Increased levels of Stress-inducible phosphoprotein-1 accelerates amyloid-β deposition in a mouse model of Alzheimer’s disease

Molecular chaperones and co-chaperones, which are part of the protein quality control machinery, have been shown to regulate distinct aspects of Alzheimer’s Disease (AD) pathology in multiple ways. Notably, the co-chaperone STI1, which presents increased levels in AD, can protect mammalian neurons from amyloid-β toxicity in vitro and reduced STI1 levels worsen Aβ toxicity in C. elegans. However, whether increased STI1 levels can protect neurons in vivo remains unknown. We determined that overexpression of STI1 and/or Hsp90 protected C. elegans expressing Aβ(3–42) against Aβ-mediated paralysis. Mammalian neurons were also protected by elevated levels of endogenous STI1 in vitro, and this effect was mainly due to extracellular STI1. Surprisingly, in the 5xFAD mouse model of AD, by overexpressing STI1, we find increased amyloid burden, which amplifies neurotoxicity and worsens spatial memory deficits in these mutants. Increased levels of STI1 disturbed the expression of Aβ-regulating enzymes (BACE1 and MMP-2), suggesting potential mechanisms by which amyloid burden is increased in mice. Notably, we observed that STI1 accumulates in dense-core AD plaques in both 5xFAD mice and human brain tissue. Our findings suggest that elevated levels of STI1 contribute to Aβ accumulation, and that STI1 is deposited in AD plaques in mice and humans. We conclude that despite the protective effects of STI1 in C. elegans and in mammalian cultured neurons, in vivo, the predominant effect of elevated STI1 is deleterious in AD

Molecular Structure of Amyloid Fibrils Controls the Relationship between Fibrillar Size and Toxicity

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP(C)) at high levels confirming that cytotoxicity was in part PrP(C)-dependent. Silencing of PrP(C) expression by small hairpin RNAs designed to silence expression of human PrP(C) (shRNA-PrP(C)) diminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP(C)-mediated and PrP(C)-independent mechanisms depends on the structure of the aggregates.This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP(C) expression can be exploited to reduce their deleterious effects

Highly Efficient Protein Misfolding Cyclic Amplification

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro

Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrPSc, suggesting that these domains of cellular prion protein (PrPC) serve as docking sites for PrPSc during prion propagation. To examine the role of polybasic domains in the context of full-length PrPC, we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrPSc molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrPSc prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrPSc molecules. Remarkably, ΔC-PrPSc and other polybasic domain PrPSc molecules displayed diminished or absent biological infectivity relative to wild-type PrPSc, despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrPSc prions interact with PrPC molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrPSc propagation. Furthermore, polybasic domain deficient PrPSc molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrPSc molecules may not depend on a single stereotypic mechanism, but that normal PrPC/PrPSc interaction through polybasic domains may be required to generate prion infectivity

Multiple molecular mechanisms form a positive feedback loop driving amyloid β42 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons

Emerging evidence supports an important role for the ROS-sensitive TRPM2 channel in mediating age-related cognitive impairment in Alzheimer’s disease (AD), particularly neurotoxicity resulting from generation of excessive neurotoxic Aβ peptides. Here we examined the elusive mechanisms by which Aβ₄₂ activates the TRPM2 channel to induce neurotoxicity in mouse hippocampal neurons. Aβ₄₂-induced neurotoxicity was ablated by genetic knockout (TRPM2-KO) and attenuated by inhibition of the TRPM2 channel activity or activation through PARP-1. Aβ₄₂-induced neurotoxicity was also inhibited by treatment with TPEN used as a Zn²⁺-specific chelator. Cell imaging revealed that Aβ₄₂-induced lysosomal dysfunction, cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS. These effects were suppressed by TRPM2-KO, inhibition of TRPM2 or PARP-1, or treatment with TPEN. Bafilomycin-induced lysosomal dysfunction also resulted in TRPM2-dependent cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, and mitochondrial generation of ROS, supporting that lysosomal dysfunction and accompanying Zn²⁺ release trigger mitochondrial Zn²⁺ accumulation and generation of ROS. Aβ₄₂-induced effects on lysosomal and mitochondrial functions besides neurotoxicity were also suppressed by inhibition of PKC and NOX. Furthermore, Aβ₄₂-induced neurotoxicity was prevented by inhibition of MEK/ERK. Therefore, our study reveals multiple molecular mechanisms, including PKC/NOX-mediated generation of ROS, activation of MEK/ERK and PARP-1, lysosomal dysfunction and Zn²⁺ release, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS, are critically engaged in forming a positive feedback loop that drives Aβ₄₂-induced activation of the TRPM2 channel and neurotoxicity in hippocampal neurons. These findings shed novel and mechanistic insights into AD pathogenesis

Selenomethionine Incorporation into Amyloid Sequences Regulates Fibrillogenesis and Toxicity

The capacity of a polypeptide chain to engage in an amyloid formation process and cause a conformational disease is contained in its sequence. Some of the sequences undergoing fibrillation contain critical methionine (Met) residues which in vivo can be synthetically substituted by selenomethionine (SeM) and alter their properties

Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and death cell in microglial cells

Excessive Zn2+ causes brain damage via promoting ROS generation. Here we investigated the role of ROS-sensitive TRPM2 channel in H2O2/Zn2+-induced Ca2+ signalling and cell death in microglial cells. H2O2/Zn2+ induced concentration-dependent increases in cytosolic Ca2+ concentration ([Ca2+]c), which was inhibited by PJ34, a PARP inhibitor, and abolished by TRPM2 knockout (TRPM2-KO). Pathological concentrations of H2O2/Zn2+ induced substantial cell death that was inhibited by PJ34 and DPQ, PARP inhibitors, 2-APB, a TRPM2 channel inhibitor, and prevented by TRPM2-KO. Further analysis indicate that Zn2+ induced ROS production, PARP-1 stimulation, increase in the [Ca2+]c and cell death, which were suppressed by chelerythrine, a protein kinase C inhibitor, DPI, a NADPH-dependent oxidase (NOX) inhibitor, GKT137831, a NOX1/4 inhibitor, and Phox-I2, a NOX2 inhibitor. Furthermore, Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]c and cell death were inhibited by PF431396, a Ca2+-sensitive PYK2 inhibitor, and U0126, a MEK/ERK inhibitor. Taken together, our study shows PKC/NOX-mediated ROS generation and PARP-1 activation as an important mechanism in Zn2+-induced TRPM2 channel activation and, TRPM2-mediated increase in the [Ca2+]c to trigger the PYK2/MEK/ERK signalling pathway as a positive feedback mechanism that amplifies the TRPM2 channel activation. Activation of these TRPM2-depenent signalling mechanisms ultimately drives Zn2+-induced Ca2+ overloading and cell death

Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold