A myosin-7B-dependent endocytosis pathway mediates cellular entry of ?-synuclein fibrils and polycation-bearing cargos.
ABSTRACT: Cell-to-cell transmission of misfolding-prone ?-synuclein (?-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when ?-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for ?-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting ?-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in ?-Syn PFFs. By contrast, MYO7B regulates ?-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to ?-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for ?-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.
Project description:BACKGROUND:Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. MATERIALS AND METHODS:Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. RESULTS:The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. CONCLUSION:Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment.
Project description:Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins (rhBMPs) from CHO cell cultures. Upon incubation of rhBMP-2 and rhBMP-7 with CHO cells at 37 °C, both rhBMP-2 and rhBMP-7 bound to the cell surface HSPGs in CHO cells, but only rhBMP-2 was actively internalized into CHO cells. Cell surface HSPGs were found to serve as the main receptor for rhBMP-2 internalization. It was also found that the cell surface HSPG-mediated endocytosis of rhBMP-2 occurred through both the clathrin- and caveolin-dependent pathways. Blockage of rhBMP-2 internalization by the addition of structural analogs of HSPGs such as dextran sulfate (DS) and heparin dramatically increased rhBMP-2 production in recombinant CHO (rCHO) cell cultures. Compared to the control cultures, addition of DS (1.0 g/L) and heparin (0.2 g/L) resulted in a 22.0- and 19.0-fold increase in the maximum rhBMP-2 concentration, respectively. In contrast, the production of rhBMP-7, which was not internalized into the rCHO cells, did not dramatically increase upon addition of DS and heparin. Taken together, rhBMPs have a different fate in terms of HSPG-mediated internalization in CHO cells. HSPG-mediated endocytosis of each rhBMP should be understood individually to increase the rhBMP yield in rCHO cell cultures.
Project description:Direct cell-to-cell transmission of proteopathic ?-synuclein (?-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of ?-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied ?-syn seed species. Herein, we employed fluorescently labeled ?-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized ?-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous ?-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous ?-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of ?-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.
Project description:Animal models that accurately recapitulate the accumulation of alpha-synuclein (?-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of ?-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of ?-syn PFFs (8 or 16??g of total protein) into striatal sites selected to concentrate ?-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an ?-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2?months and bilateral loss of nigral dopamine neurons at 6?months. Unilateral 16??g PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6?months. Bilateral injection of 16??g ?-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16??g PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These ?-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.
Project description:Alpha-synuclein (?syn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies. ?Syn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of ?syn necessitates the release of misfolded ?syn from one cell and the uptake of that ?syn by another, in which it can template the misfolding of endogenous ?syn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3? acts as a chaperone to reduce ?syn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on ?syn toxicity in the in vivo ?syn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, ?syn aggregation, and neuronal counts in the PFF model. 14-3-3? transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3? overexpression was associated with delayed ?syn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated ?syn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3? overexpression by AAV in the substantia nigra (SN) also delayed ?syn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral ?syn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3? against ?syn toxicity in vivo.
Project description:BACKGROUND:Synucleinopathy is a group of neurodegenerative disorders characterized by neurodegeneration and accumulation of alpha-synuclein (?-syn) aggregates in various brain regions. The detailed mechanism of ?-syn-caused neurotoxicity remains obscure, which is partly due to the lack of a suitable model that retains the in vivo three-dimensional cellular network and allows a convenient dissection of the neurotoxic pathways. Recent studies revealed that the pre-formed recombinant ?-syn amyloid fibrils (PFFs) induce a robust accumulation of pathogenic ?-syn species in cultured cells and animals. OBJECTIVE:Our goal is to determine whether PFFs are able to induce the pathogenic ?-syn accumulation and neurotoxicity in organotypic brain slice culture, an ex vivo system that retains the in vivo three-dimensional cell-cell connections. METHODS/RESULTS:Adding PFFs to cultured wild-type rat or mouse brain slices induced a time-dependent accumulation of pathogenic ?-syn species, which was indicated by ?-syn phosphorylated at serine 129 (p?-syn). The PFF-induced p?-syn was abolished in brain slices prepared from ?-syn null mice, suggesting that the p?-syn is from the phosphorylation of endogenous ?-syn. Human PFFs also induced p?-syn in brain slices prepared from mice expressing human ?-syn on a mouse ?-syn-null background. Furthermore, the synaptophysin immunoreactivity was inversely associated with p?-syn accumulation and an increase of neuronal loss was detected. CONCLUSION:PFF-treatment of brain slices is able to induce key pathological features of synucleinopathy: p?-syn accumulation and neurotoxicity. This model will be useful for investigating the neurotoxic mechanism and evaluating efficacy of therapeutic approaches.
Project description:Accumulation of misfolded alpha-synuclein (?-syn) into Lewy bodies (LBs) and Lewy neurites (LNs) is a major hallmark of Parkinson's disease (PD) and dementia with LBs (DLB). Recent studies showed that synthetic preformed fibrils (pffs) recruit endogenous ?-syn and induce LB/LN pathology in vitro and in vivo, thereby implicating propagation and cell-to-cell transmission of pathological ?-syn as mechanisms for the progressive spread of LBs/LNs. Here, we demonstrate that ?-syn monoclonal antibodies (mAbs) reduce ?-syn pff-induced LB/LN formation and rescue synapse/neuron loss in primary neuronal cultures by preventing both pff uptake and subsequent cell-to-cell transmission of pathology. Moreover, intraperitoneal (i.p.) administration of mAb specific for misfolded ?-syn into nontransgenic mice injected intrastriatally with ?-syn pffs reduces LB/LN pathology, ameliorates substantia nigra dopaminergic neuron loss, and improves motor impairments. We conclude that ?-syn antibodies could exert therapeutic effects in PD/DLB by blocking entry of pathological ?-syn and/or its propagation in neurons.
Project description:MHCII molecules, expressed by professional antigen-presenting cells (APCs) such as T cells and B cells, are hypothesized to play a key role in the response of cellular immunity to α-synuclein (α-syn). However, the role of cellular immunity in the neuroanatomic transmission of α-syn pre-formed fibrillar (PFF) seeds is undetermined. To illuminate whether cellular immunity influences the transmission of α-syn seeds from the periphery into the CNS, we injected preformed α-syn PFFs in the hindlimb of the Line M83 transgenic mouse model of synucleinopathy lacking MhcII. We showed that a complete deficiency in MhcII accelerated the appearance of seeded α-syn pathology and shortened the lifespan of the PFF-seeded M83 mice. To characterize whether B-cell and T-cell inherent MhcII function underlies this accelerated response to PFF seeding, we next injected α-syn PFFs in Rag1-/- mice which completely lacked these mature lymphocytes. There was no alteration in the lifespan or burden of endstage α-syn pathology in the PFF-seeded, Rag1-deficient M83+/- mice. Together, these results suggested that MhcII function on immune cells other than these classical APCs is potentially involved in the propagation of α-syn in this model of experimental synucleinopathy. We focused on microglia next, finding that while microglial burden was significantly upregulated in PFF-seeded, MhcII-deficient mice relative to controls, the microglial activation marker Cd68 was reduced in these mice, suggesting that these microglia were not responsive. Additional analysis of the CNS showed the early appearance of the neurotoxic astrocyte A1 signature and the induction of the Ifnγ-inducible anti-viral response mediated by MhcI in the MhcII-deficient, PFF-seeded mice. Overall, our data suggest that the loss of MhcII function leads to a dysfunctional response in non-classical APCs and that this response could potentially play a role in determining PFF-induced pathology. Collectively, our results identify the critical role of MhcII function in synucleinopathies induced by α-syn prion seeds.
Project description:The process of heparan sulfate proteoglycan (HSPG) internalization has been described as following different pathways. The tumor-specific branched NT4 peptide has been demonstrated to bind HSPGs on the plasma membrane and to be internalized in tumor cell lines. The polycationic peptide has been also shown to impair migration of different cancer cell lines in 2D and 3D models. Our hypothesis was that HSPG endocytosis could affect two important phenomena of cancer development: cell migration and nourishment. Using NT4 as an experimental tool mimicking heparin-binding ligands, we studied endocytosis and trafficking of HSPGs in a triple-negative human breast cancer cell line, MDA-MB-231. The peptide entered cells employing caveolin- or clathrin-dependent endocytosis and macropinocytosis, in line with what is already known about HSPGs. NT4 then localized in early and late endosomes in a time-dependent manner. The peptide had a negative effect on CDC42-activation triggered by EGF. The effect can be explained if we consider NT4 a competitive inhibitor of EGF on HS that impairs the co-receptor activity of the proteoglycan, reducing EGFR activation. Reduction of the invasive migratory phenotype of MDA-MB-231 induced by NT4 can be ascribed to this effect. RhoA activation was damped by EGF in MDA-MB-231. Indeed, EGF reduced RhoA-GTP and NT4 did not interfere with this receptor-mediated signaling. On the other hand, the peptide alone determined a small but solid reduction in active RhoA in breast cancer cells. This result supports the observation of few other studies, showing direct activation of the GTPase through HSPG, not mediated by EGF/EGFR.
Project description:Parkinson's disease is a debilitating neurodegenerative disorder that is pathologically characterized by intracellular inclusions comprised primarily of alpha-synuclein (?Syn) that can also be transmitted from neuron to neuron. Several lines of evidence suggest that these inclusions cause neurodegeneration. Thus exploring strategies to improve neuronal survival in neurons with ?Syn aggregates is critical. Previously, exposure to ?Syn pre-formed fibrils (PFFs) has been shown to induce aggregation of endogenous ?Syn resulting in cell death that is exacerbated by either starvation or inhibition of mTOR by rapamycin, both of which are able to induce autophagy, an intracellular protein degradation pathway. Since mTOR inhibition may also inhibit protein synthesis and starvation itself can be detrimental to neuronal survival, we investigated the effects of autophagy induction on neurons with ?Syn inclusions by a starvation and mTOR-independent autophagy induction mechanism. We exposed mouse primary cortical neurons to PFFs to induce inclusion formation in the presence and absence of the disaccharide trehalose, which has been proposed to induce autophagy and stimulate lysosomal biogenesis. As expected, we observed that on exposure to PFFs, there was increased abundance of pS129-?Syn aggregates and cell death. Trehalose alone increased LC3-II levels, consistent with increased autophagosome levels that remained elevated with PFF exposure. Interestingly, trehalose alone increased cell viability over a 14-d time course. Trehalose was also able to restore cell viability to control levels, but PFFs still exhibited toxic effects on the cells. These data provide essential information regarding effects of trehalose on ?Syn accumulation and neuronal survival on exposure to PFF.