Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
2.
Nat Commun ; 14(1): 3939, 2023 07 04.
Article in English | MEDLINE | ID: mdl-37402718

ABSTRACT

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.


Subject(s)
Protein Aggregation, Pathological , tau Proteins , tau Proteins/chemistry , Heparin/chemistry , Humans , Cell Line , Biosensing Techniques , Pluripotent Stem Cells , Neurons , Protein Isoforms , Cryoelectron Microscopy
3.
Neurobiol Dis ; 182: 106126, 2023 06 15.
Article in English | MEDLINE | ID: mdl-37086756

ABSTRACT

Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo.


Subject(s)
Caspase 2 , tau Proteins , Humans , Male , Female , Animals , Mice , Disease Models, Animal , tau Proteins/chemistry , tau Proteins/genetics , tau Proteins/metabolism , Caspase 2/metabolism , Brain/metabolism , Chromatin Immunoprecipitation , Ubiquitination
4.
Mol Neurodegener ; 16(1): 46, 2021 07 02.
Article in English | MEDLINE | ID: mdl-34215303

ABSTRACT

BACKGROUND: Human tauopathies including Alzheimer's disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. METHODS: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. RESULTS: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. CONCLUSIONS: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.


Subject(s)
Alzheimer Disease/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Protein Processing, Post-Translational/physiology , tau Proteins/metabolism , Animals , Humans , Lysine/metabolism , Methylation , Mice , Mice, Transgenic
5.
Nat Commun ; 11(1): 5163, 2020 10 14.
Article in English | MEDLINE | ID: mdl-33057020

ABSTRACT

Parkinson's disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson's disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson's disease.


Subject(s)
Dopaminergic Neurons/immunology , Interferon-gamma/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Microglia/immunology , Parkinson Disease/immunology , Calcium Signaling/genetics , Cell Differentiation , Cytokines/metabolism , Dopaminergic Neurons/metabolism , Gene Knockout Techniques , Glycolysis/genetics , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/physiology , Interferon-gamma/immunology , Intravital Microscopy , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Microglia/metabolism , Microtubules/metabolism , Mutation , NFATC Transcription Factors/metabolism , Parkinson Disease/genetics , Parkinson Disease/pathology , Primary Cell Culture , Signal Transduction/genetics , Signal Transduction/immunology , THP-1 Cells
6.
Acta Neuropathol Commun ; 7(1): 192, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31796124

ABSTRACT

Tau is a microtubule-binding protein that can receive various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Hyperphosphorylation of tau is linked to its aggregation and the formation of neurofibrillary tangles (NFTs), which are a hallmark of Alzheimer's disease (AD). While more than 70 phosphorylation sites have been detected previously on NFT tau, studies of oligomeric and detergent-soluble tau in human brains during the early stages of AD are lacking. Here we apply a comprehensive electrochemiluminescence ELISA assay to analyze twenty-five different PTM sites as well as tau oligomerization in control and sporadic AD brain. The samples were classified as Braak stages 0-I, II or III-IV, corresponding to the progression of microscopically detectable tau pathology throughout different brain regions. We found that soluble tau multimers are strongly increased at Braak stages III-IV in all brain regions under investigation, including the temporal cortex, which does not contain NFTs or misfolded oligomers at this stage of pathology. We additionally identified five phosphorylation sites that are specifically and consistently increased across the entorhinal cortex, hippocampus and temporal cortex in the same donors. Three of these sites correlate with tau multimerization in all three brain regions, but do not overlap with the epitopes of phospho-sensitive antibodies commonly used for the immunohistochemical detection of NFTs. Our results thus suggest that soluble multimers are characterized by a small set of specific phosphorylation events that differ from those dominating in mature NFTs. These findings shed light on early PTM changes of tau during AD pathogenesis in human brains.


Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Induced Pluripotent Stem Cells/metabolism , Protein Processing, Post-Translational/physiology , tau Proteins/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Brain/pathology , Female , Humans , Induced Pluripotent Stem Cells/pathology , Male , Phosphorylation/physiology , tau Proteins/genetics
7.
Neurobiol Dis ; 130: 104518, 2019 10.
Article in English | MEDLINE | ID: mdl-31229689

ABSTRACT

Tau cleavage by different proteolytic enzymes generates short, aggregation-prone fragments that have been implicated in the pathogenesis of Alzheimer's disease (AD). Asparagine endopeptidase (AEP) activity in particular has been associated with tau dysfunction and aggregation, and the activity of the protease is increased in both aging and AD. Using a mass spectrometry approach, we identified a novel tau cleavage site at N167 and confirmed its processing by AEP. In combination with the previously known site at N368, we show that AEP cleavage yields a tau fragment that is present in both control and AD brains at similar levels. AEP is a lysosomal enzyme, and our data suggest that it is expressed in microglia rather than in neurons. Accordingly, we observe tau cleavage at N167 and N368 after endocytotic uptake into microglia, but not neurons. However, tau168-368 does not accumulate in microglia and we thus conclude that the fragment is part of a proteolytic cascade leading to tau degradation. While we confirm previous studies showing increased overall AEP activity in AD brains, our data suggests that AEP-mediated cleavage of tau is a physiological event occurring during microglial degradation of the secreted neuronal protein. As a consequence, we caution against preventing AEP-mediated tau cleavage as a therapeutic approach in AD.


Subject(s)
Brain/metabolism , Cysteine Endopeptidases/metabolism , Microglia/physiology , tau Proteins/metabolism , HEK293 Cells , Humans , Mass Spectrometry , Neurons/physiology , Proteolysis
8.
Stem Cell Res ; 34: 101351, 2019 01.
Article in English | MEDLINE | ID: mdl-30611016

ABSTRACT

Alzheimer's Disease (AD) is the major cause of dementia in the elderly, and cortical neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) can recapitulate disease phenotypes such as tau phosphorylation or amyloid beta (Aß) deposition. Here we describe the generation of an iPSC cohort consisting of 2 sporadic AD cases and 3 controls, derived from dermal fibroblasts. All lines were karyotypically normal, showed expression of stem cell markers and efficiently differentiated into cells of all three germ layers.


Subject(s)
Alzheimer Disease/pathology , Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/pathology , Aged , Aged, 80 and over , Cell Line , Female , Humans , Male , Middle Aged
9.
Cell Rep ; 23(10): 2976-2988, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29874584

ABSTRACT

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.


Subject(s)
Drosophila melanogaster/physiology , Induced Pluripotent Stem Cells/pathology , Mitochondria/pathology , NAD/metabolism , Neurons/metabolism , Neurons/pathology , Niacinamide/analogs & derivatives , Parkinson Disease/pathology , Animals , Autophagy , Disease Models, Animal , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Endoplasmic Reticulum Stress , Glucosylceramidase/metabolism , Humans , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Dynamics , Motor Activity , Niacinamide/metabolism , Parkinson Disease/physiopathology , Pyridinium Compounds , Unfolded Protein Response
10.
Cell Syst ; 4(2): 157-170.e14, 2017 02 22.
Article in English | MEDLINE | ID: mdl-28131822

ABSTRACT

Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy.


Subject(s)
Neurodegenerative Diseases/pathology , alpha-Synuclein/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Ataxin-2/chemistry , Ataxin-2/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Susceptibility , Endoplasmic Reticulum/metabolism , Eukaryotic Initiation Factor-4G/chemistry , Eukaryotic Initiation Factor-4G/metabolism , Gene Regulatory Networks/genetics , Genome, Fungal , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Neurodegenerative Diseases/genetics , Neurons/cytology , Neurons/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , alpha-Synuclein/genetics
11.
Nat Commun ; 5: 4028, 2014 Jun 06.
Article in English | MEDLINE | ID: mdl-24905578

ABSTRACT

Mutations in the acid ß-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher's disease (GD), are the strongest genetic risk factor for Parkinson's disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects. Quantitative proteomic profiling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These findings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.


Subject(s)
Autophagy , Calcium/metabolism , Glucosylceramidase/genetics , Homeostasis , Induced Pluripotent Stem Cells/pathology , Neurons/pathology , Parkinson Disease/pathology , Cell Differentiation , Glycoside Hydrolases/metabolism , Humans , Induced Pluripotent Stem Cells/enzymology , Neurons/enzymology , Parkinson Disease/immunology , Parkinson Disease/metabolism
12.
BMC Neurol ; 14: 118, 2014 Jun 03.
Article in English | MEDLINE | ID: mdl-24894446

ABSTRACT

BACKGROUND: Charcot-Marie-Tooth disease (CMT) refers to a heterogeneous group of genetic motor and sensory neuropathies. According to the primary site of damage, a distinction is made between demyelinating and axonal forms (CMT1 and 2, respectively, when inherited as an autosomal dominant trait). Leucine-rich repeat and sterile alpha motif-containing protein 1 (LRSAM1) is a ubiquitin-protein ligase with a role in sorting internalised cell-surface receptor proteins. So far, mutations in the LRSAM1 gene have been shown to cause axonal CMT in three different families and can confer either dominant or recessive transmission of the disease. CASE PRESENTATION: We have identified a novel mutation in LRSAM1 in a small family with dominant axonal CMT. Electrophysiological studies show evidence of a sensory axonal neuropathy and are interesting in so far as giant motor unit action potentials (MUAPs) are present on needle electromyography (EMG), while motor nerve conduction studies including compound motor action potential (CMAP) amplitudes are completely normal. The underlying mutation c.2046+1G >T results in the loss of a splice donor site and the inclusion of 63 additional base pairs of intronic DNA into the aberrantly spliced transcript. This disrupts the catalytically active RING (Really Interesting New Gene) domain of LRSAM1. CONCLUSIONS: Our findings suggest that, beyond the typical length-dependent degeneration of motor axons, damage of cell bodies in the anterior horn might play a role in LRSAM1-associated neuropathies. Moreover, in conjunction with other data in the literature, our results support a model, by which disruption of the C-terminal RING domain confers dominant negative properties to LRSAM1.


Subject(s)
Axons/pathology , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Ubiquitin-Protein Ligases/genetics , Adolescent , Adult , Aged , Child , Exome/genetics , Female , Genes, Dominant , Humans , Male , Middle Aged , Muscle, Skeletal/pathology , Mutation/genetics , Pedigree , Young Adult
13.
Cell Stem Cell ; 12(3): 354-67, 2013 Mar 07.
Article in English | MEDLINE | ID: mdl-23472874

ABSTRACT

The LRRK2 mutation G2019S is the most common genetic cause of Parkinson's disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Induced Pluripotent Stem Cells/metabolism , Parkinson Disease/genetics , Parkinson Disease/metabolism , Protein Serine-Threonine Kinases/genetics , Benzamides/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Dopamine/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mutation , Neurons/cytology , Neurons/drug effects , Oxidopamine/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Rotenone/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...