The head mesodermal cell couples FMRFamide neuropeptide signaling with rhythmic muscle contraction in C. elegans.

FMRFamides are evolutionarily conserved neuropeptides that play critical roles in behavior, energy balance, and reproduction. Here, we show that FMRFamide signaling from the nervous system is critical for the rhythmic activation of a single cell of previously unknown function, the head mesodermal cell (hmc) in C. elegans. Behavioral, calcium imaging, and genetic studies reveal that release of the FLP-22 neuropeptide from the AVL neuron in response to pacemaker signaling activates hmc every 50 s through an frpr-17 G protein-coupled receptor (GPCR) and a protein kinase A signaling cascade in hmc. hmc activation results in muscle contraction through coupling by gap junctions composed of UNC-9/Innexin. hmc activation is inhibited by the neuronal release of a second FMRFamide-like neuropeptide, FLP-9, which functions through its GPCR, frpr-21, in hmc. This study reveals a function for two opposing FMRFamide signaling pathways in controlling the rhythmic activation of a target cell through volume transmission.

Presynaptic coupling by electrical synapses coordinates a rhythmic behavior by synchronizing the activities of a neuron pair.

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

Mitochondrial hydrogen peroxide positively regulates neuropeptide secretion during diet-induced activation of the oxidative stress response.

Mitochondria play a pivotal role in the generation of signals coupling metabolism with neurotransmitter release, but a role for mitochondrial-produced ROS in regulating neurosecretion has not been described. Here we show that endogenously produced hydrogen peroxide originating from axonal mitochondria (mtH2O2) functions as a signaling cue to selectively regulate the secretion of a FMRFamide-related neuropeptide (FLP-1) from a pair of interneurons (AIY) in C. elegans. We show that pharmacological or genetic manipulations that increase mtH2O2 levels lead to increased FLP-1 secretion that is dependent upon ROS dismutation, mitochondrial calcium influx, and cysteine sulfenylation of the calcium-independent PKC family member PKC-1. mtH2O2-induced FLP-1 secretion activates the oxidative stress response transcription factor SKN-1/Nrf2 in distal tissues and protects animals from ROS-mediated toxicity. mtH2O2 levels in AIY neurons, FLP-1 secretion and SKN-1 activity are rapidly and reversibly regulated by exposing animals to different bacterial food sources. These results reveal a previously unreported role for mtH2O2 in linking diet-induced changes in mitochondrial homeostasis with neuropeptide secretion.

FSHR-1/GPCR Regulates the Mitochondrial Unfolded Protein Response in Caenorhabditis elegans.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In Caenorhabditis elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPRmt The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues are not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein-coupled receptor, FSHR-1, in promoting UPRmt activation. Genetic deficiency of fshr-1 severely attenuates UPRmt activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPRmt activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPRmt, to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell nonautonomously in neurons to activate UPRmt upstream of SPHK-1 signaling in the intestine.

A Receptor Tyrosine Kinase Network Regulates Neuromuscular Function in Response to Oxidative Stress in Caenorhabditis elegans.

The transcription factor Nrf2 plays a critical role in the organism-wide regulation of the antioxidant stress response. The Nrf2 homolog SKN-1 functions in the intestinal cells nonautonomously to negatively regulate neuromuscular junction (NMJ) function in Caenorhabditis elegans To identify additional molecules that mediate SKN-1 signaling to the NMJ, we performed a candidate screen for suppressors of aldicarb resistance caused by acute treatment with the SKN-1 activator arsenite. We identified two receptor tyrosine kinases, EGL-15 (fibroblast growth factor receptor, FGFR) and DAF-2 (insulin-like peptide receptor), that are required for NMJ regulation in response to stress. Through double-mutant analysis, we found that EGL-15 functions downstream of, or parallel to, SKN-1 and SPHK-1 (sphingosine kinase), and that the EGL-15 ligand EGL-17 FGF and canonical EGL-15 effectors are required for oxidative stress-mediated regulation of NMJ function. DAF-2 also functions downstream of or parallel to SKN-1 to regulate NMJ function. Through tissue-specific rescue experiments, we found that FGFR signaling functions primarily in the hypodermis, whereas insulin-like peptide receptor signaling is required in multiple tissues. Our results support the idea that the regulation of NMJ function by SKN-1 occurs via a complex organism-wide signaling network involving receptor tyrosine kinase signaling in multiple tissues.

Sphingosine Kinase Activates the Mitochondrial Unfolded Protein Response and Is Targeted to Mitochondria by Stress.

The mitochondrial unfolded protein response (UPRmt) is critical for maintaining mitochondrial protein homeostasis in response to mitochondrial stress, but early steps in UPRmt activation are not well understood. Here, we report a function for SPHK-1 sphingosine kinase in activating the UPRmt in C. elegans. Genetic deficiency of sphk-1 in the intestine inhibits UPRmt activation, whereas selective SPHK-1 intestinal overexpression is sufficient to activate the UPRmt. Acute mitochondrial stress leads to rapid, reversible localization of SPHK-1::GFP fusion proteins with mitochondrial membranes before UPRmt activation. SPHK-1 variants lacking kinase activity or mitochondrial targeting fail to rescue the stress-induced UPRmt activation defects of sphk-1 mutants. Activation of the UPRmt by the nervous system requires sphk-1 and elicits SPHK-1 mitochondrial association in the intestine. We propose that stress-regulated mitochondrial recruitment of SPHK-1 and subsequent S1P production are critical early events for both cell autonomous and cell non-autonomous UPRmt activation.

Sphingosine Kinase Regulates Neuropeptide Secretion During the Oxidative Stress-Response Through Intertissue Signaling.

The Nrf2 antioxidant transcription factor promotes redox homeostasis in part through reciprocal signaling between neurons and neighboring cells, but the signals involved in intertissue signaling in response to Nrf2 activation are not well defined. In Caenorhabditis elegans, activation of SKN-1/Nrf2 in the intestine negatively regulates neuropeptide secretion from motor neurons. Here, we show that sphingosine kinase (SPHK-1) functions downstream of SKN-1/Nrf2 in the intestine to regulate neuropeptide secretion from motor neurons during the oxidative stress response in C. elegans hermaphrodites. SPHK-1 localizes to mitochondria in the intestine and SPHK-1 mitochondrial localization and kinase activity are essential for its function in regulating motor neuron function. SPHK-1 is recruited to mitochondria from cytosolic pools and its mitochondrial abundance is negatively regulated by acute or chronic SKN-1 activation. Finally, the regulation of motor function by SKN-1 requires the activation of the p38 MAPK cascade in the intestine and occurs through controlling the biogenesis or maturation of dense core vesicles in motor neurons. These findings show that the inhibition of SPHK-1 in the intestine by SKN-1 negatively regulates neuropeptide secretion from motor neurons, revealing a new mechanism by which SPHK-1 signaling mediates its effects on neuronal function in response to oxidative stress.SIGNIFICANCE STATEMENT Neurons are highly susceptible to damage by oxidative stress, yet have limited capacity to activate the SKN-1/Nrf2 oxidative stress response, relying instead on astrocytes to provide redox homeostasis. In Caenorhabditis elegans, intertissue signaling from the intestine plays a key role in regulating neuronal function during the oxidative stress response. Here, through a combination of genetic, behavioral, and fluorescent imaging approaches, we found that sphingosine kinase functions in the SKN-1/Nrf2 pathway in the intestine to regulate neuropeptide biogenesis and secretion in motor neurons. These results implicate sphingolipid signaling as a new component of the oxidative stress response and suggest that C. elegans may be a genetically tractable model to study non-cell-autonomous oxidative stress signaling to neurons.

Aging and SKN-1-dependent Loss of 20S Proteasome Adaptation to Oxidative Stress in C. elegans.

Aging is marked by a collapse of protein homeostasis and deterioration of adaptive stress responses that often lead to disease. During aging, the induction of stress responses decline along with protein quality control. Here, we have shown that the ability to mount an adaptive response by pretreatment with minor oxidative stress is abrogated in aged Caenorhabditis elegans We have identified a defect in SKN-1 signaling sensitivity during aging and have also found an aging-related increase in basal proteasome expression and in vitro activity, however, adaptation of the 20S proteasome in response to stress is lost in old animals. Interestingly, increased activation of SKN-1 promotes stress resistance, but is unable to rescue declining adaptation during aging. Our data demonstrate that the aging-dependent decline in SKN-1 signaling negatively impacts adaptation of the 20S proteasome in response to acute oxidative stress.

Coupling between endocytosis and sphingosine kinase 1 recruitment.

Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

Regulation of synaptic nlg-1/neuroligin abundance by the skn-1/Nrf stress response pathway protects against oxidative stress.

The Nrf family of transcription factors mediates adaptive responses to stress and longevity, but the identities of the crucial Nrf targets, and the tissues in which they function in multicellular organisms to promote survival, are not known. Here, we use whole transcriptome RNA sequencing to identify 810 genes whose expression is controlled by the SKN-1/Nrf2 negative regulator WDR-23 in the nervous system of Caenorhabditis elegans. Among the genes identified is the synaptic cell adhesion molecule nlg-1/neuroligin. We find that the synaptic abundance of NLG-1 protein increases following pharmacological treatments that generate oxidative stress or by the genetic activation of skn-1. Increasing nlg-1 dosage correlates with increased survival in response to oxidative stress, whereas genetic inactivation of nlg-1 reduces survival and impairs skn-1-mediated stress resistance. We identify a canonical SKN-1 binding site in the nlg-1 promoter that binds to SKN-1 in vitro and is necessary for SKN-1 and toxin-mediated increases in nlg-1 expression in vivo. Together, our results suggest that SKN-1 activation in the nervous system can confer protection to organisms in response to stress by directly regulating nlg-1/neuroligin expression.

PKA controls calcium influx into motor neurons during a rhythmic behavior.

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.

Extrasynaptic muscarinic acetylcholine receptors on neuronal cell bodies regulate presynaptic function in Caenorhabditis elegans.

Acetylcholine (ACh) is a potent neuromodulator in the brain, and its effects on cognition and memory formation are largely performed through muscarinic acetylcholine receptors (mAChRs). mAChRs are often preferentially distributed on specialized membrane regions in neurons, but the significance of mAChR localization in modulating neuronal function is not known. Here we show that the Caenorhabditis elegans homolog of the M1/M3/M5 family of mAChRs, gar-3, is expressed in cholinergic motor neurons, and GAR-3-GFP fusion proteins localize to cell bodies where they are enriched at extrasynaptic regions that are in contact with the basal lamina. The GAR-3 N-terminal extracellular domain is necessary and sufficient for this asymmetric distribution, and mutation of a predicted N-linked glycosylation site within the N-terminus disrupts GAR-3-GFP localization. In transgenic animals expressing GAR-3 variants that are no longer asymmetrically localized, synaptic transmission at neuromuscular junctions is impaired and there is a reduction in the abundance of the presynaptic protein sphingosine kinase at release sites. Finally, GAR-3 can be activated by endogenously produced ACh released from neurons that do not directly contact cholinergic motor neurons. Together, our results suggest that humoral activation of asymmetrically localized mAChRs by ACh is an evolutionarily conserved mechanism by which ACh modulates neuronal function.

The conserved SKN-1/Nrf2 stress response pathway regulates synaptic function in Caenorhabditis elegans.

The Nrf family of transcription factors plays a critical role in mediating adaptive responses to cellular stress and defends against neurodegeneration, aging, and cancer. Here, we report a novel role for the Caenorhabditis elegans Nrf homolog SKN-1 in regulating synaptic transmission at neuromuscular junctions (NMJs). Activation of SKN-1, either by acute pharmacological treatment with the mitochondrial toxin sodium arsenite or by mutations that cause constitutive SKN-1 activation, results in defects in neuromuscular function. Additionally, elimination of the conserved WD40 repeat protein WDR-23, a principal negative regulator of SKN-1, results in impaired locomotion and synaptic vesicle and neuropeptide release from cholinergic motor axons. Mutations that abolish skn-1 activity restore normal neuromuscular function to wdr-23 mutants and animals treated with toxin. We show that negative regulation of SKN-1 by WDR-23 in the intestine, but not at neuromuscular junctions, is necessary and sufficient for proper neuromuscular function. WDR-23 isoforms differentially localize to the outer membranes of mitochondria and to nuclei, and the effects of WDR-23 on neuromuscular function are dependent on its interaction with cullin E3 ubiquitin ligase. Finally, whole-transcriptome RNA sequencing of wdr-23 mutants reveals an increase in the expression of known SKN-1/Nrf2-regulated stress-response genes, as well as neurotransmission genes not previously implicated in SKN-1/Nrf2 responses. Together, our results indicate that SKN-1/Nrf2 activation may be a mechanism through which cellular stress, detected in one tissue, affects cellular function of a distal tissue through endocrine signaling. These results provide insight into how SKN-1/Nrf2 might protect the nervous system from damage in response to oxidative stress.

Neuropeptide secreted from a pacemaker activates neurons to control a rhythmic behavior.

BACKGROUND: Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 s, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. RESULTS: Here, we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G-protein-coupled receptor on the GABAergic neurons, as the receptor for NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo. CONCLUSIONS: Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors.

A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster.

In mammalian cells, hydrogen peroxide (H(2)O(2))-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H(2)O(2) pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity.

Localized sphingolipid signaling at presynaptic terminals is regulated by calcium influx and promotes recruitment of priming factors.

Activity-dependent changes in presynaptic function represent a critical mechanism by which synaptic strength is controlled. However, how changes in synaptic activity couple to presynaptic components to control synaptic vesicle release and recycling are poorly understood. Sphingosine kinase (SphK) is a sphingolipid metabolic enzyme whose activity-dependent recruitment to membrane regions within presynaptic terminals promotes neurotransmitter release. Here, we show that synaptic recruitment of SPHK-1, the SphK ortholog in Caenorhabditis elegans, is mediated by presynaptic calcium influx. Quantitative fluorescence imaging of live presynaptic terminals reveals that blocking presynaptic calcium influx reduces synaptic SPHK-1 abundance whereas increasing calcium influx increases SPHK-1 synaptic abundance. CALM-1, the calcium and integrin binding protein ortholog, colocalizes with SPHK-1 at release sites and regulates muscarinic-mediated synaptic SPHK-1 recruitment. We identify two additional sphingolipid metabolic enzymes that are concentrated at presynaptic terminals, and mutants lacking one of these, HYL-1/ceramide synthase, have defects in synaptic transmission and in synaptic vesicle cycling. Finally, we show that SPHK-1 activity is required for the recruitment of the priming protein UNC-13/Munc13 to presynaptic terminals following activation by muscarinic signaling. These findings suggest that calcium-dependent regulation of local S1P metabolism at synapses may be an important mechanism by which synaptic vesicle priming factors are recruited to release sites to promote synaptic transmission.

Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release.

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.

RIC-7 promotes neuropeptide secretion.

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV-mediated secretion.

Annexin A5 directly interacts with amyloidogenic proteins and reduces their toxicity.

Protein misfolding is a central mechanism for the development of neurodegenerative diseases and type 2 diabetes mellitus. The accumulation of misfolded alpha-synuclein protein inclusions in the Lewy bodies of Parkinson's disease is thought to play a key role in pathogenesis and disease progression. Similarly, the misfolding of the beta-cell hormone human islet amyloid polypeptide (h-IAPP) into toxic oligomers plays a central role in the induction of beta-cell apoptosis in the context of type 2 diabetes. In this study, we show that annexin A5 plays a role in interacting with and reducing the toxicity of the amyloidogenic proteins, h-IAPP and alpha-synuclein. We find that annexin A5 is coexpressed in human beta-cells and that exogenous annexin A5 reduces the level of h-IAPP-induced apoptosis in human islets by approximately 50% and in rodent beta-cells by approximately 90%. Experiments with transgenic expression of alpha-synuclein in Caenorhabditis elegans show that annexin A5 reduces alpha-synuclein inclusions in vivo. Using thioflavin T fluorescence, electron microscopy, and electron paramagnetic resonance, we provide evidence that substoichiometric amounts of annexin A5 inhibit h-IAPP and alpha-synuclein misfolding and fibril formation. We conclude that annexin A5 might act as a molecular safeguard against the formation of toxic amyloid aggregates.