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52 Publications visible to you, out of a total of 52
Neuronal Differentiation of LUHMES Cells Induces Substantial Changes of the Proteome.

Abstract (Expand)

Neuronal cell lines are important model systems to study mechanisms of neurodegenerative diseases. One example is the Lund Human Mesencephalic (LUHMES) cell line, which can differentiate into … dopaminergic-like neurons and is frequently used to study mechanisms of Parkinson's disease and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified with selected neuronal markers, but little is known about the proteome-wide protein abundance changes during differentiation. Using mass spectrometry and label-free quantification (LFQ), the proteome of differentiated and undifferentiated LUHMES cells and of primary murine midbrain neurons are compared. Neuronal differentiation induced substantial changes of the LUHMES cell proteome, with proliferation-related proteins being strongly down-regulated and neuronal and dopaminergic proteins, such as L1CAM and alpha-synuclein (SNCA) being up to 1,000-fold up-regulated. Several of these proteins, including MAPT and SYN1, may be useful as new markers for experimentally validating neuronal differentiation of LUHMES cells. Primary midbrain neurons are slightly more closely related to differentiated than to undifferentiated LUHMES cells, in particular with respect to the abundance of proteins related to neurodegeneration. In summary, the analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on neurodegeneration and provides a resource of the proteome-wide changes during neuronal differentiation. (ProteomeXchange identifier PXD020044).

Authors: J. Tushaus, E. S. Kataka, J. Zaucha, D. Frishman, S. A. Muller, S. F. Lichtenthaler

Date Published: 21st Sep 2020

Publication Type: Journal

ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis.

Abstract (Expand)

While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins … (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

Authors: F. Eck, S. Phuyal, M. D. Smith, M. Kaulich, S. Wilkinson, H. Farhan, C. Behrends

Date Published: 16th Sep 2020

Publication Type: Journal

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

Abstract (Expand)

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and … inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Authors: C. Z. Koo, N. Harrison, P. J. Noy, J. Szyroka, A. L. Matthews, H. E. Hsia, S. A. Muller, J. Tushaus, J. Goulding, K. Willis, C. Apicella, B. Cragoe, E. Davis, M. Keles, A. Malinova, T. A. McFarlane, P. R. Morrison, H. T. H. Nguyen, M. C. Sykes, H. Ahmed, A. Di Maio, L. Seipold, P. Saftig, E. Cull, C. Pliotas, E. Rubinstein, N. S. Poulter, S. J. Briddon, N. D. Holliday, S. F. Lichtenthaler, M. G. Tomlinson

Date Published: 4th Sep 2020

Publication Type: Journal

Basic Fibroblast Growth Factor 2-Induced Proteome Changes Endorse Lewy Body Pathology in Hippocampal Neurons.

Abstract (Expand)

Hippocampal Lewy body pathology (LBP) is associated with changes in neurotrophic factor signaling and neuronal energy metabolism. LBP progression is attributed to the aggregation of alpha-synuclein … (alpha-Syn) and its cell-to-cell transmission via extracellular vehicles (EVs). We recently discovered an enhanced EV release in basic fibroblast growth factor (bFGF)-treated hippocampal neurons. Here, we examined the EV and cell lysate proteome changes in bFGF-treated hippocampal neurons. We identified n = 2,310 differentially expressed proteins (DEPs) induced by bFGF. We applied weighted protein co-expression network analysis (WPCNA) to generate protein modules from DEPs and mapped them to published LBP datasets. This approach revealed n = 532 LBP-linked DEPs comprising key alpha-Syn-interacting proteins, LBP-associated RNA-binding proteins (RBPs), and neuronal ion channels and receptors that can impact LBP onset and progression. In summary, our deep proteomic analysis affirms the potential influence of bFGF signaling on LBP-related proteome changes and associated molecular interactions.

Authors: R. Kumar, S. Donakonda, S. A. Muller, S. F. Lichtenthaler, K. Botzel, G. U. Hoglinger, T. Koeglsperger

Date Published: 21st Aug 2020

Publication Type: Journal

Fibrillar Abeta triggers microglial proteome alterations and dysfunction in Alzheimer mouse models.

Abstract (Expand)

Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. … Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid beta (Abeta) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial Abeta Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of Abeta deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar Abeta, rather than dystrophic neurites, suggesting that fibrillar Abeta may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy.

Authors: L. Sebastian Monasor, S. A. Muller, A. V. Colombo, G. Tanrioever, J. Konig, S. Roth, A. Liesz, A. Berghofer, A. Piechotta, M. Prestel, T. Saito, T. C. Saido, J. Herms, M. Willem, C. Haass, S. F. Lichtenthaler, S. Tahirovic

Date Published: 8th Jun 2020

Publication Type: Journal

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis.

Abstract (Expand)

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked … whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-kappaB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-alpha in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

Authors: M. I. Cunha, M. Su, L. Cantuti-Castelvetri, S. A. Muller, M. Schifferer, M. Djannatian, I. Alexopoulos, F. van der Meer, A. Winkler, T. J. van Ham, B. Schmid, S. F. Lichtenthaler, C. Stadelmann, M. Simons

Date Published: 4th May 2020

Publication Type: Journal

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

Abstract (Expand)

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel … lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

Authors: J. Jia, B. Bissa, L. Brecht, L. Allers, S. W. Choi, Y. Gu, M. Zbinden, M. R. Burge, G. Timmins, K. Hallows, C. Behrends, V. Deretic

Date Published: 5th Mar 2020

Publication Type: Journal

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