dementias.

The peptidyl-prolyl cis-trans isomerase (PPIase) Pin1 modulates the activity of a range of target proteins involved in the cell cycle, transcription, translation, endocytosis, and apoptosis by facilitating dephosphorylation of phosphorylated serine or threonine residue preceding a proline (p-Ser/Thr-Pro) motifs catalyzed by phosphatases specific for the trans conformations. Pin1 targets include the neuronal microtubule-associated protein tau, whose dephosphorylation restores its ability to stabilize microtubules. We, and others, have shown that tau hyperphosphorylation in the neurofibrillary tangles (NFTs) of Alzheimer disease (AD) is associated with redirection of the predominantly nuclear Pin1 to the cytoplasm and with Pin1 shortfalls throughout subcellular compartments. As nuclear Pin1 depletion causes apoptosis, shortfalls in regard to both nuclear and p-tau targets may contribute to neuronal dysfunction. We report here that similar Pin1 redistribution and shortfalls occur in frontotemporal dementias (FTDs) characterized by abnormal protein aggregates of tau and other cytoskeletal proteins. This may be a unifying, contributory factor towards neuronal death in these dementias.