Hans Wils

Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

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.

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

Intraneuronal amyloid β and reduced brain volume in a novel APP T714I mouse model for Alzheimer's disease

Transgenic mouse models of Alzheimers disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.

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

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.

The role of mutant TAR DNA-binding protein 43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

TDP-43 (TAR DNA-binding protein 43) has been identified as a key protein of ubiquitinated inclusions in brains of patients with ALS (amyotrophic lateral sclerosis) or FTLD (frontotemporal lobar degeneration), defining a new pathological disease spectrum. Recently, coding mutations have been identified in the TDP-43 gene (TARDBP), which further confirmed the pathogenic nature of the protein. Today, several animal models have been generated to gain more insight into the disease-causing pathways of the FTLD/ALS spectrum. This mini-review summarizes the current status of TDP-43 models, with a focus on mutant TDP-43.

Methods to Investigate the Molecular Basis of Progranulin Actions on Brain and Behavior In Vivo Using Knockout Mice

Currently one of the few molecules that equally excites a neuroscientist, a cancer biologist, an immunologist, and a developmental biologist is progranulin (GRN/Grn)-a pluripotent growth factor that plays key roles in cell survival, proliferation, development, tissue regeneration, inflammation, wound healing, and angiogenesis. However, the molecular pathways associated with GRN signaling involved in these varied physiological processes are not understood. Gene inactivation has been considered as one of the best methods to delineate the biological role of a protein, and gene targeting is a direct means to disrupt a genes open reading frame and block its expression, for instance, in a mouse. Such a gene knockout animal model also served as an in vivo disease model where loss of gene or its function is thought to be the primary disease mechanism, as is the case with progranulin loss of function in frontotemporal lobar degeneration (FTLD). It is estimated that up to half of the cases of familial, dominant FTLD might be due to GRN haploinsufficiency. To understand the molecular pathways associated with GRN loss, constitutive and conditional progranulin knockout (Grn-/-) mice have also been constructed in several laboratories, including ours. These mice show several disease-characteristic features and suggest that continued studies on the Grn-/- mice would be instructive in the understanding of complex GRN biology in health and disease.

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.