Progranulin does not affect motor neuron degeneration in mutant SOD1 mice and rats

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is familial in 10% of patients, with mutations in SOD1 and C9orf72 being the most frequent cause. There is convincing evidence for overlap between ALS and frontotemporal lobar degeneration at the genetic, pathological, and clinical level. Null mutations in progranulin (PGRN) are a frequent cause of familial frontotemporal lobar degeneration. PGRN exerts neurotrophic properties on motor neurons in vitro and in vivo. We therefore examined whether PGRN could affect disease progression in mutant SOD1 mice and rats, both established models for ALS. Overexpression of PGRN in mice and intracerebroventricular delivery of PGRN in rats did not affect onset or progression of motor neuron degeneration.     

Progranulin in frontotemporal lobar degeneration and neuroinflammation

Progranulin (PGRN) is a pleiotropic protein that has gained the attention of the neuroscience community with recent discoveries of mutations in the gene for PGRN that cause frontotemporal lobar degeneration (FTLD). Pathogenic mutations in PGRN result in null alleles, and the disease is likely the result of haploinsufficiency. Little is known about the normal function of PGRN in the central nervous system apart from a role in brain development. It is expressed by microglia and neurons. In the periphery, PGRN is involved in wound repair and inflammation. High PGRN expression has been associated with more aggressive growth of various tumors. The properties of full length PGRN are distinct from those of proteolytically derived peptides, referred to as granulins (GRNs). While PGRN has trophic properties, GRNs are more akin to inflammatory mediators such as cytokines. Loss of the neurotrophic properties of PGRN may play a role in selective neuronal degeneration in FTLD, but neuroinflammation may also be important. Gene expression studies suggest that PGRN is up-regulated in a variety of neuroinflammatory conditions, and increased PGRN expression by microglia may play a pivotal role in the response to brain injury, neuroinflammation and neurodegeneration.     

Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling

To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations needs full expression of functional PGRN. Here we show that extracellular PGRN stimulates phosphorylation/activation of the neuronal MEK/extracellular regulated kinase (ERK)/p90 ribosomal S6 kinase (p90RSK) and phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stress. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity while inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP⁺. Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP⁺ toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PGRN acts as a neuroprotective factor and support the hypothesis that in FTLD reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.     

Granulin mutation drives brain damage and reorganization from preclinical to symptomatic FTLD

Granulin (GRN) mutations have been identified as a major cause of frontotemporal lobar degeneration (FTLD) by haploinsufficiency mechanism, although their effects on brain tissue dysfunction and damage still remain to be clarified. In this study, we investigated the pattern of neuroimaging abnormalities in FTLD patients, carriers and noncarriers of GRN Thr272fs mutation, and in presymptomatic carriers. We assessed regional gray matter (GM) atrophy, and resting (RS)-functional magnetic resonance imaging (fMRI). The functional connectivity maps of the salience (SN) and the default mode (DMN) networks were considered. Frontotemporal gray matter atrophy was found in all FTLD patients (more remarkably in those GRN Thr272fs carriers), but not in presymptomatic carriers. Functional connectivity within the SN was reduced in all FTLD patients (again more remarkably in those mutation carriers), while it was enhanced in the DMN. Conversely, presymptomatic carriers showed increased connectivity in the SN, with no changes in the DMN. Our findings suggest that compensatory mechanisms of brain plasticity are present in GRN-related FTLD, but with different patterns at a preclinical and symptomatic disease stage.     

Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration

Null mutations in the progranulin gene (PGRN) were recently reported to cause tau-negative frontotemporal dementia linked to chromosome 17. We assessed the genetic contribution of PGRN mutations in an extended population of patients with frontotemporal lobar degeneration (FTLD) (N=378). Mutations were identified in 10% of the total FTLD population and 23% of patients with a positive family history. This mutation frequency dropped to 5% when analysis was restricted to an unbiased FTLD subpopulation (N=167) derived from patients referred to Alzheimer's Disease Research Centers (ADRC). Among the ADRC patients, PGRN mutations were equally frequent as mutations in the tau gene (MAPT). We identified 23 different pathogenic PGRN mutations, including a total of 21 nonsense, frameshift and splice-site mutations that cause premature termination of the coding sequence and degradation of the mutant RNA by nonsense-mediated decay. We also observed an unusual splice-site mutation in the exon 1 5' splice site, which leads to loss of the Kozac sequence, and a missense mutation in the hydrophobic core of the PGRN signal peptide. Both mutations revealed novel mechanisms that result in loss of functional PGRN. One mutation, c.1477C>T (p.Arg493X), was detected in eight independently ascertained familial FTLD patients who were shown to share a common extended haplotype over the PGRN genomic region. Clinical examination of patients with PGRN mutations revealed highly variable onset ages with language dysfunction as a common presenting symptom. Neuropathological examination showed FTLD with ubiquitin-positive cytoplasmic and intranuclear inclusions in all PGRN mutation carriers.     

Genetic and neuroanatomic associations in sporadic frontotemporal lobar degeneration

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that are sensitive for tau or TDP-43 pathology in frontotemporal lobar degeneration (FTLD). Neuroimaging analyses have revealed distinct distributions of disease in FTLD patients with genetic mutations. However, genetic influences on neuroanatomic structure in sporadic FTLD have not been assessed. In this report, we use novel multivariate tools, Eigenanatomy, and sparse canonical correlation analysis to identify associations between SNPs and neuroanatomic structure in sporadic FTLD. Magnetic resonance imaging analyses revealed that rs8070723 (MAPT) was associated with gray matter variance in the temporal cortex. Diffusion tensor imaging analyses revealed that rs1768208 (MOBP), rs646776 (near SORT1), and rs5848 (PGRN) were associated with white matter variance in the midbrain and superior longitudinal fasciculus. In an independent autopsy series, we observed that rs8070723 and rs1768208 conferred significant risk of tau pathology relative to TDP-43, and rs646776 conferred increased risk of TDP-43 pathology relative to tau. Identified brain regions and SNPs may help provide an in vivo screen for underlying pathology in FTLD and contribute to our understanding of sporadic FTLD.     

Molecular signature of disease onset in Granulin mutation carriers: a gene expression analysis study

Mutations within Granulin (GRN) gene are causative of autosomal dominant frontotemporal lobar degeneration (FTLD). Though GRN mutations are inherited at birth, the disease onset usually occurs in the sixth decade of life. The objective of this study was to identify new genetic pathways linked to inherited GRN disease and involved in the shift from asymptomatic to symptomatic stages. Microarray gene expression analysis on leukocytes was carried out on 15 patients carrying GRN T272SfsX10 mutation, and their asymptomatic siblings with (n = 14) or without (n = 11) GRN mutation. The results were then validated by real-time polymerase chain reaction, and compared with those obtained in a cohort of FTLD without GRN mutation (n = 16). The association between candidate genes and damage of specific brain areas was investigated by voxel-based morphometry on magnetic resonance imaging scans (family-wise error-corrected). Leukocytes mRNA levels of TMEM40 and LY6G6F and other genes mainly involved in inflammation were significantly higher in patients carrying GRN mutations compared with asymptomatic carriers and other FTLD. The higher the levels of TMEM40 the greater is the damage of parietal lobule; the higher the LY6G6F gene expression the greater is the atrophy in superior frontal gyrus. Enhanced inflammation associated with the onset of GRN disease might be either related to disease pathogenetic mechanism leading to neurodegeneration or to a compensatory pathway that counteracts disease progression. The identification of specific molecular targets of GRN-FTLD disease is essential when considering future disease-modifying therapies.     

PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers

Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle–related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca²⁺, protein kinase C–dependent, and pertussis toxin–sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca²⁺/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.     

Mutations in the PFN1 gene are not a common cause in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration in France

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are 2 adult onset neurological disorders with overlapping symptoms and clinical characteristics. It is well established that they share a common pathologic and genetic background. Recently, mutations in profilin 1 gene (PFN1) have been identified in patients with familial ALS, suggesting a role for this gene in the pathogenesis of the disease. Based on this, we hypothesized that mutations in PFN1 might also contribute to FTLD disease. We studied a French cohort of 165 ALS/FTLD patients, without finding any variant. We conclude that mutations in PFN1 are not a common cause for ALS/FTLD in France.     

Homozygous TREM2 mutation in a family with atypical frontotemporal dementia

TREM2 mutations were first identified in Nasu-Hakola disease, a rare autosomal recessive disease characterized by recurrent fractures because of bone cysts and presenile dementia. Recently, homozygous and compound heterozygous TREM2 mutations were identified in rare families with frontotemporal lobar degeneration (FTLD) but without bone involvement. We identified a p.Thr66Met heterozygous mutation in a new consanguineous Italian family. Two sibs had early onset autosomal recessive FTLD without severe bone disorders. Atypical signs were present in this family: early parietal and hippocampus involvement, parkinsonism, epilepsy, and corpus callosum thickness on brain magnetic resonance imaging. This study further demonstrates the implication of TREM2 mutations in FTLD phenotypes. It illustrates the variability of bone phenotype and underlines the frequency of atypical signs in TREM2 carriers. This and previous studies evidence that TREM2 mutation screening should be limited to autosomal recessive FTLD with atypical phenotypes characterized by: (1) a very young age at onset (20–50 years); (2) early parietal and hippocampal deficits; (3) the presence of seizures and parkinsonism; (4) suggestive extensive white matter lesions and corpus callosum thickness on brain magnetic resonance imaging.     

Genetic analysis of matrin 3 gene in French amyotrophic lateral sclerosis patients and frontotemporal lobar degeneration with amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are adult-onset neurodegenerative diseases with overlapping clinical characteristics. They share common genetic causes and pathologic hallmarks such as TDP-43 neuronal accumulations. Recently, exome analysis identified mutations in matrin 3 (MATR3) gene in patients with familial ALS, suggesting a role for this gene in the pathogenesis of the disease. MATR3 is a nuclear matrix protein with DNA and RNA binding domains that interacts with TDP-43. To confirm the contribution of MATR3 to ALS, we studied a French cohort of 153 familial ALS or ALS/FTLD patients, without finding any variant. We conclude that mutations in MATR3 are rare in French familial ALS and ALS with FTLD patients.     

MAPT gene duplications are not a cause of frontotemporal lobar degeneration

Recurrent deletions of the 17q21.31 region encompassing the microtubule-associated protein tau (MAPT) gene have recently been described in patients with mental retardation. This region is flanked by segmental duplications that make it prone to inversions, deletions and duplications. Since gain-of-function mutations of the MAPT gene cause frontotemporal lobar degeneration (FTLD) characterized by deposition of tau protein, we hypothesize that MAPT duplication affecting gene dosage could also lead to disease. Gene dosage alterations have already been found to be involved in the etiology of neurodegenerative disorders caused by protein or peptide accumulation, such as Alzheimer's and Parkinson's diseases. To determine whether MAPT gene copy number variation is involved in FTLD, 70 patients with clinical diagnosis of FTLD and no MAPT mutation (including 12 patients with pathologically proven tau-positive FTLD) were screened by using multiplex ligation probe amplification (MLPA) with specific oligonucleotide probes. No copy number variation in the MAPT gene was observed in cases. Although our study was limited by the relatively small number of patients, it does not support the theory that chromosomal rearrangements in this region are a cause of FTLD.     

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia

Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G₁/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.     

Analysis of optineurin in frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) can occur jointly with amyotrophic lateral sclerosis (ALS), and these 2 conditions share a genetic risk factor on chromosome 9. It has been reported that mutations in optineurin (OPTN) can cause ALS. Therefore, we sequenced OPTN in 371 FTLD cases but no mutations were detected, suggesting changes in OPTN do not cause FTLD.     

Novel progranulin variants do not disrupt progranulin secretion and cleavage

A subset of frontotemporal dementia cases are neuropathologically defined by tau-negative, TAR DNA-binding protein-43, and ubiquitin-positive inclusions in the brain and are associated with mutations in the progranulin gene (GRN). Deep sequencing of families exhibiting late-onset dementia revealed several novel variants in GRN. Because of the small size of these families and limited availability of samples, it was not possible to determine whether the variants segregated with the disease. Furthermore, none of these families had autopsy confirmation of diagnosis. We sought to determine if these novel GRN variants alter progranulin (PGRN) protein stability, PGRN secretion, and PGRN cleavage in cultured cells. All the novel GRN variants behave like PGRN wild-type protein, suggesting that these variants represent rare polymorphisms. However, it remains possible that these variants affect other aspects of PGRN function or represent risk factors for dementia when combined with other modifying genes.     

Alzheimer neuropathology without frontotemporal lobar degeneration hallmarks (TAR DNA‐binding protein 43 inclusions) in missense progranulin mutation Cys139Arg

Null mutations in progranulin gene (GRN) reduce the progranulin production resulting in haploinsufficiency and are tightly associated with tau‐negative frontotemporal lobar degeneration with TAR DNA‐binding protein 43‐positive inclusions (FTLD‐TDP). Missense mutations of GRN were also identified, but their effects are not completely clear, in particular unanswered is the question of what neuropathology they elicit, also considering that their occurrence has been reported in patients with typical clinical features of Alzheimer disease. They describe two fraternal twins carrying the missense GRN Cys139Arg mutation affected by late‐onset dementia and we report the neuropathological study of one of them. Both patients were examined by neuroimaging, neuropsychological assessment and genetic analysis of GRN and other genes associated with dementia. The brain of one was obtained at autopsy and examined neuropathologically. One sister presented clinical and MRI features leading to the diagnosis of Alzheimer disease. The other underwent autopsy and the brain showed neuropathological hallmarks of Alzheimer disease with abundant Aβ‐amyloid deposition and Braak stage V of neurofibrillary pathology, in the absence of the hallmark lesions of FTLD‐TDP. Their findings may contribute to better clarify the role of progranulin in neurodegenerative diseases indicating that some GRN mutations, in particular missense ones, may act as strong risk factor for Alzheimer disease rather than induce FTLD‐TDP.     

Granulin mutations associated with frontotemporal lobar degeneration and related disorders: an update

Mutations in the gene encoding granulin (HUGO gene symbol GRN, also referred to as progranulin, PGRN), located at chromosome 17q21, were recently linked to tau-negative ubiquitin-positive frontotemporal lobar degeneration (FTLDU). Since then, 63 heterozygous mutations were identified in 163 families worldwide, all leading to loss of functional GRN, implicating a haploinsufficiency mechanism. Together, these mutations explained 5 to 10% of FTLD. The high mutation frequency, however, might still be an underestimation because not all patient samples were examined for all types of loss-of-function mutations and because several variants, including missense mutations, have a yet uncertain pathogenic significance. Although the complete phenotypic spectrum associated with GRN mutations is not yet fully characterized, it was shown that it is highly heterogeneous, suggesting the influence of modifying factors. A role of GRN in neuronal survival was suggested but the exact mechanism by which neurodegeneration and deposition of pathologic brain inclusions occur still has to be clarified.     

Anatomic correlates of stereotypies in frontotemporal lobar degeneration

Stereotypies are common in frontotemporal lobar degeneration (FTLD) however the anatomical correlates of stereotypies are unknown. We therefore set out to compare patterns of grey matter volume loss in FTLD subjects with and without stereotypies. Subjects with a diagnosis of FTLD that met international consensus criteria were prospectively recruited and separated into those with and without stereotypies. MRI and cognitive measures were obtained and voxel-based morphometry was used to assess the patterns of grey matter volume loss in those with and without stereotypies, compared to a group of age- and gender-matched controls. Demographic and clinical features were similar between subjects with and without stereotypies. FTLD subjects with stereotypies had greater volume loss in the striatum compared to those without stereotypies. Those without stereotypies showed a more widespread and typical pattern of cortical frontotemporal loss. Stereotypies in FTLD are therefore associated with a greater proportion of striatal to cortical volume loss than those without stereotypies.     

The neurotrophic properties of progranulin depend on the granulin E domain but do not require sortilin binding

Progranulin (PGRN) is a growth factor involved in wound healing, inflammation, tumor growth, and neurodegeneration. Mutations in the gene encoding PGRN give rise to shortage of PGRN and cause familial frontotemporal lobar degeneration. PGRN exerts neurotrophic functions and binding of PGRN to the membrane receptor sortilin (SORT1) mediates the endocytosis of PGRN. SORT1-mediated uptake plays an important role in the regulation of extracellular PGRN levels. We studied the role of SORT1 in PGRN-mediated neuroprotection in vitro and in vivo. The survival-enhancing effect of PGRN seemed to be dependent on the granulin E (GRN E) domain. Pharmacologic inhibition of the GRN E–SORT1 interaction or deletion of the SORT1 binding site of GRN E did not abolish its neurotrophic function. In addition, the in vivo phenotype of PGRN knockdown in zebrafish embryos was not phenocopied by SORT1 knockdown. These results suggest that GRN E mediates the neurotrophic properties of PGRN and that binding to SORT1 is not required for this effect.     

TDP-43 gene analysis in frontotemporal lobar degeneration

It has recently been established that the ubiquitinated neuronal inclusions and neurites observed in frontotemporal lobar degeneration (FTLD) contain the TAR DNA-binding protein, TDP-43. It is not uncommon for genetic variation of genes that encode proteins that accumulate in neurodegenerative conditions to increase risk for disease. We therefore examined whether variation of the TDP-43 locus was associated with an increased risk of disease in the Manchester FTLD cohort. We found no evidence of TDP-43 variation increasing risk for FTLD in this cohort. These data suggest that TDP-43 accumulation is a consequence of the disease process underlying FTLD.