SorLA Protective Function Is Restored by Improving SorLA Protein Maturation in a Subset of Alzheimer's Disease-Associated SORL1 Missense Variants.

SORL1 loss of function is associated with Alzheimer's disease (AD) risk through increased Aß peptide secretion. We expressed 10 maturation-defective rare missense SORL1 variants in HEK cells and showed that decreasing growing temperature led to a significant increase in the maturation of the encoded protein SorLA for 6/10. In edited hiPSC carrying two of these variants, maturation of the protein was restored partially by decreasing the culture temperature and was associated with concomitant decrease in Aß secretion. Correcting SorLA maturation in the context of maturation-defective missense variants could thus be a relevant strategy to improve SorLA protective function against AD.

Generation of 17q21.31 duplication iPSC-derived neurons as a model for primary tauopathies.

Tau proteins belong to the microtubule associated protein family and are mainly expressed in neurons. Tau accumulates in patients' brain in several neurodegenerative diseases, including Fronto-temporal dementia and Alzheimer's disease. Recently, we described a 17q21.31 duplication in patients presenting different cognitive or motor symptoms and characterized by the accumulation of different Tau isoforms. This duplication involves four genes, including the MAPT gene that encodes the Tau protein. The main pathophysiological consequence associated with this duplication was a 1.6-1.9-fold increase in the MAPT messenger RNA as measured in blood samples of duplication carriers. However, the pathophysiological consequences of this duplication in a cell type relevant for neurodegenerative diseases have never been explored so far. In this study, we developed the first model of primary tauopathy linked to a 17q21.31 duplication in iPSC-induced neurons from 2 unrelated carriers. As in patients' blood, we demonstrated that this duplication was associated with an increase in MAPT mRNA resulting in elevated Tau protein levels in iPSC-derived cortical neurons. We believe that these iPSC lines will be a pertinent tool to elucidate how a same genetic cause could lead to distinct types of tauopathies and the pathophysiological mechanisms associated with Tau-mediated neurodegeneration in the 17q21.31 duplication context.

Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease.

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer's disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aß peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aß secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

Clinical and neuropathological diversity of tauopathy in MAPT duplication carriers.

Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.

Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-ß-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization.

Alzheimer's disease (AD) is neuropathologically defined by two key hallmarks: extracellular senile plaques composed primarily of amyloid-ß (Aß) peptide and intraneuronal neurofibrillary tangles, containing abnormally hyperphosphorylated tau protein. The tau protein is encoded by the MAPT gene. Recently, the H1 and H2 haplotypes of the MAPT gene were associated with AD risk. The minor MAPT H2 haplotype has been linked with a decreased risk of developing late-onset AD (LOAD). MAPT haplotypes show different levels of MAPT/Tau expression with H1 being ∼1.5-fold more expressed than H2, suggesting that MAPT expression level could be related to LOAD risk. In this study, we investigated whether this moderate difference in MAPT/Tau expression could influence Aß-induced toxicity in vivo. We show that modest overexpression of tau protein in Drosophila exacerbates neuronal phenotypes in AßPP/BACE1 flies. The exacerbation of neuronal defects correlates with the accumulation of insoluble dTau oligomers, suggesting that the moderate difference in level of tau expression observed between H1 and H2 haplotypes could influence Aß toxicity through the production of oligomeric tau insoluble species.

Detection of all adult Tau isoforms in a 3D culture model of iPSC-derived neurons.

Tauopathies are a class of neurodegenerative diseases characterized by the presence of pathological intracellular deposits of Tau proteins. Six isoforms of Tau are expressed in the adult human brain, resulting from alternative splicing of the MAPT gene. Tau splicing is developmentally regulated such that only the smallest Tau isoform is expressed in fetal brain, contrary to the adult brain showing the expression of all 6 isoforms. Induced Pluripotent Stem Cell (iPSC) technology has opened up new perspectives in human disease modeling, including tauopathies. However, a major challenge to in vitro recapitulation of Tau pathology in iPSC-derived neurons is their relative immaturity. In this study, we examined the switch in Tau splicing from fetal-only to all adult Tau isoforms during the differentiation of iPSC-derived neurons in a new 3D culture system. First, we showed that iPSC-induced neurons inside Matrigel-coated alginate capsules were able to differentiate into cortical neurons. Then, using a new assay that allowed both the qualitative and the quantitative analysis of all adult MAPT mRNA isoforms individually, we demonstrated that BrainPhys-maintained neurons expressed the 6 adult MAPT mRNA transcripts from 25 weeks of maturation, making this model highly suitable for modeling Tau pathology and therapeutic purposes.

Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila.

TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.

FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression in Drosophila.

TDP-43 is a major disease-causing protein in amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Today, >50 missense mutations in the TARDBP/TDP-43 gene have been described in patients with FTLD/ALS. However, the functional consequences of FTLD/ALS-linked TDP-43 mutations are not fully elucidated. In the physiological state, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. In the present study, we investigated whether the FTLD/ALS-associated mutations could interfere with TDP-43 protein's capacity to modulate its own protein levels using Drosophila as an experimental model. Our data show that FTLD/ALS-associated mutant proteins regulate TDP-43 production with the same efficiency as the wild-type form of the protein. Thus, FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.

Splicing factors act as genetic modulators of TDP-43 production in a new autoregulatory TDP-43 Drosophila model.

TDP-43 is a critical RNA-binding factor associated with RNA metabolism. In the physiological state, maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. As such, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. TDP-43 is a major disease-causing protein in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several studies argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulating the cycle of TDP-43 production might therefore provide a new therapeutic strategy. In this study, we developed a new transgenic Drosophila model mimicking the TDP-43 autoregulatory feedback loop in order to identify genetic modulators of TDP-43 protein steady-state levels in vivo. First, we showed that our TDP-43_TDPBR Drosophila model recapitulates key features of the TDP-43 autoregulatory processes previously described in mammalian and cellular models, namely alternative splicing events, differential usage of polyadenylation sites, nuclear retention of the transcript and a decrease in steady-state mRNA levels. Using this new Drosophila model, we identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. Interestingly, our data indicate that these three RNA-binding proteins regulate TDP-43 protein production, at least in part, by controlling mRNA steady-state levels.

Enhancing Mitofusin/Marf ameliorates neuromuscular dysfunction in Drosophila models of TDP-43 proteinopathies.

Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, we examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43-induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. We show here for the first time that enhancing the profusion gene mitofusin/marf is beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target.

Protein kinase CK-1 inhibitors as new potential drugs for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons in cortex, brain stem, and spinal cord die progressively, resulting in muscle wasting, paralysis, and death. Currently, effective therapies for ALS are lacking; however, identification of pathological TAR DNA-binding protein 43 (TDP-43) as the hallmark lesion in sporadic ALS suggests new therapeutic targets for pharmacological intervention. Pathological TDP-43 phosphorylation appears to drive the onset and progression of ALS and may result from upregulation of the protein kinase CK-1 in affected neurons, resulting in postranslational TDP-43 modification. Consequently, brain penetrant specific CK-1 inhibitors may provide a new therapeutic strategy for treating ALS and other TDP-43 proteinopathies. Using a chemical genetic approach, we report the discovery and further optimization of a number of potent CK-1δ inhibitors. Moreover, these small heterocyclic molecules are able to prevent TDP-43 phosphorylation in cell cultures, to increase Drosophila lifespan by reduction of TDP-43 neurotoxicity, and are predicted to cross the blood-brain barrier. Thus, N-(benzothiazolyl)-2-phenyl-acetamides are valuable drug candidates for further studies and may be a new therapeutic approach for ALS and others pathologies in which TDP-43 is involved.

Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila.

Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.

Both cytoplasmic and nuclear accumulations of the protein are neurotoxic in Drosophila models of TDP-43 proteinopathies.

Recently, the TAR DNA-binding protein-43 (TDP-43) has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patient with amyotrophic lateral sclerosis or frontotemporal lobar degeneration. Pathological proteins are abnormally hyperphosphorylated and partially cleaved to generate C-terminal fragments. In this issue, we addressed the mechanism underlying TDP-43 toxicity in vivo, using Drosophila as an experimental model. We developed new Drosophila transgenic models expressing different variants of full-length human TDP-43 proteins presenting different subcellular localizations: a wild-type form of hTDP-43 and two mutants forms of the protein, hTDP-43mutNLS and hTDP43mutNES, which lack nuclear localization signals (NLS) and nuclear export signals (NES), respectively. Using an inducible GAL4 system, we found that both nuclear and cytoplasmic accumulations of TDP-43 in adult neurons lead to reduction of lifespan in Drosophila, the gradient of toxicity being hTDP-43>hTDP-43mutNLS>hTDP43mutNES. This toxicity occurs regardless of inclusions formation. In the other hand, in retina, muscle and glial cells, only the accumulation of cytoplasmic species of TDP-43 was toxic. Biochemical data showed that human TDP-43 proteins expressed in adult fly neurons are abnormally phosphorylated on the disease-specific Ser409/Ser410 site and processed. In conclusion, our data show that TDP-43 expression in flies recapitulates several biochemical key features of human TDP-43 proteinopathies, including abnormal phosphorylation on a disease-specific site and processing of the protein. Moreover, our TDP-43 Drosophila models indicate that distinct pathways of TDP-43 toxicity might operate depending on the cell type.

Drosophila models of human tauopathies indicate that Tau protein toxicity in vivo is mediated by soluble cytosolic phosphorylated forms of the protein.

Tau is a neuronal microtubule-associated protein involved in microtubules assembly and stabilization. Tauopathies, including Alzheimer's disease and fronto-temporal dementia with parkinsonism linked to chromosome 17, are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally and hyperphosphorylated Tau. Currently, the molecular mechanisms underlying Tau-mediated cellular toxicity remain elusive. To address the determinants of Tau neurotoxicity, we used Drosophila models of human tauopathies to study the microtubule-binding properties of human Tau proteins in vivo. We showed that, in contrast to endogenous Drosophila Tau, human Tau proteins bind very poorly to microtubules in Drosophila, and are mostly recovered as soluble cytosolic hyperphosphorylated species. This weak binding of human Tau to microtubules is neither because of microtubules saturation nor competition with endogenous Drosophila Tau, but clearly depends on its phosphorylation degree. We also reported that accumulation of cytosolic hyperphosphorylated forms of human Tau proteins correlates with human Tau-mediated neurodegeneration in flies, supporting the key role of soluble cytosolic hyperphosphorylated Tau proteins as toxic species in vivo.