Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’s disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of Aβ1–42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of Aβ1–42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of Aβ1–42 the most. Kinetics of Aβ1–42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased Aβ1–42 uptake from the earliest time points, while other syndecans facilitated Aβ1–42 internalization at a slower pace. Internalized Aβ1–42 colocalized with syndecans and flotillins, highlighting the role of lipid-rafts in syndecan-mediated uptake. Syndecan-3 and 4 also triggered fibrillation of Aβ1–42, further emphasizing the pathophysiological relevance of syndecans in plaque formation. Overall our data highlight syndecans, especially the neuron-specific syndecan-3 isoform, as important players in amyloid pathology and show that syndecans, regardless of cell type, facilitate key molecular events in neurodegeneration.
Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-like spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of α-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of α-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both α-synuclein and tau. Syndecan-mediated internalization of α-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monomeric α-synuclein and tau, yet exerts a fibril forming effect on both proteins. Data obtained from syndecan overexpressing cellular models presents syndecans, especially the neuron predominant syndecan-3, as important mediators of seeding and spreading of α-synuclein and tau and reveal how syndecans contribute to fundamental molecular events of α-synuclein and tau pathology.
Cell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies.
BackgroundUse of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects.MethodsTo address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies.ResultsAlthough both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes.ConclusionTRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials.
Hyperthermia is a promising treatment modality for cancer in combination both with radio-and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo-or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies.
As endocytic uptake of the Antennapedia homeodomain-derived penetratin peptide (RQIKIWFQNRRMKWKK) is finally being revealed, some of the early views about penetratin need to be reconsidered. Endocytic uptake seems to contradict the indispensability of tryptophans and also the minimum length of 16 amino acid residues for efficient internalization. To revise the membrane translocation of penetratin, two penetratin analogs were designed and synthesized: a peptide in which tryptophans were replaced by phenylalanines (Phe(6,14)-penetratin, RQIKIFFQNRRMKFKK) and a shortened analog (dodeca-penetratin, RQIKIWF-R-KWKK) made up of only 12 residues. The peptides were fluorescently labeled and applied to live, unfixed cells from various lines. Cellular uptake was analysed by confocal microscopy and flow cytometry. Low temperature or ATP-depletion blocked the intracellular entry of all three penetratin peptides. A decrease in membrane fluidity or cholesterol depletion with methyl-beta-cyclodextrin greatly inhibited peptide uptake, showing the involvement of cholesterol-rich lipid rafts in internalization. Exogenous heparan sulfate also diminished the internalization of penetratin and its derivatives, reflecting the paramount importance of electrostatic interactions with polyanionic cell-surface proteoglycans. The beneficial presence of tryptophans is supported by observations on the decreased cellular uptake of Phe(6, 14)-penetratin. The maintained translocational efficiency of dodeca-penetratin demonstrates that a thorough understanding of penetratin internalization can yield new penetratin analogs with unaltered translocational abilities. This study provides evidence on the energy-dependent and lipid raft-mediated endocytic uptake of penetratin and highlights the necessity of revealing those pathways that cationic cell-penetrating peptides employ to enter live cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.