WVE‐005, a stereopure antisense oligonucleotide for MAPT silencing in tauopathies

Tau Pathology Generated by Overexpression of Tau

FTDP-17: 2-Year Follow-Up of Motor and Cognitive Features After Autologous Stem-Cell Transplantation

Microtubule-associated protein tau genetic variations are uncommon cause of frontotemporal dementia in south India

Accumulating evidence suggests that misfolded MAPT (microtubule associated protein tau), the main component of neurofibrillary tangles in tauopathies, is subject to degradation by the autophagy-lysosomal pathway. Selective autophagy is a subtype of macroautophagy that requires cargo receptors, such as OPTN (optineurin) or SQSTM1, to recognize specific targets for their sequestration within the autophagosome and their eventual degradation by the lysosome, although their roles in targeting distinct MAPT species have not been fully investigated. Using cargo receptor knockout cell lines and a seeding-based cellular assay in which neurofibrillary tangle pathology can be modeled in vitro, we reveal that while OPTN primarily targets soluble MAPT expressed in physiological conditions, SQSTM1 predominantly degrades insoluble but not soluble mutant MAPT. Endogenous SQSTM1 colocalizes with misfolded and aggregated MAPT species in vitro and in vivo, and both this colocalization and its function in MAPT clearance require both the LC3-interacting region (LIR) motif and also the PB1 self-polymerization domain of SQSTM1. Further, pathogenic MAPT accumulation reduces basal macroautophagy/autophagy in vitro and is associated with a compensatory upregulation of the lysosomal pathway in vivo. Finally, increased expression of SQSTM1 in MAPT transgenic mouse brains ameliorates MAPT pathology and prion-like spreading. Our results uncover distinct properties of selective autophagy receptors in targeting different MAPT species, implicate compromised autophagy as a potential underlying factor in mutant MAPT deposition, and demonstrate a potent and specific role of SQSTM1 in targeted clearance of pathogenic MAPT, through which it blocks neurofibrillary tangle accumulation and pathological spreading. Abbreviations: AAV: adeno-associated virus; AD: Alzheimer disease; ALP: autophagy-lysosomal pathway; ALS: amyotrophic lateral sclerosis; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; FTD: frontotemporal dementias; HD: Huntington disease; HTT: huntingtin; LIR: LC3-interacting region; NBR1: autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NFTs: neurofibrillary tangles; MAPT: microtubule associated protein tau; OPTN: optineurin; p-MAPT: hyperphosphorylated MAPT; PFA: paraformaldehyde; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1 Tax1: binding protein 1; ThioS: thioflavin-S; UBA: ubiquitin-associated

The identification of tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has revealed invaluable information regarding the role of the tau protein in neurodegenerative disease. Over the past year several new mutations have been identified, and experimental studies have provided further insight into the mechanism of neurodegeneration due to tau mutations and possible interactions with amyloid pathology. Extensive clinical and pathological variation is seen in patients with different types of mutation, as well as in patients with the same mutation. Mutations may be found in patients with frontotemporal dementia (FTD), parkinsonism, progressive supranuclear palsy and corticobasal degeneration, justifying mutation analysis in familial cases of these disorders. Genetic heterogeneity exists in frontotemporal dementia, because a number of FTDP-17 families have neither tau mutations nor tau pathology. Genetic linkage has been found in familial FTD (chromosome 3), FTD with amyotrophic lateral sclerosis (9q21-q22), and FTD with inclusion body myopathy (9q13.3-p12). Tau deposits may consist of mainly mutated protein, or of mutated and wild-type protein in equal amounts, depending on the mutation. Recent animal studies show that amyloid-beta deposition may accelerate formation of neurofibrillary tangles. Hopefully, the identification of responsible genetic defects and associated proteins will be helpful in improving our understanding of the role of the tau protein in the common neurodegenerative process of frontotemporal degeneration.

BackgroundTauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s dimentia (AD), are neurodegenerative diseases characterized by the pathological aggregation of paired helical filaments (PHFs) or neurofibrillary tangles (NFTs) within neurons and glia, leading to cell death1. PHFs and NFTs are formed by aggregation of hyperphosphorylated tau1. Mutations in the microtubule‐associated protein tau (MAPT) gene result in tauopathies. Moreover, FTD is a common clinical syndrome of 4 repeat (4R) tauopathies, as defined by aggregation of tau protein isoforms with four microtubule binding domains2. Here, we aimed to develop and characterize a physiologically relevant and robust in vitro FTD model, to aid the future development of FTD disease therapeutics.MethodUsing CRISPR‐Cas9 gene editing technology, familial mutations MAPT P301S and N279K underlying AD/FTD3 were engineered into an iPSC line that carries the opti‐oxTM technology and can rapidly be reprogrammed into glutamatergic neurons4. By means of immunocytochemistry we characterized b‐amyloid oligomer treated neurons, and neurons with the MAPT mutations to assess tau hyperphosphorylation.ResultThe CRISPR‐edited MAPT neurons can mature and show a healthy morphology. b‐amyloid oligomers (dose dependently) induce tau hyperphosphorylation in wild type glutamatergic neurons. Total tau expression is reduced in MAPT P301S homozygous and N279K heterozygous cells when compared to wild type cells. The MAPT P301S cells also show hyperphosphorylation for p‐tau 217, p‐tau202/205, and p‐tau404, and the N279K cells show hyperphosphorylation for p‐tau202/205, and p‐tau404.ConclusionThe elevated p‐tau to total tau ratio measured by the immunocytochemistry assay indicates the potential of at least two cell lines as possible disease models to aid future research into developing AD/FTD disease therapeutics.

Progressive supranuclear palsy (PSP) is a progressive degenerative disorder characterized by neuronal loss, gliosis and abnormal fibril formation of abnormally phosphorylated tau protein in neurons and glia cells, but the cause is not clear at present. For the purpose of clarifying the pathological significance of accumulation of tau protein in neurons and oligodendroglia in PSP, we morphologically classified neurofibrillary tangles (NFT) and coiled bodies (CB) in oligodendroglia in three PSP brains into four stages, using double staining for immunohistochemistry with AT8 antibody and modified Gallyas-Braak (GB) staining. AT8-positive neurons without abnormal fibril structure with GB staining were classified as stage I, AT8-positive neurons containing a few fibril structures with GB staining were classified stage II, AT8-positive neurons containing mature fibril structures were classified as stage III, and AT8 negative neurons containing abnormal fibril structures stained only with GB staining were classified as stage IV (ghost tangles). These stages were also assessed for CB. Then we counted the number of cells of each stage in various brain regions to investigate the relationship of NFT and CB with neuronal loss and gliosis. The results showed that there were very few stage IV NFT and CB, which reflect cell death, but that stage III NFT and CB were abundant. Moreover, CB were abundant in regions with severe neuronal loss. These results suggest that appearance of CB is closely associated with degenerative regions.

Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimer's disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Tau's molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tau-specific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear palsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

ImportanceAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by loss of upper and lower motor neurons. Although novel ALS genetic variants have been identified, the shared genetic risk between ALS and other neurodegenerative disorders remains poorly understood.ObjectivesTo examine whether there are common genetic variants that determine the risk for ALS and other neurodegenerative diseases and to identify their functional pathways.Design, Setting, and ParticipantsIn this study conducted from December 1, 2016, to August 1, 2017, the genetic overlap between ALS, sporadic frontotemporal dementia (FTD), FTD with TDP-43 inclusions, Parkinson disease (PD), Alzheimer disease (AD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP) were systematically investigated in 124 876 cases and controls. No participants were excluded from this study. Diagnoses were established using consensus criteria.Main Outcomes and MeasuresThe primary outcomes were a list of novel loci and their functional pathways in ALS, FTD, PSP, and ALS mouse models.ResultsAmong 124 876 cases and controls, genome-wide conjunction analyses of ALS, FTD, PD, AD, CBD, and PSP revealed significant genetic overlap between ALS and FTD at known ALS loci: rs13302855 and rs3849942 (nearest gene, C9orf72; P = .03 for rs13302855 and P = .005 for rs3849942) and rs4239633 (nearest gene, UNC13A; P = .03). Significant genetic overlap was also found between ALS and PSP at rs7224296, which tags the MAPT H1 haplotype (nearest gene, NSF; P = .045). Shared risk genes were enriched for pathways involving neuronal function and development. At a conditional FDR P < .05, 22 novel ALS polymorphisms were found, including rs538622 (nearest gene, ERGIC1; P = .03 for ALS and FTD), which modifies BNIP1 expression in human brains (35 of 137 females; mean age, 59 years; P = .001). BNIP1 expression was significantly reduced in spinal cord motor neurons from patients with ALS (4 controls: mean age, 60.5 years, mean [SE] value, 3984 [760.8] arbitrary units [AU]; 7 patients with ALS: mean age, 56 years, mean [SE] value, 1999 [274.1] AU; P = .02), in an ALS mouse model (mean [SE] value, 13.75 [0.09] AU for 2 SOD1 WT mice and 11.45 [0.03] AU for 2 SOD1 G93A mice; P = .002) and in brains of patients with PSP (80 controls: 39 females; mean age, 82 years, mean [SE] value, 6.8 [0.2] AU; 84 patients with PSP: 33 females, mean age 74 years, mean [SE] value, 6.8 [0.1] AU; β = –0.19; P = .009) or FTD (11 controls: 4 females; mean age, 67 years; mean [SE] value, 6.74 [0.05] AU; 17 patients with FTD: 10 females; mean age, 69 years; mean [SE] value, 6.53 [0.04] AU; P = .005).Conclusions and RelevanceThis study found novel genetic overlap between ALS and diseases of the FTD spectrum, that the MAPT H1 haplotype confers risk for ALS, and identified the mitophagy-associated, proapoptotic protein BNIP1 as an ALS risk gene. Together, these findings suggest that sporadic ALS may represent a selectively pleiotropic, polygenic disorder.

Fluorine-18 labeled PI-2620 tau positron emission tomography and quantitative susceptibility mapping reveal spatiotemporal interactions between tau deposition and iron accumulation in progressive supranuclear palsy

9 - Neurobiology of progressive supranuclear palsy

An inverted region on chromosome 17 has been previously linked to many Pick complex diseases. Due to the inversion, an exact causal locus has been difficult to identify, but the microtubule-associated protein tau gene is a likely candidate gene for its involvement in these diseases with tau inclusion. To search for variants that confer susceptibility to 4 tauopathies and clinically related disorders. Genomewide association study. University research laboratory. A total of 231 samples were genotyped from an unrelated white population of patients with progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia, and frontotemporal dementia with amyotrophy. Unaffected individuals from the same population were used as controls. The results from an inverted region of chromosome 17 that contains the MAPT gene. Genotypes of cases and controls were compared using a Fisher exact test on a marker-by-marker basis. Haplotypes were determined by visually inspecting genotypes. Comparing any particular disease and controls, the association was constant across the inverted chromosome segment. Significant associations were seen for PSP and PSP combined with CBD. Of the 2 haplotypes seen in the region, H1 was overrepresented in PSP and CBD cases compared with controls. As expected, the markers are highly correlated and the association is seen across the entire region, which makes it difficult to narrow down a disease-causing variant or even a possible candidate gene. However, considering the pathologic abnormalities of these diseases and the involvement of tau mutations seen in familial forms, the MAPT gene represents the most likely cause driving the association.

Distinct neuronal and glial tau pathologies define corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). Additional Alzheimer disease, TDP-43, and Lewy body copathologies are also common. The interplay of these pathologies with clinical symptoms remains unclear as individuals can present with corticobasal syndrome, frontotemporal dementia, PSP, or atypical Parkinsonism and may have additional secondary impairments. We report clinical, pathological, and genetic interactions in a cohort of CBD and PSP cases. Neurofibrillary tangles and plaques were common. Apolipoprotein E (APOE)ε4 carriers had more plaques while PSP APOEε2 carriers had fewer plaques. TDP-43 copathology was present and age-associated in 14% of PSP, and age-independent in 33% of CBD. Lewy body copathology varied from 9% to 15% and was not age-associated. The primary FTD-Tau burden-a sum of the neuronal, astrocytic and oligodendrocytic tau-was not age-, APOE-, or MAPT-related. In PSP, FTD-Tau, independent of copathology, associated with executive, language, motor, and visuospatial impairments, while PSP with Parkinsonism had a lower FTD-Tau burden, but this was not the case in CBD. Taken together, our results indicate that the primary tauopathy burden is the strongest correlate of clinical PSP, while copathologies are principally determined by age and genetic risk factors.

The hallmark of Alzheimer's disease (AD) pathology is an accumulation of amyloid β (Aβ) and phosphorylated tau, which are encoded by the amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT) genes, respectively. Less than 5% of all AD cases are familial in nature, i.e. caused by mutations in APP, PSEN1 or PSEN2. Almost all mutations found in them are related to an overproduction of Aβ1-42, which is prone to aggregation. While these genes are mutation free, their function, or those of related genes, could be compromised in sporadic AD as well. In this study, pyrosequencing analysis of post-mortem brains revealed aberrant CpG methylation in APP, MAPT and GSK3B genes of the AD brain. These changes were further evaluated by a newly developed in vitro-specific DNA methylation system, which in turn highlighted an enhanced expression of APP and MAPT. Cell nucleus sorting of post-mortem brains revealed that the methylation changes of APP and MAPT occurred in both neuronal and non-neuronal cells, whereas GSK3B was abnormally methylated in non-neuronal cells. Further analysis revealed an association between abnormal APP CpG methylation and apolipoprotein E ε4 allele (APOE ε4)-negative cases. The presence of a small number of highly methylated neurons among normal neurons contribute to the methylation difference in APP and MAPT CpGs, thus abnormally methylated cells could compromise the neural circuit and/or serve as 'seed cells' for abnormal protein propagation. Our results provide a link between familial AD genes and sporadic neuropathology, thus emphasizing an epigenetic pathomechanism for sporadic AD.