Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and β-amyloid aggregates

Transcellular propagation of protein aggregate “seeds” has been proposed to mediate the progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We previously reported that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface, promoting cellular uptake and intracellular seeding. However, the specificity and binding mode of these protein aggregates to HSPGs remain unknown. Here, we measured direct interaction with modified heparins to determine the size and sulfation requirements for tau, α-synuclein, and β-amyloid (Aβ) aggregate binding to glycosaminoglycans (GAGs). Varying the GAG length and sulfation patterns, we next conducted competition studies with heparin derivatives in cell-based assays. Tau aggregates required a precise GAG architecture with defined sulfate moieties in the N- and 6-O-positions, whereas the binding of α-synuclein and Aβ aggregates was less stringent. To determine the genes required for aggregate uptake, we used CRISPR/Cas9 to individually knock out the major genes of the HSPG synthesis pathway in HEK293T cells. Knockouts of the extension enzymes exostosin 1 (EXT1), exostosin 2 (EXT2), and exostosin-like 3 (EXTL3), as well as N-sulfotransferase (NDST1) or 6-O-sulfotransferase (HS6ST2) significantly reduced tau uptake, consistent with our biochemical findings, and knockouts of EXT1, EXT2, EXTL3, or NDST1, but not HS6ST2 reduced α-synuclein uptake. In summary, tau aggregates display specific interactions with HSPGs that depend on GAG length and sulfate moiety position, whereas α-synuclein and Aβ aggregates exhibit more flexible interactions with HSPGs. These principles may inform the development of mechanism-based therapies to block transcellular propagation of amyloid protein–based pathologies

Biochemische, biophysikalische und histologische Analysen der Aktin- Nukleationsfaktoren FHOD1 und ForC

Ziel der Studie war die Analyse der Aktin-Nukleationsfaktoren FHOD1 und ForC. Eine Bindung von Rac1 an die FHOD1- bzw. ForC-GBD konnte mit Hilfe der analytischen Gelfiltration und der isothermalen Titrationskalorimetrie nicht nachgewiesen werden. Anhand von Lipidblots ergaben sich erste Hinweise auf eine Bindung von phosphoryliertem FHOD1-Volllängenprotein an Phosphatidylsäure, möglicherweise unter Vermittlung des FHOD1-N-Terminus. In zweidimensionalen NMR-Spektren der ForC-GBD fand sich eine hochgeordnete Strukturfaltung der Domäne. In Zusammenarbeit mit Frau PD Dr. S. Dames konnte die Domänenstruktur einer Ubiquitin-Faltung für die ForC-GBD ermittelt werden. In immunhistochemischen Färbungen mit einem anti-FHOD1-Antikörper (Prof. Dr. H.-G. Mannherz) ließ sich eine hohe FHOD1-Expression in proliferierenden B-Lymphozyten in den Keimzentren der Milz-Lymphfollikel der Maus darstellen, die auf eine mögliche Funktion von FHOD1 für die humorale Immunantwort hinweist

Cognitive Decline in Older People with Multiple Sclerosis—A Narrative Review of the Literature

Several important questions regarding cognitive aging and dementia in older people with multiple sclerosis (PwMS) are the focus of this narrative review: Do older PwMS have worse cognitive decline compared to older people without MS? Can older PwMS develop dementia or other neurodegenerative diseases such as Alzheimer’s disease (AD) that may be accelerated due to MS? Are there any potential biomarkers that can help to determine the etiology of cognitive decline in older PwMS? What are the neural and cellular bases of cognitive aging and neurodegeneration in MS? Current evidence suggests that cognitive impairment in MS is distinguishable from that due to other neurodegenerative diseases, although older PwMS may present with accelerated cognitive decline. While dementia is prevalent in PwMS, there is currently no consensus on defining it. Cerebrospinal fluid and imaging biomarkers have the potential to identify disease processes linked to MS and other comorbidities—such as AD and vascular disease—in older PwMS, although more research is required. In conclusion, one should be aware that multiple underlying pathologies can coexist in older PwMS and cause cognitive decline. Future basic and clinical research will need to consider these complex factors to better understand the underlying pathophysiology, and to improve diagnostic accuracy

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitin-like α/β-roll fold with an unusually long loop between β-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of α-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the β1-β2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-l-serine) and 1,2-dihexanoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes

Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres

Summary The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1-339) and (ii) a peptide corresponding the sequence from position 358 to 371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibres and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localisation to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-�1 indicated the additional localisation of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also ist presence in costameres

Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and β-amyloid aggregates

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