CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Retiring the term FTDP-17 as MAPT mutations are genetic forms of sporadic frontotemporal tauopathies.

See Josephs (doi:10.1093/brain/awx367) for a scientific commentary on this article.In many neurodegenerative disorders, familial forms have provided important insights into the pathogenesis of their corresponding sporadic forms. The first mutations associated with frontotemporal lobar degeneration (FTLD) were found in the microtubule-associated protein tau (MAPT) gene on chromosome 17 in families with frontotemporal degeneration and parkinsonism (FTDP-17). However, it was soon discovered that 50% of these families had a nearby mutation in progranulin. Regardless, the original FTDP-17 nomenclature has been retained for patients with MAPT mutations, with such patients currently classified independently from the different sporadic forms of FTLD with tau-immunoreactive inclusions (FTLD-tau). The separate classification of familial FTLD with MAPT mutations implies that familial forms cannot inform on the pathogenesis of the different sporadic forms of FTLD-tau. To test this assumption, this study pathologically assessed all FTLD-tau cases with a known MAPT mutation held by the Sydney and Cambridge Brain Banks, and compared them to four cases of four subtypes of sporadic FTLD-tau, in addition to published case reports. Ten FTLD-tau cases with a MAPT mutation (K257T, S305S, P301L, IVS10+16, R406W) were screened for the core differentiating neuropathological features used to diagnose the different sporadic FTLD-tau subtypes to determine whether the categorical separation of MAPT mutations from sporadic FTLD-tau is valid. Compared with sporadic cases, FTLD-tau cases with MAPT mutations had similar mean disease duration but were younger at age of symptom onset (55 ± 4 years versus 70 ± 6 years). Interestingly, FTLD-tau cases with MAPT mutations had similar patterns and severity of neuropathological features to sporadic FTLD-tau subtypes and could be classified into: Pick's disease (K257T), corticobasal degeneration (S305S, IVS10‰+‰16, R406W), progressive supranuclear palsy (S305S) or globular glial tauopathy (P301L, IVS10‰+‰16). The finding that the S305S mutation could be classified into two tauopathies suggests additional modifying factors. Assessment of our cases and previous reports suggests that distinct MAPT mutations result in particular FTLD-tau subtypes, supporting the concept that they are likely to inform on the varied cellular mechanisms involved in distinctive forms of sporadic FTLD-tau. As such, FTLD-tau cases with MAPT mutations should be considered familial forms of FTLD-tau subtypes rather than a separate FTDP-17 category, and continued research on the effects of different mutations more focused on modelling their impact to produce the very different sporadic FTLD-tau pathologies in animal and cellular models.

DNA methylation of the MAPT gene in Parkinson's disease cohorts and modulation by vitamin E in vitro.

Parkinson's disease (PD) is a neurodegenerative disorder for which environmental factors influence disease risk and may act via an epigenetic mechanism. The microtubule-associated protein tau (MAPT) is a susceptibility gene for idiopathic PD. Methylation levels were determined by pyrosequencing of bisulfite-treated DNA in a leukocyte cohort (358 PD patients and 1084 controls) and in two brain cohorts (Brain1, comprising 69 cerebellum controls; and Brain2, comprising 3 brain regions from 28 PD patients and 12 controls). In vitro assays involved the transfection of methylated promoter-luciferase constructs or treatment with an exogenous micronutrient. In normal leukocytes, the MAPT H1/H2 diplotype and sex were predictors of MAPT methylation. Haplotype-specific pyrosequencing confirmed that the H1 haplotype had higher methylation than the H2 haplotype in normal leukocytes and brain tissues. MAPT methylation was negatively associated with MAPT expression in the Brain1 cohort and in transfected cells. Methylation levels differed between three normal brain regions (Brain2 cohort, putamen < cerebellum < anterior cingulate cortex). In PD samples, age at onset was positively associated with MAPT methylation in leukocytes. Moreover, there was hypermethylation in the cerebellum and hypomethylation in the putamen of PD patients compared with controls (Brain2 cohort). Finally, leukocyte methylation status was positively associated with blood vitamin E levels, and the effect was more significant in H2 haplotype carriers; this result was confirmed in cells that were exposed to 100 µM vitamin E. The significant effects of sex, diplotype, and brain region suggest that hypermethylation of the MAPT gene is neuroprotective by reducing MAPT expression. The effect of vitamin E on MAPT represents a possible gene-environment interaction.

Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis.

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

Glycogen synthase kinase-3beta and tau genes interact in Alzheimer's disease.

OBJECTIVE: We examined the epistatic effect between haplotypes of glycogen synthase kinase-3beta (GSK3B) gene and microtubule-associated protein Tau (MAPT) gene in Alzheimer's disease (AD). METHODS: A genetic association study of three AD cohorts was made. Linear regression analyses were used to examine effects of MAPT polymorphisms on gene expression and alternative splicing. beta-Catenin levels and signaling were determined using Western blot and luciferase reporter assays in cells transfected with a combination of GSK3B and MAPT complementary DNA. RESULTS: Consistent interaction between GSK3B and MAPT genes in three late-onset AD cohorts was observed, with the GSK3B haplotype (T-T) significantly increasing the risk for AD in individuals with at least one H2 haplotype (odds ratio, 1.68-2.33; p = 0.005-0.036). The GSK3B haplotype was significantly protective in the Chinese cohort (odds ratio, 0.33; p = 0.016), after adjusting for the effect of age and sex. There are significant differences in in vivo transcriptional efficiency between the two MAPT haplotypes (H1 and H2) as determined by measurement of cerebellar transcripts (p < 0.001). Overexpression of either MAPT or GSK3B resulted in decreased beta-catenin levels compared with a control vector (p < 0.001). Conversely, cotransfection of both of these molecules increased beta-catenin signaling. INTERPRETATION: Our genetic and biochemical analyses have identified a novel interaction between Tau and GSK-3beta in late-onset AD causative factors. Our data are consistent with an epistatic model of interaction where discordant levels of GSK3B and MAPT gene expression can lead to altered beta-catenin levels and pathogenicity.

Effect of Fluvoxamine on Amyloid-ß Peptide Generation and Memory.

Alzheimer's disease is characterized by abnormal amyloid-ß (Aß) peptide accumulation beginning decades before symptom onset. An effective prophylactic treatment aimed at arresting the amyloidogenic pathway would therefore need to be initiated prior to the occurrence of Aß pathology. The SIGMAR1 gene encodes a molecular chaperone that modulates processing of the amyloid-ß protein precursor (AßPP). Fluvoxamine is a selective serotonin reuptake inhibitor and a potent SIGMAR1 agonist. We therefore hypothesized that fluvoxamine treatment would reduce Aß production and improve cognition. We firstly investigated the impact of SIGMAR1 on AßPP processing, and found that overexpression and knockdown of SIGMAR1 significantly affected γ-secretase activity in SK-N-MC neuronal cells. We then tested the impact of fluvoxamine on Aß production in an amyloidogenic cell model, and found that fluvoxamine significantly reduced Aß production by inhibiting γ-secretase activity. Finally, we assessed the efficacy of long-term treatment (i.e., ∼8 months) of 10 mg/kg/day fluvoxamine in the J20 amyloidogenic mouse model; the treatment was initiated prior to the occurrence of predicted Aß pathology. Physical examination of the animals revealed no overt pathology or change in weight. We conducted a series of behavioral tests to assess learning and memory, and found that the fluvoxamine treatment significantly improved memory function as measured by novel object recognition task. Two other tests revealed no significant change in memory function. In conclusion, fluvoxamine has a clear impact on γ-secretase activity and AßPP processing to generate Aß, and may have a protective effect on cognition in the J20 mice.

No association of spastic paraparesis genes in PSEN1 Alzheimer's disease with spastic paraparesis.

Familial Alzheimer's disease due to presenilin 1 (PSEN1) mutations shows considerable phenotypic variability with differences in neuropathology and neurological symptoms. Spastic paraparesis is a common neurological phenotype associated with Alzheimer's disease arising from PSEN1 mutations. To investigate whether known genes that cause spastic paraparesis could act as Alzheimer's disease-modifier genes, we sequenced nine spastic paraparesis genes in three Alzheimer's disease families with PSEN1 exon 9 deletions. We did not observe any correlation of polymorphisms or mutations in the nine spastic paraparesis genes with the variable phenotype seen in families with Alzheimer's disease and spastic paraparesis. These results suggest a need for a continuing search for genes that cause the phenotypic variation in Alzheimer's disease and spastic paraparesis.

Role of the Long Non-Coding RNA MAPT-AS1 in Regulation of Microtubule Associated Protein Tau (MAPT) Expression in Parkinson's Disease.

Studies investigating the pathogenic role of the microtubule associated protein tau (MAPT) gene in Parkinson's disease (PD) have indicated that DNA methylation of the promoter region is aberrant in disease, leading to dysregulated MAPT expression. We examined two potential regulators of MAPT gene expression in respect to PD, a promoter-associated long non-coding RNA MAPT-AS1, and DNA methyltransferases (DNMTs), enzymes responsible for new and maintenance of DNA methylation. We assessed the relationship between expression levels of MAPT and the candidate MAPT-AS1, DNMT1, DNMT3A and DNMT3B transcripts in four brain regions with varying degrees of cell loss and pathology (putamen, anterior cingulate cortex, visual cortex and cerebellum) in N = 10 PD and N = 10 controls. We found a significant decrease in MAPT-AS1 expression in PD (p = 7.154 x 10-6). The transcript levels of both MAPT-AS1 (p = 2.569 x 10-4) and DNMT1 (p = 0.001) correlated with those of MAPT across the four brain regions, but not with each other. Overexpression of MAPT-AS1 decreased MAPT promoter activity by ∼2.2 to 4.3 fold in an in vitro luciferase assay performed in two cell lines (p ≤ 2.678 x 10-4). Knock-down expression of MAPT-AS1 led to a 1.3 to 6.3 fold increase in methylation of the endogenous MAPT promoter (p ≤ 0.011) and a 1.2 to 1.5 fold increased expression of the 4-repeat MAPT isoform transcript (p ≤ 0.013). In conclusion, MAPT-AS1 and DNMT1 have been identified as potential epigenetic regulators of MAPT expression in PD across four different brain regions. Our data also suggest that increased MAPT expression could be associated with disease state, but not with PD neuropathology severity.