α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol

Alzheimer’s disease (AD) is characterized by the presence of amyloid β (Aβ) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic risk factor for sporadic AD. In vitro evidence suggests that apoE links to Aβ production through nanoscale lipid compartments (lipid clusters), but its regulation in vivo is unclear. Here, we use superresolution imaging in the mouse brain to show that apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) in and out of lipid clusters, where it interacts with β- and γ-secretases to generate Aβ-peptide. We find that the targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid clusters, where it interacts with α-secretase and gives rise to soluble APP-α (sAPP-α), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and γ-secretase trafficking, suggesting that the ratio of Aβ to sAPP-α is regulated by the trafficking of the substrate, not the enzymes. We conclude that cholesterol is kept low in neurons, which inhibits Aβ accumulation and enables the astrocyte regulation of Aβ accumulation by cholesterol signaling.

Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, is characterized by the progressive loss of cognitive function and the accumulation of amyloid β (Aβ) peptide and phosphorylated tau (1). Amyloid plaques are composed of aggregates of Aβ peptide, a small hydrophobic protein excised from the transmembrane domain of amyloid precursor protein (APP) by proteases known as beta- (β-) and gamma- (γ-) secretases (SI Appendix, Fig. S1A). In high concentrations, Aβ peptide can aggregate to form Aβ plaques (2–4). The nonamyloidogenic pathway involves a third enzyme, alpha- (α-) secretase, which generates a soluble APP fragment (sAPP-α), helps set neuronal excitability in healthy individuals (5), and does not contribute to the generation of amyloid plaques. Therefore, by preventing Aβ production, α-secretase–mediated APP cleavage reduces plaque formation. Strikingly, both pathways are finely regulated by cholesterol (6) (SI Appendix, Fig. S1B).In cellular membranes, cholesterol regulates the formation of lipid clusters (also known as lipid rafts) and the affinity of proteins to lipid clusters (7), including β-secretase and γ-secretase (8–10). α-secretase does not reside in lipid clusters; rather, α-secretase is thought to reside in a region made up of disordered polyunsaturated lipids (11). The location of APP is less clear. In detergent-resistant membrane (DRM) studies, it primarily associates with lipid from the disordered region, although not exclusively (8, 10, 12–14). Endocytosis is thought to bring APP in proximity to β-secretase and γ-secretase, and this correlates with Aβ production. Cross-linking of APP with β-secretase on the plasma membrane also increases Aβ production, leading to a hypothesis that lipid clustering in the membrane contributes to APP processing (11, 14, 15) (SI Appendix, Fig. S1A). Testing this hypothesis in vivo has been hampered by the small size and transient nature of lipid clusters (often <100 nm), which is below the resolution of light microscopy.Superresolution imaging has emerged as a complimentary technique to DRMs, with the potential to interrogate cluster affinity more directly in a native cellular environment (16). We recently employed superresolution imaging to establish a membrane-mediated mechanism of general anesthesia (17). In that mechanism, cholesterol causes lipid clusters to sequester an enzyme away from its substrate. Removal of cholesterol then releases and activates the enzyme by giving it access to its substrate (SI Appendix, Fig. S1C) (7, 18). A similar mechanism has been proposed to regulate the exposure of APP to its cutting enzymes (11, 15, 19–21).Neurons are believed to be the major source of Aβ in normal and AD brains (22, 23). In the adult brain, the ability of neurons to produce cholesterol is impaired (24). Instead, astrocytes make cholesterol and transport it to neurons with apolipoprotein E (apoE) (25–27). Interestingly, apoE, specifically the e4 subtype (apoE4), is the strongest genetic risk factor associated with sporadic AD (28, 29). This led to the theory that astrocytes may be controlling Aβ accumulation through regulation of the lipid cluster function (11, 15, 19), but this has not yet been shown in the brain of an animal. Here, we show that astrocyte-derived cholesterol controls Aβ accumulation in vivo and links apoE, Aβ, and plaque formation to a single molecular pathway.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

Mechanistic basis for receptor-mediated pathological α-synuclein fibril cell-to-cell transmission in Parkinson's disease

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson’s disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

Aggregation and the spread of amyloid proteins, such as α-synuclein (α-syn), amyloid-β, Tau, and TDP43, are critical events in the pathogenesis of neurodegenerative disorders, including Parkinson''s disease (PD), Alzheimer’s disease, and amyotrophic lateral sclerosis, respectively (1, 2). As the hallmark of PD and other α-synucleinopathies, α-syn aggregation spreads in a prion-like progressive and stepwise manner both within the brain and from other organs to the brain during disease progression (3–7). Pathological α-syn aggregation can template monomeric α-syn to aggregate and participate in disease pathogenesis. Pathological α-syn inclusion can spread in the grafted neurons of PD patients (4, 8). Brain extracts from patients with multiple system atrophy can transmit neurodegeneration to genetically engineered mice (9). A single administration of α-syn preformed fibrils (PFFs) in mouse brains can recapitulate the pathological phenotypes of α-synucleinopathies (10–13).Selected cell surface proteins, such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1), have been found to serve as receptors for α-syn PFF internalization and transmission (10, 14, 15). Intriguingly, these receptors preferentially recognize α-syn PFFs rather than the monomer (10). The α-syn monomer is intrinsically disordered and forms α-helical conformation upon membrane binding as involved in synaptic vesicle trafficking (16–19). Cryogenic electron microscopic (cryo-EM) structures of full-length α-syn amyloid fibrils show that the central region of α-syn, approximately covering residues 37 to 99, is involved in the formation of a cross-β fibril core (termed as FC region), while the remaining N and C termini remain flexible (20–24). Despite the recent successes in the structural determination of α-syn amyloid fibrils, considerable challenges remain in linking the structural information to α-syn pathology. The structural basis underlying α-syn transmission, specifically the interplay between α-syn PFFs and receptors, is unknown. It also remains unclear how receptors, for example, LAG3 and APLP1, selectively recognize α-syn PFFs over monomers, nor do we know the role of posttranslational modification of α-syn in this process.In this work, we combined multiple biophysical, cellular, and in vivo approaches to reveal the structural basis underlying the receptor binding of α-syn amyloid fibrils during cell-to-cell transmission. We found that the D1 domain of LAG3 utilizes a positively charged surface to capture the acidic C terminus of α-syn, which is exposed and concentrated on the surface of α-syn fibrils. In contrast, α-syn monomers adopt a self-shielded conformation to impede the exposure of the C terminus. Phosphorylation at serine 129 (pS129) of α-syn, a pathological biomarker in PD (25–27), significantly enhances the binding of α-syn PFFs to LAG3 and APLP1 and promotes the cell-to-cell transmission in vitro and in vivo. Our work provides the structural basis for the receptor-mediated neuronal internalization and transmission of α-syn fibrils and suggests that the C terminus, specifically residues 118 to 140, is a pathological epitope of α-syn for receptor binding and thus may serve as a promising target for the therapeutic drug development to block PD progression.     

Cysteine cathepsins are essential in lysosomal degradation of α-synuclein

A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.A presynaptic neuronal protein, α-synuclein (α-syn), is linked genetically and neuropathologically to Parkinson’s disease (PD) (1). The protein exists in a multitude of conformations that likely dictates the physiological function of α-syn (2–6). Upon membrane association, α-syn adopts an α-helical structure (7), whereas in solution, the protein is disordered (3, 6, 8). Elucidation of molecular mechanisms underlying the transformation of α-syn to a disease-associated species is still the subject of intense research. However, the deposition of insoluble α-syn fibrils rich in β-sheet structure, generally referred to as amyloid is a hallmark of the disease (9, 10).Understanding the cellular pathways that control α-syn aggregation is critical in establishing ways to circumvent the progression of PD. A primary cause leading to PD is associated with impaired α-syn turnover (11–13). Hence, boosting the degradation of α-syn could be an effective way to alleviate this burden. Both in vitro and in vivo data support the involvement of the proteasome and lysosome in α-syn degradation (14, 15). The proteasome pathway is generally considered to be responsible for removal of soluble α-syn, whereas the lysosome eliminates aggregation-prone species or excess levels of α-syn. Dysfunction of either system has been shown to increase α-syn levels (16).α-syn enters via two main autophagic pathways (17), either through macroautophagy (14) or chaperone-mediated autophagy (18) into the lysosome, where the protein is degraded. The main class of lysosomal proteases is the cathepsins (Cts), which are subdivided based on the active-site amino acids that confer catalytic activity. These are cysteine (CtsB, CtsC, CtsF, CtsH, CtsK, CtsL, CtsS, CtsV, and CtsX), serine (CtsA and CtsG), and aspartyl (CtsD and CtsE) proteases (19). Activity of cathepsins is optimal in acidic pH, and most are inactive at neutral pH. CtsD (20) is the only protease implicated to date in the lysosomal degradation of α-syn (21–24).Although prior literature supports CtsD involvement, the question of whether other lysosomal protease(s) are involved is also raised. For example, a strong correlation exists in vivo between overexpression of CtsD and α-syn levels as well as the neurotoxic potential of overexpressed α-syn (21, 22). However, in vitro CtsD activity yields incomplete proteolysis of α-syn and generates truncated C-terminal (α-synΔC) species (24). More concerning is that both α-synΔC peptides, such as residues 1–87 (10), 1–103 (25), and 1–119 (26) of α-syn (residues 1–140), and the acidic environment of the lysosomal lumen (27) are known to enhance amyloid formation. Interestingly, human tissue samples contain an abundance of α-synΔC species under normal physiological conditions (28). Some of these α-syn fragments (1–115, 1–119, 1–122, 1–133, and 1–135) have also been isolated from Lewy bodies (29), classic PD hallmarks. The significance of these truncations remain ill-defined, but one could speculate the involvement of CtsD and partial lysosomal degradation of α-syn (24).Here, we sought to identify other lysosomal cathepsins and environmental factors needed to facilitate α-syn degradation and to avoid aggregation. Direct interaction between α-syn and lysosomal proteases was shown by purified mouse brain and liver lysosomal extracts as well as purified human cathepsins and was characterized by peptide mapping using liquid chromatography–mass spectrometry (LC-MS). We found that mouse brain and liver lysosomal extracts harbor mostly cysteine cathepsin activity (namely CtsB and CtsL) that efficiently degrades recombinant α-syn. Lysosomal and individual cathepsin activities on soluble, membrane-bound, and fibrillar α-syn as well as their impact on α-syn amyloid formation have been evaluated.     

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ₁34.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ₁34.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ₁34.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ₁34.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Herpes simplex viruses (HSV) are human pathogens that switch between lytic and latent infections intermittently (1, 2). This is a lifelong source of infectious viruses (1, 2), in which immediate early proteins drive the onset of HSV replication. Among them, ICP0 enables viral gene expression or reactivation from latency (2–4), which involves chromatin remodeling of the HSV genome, resulting in de novo virus production. In this process, the accessory factor γ₁34.5 of HSV is thought to govern viral protein synthesis (5, 6). It has long been known that γ₁34.5 precludes translation arrest mediated by double-stranded RNA–dependent protein kinase PKR (7–9). The γ₁34.5 protein has also been shown to dampen intracellular nucleic acid sensing, inhibit autophagy, and facilitate virus nuclear egress (10–17). In experimental animal models, wild-type HSV, but not HSV that lacks the γ₁34.5 gene, replicates competently, penetrates from the peripheral tissues to the nervous system and reactivates from latency (18–23). Despite these observations, active HSV replication or reactivation from latency is not readily reconciled by the currently known functions of the γ₁34.5 protein (8–13, 16, 17).Several lines of work demonstrate that tripartite motif (TRIM) proteins regulate innate immune signaling and cell intrinsic resistance to virus infections (24, 25). These host factors typically work as E3 ubiquitin ligases that can synthesize degradative or nondegradative ubiquitination on viral or host proteins. A number of TRIM proteins, for example TRIM5α, TRIM19, TRIM21, TRIM22, and TRIM43, act at different steps of virus replication and subsequently inhibit viral production (26–32). Recent evidence indicates that TRIM23 limits the replication of certain RNA viruses and DNA viruses, including HSV-1 (33). In doing so, TRIM23 recruits TANK-binding kinase 1 (TBK1) to autophagosomes, thus promoting TBK1-mediated phosphorylation and activation of the autophagy receptor p62 and ultimately leading to autophagy. It is unknown whether TRIM23 plays an additional role(s) in HSV infection.Here, we report that ICP0 expression is regulated by the γ₁34.5 protein and TRIM23 during HSV-1 infection. We show that TRIM23 facilitates the proteasomal degradation of ICP0, whereas viral γ₁34.5 maintains steady-state ICP0 expression by preventing K27-linked TRIM23 autoubiquitination that is required for TRIM23 activation. The γ₁34.5 protein also interacts with and stabilizes ICP0, enabling productive infection. Furthermore, we provide evidence that TRIM23 binds to ICP0 and induces its K11-linked polyubiquitination, which triggers K48-linked polyubiquitin-dependent proteasomal degradation of ICP0. These insights establish a model of posttranslational networks in which virus- and host-mediated mechanisms regulate immediate early protein ICP0 stability and thereby lytic HSV replication.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell–killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy.

Macrophages are innate immune cells that show phenotypic heterogeneity and functional diversity; and they play key roles in development, tissue homeostasis and repair, and in cancer, as well as in defense against pathogens (1–3). In the tumor microenvironment (TME), macrophages are exposed to a variety of stimuli, including cell–cell contact, hypoxia, as well as soluble and insoluble factors such as cytokines, chemokines, metabolites, and extracellular matrix components (2, 4). These environmental cues promote the acquisition by macrophages of protumorigenic phenotypes that facilitate tumor development, progression, and metastasis as well as suppress antitumor immune responses (2, 4). A high density of macrophages within tumor tissue is associated with poor prognosis in patients with various types of cancer, including that of the bladder or breast (5–7). Depletion of macrophages in the TME or the reprogramming of these cells to acquire antitumorigenic phenotypes has been shown to ameliorate the immunosuppressive condition and result in a reduction in tumor burden in both preclinical and clinical studies (2, 4, 8, 9). Macrophages within the TME have therefore attracted much attention as a potential therapeutic target for cancer immunotherapy.Signal regulatory protein α (SIRPα) is a transmembrane protein that possesses one NH₂-terminal immunoglobulin (Ig)-V–like and two Ig-C domains in its extracellular region, as well as immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic region (10, 11). The extracellular region of SIRPα interacts with that of CD47, another member of the Ig superfamily of proteins, with this interaction constituting a means of cell–cell communication. The expression of SIRPα in hematopoietic cells is restricted to the myeloid compartment—including macrophages, neutrophils, and dendritic cells (DCs)—whereas CD47 is expressed in most normal cell types as well as cancer cells (12, 13). The interaction of SIRPα on macrophages with CD47 on antibody (Ab)-opsonized viable cells such as blood cells or cancer cells prevents phagocytosis of the latter cells by the former (13–15), with this negative regulation of macrophages being thought to be mediated by SHP1, a protein tyrosine phosphatase that binds to the cytoplasmic region of SIRPα (14). Indeed, blockade of the CD47–SIRPα interaction by Abs to either SIRPα or CD47, in combination with a tumor-targeting Ab such as rituximab (anti-CD20), was found to enhance the Ab-dependent cellular phagocytosis (ADCP) activity of macrophages for cancer cells that do not express SIRPα, resulting in marked suppression of tumor formation in mice (15–19). Targeting of SIRPα in combination with a tumor-targeting Ab therefore provides a potential approach to cancer immunotherapy dependent on enhancement of the ADCP activity of macrophages for cancer cells. In contrast, the effect of Abs to SIRPα in the absence of a tumor-targeting Ab on the phagocytosis by macrophages of, as well as on tumor formation by, cancer cells that do not express SIRPα was minimal or limited.We have now further examined the antitumor efficacy of a monoclonal Ab (mAb) to mouse SIRPα (MY-1) (20) in immunocompetent mice transplanted subcutaneously with several types of murine cancer cells that do not express SIRPα. This Ab prevents the binding of mouse CD47 to SIRPα and cross-reacts with mouse SIRPβ1 (15). We found that monotherapy with MY-1 efficiently attenuated the growth of tumors formed by bladder or mammary cancer cells. In addition, MY-1 markedly promoted the induction of antitumorigenic macrophages able to target these cancer cells. Furthermore, our results suggest that SIRPβ1 on macrophages likely participated in the antitumorigenic effect of MY-1.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Oncogenic activity of the regulatory subunit p85β of phosphatidylinositol 3-kinase (PI3K)

Expression of the regulatory subunit p85β of PI3K induces oncogenic transformation of primary avian fibroblasts. The transformed cells proliferate at an increased rate compared with nontransformed controls and show elevated levels of PI3K signaling. The oncogenic activity of p85β requires an active PI3K-TOR signaling cascade and is mediated by the p110α and p110β isoforms of the PI3K catalytic subunit. The data suggest that p85β is a less effective inhibitor of the PI3K catalytic subunit than p85α and that this reduced level of p110 inhibition accounts for the oncogenic activity of p85β.Class IA PI3Ks (phosphatidylinositol 3-kinase) are dimeric enzymes consisting of a catalytic subunit and a regulatory subunit. The two major regulatory subunits are p85α and p85β (1). They stabilize and inhibit the catalytic subunit p110 by domain-specific interactions (2–6). The p85α and p85β proteins share core functions, but also display unique activities (7–10). Both p85α and p85β are found mutated in several cancers. These mutants show oncogenic activity; most of the p85α mutations disrupt inhibitory interactions between p85 and p110 or destabilize PTEN (phosphatase and tensin homolog) and result in increased PI3K signaling (5, 11–14). The molecular mechanisms by which p85β mutations activate PI3K signaling have not been fully explored. Elevated expression of wild-type p85β is found in several cancers, and in an experimental setting drives tumor progression (15). A recent study has revealed a role of p85β in the formation of invadopodia with possible effects on metastatic cellular behavior (16). Here we show that expression of p85β induces cellular transformation of primary fibroblasts, increased cell proliferation and elevated PI3K signaling. The oncogenic activity of p85β depends on active PI3K and TOR (target of rapamycin) signaling and is mediated by two PI3K catalytic isoforms, p110α and p110β. Our data are compatible with the conclusion that p85β exerts a reduced inhibitory activity on p110 compared with p85α.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson’s disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder caused by loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase). GCase degrades glycosphingolipids (GSLs), including glucosylceramides (GluCers), into glucose and ceramide, and GCase mutations result in the accumulation of GluCer in lysosomes of various tissues. Heterozygote carriers of the same loss-of-function GCase mutations are estimated to be at 5- to 10-fold higher risk for developing Parkinson’s disease (PD) or Lewy body dementia (1). In GD, significant variability exists in the clinical and pathological presentation, resulting in three main GD subtypes (2). Type 1 GD is characterized by visceral abnormalities, including enlarged liver and spleen and bone marrow dysfunction, leading to thrombocytopenia but without neurodegeneration and α-synuclein (α-syn) pathology (3). Types 2 and 3 demonstrate similar visceral symptoms but with additional extensive neuronal loss, α-syn pathology in the form of classical Lewy bodies, and neurological dysfunction (3, 4). As life expectancy of type 1 GD has increased because of enzyme replacement therapy, a higher percentage of patients develop PD symptoms with age (5), suggesting that aging could contribute to the penetrance of GBA1 mutations. The dramatic phenotypic heterogeneity suggests that GD is not a simple, monogenic disease but a complex disorder that is influenced by both genetic and nongenetic modifiers. Although the factors that contribute to clinical and pathological variability in GD are not known, genetic modifiers have been identified that associate with GD severity, including CLN8 and SCARB2 (6, 7). Within PD patients that harbor GBA1 mutations (GBA-PD), the search for genetic modifiers has shown that synergism may exist with the SNCA gene that encodes α-syn and CTSB that encodes lysosomal cathepsin B (8). Variants in lysosomal cathepsins could influence the severity of α-syn accumulation, since, under physiological or pathological conditions, α-syn can be degraded by the lysosome (9–11) and is a direct substrate of cathepsin B and L (12).An additional factor that may contribute to phenotypic variability in GD is the accumulation of specific GluCer subtypes with particular acyl chain lengths. GluCer and other GSLs exist as a family of lipid isoforms differentiated by the length of the N-acyl fatty acid moiety linked to the sphingoid base. GluCer chains range from C14 to C26 in the brain; however, C18 and C24:1 are the predominant species (13). Studies of neuronopathic GD (nGD) brain or mouse models showed intraneuronal accumulation of multiple GluCer species that correlated with neuroinflammation (14–19), and some cases demonstrate selective accumulation of long-chain GluCers in nGD (20). Our recent work in PD patient midbrain neurons showed that inhibition of wild-type (wt) GCase, caused by α-syn, resulted in the selective accumulation of long-chain-length GluCers, including C22 and C24:1, while C14, 16, and C18 were unchanged (21). Together, these data indicate that GluCer accumulation plays an important role in neurodegeneration induced by GBA1 mutations; however, the specific contributions of distinct GluCer species have not been examined.Here, we extend our studies on the role of GSLs in α-syn aggregation to further define conditions that are required to induce pathology and neurological dysfunction. We previously showed that α-syn exists as monomers and high–molecular weight (HMW) oligomers under physiological conditions in human midbrain cultures (22). In vitro, we found that GluCer mildly induced aggregation of α-syn monomers but primarily acted on physiological oligomers to convert them into toxic oligomers and fibrillar inclusions (22). α-syn accumulation can be prevented or reversed by reducing GSLs with GluCer synthase inhibitors (GCSi) in both GD and PD patient cultures, as well as in mouse models (22–24). While this work suggests a close relationship between GCase function and α-syn pathology, additional factors must exist that create a permissive environment for α-syn accumulation. Indeed, studies that used newborn mice or embryonic primary neuron cultures treated with the GCase inhibitor, conduritol beta epoxide (CBE), have shown no changes in α-syn despite reduced GCase activity (25–27). However, other studies that use matured neuron cultures, neuronal cell lines, or adult mice have shown that CBE dramatically induces α-syn aggregates (22, 28–31). We used an in vivo GD model and induced pluripotent stem cell (iPSC)–derived patient midbrain cultures to identify specific conditions that are required to induce α-syn pathology, providing possible explanations for the variable neurological penetrance in patients that harbor GBA1 mutations.     

Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio,reduces lipid-rich aggregates,and attenuates motor and cognitive deficits in mice

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

GBA1 gene mutations in Gaucher’s disease carriers are recognized as the most important risk factors for developing Parkinson’s disease (PD), since large multicenter patient cohorts identified GBA variants in PD, including in ∼3% of sporadic PD patients and up to ∼15% of the Ashkenazi Jewish population with PD (1). Homozygous and heterozygous GBA1 mutation carriers display a similar risk (∼20%) of developing PD (2). GBA1 mutations can impact the activity of its gene product, the lysosomal lipid metabolism enzyme glucocerebrosidase (GCase), leading to changes in cellular lipid content and lipid membrane morphologies (3, 4). Clinically, PD patients with GBA1 mutations are largely indistinguishable from the idiopathic form. Both populations exhibit widespread α-synuclein (αS)+ Lewy bodies (LBs), including in the hippocampus and other brain regions, and these are associated with motor deficits and cognitive decline (2). PD-GBA1 mutation carriers are at a greater risk of cognitive impairments, and this finding is consistent with a higher incidence of GBA1 mutations in =DLB patients (5, 6). Recent morphological analyses of “sporadic” PD brain tissues have revealed that Lewy-type inclusions also contain substantial amounts of lipid-rich membranes and vesicles, including lysosomes (7). Additional evidence for the role of GCase in αS homeostasis has been generated in mouse studies and in GBA1-mutant neural cells, suggesting increased accumulation of αS secondary to different pathogenic GBA1 mutations (8–11).Accumulating evidence from our laboratory (12–14) and others (15–18) shows that αS normally occurs in a dynamic equilibrium between helically folded tetramers and “natively unfolded” monomers. Regarding the relevance of αS tetramers to disease, we found that all familial PD (fPD)–causing αS mutations decrease the physiological tetramer–monomer (T:M) ratio and some induce cytoplasmic inclusions and neurotoxicity in human (hu) and rodent cell culture (13). Supporting these findings, neurons harboring PD-causing GBA1 mutations shifted endogenous wild-type (wt) αS tetramers to monomers that lead to abnormal phosphorylated serine 129 αS (pS129) + αS accumulation (18), indicating lipid metabolism can impact physiological αS homeostasis. Mechanistic studies have shown that saturated fatty acids (SFAs) stabilize normal tetramers, while unsaturated FAs, such as oleic acid, decrease the T:M ratio (19, 20). Accordingly, decreasing stearoyl-CoA desaturase (SCD) activity, the rate-limiting enzyme for generating monounsaturated (MU) FA, decreases αS+ neuronal inclusions in yeast, rat cortical neurons, hu wt, fPD E46K–induced neurons, and in 3K cell culture models (19–21).Our recent approach to treating hu wt or 3K αS mutant mice with SCD inhibitors showed that the prolonged increases in the T:M ratio can reduce excess triacylglycerides (TAGs), lipid droplets (LDs) (rich in TAGs), and pS129 αS+ aggregates, aiding multiple PD motor phenotypes (22). Intriguingly, overexpressing hu wtGBA increased the αS T:M ratio in Gaucher’s GBA1-mutant neuronal culture (18).Whether early transduction and prolonged increase of hu wtGCase can enhance αS T:M homeostasis in vivo has yet not been examined. To begin investigating this question, we used the tetramer-abrogating “3K” αS mutant mouse line that is a biochemical amplification of the E46K mutation-causing PD. The 3K mutation shifts the normally aggregation-resistant αS tetramers (12) to increased levels of monomers that then cluster with vesicle membranes and form sizeable aggregates, thereby producing multiple PD-like motor phenotypes by the age of 6 mo (23). The Thy1.2 promotor that drives the 3K transgene reaches stable expression from postnatal day 7 onwards (24), thereby enabling us to study whether GBA1 effects the onset of αS dyshomeostasis in mouse brain when injecting it into 3K neonates. Here, we transduced an adeno-associated virus (AAV)–wtGBA1 vector by intracerebroventricular (ICV) injections in 3K and control littermate pups at P1 and then, 6 mo later, performed motor and cognitive testing and examined the brains for αS species, GCase activity, lysosomal abnormalities, and lipid aggregation patterns.