Geert Joris

A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study

BACKGROUND Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region. METHODS We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers. FINDINGS In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5-4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes. INTERPRETATION We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum. FUNDING Full funding sources listed at end of paper (see Acknowledgments).

TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration.

Neuronal cytoplasmic and intranuclear aggregates of RNA-binding protein TDP-43 are a hallmark feature of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ALS and FTLD show a considerable clinical and pathological overlap and occur as both familial and sporadic forms. Though missense mutations in TDP-43 cause rare forms of familial ALS, it is not yet known whether this is due to loss of TDP-43 function or gain of aberrant function. Moreover, the role of wild-type (WT) TDP-43, associated with the majority of familial and sporadic ALS/FTLD patients, is also currently unknown. Generating homozygous and hemizygous WT human TDP-43 transgenic mouse lines, we show here a dose-dependent degeneration of cortical and spinal motor neurons and development of spastic quadriplegia reminiscent of ALS. A dose-dependent degeneration of nonmotor cortical and subcortical neurons characteristic of FTLD was also observed. Neurons in the affected spinal cord and brain regions showed accumulation of TDP-43 nuclear and cytoplasmic aggregates that were both ubiquitinated and phosphorylated as observed in ALS/FTLD patients. Moreover, the characteristic ≈25-kDa C-terminal fragments (CTFs) were also recovered from nuclear fractions and correlated with disease development and progression in WT TDP-43 mice. These findings suggest that ≈25-kDa TDP-43 CTFs are noxious to neurons by a gain of aberrant nuclear function.

TMEM106B is associated with frontotemporal lobar degeneration in a clinically diagnosed patient cohort

In a genome-wide association study of frontotemporal lobar degeneration with pathological inclusions of TAR DNA-binding protein, significant association was obtained with three single nucleotide polymorphisms at 7p21.3, in a region encompassing the gene TMEM106B. This study also suggested a potential modifying effect of TMEM106B on disease since the association was strongest in progranulin mutation carriers. Further, the risk effect seemed to correlate with increased TMEM106B expression in patients. In the present study, we sought to replicate these three findings using an independent Flanders–Belgian cohort of primarily clinically diagnosed patients with frontotemporal lobar degeneration (n = 288). We were able to confirm the association with TMEM106B with a P-value of 0.008 for rs1990622, the top marker from the genome-wide association study [odds ratio 0.75 (95% confidence interval 0.61–0.93)]. Further, high-density single nucleotide polymorphism mapping suggested that the association was solely driven by the gene TMEM106B. Homozygous carriers of the TMEM106B protective alleles had a 50% reduced risk of developing frontotemporal lobar degeneration. However, we were unable to detect a modifying effect of the TMEM106B single nucleotide polymorphisms on onset age in progranulin mutation carriers belonging to an extended, clinical and pathological well-documented founder family segregating a progranulin null mutation. Also, we could not observe significant differences in messenger RNA expression between patients and control individuals in lymphoblast cell lines and in brain frontal cortex. In conclusion, we replicated the genetic TMEM106B association in a primarily clinically diagnosed cohort of patients with frontotemporal lobar degeneration from Flanders–Belgium. Additional studies are needed to unravel the molecular role of TMEM106B in disease onset and pathogenesis.

Cellular ageing, increased mortality and FTLD-TDP-associated neuropathology in progranulin knockout mice†

Loss‐of‐function mutations in progranulin (GRN) are associated with frontotemporal lobar degeneration with intraneuronal ubiquitinated protein accumulations composed primarily of hyperphosphorylated TDP‐43 (FTLD‐TDP). The mechanism by which GRN deficiency causes TDP‐43 pathology or neurodegeneration remains elusive. To explore the role of GRN in vivo, we established Grn knockout mice using a targeted genomic recombination approach and Cre‐LoxP technology. Constitutive Grn homozygous knockout (Grn−/−) mice were born in an expected Mendelian pattern of inheritance and showed no phenotypic alterations compared to heterozygous (Grn+/−) or wild‐type (Wt) littermates until 10 months of age. From then, Grn−/− mice showed reduced survival accompanied by significantly increased gliosis and ubiquitin‐positive accumulations in the cortex, hippocampus, and subcortical regions. Although phosphorylated TDP‐43 could not be detected in the ubiquitinated inclusions, elevated levels of hyperphosphorylated full‐length TDP‐43 were recovered from detergent‐insoluble brain fractions of Grn−/− mice. Phosphorylated TDP‐43 increased with age and was primarily extracted from the nuclear fraction. Grn−/− mice also showed degenerative liver changes and cathepsin D‐positive foamy histiocytes within sinusoids, suggesting widespread defects in lysosomal turnover. An increase in insulin‐like growth factor (IGF)‐1 was observed in Grn−/− brains, and increased IGF‐1 signalling has been associated with decreased longevity. Our data suggest that progranulin deficiency in mice leads to reduced survival in adulthood and increased cellular ageing accompanied by hyperphosphorylation of TDP‐43, and recapitulates key aspects of FTLD‐TDP neuropathology. Copyright

Progranulin expression correlates with dense-core amyloid plaque burden in Alzheimer disease mouse models.

Amyloid‐β (Aβ) plaques are pathological hallmarks of Alzheimer disease (AD). In addition, innate inflammatory responses, such as those mediated by microglia, are integral to the pathogenesis of AD. Interestingly, only dense‐core plaques and not diffuse plaques are associated with neuritic and inflammatory pathology in AD patients as well as in mouse AD models. However, the precise neuropathological changes that occur in the brain in response to amyloid deposition are largely unknown. To study the molecular mechanism(s) responsible for Aβ‐mediated neuropathology, we performed a gene expression analysis on laser‐microdissected brain tissue of Tg2576 and APPPS1 mice that are characterized by different types of amyloid plaques and genetic backgrounds. Data were validated by image and biochemical analyses on different ages of Tg2576, APPPS1, and Aβ42‐depositing BRI‐Aβ42 mice. Consistent with an important role of inflammatory responses in AD, we identified progranulin (mouse Grn; human GRN) as one of the top ten up‐regulated molecules in Tg2576 (≈8‐fold increased) and APPPS1 (≈2‐fold increased) mice compared to littermate controls, and among the eight significantly up‐regulated molecules common to both mouse models. In addition, Grn levels correlated significantly with amyloid load, especially the dense‐core plaque pathology (p < 0.001). We further showed that Grn is up‐regulated in microglia and neurons and neurites around dense‐core plaques, but not in astrocytes or oligodendrocytes, as has been shown in AD patients. Our data therefore support the ongoing use of these mouse models in drug trials, especially those with anti‐inflammatory compounds. Moreover, the correlation of Grn with increasing disease severity in AD mouse models prompts human studies exploring the viability of GRN as a disease biomarker. Because loss of GRN has recently been shown to cause frontotemporal dementia and serves as a risk factor for AD, the strong GRN reactivity around dense‐core plaques is consistent with an important role of this factor in AD pathogenesis. Copyright

Identification of 2 Loci at Chromosomes 9 and 14 in a Multiplex Family With Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

BACKGROUND Frontotemporal lobar degeneration (FTLD) is a neurodegenerative brain disorder that can be accompanied by signs of amyotrophic lateral sclerosis (ALS). OBJECTIVE To identify a novel gene for FTLD-ALS. DESIGN Genome-wide linkage study in a multiplex family with FTLD-ALS with subsequent fine mapping and mutation analyses. SETTING Memory Clinic of the Middelheim General Hospital. PATIENTS An extended Belgian family with autosomal dominant FTLD-ALS, DR14, with a mean age at onset of 58.1 years (range, 51-65 years [n = 9]) and mean disease duration of 6.4 years (range, 1-17 years [n = 9]). The proband with clinical FTLD showed typical FTLD pathology with neuronal ubiquitin-immunoreactive inclusions that were positive for the transactivation response DNA-binding protein 43 (TDP-43). MAIN OUTCOME MEASURE Linkage to chromosome 9 and 14. RESULTS We found significant linkage to chromosome 9p23-q21 (multipoint logarithm of odds [LOD] score = 3.38) overlapping with a known FTLD-ALS locus (ALSFTD2) and nearly significant linkage to a second locus at chromosome 14q31-q32 (multipoint LOD score = 2.79). Obligate meiotic recombinants defined candidate regions of 74.7 megabase pairs (Mb) at chromosome 9 and 14.6 Mb near the telomere of chromosome 14q. In both loci, the disease haplotype segregated in all patients in the family. Mutation analysis of selected genes and copy number variation analysis in both loci did not reveal segregating pathogenic mutations. CONCLUSIONS Family DR14 provides additional significant evidence for the importance of the chromosome 9 gene to FTLD-ALS and reveals a possible novel locus for FTLD-ALS at chromosome 14. The identification of the underlying genetic defect(s) will significantly contribute to the understanding of neurodegenerative disease mechanisms in FTLD, ALS, and associated neurodegenerative disorders.

Increased caspase activation and decreased TDP‐43 solubility in progranulin knockout cortical cultures

J. Neurochem. (2010) 115, 735–747.

Overexpression of ALS-Associated p.M337V Human TDP-43 in Mice Worsens Disease Features Compared to Wild-type Human TDP-43 Mice

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis (ALS), while wild-type TDP-43 is a pathological hallmark of patients with sporadic ALS and frontotemporal lobar degeneration (FTLD). Various in vitro and in vivo studies have also demonstrated toxicity of both mutant and wild-type TDP-43 to neuronal cells. To study the potential additional toxicity incurred by mutant TDP-43 in vivo, we generated mutant human TDP-43 (p.M337V) transgenic mouse lines driven by the Thy-1.2 promoter (Mt-TAR) and compared them in the same experimental setting to the disease phenotype observed in wild-type TDP-43 transgenic lines (Wt-TAR) expressing comparable TDP-43 levels. Overexpression of mutant TDP-43 leads to a worsened dose-dependent disease phenotype in terms of motor dysfunction, neurodegeneration, gliosis, and development of ubiquitin and phosphorylated TDP-43 pathology. Furthermore, we show that cellular aggregate formation or accumulation of TDP-43 C-terminal fragments (CTFs) are not primarily responsible for development of the observed disease phenotype in both mutant and wild-type TDP-43 mice.

Overexpression of Wild-type TDP-43 Leads to Motor Neuron Degeneration and Spastic Quadriplegia in Germline Transgenic Mice

Background: Frontotemporal dementia (FTD) is a clinical syndrome with heterogeneous molecular basis. Although the neuropathology associated with most FTD is characterized by abnormal cellular aggregates of either TDP-43 or tau protein, there remains a significant subgroup (w15%) characterized by ubiquitin-immunoreactive (ub-ir) inclusions that are negative for both tau and TDP-43. Missense mutations in the gene encoding the fused in sarcoma (FUS) protein (also known as translated in liposarcoma, TLS), on chromosome 16, have recently been identified as a cause of familial amyotrophic lateral sclerosis (ALS). The associated pathology is described as including neuronal inclusion bodies that are immunoreactive for FUS (FUS-ir) but negative for TDP-43. Objective: Because of the recognized clinical, genetic and pathological overlap between ALS and FTD, we investigated the possible role of FUS in FTD. Methods: Immunohistochemistry, double label immunofluorescence, immunoblotting, and molecular genetic analysis. Results: In all cases, FUS immunohistochemistry (IHC) demonstrated normal physiological staining of neuronal nuclei and cytoplasm and some glial nuclei. No FUS-ir pathology was identified in cases of FTD with TDP-43 or tau pathology, or TDP-43-positive ALS. However, in a significant proportion of cases with tau/TDP-43-negative FTD, FUS IHC labeled neuronal cytoplasmic and intranuclear inclusions of similar morphology, number and anatomical distribution as were demonstrated with ubiquitin IHC. The co-localization of FUS and ubiquitin in neuronal inclusions was confirmed with double label immunofluorescence. Neurons that contained inclusions retained at least some normal physiological FUS staining. FUS IHC also demonstrated previously unrecognized inclusions in glial cells. The pathological changes were demonstrated with multiple antibodies that recognize different epitopes across the entire FUS protein. Immunoblot analysis confirmed increased amounts of insoluble FUS in post-mortem brain tissue from these cases. All cases of FTD with FUS pathology were sporadic and molecular genetic analysis did not identify any mutations in the FUS gene or abnormal levels of FUS mRNA expression. Conclusion: These findings suggest that FUS is the pathological protein in a significant subgroup of sporadic FTD and reinforce the concept that FTD and ALS are closely related conditions.