Rare variants in IFFO1, DTNB, NLRC3 and SLC22A10 associate with Alzheimer's disease CSF profile of neuronal injury and inflammation.

Alzheimer's disease (AD) biomarkers represent several neurodegenerative processes, such as synaptic dysfunction, neuronal inflammation and injury, as well as amyloid pathology. We performed an exome-wide rare variant analysis of six AD biomarkers (ß-amyloid, total/phosphorylated tau, NfL, YKL-40, and Neurogranin) to discover genes associated with these markers. Genetic and biomarker information was available for 480 participants from two studies: EMIF-AD and ADNI. We applied a principal component (PC) analysis to derive biomarkers combinations, which represent statistically independent biological processes. We then tested whether rare variants in 9576 protein-coding genes associate with these PCs using a Meta-SKAT test. We also tested whether the PCs are intermediary to gene effects on AD symptoms with a SMUT test. One PC loaded on NfL and YKL-40, indicators of neuronal injury and inflammation. Four genes were associated with this PC: IFFO1, DTNB, NLRC3, and SLC22A10. Mediation tests suggest, that these genes also affect dementia symptoms via inflammation/injury. We also observed an association between a PC loading on Neurogranin, a marker for synaptic functioning, with GABBR2 and CASZ1, but no mediation effects. The results suggest that rare variants in IFFO1, DTNB, NLRC3, and SLC22A10 heighten susceptibility to neuronal injury and inflammation, potentially by altering cytoskeleton structure and immune activity disinhibition, resulting in an elevated dementia risk. GABBR2 and CASZ1 were associated with synaptic functioning, but mediation analyses suggest that the effect of these two genes on synaptic functioning is not consequential for AD development.

Whole-exome rare-variant analysis of Alzheimer's disease and related biomarker traits.

INTRODUCTION: Despite increasing evidence of a role of rare genetic variation in the risk of Alzheimer's disease (AD), limited attention has been paid to its contribution to AD-related biomarker traits indicative of AD-relevant pathophysiological processes. METHODS: We performed whole-exome gene-based rare-variant association studies (RVASs) of 17 AD-related traits on whole-exome sequencing (WES) data generated in the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study (n = 450) and whole-genome sequencing (WGS) data from ADNI (n = 808). RESULTS: Mutation screening revealed a novel probably pathogenic mutation (PSEN1 p.Leu232Phe). Gene-based RVAS revealed the exome-wide significant contribution of rare coding variation in RBKS and OR7A10 to cognitive performance and protection against left hippocampal atrophy, respectively. DISCUSSION: The identification of these novel gene-trait associations offers new perspectives into the role of rare coding variation in the distinct pathophysiological processes culminating in AD, which may lead to identification of novel therapeutic and diagnostic targets.

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.

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 approximately 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 approximately 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.

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

Null mutations in progranulin (GRN) are associated with frontotemporal lobar degeneration characterized by intraneuronal accumulation of TAR DNA-binding protein-43 (TDP-43). However, the mechanism by which GRN deficiency leads to neurodegeneration remains largely unknown. In primary cortical neurons derived from Grn knockout (Grn(-/-) ) mice, we found that Grn-deficiency causes significantly reduced neuronal survival and increased caspase-mediated apoptosis, which was not observed in primary mouse embryonic fibroblasts derived from Grn(-/-) mice. Also, neurons derived from Grn(-/-) mice showed an increased amount of pTDP-43 accumulations. Furthermore, proteasomal inhibition with MG132 caused increased caspase-mediated TDP-43 fragmentation and accumulation of detergent-insoluble 35- and 25-kDa C-terminal fragments in Grn(-/-) neurons and mouse embryonic fibroblasts. Interestingly, full-length TDP-43 also accumulated in the detergent-insoluble fraction, and caspase-inhibition prevented MG132-induced generation of TDP-43 C-terminal fragments but did not block the pathological conversion of full-length TDP-43 from soluble to insoluble species. These data suggest that GRN functions as a survival factor for cortical neurons and GRN-deficiency causes increased susceptibility to cellular stress. This leads to increased aggregation and accumulation of full-length TDP-43 along with its C-terminal derivatives by both caspase-dependent and independent mechanisms.

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

Amyloid-beta (Abeta) 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 Abeta-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 Abeta42-depositing BRI-Abeta42 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 ( approximately 8-fold increased) and APPPS1 ( approximately 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.

Reduced brain volumes in mice expressing APP-Austrian mutation but not in mice expressing APP-Swedish-Austrian mutations.

We previously described two transgenic mouse lines expressing sub-endogenous levels of the 'Austrian' APP-T714I mutation (driven by the prenatally active PDGF-beta promoter; APP-Au mice) and showing intraneuronal Abeta pathology and reduced brain volumes on MRI at 12 and 20 months of age. To further investigate whether reduced brain sizes were caused by neurodegeneration or a neurodevelopmental defect, we now measured brain volumes as early as postnatal day 10. At this age, a distinguishable reduction in brain volumes was absent, indicating that brain volume deficits in APP-Au mice are not caused by a neurodevelopmental defect. To further study the association between intraneuronal Abeta and reduced brain volumes, we further generated and analyzed an APP transgenic mouse model expressing both Austrian and Swedish (K670N/M671L) mutations (APP-SwAu mice). APP-Swedish mutation is known to lead to altered APP processing in the secretory pathway, precluding its later processing in endosomal-lysosomal compartments, the site of intraneuronal Abeta accumulation. Also, to have higher levels of transgene expression only after birth, a murine Thy-1 promoter was utilized for APP-SwAu mouse lines. Despite having five times higher transgene APP levels compared to APP-Au mice, APP-SwAu mice showed significantly lower intraneuronal Abeta levels in the absence of reduced brain volumes, suggesting that intraneuronal Abeta accumulation is related to reduced brain volumes in APP-Au mice. These data also provide a first in vivo indication of altered processing of APP-Swedish at sub-endogenous levels, an effect not observed in mouse models expressing the APP-Swedish mutation in high amounts.

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.

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).

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.