J. van der Zee

TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis

Loss of TREM2 function impairs phagocytosis and correlates with decreased soluble TREM2 in biological fluids of patients with neurodegenerative disorders. TREM2 and Neurodegeneration Little is known about how risk factors facilitate initiation and propagation of neurodegenerative disorders. Rare mutations in TREM2 increase the risk for several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease, and frontotemporal dementia (FTD). Kleinberger et al. now show that mutations associated with neurodegenerative diseases interfere with TREM2 function by preventing its maturation, transport to the cell surface, and shedding. Expression of mutant TREM2 led to reduced phagocytic activity by different cell types, suggesting that removal of cellular debris by, for example, microglia in the brain might be affected in patients with TREM2 mutations. In a patient with FTD-like syndrome carrying a homozygous TREM2 mutation, no soluble TREM2 was detected in the cerebrospinal fluid (CSF) and plasma. Patients with sporadic FTD and AD showed slightly reduced concentrations of soluble TREM2 in their CSF. Although much further testing and validation are needed, soluble TREM2 might be useful as a marker of neurodegeneration. Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to Nasu-Hakola disease, Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and FTD-like syndrome without bone involvement. TREM2 is an innate immune receptor preferentially expressed by microglia and is involved in inflammation and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 function is unclear. We report that missense mutations associated with FTD and FTD-like syndrome reduce TREM2 maturation, abolish shedding by ADAM proteases, and impair the phagocytic activity of TREM2-expressing cells. As a consequence of reduced shedding, TREM2 is virtually absent in the cerebrospinal fluid (CSF) and plasma of a patient with FTD-like syndrome. A decrease in soluble TREM2 was also observed in the CSF of patients with AD and FTD, further suggesting that reduced TREM2 function may contribute to increased risk for two neurodegenerative disorders.

Genetic contribution of FUS to frontotemporal lobar degeneration

Background: Recently, the FUS gene was identified as a new causal gene for amyotrophic lateral sclerosis (ALS) in ∼4% of patients with familial ALS. Since ALS and frontotemporal lobar degeneration (FTLD) are part of a clinical, pathologic, and genetic disease spectrum, we investigated a potential role of FUS in FTLD. Methods: We performed mutational analysis of FUS in 122 patients with FTLD and 15 patients with FTLD-ALS, as well as in 47 patients with ALS. Mutation screening was performed by sequencing of PCR amplicons of the 15 FUS exons. Results: We identified 1 patient with FTLD with a novel missense mutation, M254V, that was absent in 638 control individuals. In silico analysis predicted this amino acid substitution to be pathogenic. The patient did not have a proven family history of neurodegenerative brain disease. Further, we observed the known R521H mutation in 1 patient with ALS. No FUS mutations were detected in the patients with FTLD-ALS. While insertions/deletions of 2 glycines (G) were suggested to be pathogenic in the initial FUS reports, we observed an identical GG-deletion in 2 healthy individuals and similar G-insertions/deletions in 4 other control individuals, suggesting that G-insertions/deletions within this G-rich region may be tolerated. Conclusions: In a first analysis of FUS in patients with frontotemporal lobar degeneration (FTLD), we identified a novel FUS missense mutation, M254V, in 1 patient with pure FTLD. At this point, the biologic relevance of this mutation remains elusive. Screening of additional FTLD patient cohorts will be needed to further elucidate the contribution of FUS mutations to FTLD pathogenesis.

Progranulin genetic variability contributes to amyotrophic lateral sclerosis.

Objectives: Null mutations in progranulin (PGRN) cause ubiquitin-positive frontotemporal dementia (FTD) linked to chromosome 17q21 (FTDU-17). Here we examined PGRN genetic variability in amyotrophic lateral sclerosis (ALS), a neurodegenerative motor neuron disease that overlaps with FTD at a clinical, pathologic, and epidemiologic level. Methods: We sequenced all exons, exon-intron boundaries, and 5′ and 3′ regulatory regions of PGRN in a Belgian sample of 230 patients with ALS. The frequency of observed genetic variants was determined in 436 healthy control individuals. The contribution of eight frequent polymorphisms to ALS risk, onset age, and survival was assessed in an association study in the Belgian sample and a replication series of 308 Dutch patients with ALS and 345 Dutch controls. Results: In patients with ALS we identified 11 mutations, 5 of which were predicted to affect PGRN protein sequence or levels (four missense mutations and one 5′ regulatory variant). Moreover, common variants (rs9897526, rs34424835, and rs850713) and haplotypes were significantly associated with a reduction in age at onset and a shorter survival after onset of ALS in both the Belgian and the Dutch studies. Conclusion: PGRN acts as a modifier of the course of disease in patients with amyotrophic lateral sclerosis, through earlier onset and shorter survival.

Genetic variability in progranulin contributes to risk for clinically diagnosed Alzheimer disease

Objective: Loss-of-function mutations in the progranulin gene (PGRN) were identified in frontotemporal lobar degeneration (FTLD) with ubiquitin-immunoreactive neuronal inclusions (FTLD-U). We assessed whether PGRN also contributes to genetic risk for Alzheimer disease (AD) in an extended Belgian AD patient group (n = 779, onset age 74.7 ± 8.7 years). Methods: A mutation analysis of the PGRN coding region was performed. The effect of missense mutations was assessed using in silico predictions and protein modeling. Risk effects of common genetic variants were estimated by logistic regression analysis and gene-based haplotype association analysis. Results: We observed seven missense mutations in eight patients (1.3%). Convincing pathogenic evidence was obtained for two missense mutations, p.Cys139Arg and p.Pro451Leu, affecting PGRN protein folding and leading to loss of PGRN by degradation of the misfolded protein. In addition, we showed that PGRN haplotypes were associated with increased risk for AD. Conclusions: Our data support a role for PGRN in patients with clinically diagnosed Alzheimer disease (AD). Further, we hypothesize that at least some PGRN missense mutations might lead to loss of functional protein. Whether the underlying pathology in our cases proves to be AD, frontotemporal lobar degeneration, or a combination of the two must await further investigations.

Clinical heterogeneity in 3 unrelated families linked to VCP p.Arg159His

Background: Families associated with missense mutations in the valosin-containing protein (VCP) present with a rare autosomal dominant multisystem disorder of frontotemporal lobar degeneration (FTLD), inclusion body myopathy (IBM), and Paget disease of bone (PDB), referred to as IBMPFD. Methods: We used exon-based genomic DNA sequencing to test for VCP mutations in 123 unrelated Belgian patients with FTLD and their relatives, and the absence of such mutations in 157 control individuals. We analyzed haplotype sharing among mutation carriers by genotyping 8 microsatellite markers in the VCP locus. We obtained family history and clinical and pathologic data using established diagnostic instruments. Results: Mutation analysis of VCP identified 2 Belgian patients with FTLD carrying the p.Arg159His mutation, which segregated in their families. In one family, patients presented with FTLD only, whereas in the other family, patients developed FTLD, PDB, or both without signs of IBM for any of the mutation carriers. We had previously identified p.Arg159His in an Austrian family with patients exhibiting both IBM and PDB. Haplotype sharing analysis indicated that the 3 p.Arg159His families are unrelated. Clinical follow-up of the Austrian family identified dementia symptoms in 1 patient. Autopsy data of 3 patients of the 2 Belgian families revealed FTLD pathology with numerous ubiquitin-immunoreactive, intranuclear inclusions and dystrophic neurites staining positive for TDP-43 protein. Conclusions: In 3 unrelated families with IBMPFD segregating VCP p.Arg159His, we observed a high degree of clinical heterogeneity and variable penetrance of the 3 cardinal clinical phenotypes: inclusion body myopathy, Paget disease of bone, and frontotemporal lobar degeneration. In contrast, the neuropathologic phenotype was consistent with FTLD-TDP type 4.

TARDBP mutation p.Ile383Val associated with semantic dementia and complex proteinopathy

Neurodegenerative diseases are morphologically and molecularly characterized by the deposition of abnormal protein aggregates in particular anatomical areas and cellular populations that are accompanied by functional alterations and consequent clinical manifestations. These depend generally on the anatomical distribution of pathology, as represented by the group of frontotemporal lobar degenerations (FTLD), where the same topography may have different clinical manifestations, diverse underlying pathologies (e.g. tauopathies, TDP-43 proteinopathies, FUSopathies), and various associated genetic defects (e.g. MAPT, GRN, C9orf72, TARDBP, VCP) that overlap with those of amyotrophic lateral sclerosis (ALS). Here we present a patient with FTLD clinically manifested by language alterations, neuropathologically by a complex combination of different proteinopathies and genetically by a TARDBP mutation not described in FTLD so far, but in ALS. The patient was a 60-year-old man who presented with slowly progressive memory problems associated with anomia, difficulties in decision making and blunted affect. There was a history of probable pathological gambling. His father had suffered from nonspecified dementia and died without autopsy at the age of 73 years. At the age of 62 years, cognitive evaluation disclosed anosognosia with preserved attention, severe deficiency in naming, semantic knowledge and in verbal abstraction. There was an important alteration of verbal and associative memory with normal visual memory, normal visuomotor speed and manipulative abstraction. Magnetic resonance imaging (MRI) showed marked left temporal atrophy (Figure 1A) associated with hypoperfusion on single-photon emission computed tomography (SPECT) (Figure 1B). During disease evolution, language alteration progressed to mutism. He also presented behavioural alterations with disinhibition, hyperphagia, hypersomnia, mental rigidity and childish behaviours. Since the age of 69 years he became progressively dependent for daily activities and personal care with sphincter incontinence and was institutionalized in a nursery home at the age of 70. He died at the age of 72 years with the clinical diagnosis of semantic dementia subtype of FTLD. Autopsied brain was donated for diagnostic and research purposes, after written informed consent was signed by a next of kin. Unfixed brain weight was 1150 g. On gross examination diffuse symmetric, temporally accentuated atrophy was observed, with narrowing of gyral pattern and widening of sulci. Coronal sections showed moderate ventricular enlargement and severe atrophy of amygdala, anterior segments of hippocampus and parahippocampal gyrus (Figure 2A–C). No alterations of basal ganglia, thalamic nuclei, pigmented brain stem nuclei or spinal cord were seen. Microscopically, there was marked neuronal loss, astrogliosis and microglial activation involving orbitofrontal cortex, anterior cingulum, insula, amygdala, and very severely the entoand transentorhinal cortices, accompanied by superficial cortical microspongiosis (Figure 2E,F). The motor cortex showed preserved motor neurones. The hippocampal formation per se, frontal (Figure 2D) and occipital cortices as well as basal ganglia, thalamic and brainstem nuclei were comparatively well preserved. Mild loss of motor neurones of the anterior horn of cervical, thoracic and lumbal spinal cord was appreciated together with single chromatolytic neurones and isolated neuronophagia (Figure 2H), without signs of corticospinal tract degeneration (Figure 2G). Immunohistochemistry revealed a moderate amount of ubiquitin (DAKO, Glostrup, Denmark) and TDP-43 (clone 2E2-D3, Abnova, Taipei, Taiwan) immunoreactive protein aggregates in form of small neuronal cytoplasmic inclusions (NCI) (Figure 2K,L) and fine and short neurites (DN) (Figure 2M), preferentially in superficial cortical areas. In the severely degenerated parahippocampal region there were surprisingly few TDP-43+ NCI and DN. No intranuclear inclusions were detected nor other protein aggregates in these areas. In spinal cord, no unequivocal ubiquitin or TDP-43+ aggregates were detected at multiple levels (Figure 2I). While difficult to classify, alterations most likely correspond to type A according to current harmonized FTLD-classification [1]. Concomitant hyperphosphorylated tau (clone AT8, ThermoScientific, Rockford, IL, USA) immunoreactive

Chromosome 17-linked frontotemporal dementia with ubiquitin-positive, tau-negative inclusions

Familial forms of frontotemporal dementia (FTD) are in 10–43% of patients, caused by mutations in the gene encoding the microtubule associated protein tau (MAPT) located at chromosome 17q21. Neuropathologically, these patients are characterized by tau-positive depositions in brain. However, autosomal dominant forms of FTD without MAPT mutations have been reported, suggesting other tauopathy-related genetic defects. One such form is FTD linked to 17q21, with tau-negative but ubiquitine-positive neuronal inclusions or FTD-U. We previously reduced the candidate chromosomal region to 4.8 cM in a Dutch FTD-U family, 1083. A mutation in MAPT was excluded by genomic sequencing. More recently, we identified three Belgian FTD families of which two, DR2 and DR8, showed linkage to the 17q21 region. Both families shared a common haplotype in an 8.04 cM region, indicating that they are genetically related to a common founder. In the third family, DR7, we obtained an autopsy confirmation of the characteristic ubiquitin-positive, tau-negative neuronal inclusions. Currently, there are 11 FTD families linked to 17q21 that do not segregate a MAPT mutation, of which five are conclusively linked (LOD score > 3). Together the data suggest that FTD-U could represent an important subtype of FTD, and that identification of the underlying gene defect might significantly contribute to our understanding of the pathomechanism leading to neurodegeneration in this dementia subtype.