The human MAPT locus generates circular RNAs

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12?→?10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.     

Progranulin Stimulates the In Vitro Maturation of Pro-Cathepsin D at Acidic pH

Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆ T_m = − 1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.     

Role of <Emphasis Type="Italic">MAPT</Emphasis> mutations and haplotype in frontotemporal lobar degeneration in Northern Finland

Background

Frontotemporal lobar degeneration (FTLD) consists of a clinically and neuropathologically heterogeneous group of syndromes affecting the frontal and temporal lobes of the brain. Mutations in microtubule-associated protein tau (MAPT), progranulin (PGRN) and charged multi-vesicular body protein 2B (CHMP2B) are associated with familial forms of the disease. The prevalence of these mutations varies between populations. The H1 haplotype of MAPT has been found to be closely associated with tauopathies and with sporadic FTLD. Our aim was to investigate MAPT mutations and haplotype frequencies in a clinical series of patients with FTLD in Northern Finland.

Methods

MAPT exons 1, 2 and 9–13 were sequenced in 59 patients with FTLD, and MAPT haplotypes were analysed in these patients, 122 patients with early onset Alzheimer's disease (eoAD) and 198 healthy controls.

Results

No pathogenic mutations were found. The H2 allele frequency was 11.0% (P = 0.028) in the FTLD patients, 9.8% (P = 0.029) in the eoAD patients and 5.3% in the controls. The H2 allele was especially clustered in patients with a positive family history (P = 0.011) but did not lower the age at onset of the disease. The ApoE4 allele frequency was significantly increased in the patients with eoAD and in those with FTLD.

Conclusion

We conclude that although pathogenic MAPT mutations are rare in Northern Finland, the MAPT H2 allele may be associated with increased risks of FTLD and eoAD in the Finnish population.

     

Jump from Pre-mutation to Pathologic Expansion in C9orf72

An expanded G₄C₂ repeat in C9orf72 represents the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the lower limit for pathological expansions is unknown (the suggested cutoff is 30 repeats). It has been proposed that the expansion might have occurred only once in human history and subsequently spread throughout the population. However, our present findings support a hypothesis of multiple origins for the expansion. We report a British-Canadian family in whom a ∼70-repeat allele from the father (unaffected by ALS or FTLD at age 89 years) expanded during parent-offspring transmission and started the first generation affected by ALS (four children carry an ∼1,750-repeat allele). Epigenetic and RNA-expression analyses further discriminated the offspring’s large expansions (which were methylated and associated with reduced C9orf72 expression) from the ∼70-repeat allele (which was unmethylated and associated with upregulation of C9orf72). Moreover, RNA foci were only detected in fibroblasts from offspring with large expansions, but not in the father, who has the ∼70-repeat allele. All family members with expansions were found to have an ancient known risk haplotype, although it was inherited on a unique 5-Mb genetic backbone. We conclude that small expansions (e.g., 70 repeats) might be considered “pre-mutations” to reflect their propensity to expand in the next generation. Follow-up studies might help explain the high frequency of ALS- or FTLD-affected individuals with an expansion but without a familial history (e.g., 21% among Finnish ALS subjects).     

Expression of C9orf72 hexanucleotide repeat expansion leads to formation of RNA foci and dipeptide repeat proteins but does not influence autophagy or proteasomal function in neuronal cells

C9orf72 hexanucleotide repeat expansion (HRE) is the major genetic cause underpinning frontotemporal lobar degeneration (FLTD) and amyotrophic lateral sclerosis (ALS). C9orf72 HRE-associated pathogenesis involves both loss-of-function, through reduced C9orf72 levels, and gain-of-function mechanisms, including formation of RNA foci and generation of dipeptide repeat (DPR) proteins. In addition, dysfunctional protein degradation pathways, i.e. autophagy and ubiquitin-proteasome system (UPS), are suggested. Our aim was to study the gain-of-function mechanisms in the context of the function of protein degradation pathways as well as the regulation of the DPR proteins through these pathways. To this end, we expressed the pathological HRE in neuronal N2a cells and mouse primary cortical neurons. Protein degradation pathways were modulated to induce or block autophagy or to inhibit UPS. In addition, proteasomal activity was assessed. The C9orf72 HRE-expressing N2a cells and neurons were confirmed to produce RNA foci and DPR proteins, predominantly the Poly-GP proteins. However, the presence of these pathological hallmarks did not result in alterations in autophagy or proteasomal activity in either of the studied cell types. In N2a cells, Poly-GP proteins appeared in soluble forms and Lactacystin-mediated UPS inhibition increased their levels, indicating proteasomal regulation. Similar effects were not observed in cortical neurons, where the Poly-GP proteins formed also higher molecular weight forms. These results suggest a cell type-specific morphology and regulation of the DPR proteins. Further studies in other model systems may shed additional light onto the effects of the C9orf72 HRE on cellular protein degradation pathways and the regulation of the DPR protein levels.     

Tau inhibits mitochondrial calcium efflux and makes neurons vulnerable to calcium-induced cell death

Aggregation or phosphorylation of the microtubule-associated protein tau is the pathological hallmark in a number of diseases termed tauopathies, which include the most common neurodegenerative disorder, Alzheimer’s disease; or frontotemporal dementia, linked to mutations in the gene MAPT encoding tau. Although misfolded tau has strong familial and histopathological (as in intracellular tangles) association with neurodegenerative disorders, the cellular mechanism of tau-induced pathology remains to be controversial. Here we studied the effect of tau on the cytosolic and mitochondrial calcium homeostasis using primary cortical cultures treated with the protein and iPSC-derived neurons bearing the 10 + 16 MAPT mutation linked to frontotemporal dementia. We found that incubation of the primary cortical co-cultures of neurons and astrocytes with tau induced spontaneous Ca²⁺ oscillations in the neurons, which were also observed in iPSC-neurons with the 10 + 16 MAPT mutation. Importantly, tau inhibited mitochondrial calcium efflux via the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) in both neurons and astrocytes. This inhibition led to mitochondrial depolarisation in response to physiological and pathological calcium stimuli and made these cells vulnerable to calcium-induced caspase 3 activation and cell death. Thus, inhibition of the mitochondrial NCLX in neurons with misfolded or mutated tau can be involved in the mechanism of neurodegeneration.     

Neuroimmune dysfunction in frontotemporal dementia: Insights from progranulin and C9orf72 deficiency

Neuroimmune dysfunction is a cardinal feature of neurodegenerative diseases. But how immune dysregulation in the brain and peripheral organs contribute to neurodegeneration remains unclear. Here, we discuss the recent advances highlighting neuroimmune dysfunction as a key disease-driving factor in frontotemporal dementia (FTD). We provide an overview of the clinical observations supporting a high prevalence of autoimmune diseases in FTD patients with mutations in GRN or C9orf72. We then focus on a myriad of evidence from human genetic studies, mouse models, in vitro assays, and multi-omics platform, which indicate that haploinsufficiency in GRN and C9orf72 promotes neuroimmune dysfunction and contributes to neurodegeneration and premature death. These compelling data provide key insights to disease mechanisms, biomarker discovery, and therapeutic interventions for FTD (120 words).     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

SQSTM1 mutations – Bridging Paget disease of bone and ALS/FTLD

Paget disease of bone (PDB) is a skeletal disorder common in Western Europe but extremely rare in the Indian subcontinent and Far East. The condition has a strong genetic element with mutations affecting the SQSTM1 gene, encoding the p62 protein, frequently identified. Recently SQSTM1 mutations have also been reported in a small number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), neurodegenerative disorders in which significant coexistence with PDB has not been previously recognized. Although several SQSTM1 mutations are common to both ALS/FTLD and PDB, many are ALS/FTLD-specific. The p62 protein regulates various cellular processes including NF-κB signaling and autophagy pathways. Here we consider how knowledge of the impact of PDB-associated SQSTM1 mutations (several of which are now known to be relevant for ALS/FTLD) on these pathways, as well as the locations of the mutations within the p62 primary sequence, may provide new insights into ALS/FTLD disease mechanisms.     

Hexanucleotide Repeat Expansion in C9ORF72 Is Not Detected in the Treatment-Resistant Schizophrenia Patients of Chinese Han

Hexanucleotide (GGGGCC) repeat expansion in C9ORF72 (HRE) causes frontotemporal lobar degeneration, frontotemporal dementia–amyotrophic lateral sclerosis, and amyotrophic lateral sclerosis. HRE was also seen in the genomes of patients suffering from several other degenerative diseases. However, whether it is present in the treatment-resistant schizophrenia patients remains unknown. Genotyping 386 patients suffering from treatment-resistant schizophrenia using the method of Repeat-Primed PCR, we reported here that no HRE was detected in the patients of Chinese Han.     

Hypermethylation of the CpG Island Near the G4C2 Repeat in ALS with a C9orf72 Expansion

The G₄C₂ repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G₄C₂ repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5′ of the G₄C₂ repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).     

Niemann-Pick C Disease Gene Mutations and Age-Related Neurodegenerative Disorders

Niemann-Pick type C (NPC) disease is a rare autosomal-recessively inherited lysosomal storage disorder caused by mutations in NPC1 (95%) or NPC2. Given the highly variable phenotype, diagnosis is challenging and particularly late-onset forms with predominantly neuropsychiatric presentations are likely underdiagnosed. Pathophysiologically, genetic alterations compromising the endosomal/lysosomal system are linked with age-related neurodegenerative disorders. We sought to examine a possible association of rare sequence variants in NPC1 and NPC2 with Parkinson''s disease (PD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), and to genetically determine the proportion of potentially misdiagnosed NPC patients in these neurodegenerative conditions. By means of high-resolution melting, we screened the coding regions of NPC1 and NPC2 for rare genetic variation in a homogenous German sample of patients clinically diagnosed with PD (n = 563), FTLD (n = 133) and PSP (n = 94), and 846 population-based controls. The frequencies of rare sequence variants in NPC1/2 did not differ significantly between patients and controls. Disease-associated NPC1/2 mutations were found in six PD patients (1.1%) and seven control subjects (0.8%), but not in FTLD or PSP. All rare variation was detected in the heterozygous state and no compound heterozygotes were observed. Our data do not support the hypothesis that rare NPC1/2 variants confer susceptibility for PD, FTLD, or PSP in the German population. Misdiagnosed NPC patients were not present in our samples. However, further assessment of NPC disease genes in age-related neurodegeneration is warranted.     

Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease, affecting the upper and/or lower motor neurons. However, extramotor symptoms can also occur; cognitive deficits are present in more than 40% of patients and 5–8% of ALS patients develop frontotemporal dementia. There is no effective treatment for ALS and median survival is 2–3 years after onset.Amyotrophic lateral sclerosis is a genetically heterogeneous disorder with monogenic forms as well as complex genetic etiology. Currently, complex genetic risk factors are of minor interest for routine diagnostic testing or counseling of patients and their families. By contrast, a monogenic cause can be identified in 70% of familial and 10% of sporadic ALS cases. The most frequent genetic cause is a noncoding hexanucleotide repeat expansion in the C9orf72 gene. In recent years, high-throughput sequencing technologies have helped to identify additional monogenic and complex risk factors of ALS.Genetic counseling should be offered to all ALS patients and their first- and possibly second-degree relatives, and should include information about the possibilities and limitations of genetic testing. Routine diagnostic testing should at least encompass the most frequently mutated disease genes (C9orf72, SOD1, TDP-43, FUS). Targeted sequencing approaches including further disease genes may be applied. Caution is warranted as the C9orf72 repeat expansion cannot be detected by routine sequencing technologies and testing by polymerase chain reaction (PCR) is failure-prone.Predictive testing is possible in families in which a genetic cause has been identified, but the limitations of genetic testing (i.?e., the problems of incomplete penetrance, variable expressivity and possible oligogenic inheritance) have to be explained to the families.     

The roles of intrinsic disorder-based liquid-liquid phase transitions in the “Dr. Jekyll–Mr. Hyde” behavior of proteins involved in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

Pathological developments leading to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are associated with misbehavior of several key proteins, such as SOD1 (superoxide dismutase 1), TARDBP/TDP-43, FUS, C9orf72, and dipeptide repeat proteins generated as a result of the translation of the intronic hexanucleotide expansions in the C9orf72 gene, PFN1 (profilin 1), GLE1 (GLE1, RNA export mediator), PURA (purine rich element binding protein A), FLCN (folliculin), RBM45 (RNA binding motif protein 45), SS18L1/CREST, HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1), ATXN2 (ataxin 2), MAPT (microtubule associated protein tau), and TIA1 (TIA1 cytotoxic granule associated RNA binding protein). Although these proteins are structurally and functionally different and have rather different pathological functions, they all possess some levels of intrinsic disorder and are either directly engaged in or are at least related to the physiological liquid-liquid phase transitions (LLPTs) leading to the formation of various proteinaceous membrane-less organelles (PMLOs), both normal and pathological. This review describes the normal and pathological functions of these ALS- and FTLD-related proteins, describes their major structural properties, glances at their intrinsic disorder status, and analyzes the involvement of these proteins in the formation of normal and pathological PMLOs, with the ultimate goal of better understanding the roles of LLPTs and intrinsic disorder in the “Dr. Jekyll–Mr. Hyde” behavior of those proteins.     

Large C9orf72 Hexanucleotide Repeat Expansions Are Seen in Multiple Neurodegenerative Syndromes and Are More Frequent Than Expected in the UK Population

Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800–4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.     

Modeling ALS and FTLD proteinopathies in yeast: An efficient approach for studying protein aggregation and toxicity

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.Key words: amyotrophic lateral sclerosis, ALS, frontotemporal lobar degeneration, FTLD, yeast, amyloid, prion, FUS, TDP-43, SOD1     

Strikingly different clinicopathological phenotypes determined by progranulin-mutation dosage

We performed hypothesis-free linkage analysis and exome sequencing in a family with two siblings who had neuronal ceroid lipofuscinosis (NCL). Two linkage peaks with maximum LOD scores of 3.07 and 2.97 were found on chromosomes 7 and 17, respectively. Unexpectedly, we found these siblings to be homozygous for a c.813_816del (p.Thr272Serfs^∗10) mutation in the progranulin gene (GRN, granulin precursor) in the latter peak. Heterozygous mutations in GRN are a major cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), the second most common early-onset dementia. Reexamination of progranulin-deficient mice revealed rectilinear profiles typical of NCL. The age-at-onset and neuropathology of FTLD-TDP and NCL are markedly different. Our findings reveal an unanticipated link between a rare and a common neurological disorder and illustrate pleiotropic effects of a mutation in the heterozygous or homozygous states.     

The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene

GGGGCC (G₄C₂) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G₄C₂ repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G₄C₂ repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G₄C₂ repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G₄C₂ repeat RNA. Urea-resistant interaction between G₄C₂ repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G₄C₂ repeat translation elongation.