John Woulfe

Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration

The aim of this study was to improve the neuropathologic recognition and provide criteria for the pathological diagnosis in the neurodegenerative diseases grouped as frontotemporal lobar degeneration (FTLD); revised criteria are proposed. Recent advances in molecular genetics, biochemistry, and neuropathology of FTLD prompted the Midwest Consortium for Frontotemporal Lobar Degeneration and experts at other centers to review and revise the existing neuropathologic diagnostic criteria for FTLD. The proposed criteria for FTLD are based on existing criteria, which include the tauopathies [FTLD with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, sporadic multiple system tauopathy with dementia, argyrophilic grain disease, neurofibrillary tangle dementia, and FTD with microtubule-associated tau (MAPT) gene mutation, also called FTD with parkinsonism linked to chromosome 17 (FTDP-17)]. The proposed criteria take into account new disease entities and include the novel molecular pathology, TDP-43 proteinopathy, now recognized to be the most frequent histological finding in FTLD. TDP-43 is a major component of the pathologic inclusions of most sporadic and familial cases of FTLD with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease (MND). Molecular genetic studies of familial cases of FTLD-U have shown that mutations in the progranulin (PGRN) gene are a major genetic cause of FTLD-U. Mutations in valosin-containing protein (VCP) gene are present in rare familial forms of FTD, and some families with FTD and/or MND have been linked to chromosome 9p, and both are types of FTLD-U. Thus, familial TDP-43 proteinopathy is associated with defects in multiple genes, and molecular genetics is required in these cases to correctly identify the causative gene defect. In addition to genetic heterogeneity amongst the TDP-43 proteinopathies, there is also neuropathologic heterogeneity and there is a close relationship between genotype and FTLD-U subtype. In addition to these recent significant advances in the neuropathology of FTLD-U, novel FTLD entities have been further characterized, including neuronal intermediate filament inclusion disease. The proposed criteria incorporate up-to-date neuropathology of FTLD in the light of recent immunohistochemical, biochemical, and genetic advances. These criteria will be of value to the practicing neuropathologist and provide a foundation for clinical, clinico-pathologic, mechanistic studies and in vivo models of pathogenesis of FTLD.

Acid β‐glucosidase mutants linked to gaucher disease, parkinson disease, and lewy body dementia alter α‐synuclein processing

Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α‐synuclein (SNCA). A loss in lysosomal acid–β‐glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the genes association with increased synucleinopathy risk.

FUS-immunoreactive intranuclear inclusions in neurodegenerative disease

Neuronal intranuclear inclusions (NIIs) are a histopathological hallmark of several neurodegenerative disorders. However, the role played by NIIs in neurodegenerative pathogenesis remains enigmatic. Defining their molecular composition represents an important step in understanding the pathophysiology of these disorders. Recently, a nuclear protein, “fused‐in‐sarcoma” (FUS) was identified as the pathological protein in two forms of frontotemporal lobar degeneration (FTLD‐IF, formerly known as neuronal intermediate filament inclusion disease, and FTLD‐UPS, formerly known as atypical FTLD‐U), both of which are characterized by the presence of NII. The objective of the present study was to determine the range of neurodegenerative disorders characterized by FUS‐positive NIIs. Immunostaining for FUS revealed intense reactivity of NIIs in FTLD‐IF and FTLD‐UPS as well as in Huntingtons disease, spinocerebellar ataxias 1 and 3, and neuronal intranuclear inclusion body disease. In contrast, there was no FUS staining of NIIs in inherited forms of FTLD‐TDP caused by GRN and VCP mutations, fragile‐X‐associated tremor ataxia syndrome, or oculopharyngeal muscular dystrophy. In a cell culture model of Huntingtons disease, NIIs were intensely FUS‐positive. NII‐bearing cells displayed loss of the normal diffuse nuclear pattern of FUS staining. This suggests that sequestration of nuclear FUS by NIIs may interfere with its normal nuclear localization.

Mutations in STAMBP, encoding a deubiquitinating enzyme, cause microcephaly-capillary malformation syndrome

Microcephaly–capillary malformation (MIC-CAP) syndrome is characterized by severe microcephaly with progressive cortical atrophy, intractable epilepsy, profound developmental delay and multiple small capillary malformations on the skin. We used whole-exome sequencing of five patients with MIC-CAP syndrome and identified recessive mutations in STAMBP, a gene encoding the deubiquitinating (DUB) isopeptidase STAMBP (STAM-binding protein, also known as AMSH, associated molecule with the SH3 domain of STAM) that has a key role in cell surface receptor–mediated endocytosis and sorting. Patient cell lines showed reduced STAMBP expression associated with accumulation of ubiquitin-conjugated protein aggregates, elevated apoptosis and insensitive activation of the RAS-MAPK and PI3K-AKT-mTOR pathways. The latter cellular phenotype is notable considering the established connection between these pathways and their association with vascular and capillary malformations. Furthermore, our findings of a congenital human disorder caused by a defective DUB protein that functions in endocytosis implicates ubiquitin-conjugate aggregation and elevated apoptosis as factors potentially influencing the progressive neuronal loss underlying MIC-CAP syndrome.

Loss of UCHL1 promotes age-related degenerative changes in the enteric nervous system

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging.

PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson’s disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+–MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport..Mutations in the mitochondrial kinase PINK1 result in familial Parkinson’s disease. Here the authors show that LETM1, a mitochondrial inner membrane protein, is a substrate of PINK1 that regulates Ca2+ handling in mitochondria in response to mitochondrial toxins.

Multisite Assessment of Aging-Related Tau Astrogliopathy (ARTAG)

Aging-related tau astrogliopathy (ARTAG) is a recently introduced terminology. To facilitate the consistent identification of ARTAG and to distinguish it from astroglial tau pathologies observed in the primary frontotemporal lobar degeneration tauopathies we evaluated how consistently neuropathologists recognize (1) different astroglial tau immunoreactivities, including those of ARTAG and those associated with primary tauopathies (Study 1); (2) ARTAG types (Study 2A); and (3) ARTAG severity (Study 2B). Microphotographs and scanned sections immunostained for phosphorylated tau (AT8) were made available for download and preview. Percentage of agreement and kappa values with 95% confidence interval (CI) were calculated for each evaluation. The overall agreement for Study 1 was >60% with a kappa value of 0.55 (95% CI 0.433-0.645). Moderate agreement (>90%, kappa 0.48, 95% CI 0.457-0.900) was reached in Study 2A for the identification of ARTAG pathology for each ARTAG subtype (kappa 0.37-0.72), whereas fair agreement (kappa 0.40, 95% CI 0.341-0.445) was reached for the evaluation of ARTAG severity. The overall assessment of ARTAG showed moderate agreement (kappa 0.60, 95% CI 0.534-0.653) among raters. Our study supports the application of the current harmonized evaluation strategy for ARTAG with a slight modification of the evaluation of its severity.

Abstract 1968: Inactivation of the tumor suppressor Lgl via PTEN loss promotes the invasiveness of glioblastoma multiforme.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma multiforme (GBM) is the most aggressive and invasive form of brain tumor. From diagnosis the average survival time is about one year. While other forms of brain cancer can be successfully removed by surgical means, the invasive nature of GBM results in frequent relapses at secondary sites within the brain. PTEN loss is very common in GBM and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Here we show that this event results in the constitutive phosphorylation and inactivation of the tumor suppressor lethal giant larvae (LGL). In Drosophila, loss of LGL causes both over-proliferation and increased invasiveness of epithelial and brain tissue. Studies in yeast and mammals have also shown a role for Lgl in vesicle trafficking and cell motility. Its role in the proliferation and invasiveness in human brain cancer has not been investigated previously. In both cell culture and in vivo subcutaneous mouse models, expression of a non-phosphorylatable, constitutively active form of LGL (LGL-3SA) did not significantly affect the proliferation of human U87MG glioblastoma cells. However, LGL-3SA expression did lead to a reduction in the invasiveness of U87MG cells in vitro. This appeared to be mediated in part by effects on matrix metalloproteinase trafficking, as LGL-3SA prevented the delivery of matrix metalloproteinase 14 (MMP14) to the leading edge of U87MG cells. The role of Lgl in primary glioblastoma stem-like cells was also assessed, as these cells retain their invasive properties in mouse xenografts and are therefore a more clinically relevant model of glioblastoma. All primary glioblastoma stem-like cells expressed Lgl. MMP14 expression was also detected in primary glioblastoma stem-like cells, and Lgl-3SA impaired its trafficking in these cells as well. Experiments are underway to assess the affects of Lgl-3SA expression on the in vivo invasiveness of intracranial xenograft tumours generated using glioblastoma stem-like cells. Our current in vitro data suggest that inactivation of Lgl is an essential downstream step mediating the increased invasiveness of PTEN negative glioblastoma. Inactivation of Lgl enhances invasiveness by increasing the delivery of MMP-14 to the cell surface, where it can promote the degradation of extracellular matrix directly and also indirectly by activation of other matrix metalloproteinases. Citation Format: Alexander Gont, Jennifer Hanson, Mathieu Soucie, Amin Kassam, Vasco DaSilva, John Woulfe, Garth Nicholas, Sylvie Lavictoire, Ian Restall, Ian A. Lorimer. Inactivation of the tumor suppressor Lgl via PTEN loss promotes the invasiveness of glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1968. doi:10.1158/1538-7445.AM2013-1968