Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

Amelioration of pathologic α-synuclein-induced Parkinson’s disease by irisin

Physical activity provides clinical benefit in Parkinson’s disease (PD). Irisin is an exercise-induced polypeptide secreted by skeletal muscle that crosses the blood–brain barrier and mediates certain effects of exercise. Here, we show that irisin prevents pathologic α-synuclein (α-syn)-induced neurodegeneration in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Intravenous delivery of irisin via viral vectors following the stereotaxic intrastriatal injection of α-syn PFF cause a reduction in the formation of pathologic α-syn and prevented the loss of dopamine neurons and lowering of striatal dopamine. Irisin also substantially reduced the α-syn PFF-induced motor deficits as assessed behaviorally by the pole and grip strength test. Recombinant sustained irisin treatment of primary cortical neurons attenuated α-syn PFF toxicity by reducing the formation of phosphorylated serine 129 of α-syn and neuronal cell death. Tandem mass spectrometry and biochemical analysis revealed that irisin reduced pathologic α-syn by enhancing endolysosomal degradation of pathologic α-syn. Our findings highlight the potential for therapeutic disease modification of irisin in PD.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive worsening of motor symptoms, including bradykinesia, resting tremor, and rigidity (1, 2). Nonmotor symptoms often precede and accompany the motor symptoms and include autonomic dysfunction and neuropsychiatric sequelae (3). The most notable loss of neurons occurs in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), although neuronal loss also occurs in the locus coeruleus, dorsal raphe nucleus, the dorsal motor nucleus of the vagus, and nucleus basalis of Meynert (4). In addition to neuronal loss, there is accumulation of misfolded pathologic α-synuclein that drives the pathogenesis of PD, including the neuronal dysfunction and the ultimate of neuronal degeneration (5, 6). Current treatments for PD include the replacement of dopamine (DA) via L-DOPA, DA agonists, and other agents to treat the nonmotor symptoms. As the disease progresses, deep brain stimulation and other neurosurgical approaches can be used to treat the side effects of DA replacement therapy. Importantly, these treatments only address the symptomology, and over time there is a progressive decline in normal function. Moreover, there are no treatments that slow the progression or inhibit the underlying drivers of PD pathogenesis. As such, treatments that result in durable arrest of PD symptoms are urgently needed.Irisin is a small polypeptide that is secreted by skeletal muscle and other tissues into the blood of mice and humans (7, 8). The amino acid sequence is conserved 100% between mice and humans, suggesting a critical, conserved function. Importantly, the expression of irisin and its precursor protein FNDC5 is increased in muscle in response to many forms of exercise, both in rodents and in humans. Irisin levels increase in the blood of humans with exercise training, as determined by tandem mass spectrometry (8). In adipose cells, osteocytes, osteoclasts, and astrocytes integrin αV/β5 is the major functioning receptor for irisin (9, 10).Physical activity can prevent and ameliorate the symptoms of multiple forms of neurodegeneration, including Alzheimer’s disease (AD) and PD (11–14). Since irisin carries some of the benefits of exercise to adipose tissues, we and others have begun to study the effects of irisin in various models of neurodegeneration. In the earliest study, we showed that elevated expression of FNDC5 in the liver via the use of adenoviral vectors, and presumptive elevations of irisin in the blood, stimulated an “exercise-like” program of gene expression in the hippocampus (15). Moreover, the expression of FNDC5 with these same viral vectors rescued memory deficits in a mouse model of AD (16). Most recently, irisin itself was shown to be the active moiety regulating cognitive function in four separate mouse models. Importantly, elevation of the blood levels of the mature, cleaved irisin using adeno-associated virus (AAV) was sufficient to improve cognitive function and reduce neuroinflammation in two distinct models of AD (9). Furthermore, irisin itself crossed the blood–brain barrier (BBB), at least when the protein was produced from the liver with these AAV vectors.In the current study, we examine the effects of irisin on the pathophysiology of PD, using the α-synuclein preformed fibril (α-syn PFF) seeding model in vitro and in vivo. Pathologic α-syn is thought to spread “prion-like” in the brains of PD patients and certain other neurological disorders, where they cause neuronal death and dysfunction. We show here that irisin has powerful effects in preventing both the accumulation of pathologic α-syn and neuronal cell death in primary cell culture. Furthermore, elevation of blood irisin levels in mice normalizes the histological manifestations in the SNpc and the PD-like symptomology involving movement and grip strength induced by intrastriatal injection of α-syn PFF. Together, these data suggest the potential therapeutic value of irisin in PD and other neurodegenerative states that involve α-syn.     

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.     

Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson’s disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Parkinson’s disease is the most common neurodegenerative movement disorder (1, 2). A distinctive pathophysiological signature of this disease is the presence of abnormal intraneuronal protein deposits known as Lewy bodies (3, 4). One of the main components of Lewy bodies is α-synuclein (5), a peripheral membrane protein highly abundant at neuronal synapses (6, 7) and genetically linked with Parkinson’s disease (8, 9). This 140-residue disordered protein can be subdivided into three domains, an amphipathic N-terminal region (amino acids 1 to 60), a central hydrophobic region (non-amyloid-β component, or NAC, amino acids 61 to 95), and an acidic proline-rich C-terminal tail (amino acids 96 to 140) (7). Although α-synuclein aggregation is characteristic of Parkinson’s disease and related synucleinopathies, the corresponding mechanism and its possible pathological role in disease are not yet fully understood.Generally, the aggregation process of proteins proceeds through a series of interconnected microscopic steps, including primary nucleation, elongation, and secondary nucleation (10, 11). During primary nucleation, the self-assembly of proteins from their native, monomeric form leads to the formation of oligomeric species, an event that may occur in solution or on surfaces including biological membranes (12, 13). The formation of these oligomers is typically a slow event governed by high kinetic barriers (10, 11). Once formed, the oligomers may convert into ordered assemblies rich in β structure, which are capable of further growth into fibrillar aggregates (14). In many cases, the surfaces of existing fibrillar aggregates then further catalyze the formation of new oligomers (15, 16). This secondary nucleation process is typically characterized by the assembly of protein monomers on the surface of fibrils that eventually nucleate into new oligomeric species (15, 16). This autocatalytic mechanism generates rapid fibril proliferation (15).In the case of the aggregation process of α-synuclein, several key questions are still open, including two that we are addressing in this study. The first concerns whether there are cellular conditions under which α-synuclein can undergo spontaneous aggregation, and the second whether the proliferation of α-synuclein fibrils by aggregate-dependent feedback processes can take place at physiological pH. These questions are relevant because according to our current knowledge, α-synuclein aggregation does not readily take place spontaneously in the absence of contributing factors such as lipid membranes. Furthermore, secondary nucleation contributes significantly to the aggregation process only at acidic pH (13, 17). It thus remains challenging to rationalize the links between α-synuclein aggregation and Parkinson’s disease.To address this problem, we investigated whether it is possible to leverage the recent finding that α-synuclein can undergo a phase separation process resulting in the formation of dense liquid condensates (18–21). Phase separation has recently emerged as a general phenomenon associated with a wide variety of cellular functions (22–25) and closely linked with human disease (23, 26–29). This process has been reported for a wide range of proteins implicated in neurodegenerative conditions, including tau, fused in sarcoma (FUS), and TAR DNA binding protein 43 (TDP-43) (30–32). Since it has also been shown that protein aggregation can take place within liquid condensates (19, 26, 32–36), we asked whether it is possible to characterize at the microscopic level the condensate-induced aggregation mechanism of α-synuclein by determining the kinetic rate constants of the corresponding microscopic processes.To enable the accurate determination of the rate constants for the microscopic steps in α-synuclein aggregation within condensates, we developed fluorescence-based aggregation assays to monitor both the spontaneous aggregation of α-synuclein and the aggregation in the presence of aggregate seeds. Using these assays within the framework of a kinetic theory of protein aggregation (10, 11, 37), we show that α-synuclein can undergo spontaneous homogenous primary nucleation and fast aggregate-dependent proliferation within condensates at physiological pH.     

The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer’s disease

Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer’s disease (AD). Interleukin (IL)-1β is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1β precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1β is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice (P = 0.001), which was completely rescued by OLT1177 fed to mice (P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD (P = 0.007 to untreated APP/PS1). In addition, microglia were less activated (P = 0.07) and the number of plaques was reduced in the cortex (P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome.

Alzheimer’s disease (AD) and other related neurodegenerative diseases leading to dementia represent an enormous burden for the society and health economies. AD patients suffer progressive cognitive and functional deficits often for many years, which result in a heavy burden to patients, families, and the public health system. In fact, in 2015 an estimated 46.8 million people worldwide were living with dementia, which could extend to 131.5 million by 2050 (1). Rising prevalence and mortality rates in combination with a lack of effective treatments lead to enormous costs to society. Research on AD in the last decades has focused on the pathological hallmarks and cellular deposits of amyloid-β (Aβ) peptides and neurofibrils (2). Recently, there has been increased evidence supporting a central role of the immune system in the progression or even the origin of the disease (3–5). In this respect, it is noteworthy that it has been known since 1989 that levels of interleukin (IL)-1β, one of the main mediators of innate immune response, are elevated in brains of patients with AD and can be associated with the progression and onset of AD (6–11). Additionally, it was shown that the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (12, 13), a multisubunit complex important for the maturation of IL-1β, is activated by Aβ peptides, leading to an overproduction of IL-1β, neuroinflammation, and cognitive impairment (14, 15). Inhibition of the NLRP3 inflammasome and the subsequent reduced IL-1β production can be linked to a change in the phenotype of microglia, the innate immune cells in the brain. Heneka et al. (16) pointed out the important role of the NLRP3 inflammasome/caspase-1 axis in AD pathogenesis by demonstrating significant improvements (e.g., in cognition) in APP/PS1 mice (a mouse model for AD) when crossed with NLRP3^−/− animals. The APP/PS1 mice express a human amyloid precursor protein (APP) and human presenilin-1 (PS1), leading to the accumulation of Aβ peptides, neuroinflammation, and cognitive impairment (17).OLT1177 (rINN: dapansutrile) is a new chemical entity small molecule that specifically targets the NLRP3 inflammasome and prevents the activation of caspase-1 and the maturation and release of IL-1β (18). OLT1177 has been shown to be well tolerated in animals and humans (18) and is currently in phase 2 clinical studies for the treatment of inflammatory conditions, such as osteoarthritis (topical gel dosage form) and inflammatory diseases, such as acute gout flare (oral capsule dosage form), among other diseases (19).In this study, we used the APP/PS1 mouse model of AD to investigate the effects of OLT1177 as an acute, oral pharmacological intervention (17). Six-month-old WT and APP/PS1ΔE9 mice consumed ad libitum OLT1177 in feed pellets (∼0, 500, or 1,000 mg/kg/d based on feed concentrations of 0, 3.75 or 7.5 g of OLT1177 per kilogram of feed; hereafter referred to as 3.75 or 7.5 g/kg OLT1177) for the treatment duration of 3 mo. APP/PS1 mice treated with OLT1177 showed rescue effects in various assessments, ranging from improved cognitive function to overall reduction in proinflammatory cytokines in the brain, suggesting the potential benefits of pharmaceutically blocking NLRP3 signaling in AD.     

The N terminus of α-synuclein dictates fibril formation

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson’s disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41–140). This behavior can be explained by the unique fibril structure that is adopted by 41–140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Amyloid formation of α-synuclein (α-syn) is a pathological feature of Parkinson’s disease (PD), multiple-system atrophy (MSA), and dementia with Lewy bodies (1, 2). An abundant presynaptic protein (3), α-syn is 140 amino acids in length with a putative biological function in aiding the exocytosis of synaptic vesicles (4–6), in which the first 89 N-terminal residues fold into a helical structure upon membrane association (7). In its disease-associated, aggregated amyloid state, residues 37 through 97 adopt β-sheet structure (8), which overlaps with the lipid-binding domain. Notably, both N- and C-terminal α-syn truncations are associated with PD (9). So far, N-terminally truncated (ΔN) α-syn variants 5‒140, 39‒140, 65‒140, 66‒140, 68‒140, and 71‒140 and C-terminally truncated (ΔC) α-syn variants, 1‒101, 1‒103, 1‒115, 1‒122, 1‒124, 1‒135, and 1‒139 have been found in brains of PD patients (10–12).α-Syn truncations originate from incomplete degradation, which has been attributed to various cytosolic (13–15) and lysosomal proteases (16, 17). In fact, ∼60% of the abovementioned truncations can be assigned to cleavages by lysosomal asparagine endopeptidase (AEP), cathepsin (Cts) D, CtsB, and CtsL (15–17). Removal of the C terminus (residues 104–140) is shown to accelerate fibril formation both in vitro and in vivo (18–25). On the other hand, perplexing behaviors of ΔN-variants have been documented; while deleting the first 20 residues has minimal perturbation, the removal of either the first 10 or 30 residues slows aggregation kinetics (26). Nevertheless, the influence of N-terminal residues on α-syn aggregation has been shown by both insertion [tandem repeat of residues 9–30 (27)] and deletion [Δ36–42 (28) and Δ52–55 (29)] mutants, in which fibril formation can be completely impeded.Recently, structure determination by cryogenic electron microscopy (cryo-EM) has revealed fibril structures for full-length α-syn (1–140) (24, 30–32), C-terminal truncations (24, 33), phosphorylated Y39 (34), and PD-related mutants, E46K (35, 36), H50Q (37), and A53T (38). One striking feature of these fibrils is the eclectic mix of structures, often termed as fibril polymorphism. In fact, it was recently shown that different conformational strains of α-syn fibrils are present in PD and MSA patients (39, 40). The outstanding question still remains as to how the same polypeptide chain can produce such a vast number of polymorphic structures. While there are significant structural differences, some features of α-syn fibrils are conserved. All fibrils are formed from a twisting pair of protofilaments with the exception of a H50Q polymorph, which is composed of a single filament. A kernel motif of a bent β-arch appears in all structures. Also, at least one inter- or intramolecular salt bridge between a Lys and Glu is revealed in each structure (24, 30–38, 40), which is not surprising given that there are numerous possibilities for salt bridges between the 14 Lys, 8 Glu, and 2 Asp residues located throughout the first 100 residues in the sequence (Fig. 1A and SI Appendix, Fig. S1). Generally, residues between 37 and 97 constitute the fibril core with a few exceptions that involve additional residues in the N terminus, which include phosphorylated Y39 fibrils with an extended core of 1–100 (34) and two polymorphs of 1–140 showing interactions of N-terminal β-strands (residues 14–24) (30). Fibrils derived from brains of MSA patients also indicate additional involvement of the N-terminal region extending to residue 14 (40). Due to the contribution of N-terminal residues in these structures and the fact that C-terminal truncations resulted in modest conformational changes, we hypothesize that N-terminal residues play a greater role in influencing fibril structure.Open in a separate windowFig. 1.Aggregation of ΔN-α-syns. (A) Schematic representation of α-syn primary sequence (residues 1–140), showing basic (blue) and acidic (red) residues. Underlined regions correspond to truncations used in this study: 14‒140 (blue), 36‒140 (magenta), and 41‒140 (green). (B and C) Comparison of aggregation kinetics monitored by ThT fluorescence at 37 °C. [α-Syn] = 35 µM (B) and 70 µM (C) with [ThT] = 10 µM in 20 mM NaPi, 140 mM NaCl, pH 7.4. The solid line and shaded region represent the mean and SD, respectively (n ≥ 4). Representative TEM images of (D) 1‒140, (E) 14‒140, (F) 36‒140, and (G) 41‒140 were taken at 35 µM. Different fibril polymorphs observed are noted. Additional fields of view are shown in SI Appendix, Figs. S3–S5.Here, we sought to understand the role of the N terminus in α-syn fibril formation by removing different N-terminal residues and evaluating their effects on aggregation kinetics, fibril structure, and propagation. Three ∆N-terminal constructs (14‒140, 36‒140, and 41‒140) have been examined, in which the first 13-, 35-, and 40-residues in the N terminus were deleted (Fig. 1A). We specifically chose these sites based on the locations of native Gly residues, which allows us to generate native sequences (i.e., no overhang) upon Tobacco Etch Virus (TEV) protease cleavage of the hexahistidine affinity tag, which facilitates facile protein purification. All three ∆N-α-syn exhibited different aggregation kinetics and distinct fibril ultrastructural features as determined by thioflavin-T (ThT) fluorescence and transmission electron microscopy (TEM), respectively. In cross-seeding experiments, both fibrillar 36‒140 and 41‒140 did not seed the full-length (1‒140) protein, while 14‒140 fared better but less efficient than self-seeding, supportive of the significant impact of removing N-terminal residues in fibril structure. The reverse reaction involving full-length seeds showed that fibril formation of 14‒140 and 36‒140 but not 41‒140 could be accelerated. This observation is explained by the fibril structure adopted by 41–140, which was determined by cryo-EM to an overall resolution of 3.2 Å. Unlike any currently known α-syn structure, the amyloid core is formed by two asymmetric protomers with different amino acid chain lengths, adopting an extended β-hairpin (E61‒D98) and the bent β-arch kernel (E46‒K96) with a large nonpolar interfilament interface (442 Ų) stabilized by an intermolecular salt bridge between K80 and E83. Collectively, these results establish the important role of N-terminal residues in fibril formation and structure.     

Ceramide accumulation induces mitophagy and impairs β-oxidation in PINK1 deficiency

Energy production via the mitochondrial electron transport chain (ETC) and mitophagy are two important processes affected in Parkinson’s disease (PD). Interestingly, PINK1, mutations of which cause early-onset PD, plays a key role in both processes, suggesting that these two mechanisms are connected. However, the converging link of both pathways currently remains enigmatic. Recent findings demonstrated that lipid aggregation, along with defective mitochondria, is present in postmortem brains of PD patients. In addition, an increasing body of evidence shows that sphingolipids, including ceramide, are altered in PD, supporting the importance of lipids in the pathophysiology of PD. Here, we identified ceramide to play a crucial role in PINK1-related PD that was previously linked almost exclusively to mitochondrial dysfunction. We found ceramide to accumulate in mitochondria and to negatively affect mitochondrial function, most notably the ETC. Lowering ceramide levels improved mitochondrial phenotypes in pink1-mutant flies and PINK1-deficient patient-derived fibroblasts, showing that the effects of ceramide are evolutionarily conserved. In addition, ceramide accumulation provoked ceramide-induced mitophagy upon PINK1 deficiency. As a result of the ceramide accumulation, β-oxidation in PINK1 mutants was decreased, which was rescued by lowering ceramide levels. Furthermore, stimulation of β-oxidation was sufficient to rescue PINK1-deficient phenotypes. In conclusion, we discovered a cellular mechanism resulting from PD-causing loss of PINK1 and found a protective role of β-oxidation in ETC dysfunction, thus linking lipids and mitochondria in the pathophysiology of PINK1-related PD. Furthermore, our data nominate β-oxidation and ceramide as therapeutic targets for PD.

Loss of PINK1 function causes autosomal recessive early-onset Parkinson’s disease (PD). Most patients present with bradykinesia, rigidity, resting tremor, and dyskinesia and are responsive to dopamine replacement therapy (1). On the cellular level, PINK1 disease mutations result in impaired energy metabolism and a variety of mitochondrial defects that can partially be alleviated by stimulation of energy metabolism (2–4). Intriguingly, abnormal mitochondrial morphology, along with lipid aggregates, was recently discovered to be present in Lewy bodies of postmortem PD patients’ brains (5), challenging the previously held notion of alpha-synuclein being the almost exclusive neuropathological correlate. This finding confirms the involvement of mitochondrial dysfunction in PD and additionally suggests a critical role of lipids in the pathogenesis of PD.PINK1 is important for the phosphorylation of the Complex I subunit NdufA10 resulting in efficient Complex I and electron transport chain (ETC) activity (6, 7). This function is evolutionarily conserved between Drosophila and humans. Hence, in both flies and humans, loss of PINK1 results in an impaired ETC, reduced ATP levels, and defective mitochondrial morphology (6, 8, 9), all of which are ubiquitously observed in the fly already at the early larval stage. Furthermore, alongside Parkin, PINK1 plays a crucial role in mitophagy to remove defective mitochondria that appears to be defective in an age-dependent fashion (10–13). Pink1-mutant Drosophila melanogaster additionally show thorax muscle degeneration and defective flying ability (8, 9). These latter defects, together with impaired mitochondrial morphology, can be rescued by expressing the fission-promoting protein Drp1 (14). However, increased fission does not improve ETC-related defects (15). Furthermore, stimulation or facilitation of the ETC rescues ETC-related phenotypes in pink1-mutant Drosophila, including ATP levels and mitochondrial morphology (3, 4, 7, 15, 16). These data collectively suggest two parallel mechanisms that converge on a shared common pathway leading to the development of PD. However, the link between these two pathways has yet to be resolved.Recently, disrupted lipid homeostasis has garnered increasing attention in PD (16–18). Furthermore, ceramide, the basic sphingolipid, is altered in several PD models and has been implicated in PD-related alpha-synuclein toxicity (17–20). Interestingly, ceramide induces mitophagy that is facilitated by Drp1 (21). Furthermore, pathogenic variants in Glucocerebrosidase (GCase), an enzyme involved in ceramide synthesis, are known to be the most common risk factor for PD (22, 23). However, the exact mechanism remains enigmatic. We found increased ceramide levels in isolated mitochondria of Pink1^−/− knockout (KO) mouse embryonic fibroblasts (MEFs) (16) and Pink1-deficient flies. Increased ceramide levels are detrimental for proper ETC function (24). Hence, we hypothesize that ceramide accumulation in PINK1 deficiency affects ETC function and mitophagy and constitutes the missing link between these two important processes affected in PD.     

Therapeutic functions of astrocytes to treat α-synuclein pathology in Parkinson’s disease

Intraneuronal inclusions of misfolded α-synuclein (α-syn) and prion-like spread of the pathologic α-syn contribute to progressive neuronal death in Parkinson’s disease (PD). Despite the pathologic significance, no efficient therapeutic intervention targeting α-synucleinopathy has been developed. In this study, we provide evidence that astrocytes, especially those cultured from the ventral midbrain (VM), show therapeutic potential to alleviate α-syn pathology in multiple in vitro and in vivo α-synucleinopathic models. Regulation of neuronal α-syn proteostasis underlies the therapeutic function of astrocytes. Specifically, VM-derived astrocytes inhibited neuronal α-syn aggregation and transmission in a paracrine manner by correcting not only intraneuronal oxidative and mitochondrial stresses but also extracellular inflammatory environments, in which α-syn proteins are prone to pathologic misfolding. The astrocyte-derived paracrine factors also promoted disassembly of extracellular α-syn aggregates. In addition to the aggregated form of α-syn, VM astrocytes reduced total α-syn protein loads both by actively scavenging extracellular α-syn fibrils and by a paracrine stimulation of neuronal autophagic clearance of α-syn. Transplantation of VM astrocytes into the midbrain of PD model mice alleviated α-syn pathology and protected the midbrain dopamine neurons from neurodegeneration. We further showed that cografting of VM astrocytes could be exploited in stem cell–based therapy for PD, in which host-to-graft transmission of α-syn pathology remains a critical concern for long-term cell therapeutic effects.

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder with movement symptoms characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta of the midbrain with the concomitant loss of nigrostriatal DA neurotransmission. A pathologic hallmark of PD is intraneuronal inclusion of α-synuclein (α-syn) aggregates, called Lewy bodies and Lewy neurites. The α-syn aggregates cause various cellular dysfunctions including mitochondrial impairment, defective endoplasmic reticulum (ER) function, autolysosomal pathways, and synaptic and nuclear dysfunctions (1, 2). Aggregated α-syn is released from neuronal cells and acts as a ligand for patterned recognition receptors, which activate inflammatory responses in glial cells (3, 4). Furthermore, the pathologic protein aggregates undergo neuron-to-neuron transmission in a prion-like fashion (reviewed in ref. 5). The α-syn propagation and neuroinflammation are closely related to disease progression and clinical severity (6).Given its pathologic significance, the α-syn proteinopathy is a major research focus to develop disease-modifying therapies for PD and other synucleinopathic disorders such as Lewy body dementia, multiple system atrophy, and certain forms of Alzheimer’s disease. However, no therapeutic intervention to effectively eliminate the pathologic α-syn has been developed to date. In addition to the diseased conditions, the aggregated species of α-syn are also accumulated in the midbrain SN during normal aging, but not in young brain tissues (7), suggesting the existence of homeostatic regulation to prevent and resolve α-syn aggregation in young and healthy brains. This suggests homeostatic functions may be useful in developing therapeutic tools. In this regard, astrocytes are a prime cell type to be studied for therapeutic applications, as this glia cell type has multiple functions related to maintaining brain homeostasis, including those for correct functioning of neurons and protecting neuronal cells from pathologic insults (reviewed in ref. 8). Recent studies have shown the capacity of astrocytes to efficiently take up and degrade α-syn (9–12). Due to the astrocyte scavenging effect, α-syn inclusions are usually not detected in astrocytes of PD patients except in advanced stages of the disease (13–18). In addition, in contrast to efficient transmission of neuronal α-syn proteins into astrocytes, α-syn transfer from astrocytes to neuronal cells is inefficient (11), collectively suggesting a role for astrocytes in scavenging α-syn rather than in spreading it. The role of homeostatic astrocytes in α-syn pathology, however, remains to be unraveled.In this study, we showed that astrocytes, especially those cultured from the ventral midbrain (VM), the brain region primarily affected in PD, substantially alleviate neuronal α-syn pathology by regulating a series of the proteostasis procedures associated with formation, transmission, disaggregation, and clearance of toxic α-syn aggregates. Upon transplantation, VM-type astrocytes efficiently eliminated pathologic α-syn accumulation and α-syn–induced DA neuron degeneration in the midbrain of PD model mice. We further show that host-to-graft propagation of toxic α-syn, reported as a critical concern in the cell-based therapeutic approach for PD (19, 20), was greatly prevented by cografting the cultured astrocytes. Based on these findings, the therapeutic actions of astrocytes are proposed for use in relieving α-syn–mediated neuronal toxicity and in setting up a desirable cell-based therapy free from host-to-graft α-syn propagation in PD.     

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.     

Soluble TREM2 inhibits secondary nucleation of Aβ fibrillization and enhances cellular uptake of fibrillar Aβ

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer’s disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-β (Aβ) into plaques, particularly Aβ40 and Aβ42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aβ fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aβ, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aβ40 and Aβ42, even at a high micromolar concentration, while it does bind to fibrillar Aβ42 and Aβ40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aβ, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2–Aβ fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake.

Alzheimer’s disease (AD) is the most common form of dementia and features the neuropathological hallmarks of extracellular Aβ plaques and intraneuronal tau neurofibrillary tangles (1, 2). Human genetic studies on heritable mutations in APP and PSEN causing early-onset familial AD (3) argue that pathogenic Aβ drives tau neurofibrillary tangle formation; in contrast, mutations in MAPT do not lead to Aβ pathology nor cause AD, but rather a rare genetic form of early-onset primary tauopathy (4). In support of the molecular genetics, a recent cross-sectional study in postmortem human AD brain samples demonstrated the presence and correlation of robust prion bioactivity for Aβ and tau proteins in nearly all cases (5), suggesting that even at death, Aβ in prion conformations are active in the late stages of disease. Together, these data establish the importance of pathogenic Aβ throughout AD progression and highlight the urgent need to better understand the cellular and molecular mechanisms that mitigate Aβ’s role in pathogenesis.Microglia are the innate immune effector cell in the brain with myriad functions in healthy aging and neurological diseases. Recent human genetic studies have discovered mutations in several genes encoding microglia-specific proteins that increase risk for AD, thus supporting the notion that microglia are central to AD pathogenesis. Genetic variants of triggering receptor expressed on myeloid cells 2 (TREM2), a cell-surface receptor expressed on myeloid cells and microglia, increase the risk of AD by threefold, implicating microglia and the innate immune system as important determinants in AD pathogenesis (6). TREM2 consists of an extracellular Ig-like domain, a transmembrane domain, and a cytoplasmic tail. Proteolytic cleavage of TREM2 at His157 releases soluble TREM2 (sTREM2) that can be detected in the cerebrospinal fluid (7). While the function of sTREM2 is uncertain, it is believed to promote microglia survival, proliferation, and phagocytosis, making it important for cell viability and innate immune functions in the brain (6, 8, 9). Full-length membrane-bound TREM2 binds to its adaptor protein, DAP12, on the surface of microglia to transmit downstream signaling in response to clustering induced by multivalent ligands (10). Most of the studied mutations are in the Ig-like domain of TREM2. Misfolding, retention, and aberrant shedding are postulated to be caused by some mutations, while other variants have altered ability to interact with their binding partners (8, 11, 12).The R47H mutation in TREM2 constitutes one of the strongest single allele genetic risk factors for AD. The R62H, D87N, and T96K mutations in TREM2 were also linked to AD after extensive analyses of TREM2 polymorphisms (13–16). Several in vivo studies show that TREM2 regulates polarization of microglial processes toward Aβ deposits, leading to plaque compaction and pacification in human AD brain samples and mouse models (17–19). Genetic deletion of TREM2 expression in transgenic mice injected with exogenous Aβ fibrils leads to accelerated amyloid plaque seeding (20). The prominent phenotype in plaque-associated microglia suggests that the effects of AD-risk mutations or genetic deletions are driven by loss of full-length TREM2 signaling. However, a recent in vivo study using exogenously injected recombinant sTREM2 showed reduced amyloid burden and behavioral rescue in mice (21). New clues for the potential importance of sTREM2 in AD have been revealed in clinical studies on living AD patients. sTREM2 can be measured in the cerebrospinal fluid (CSF) and it increases during early stages of AD symptomology (22, 23), suggesting that sTREM2 may be a biomarker for microglia activation. Recent studies indicate that AD patients with relatively high levels of sTREM2 in the CSF have slower rates of amyloid accumulation and reduced cognitive decline (24, 25). These human data support the hypothesis that microglia and sTREM2 play a protective role in early stages of AD progression.While most risk variants of TREM2 exist in the ligand-binding Ig-like domain, the AD-associated point mutation H157Y falls within the stalk region and is known to increase the shedding of full-length TREM2, which possibly results in higher titers of sTREM2 (6). Elevated ectodomain shedding reduces cell-surface full-length TREM2 available for TREM2-mediated phagocytosis and plaque compaction as well as down-stream signal transduction. Although more work is needed, such data begin to suggest there is a delicate balance between the functions of membrane-bound and secreted TREM2, and hence opposing cellular effects of TREM2 variants can emerge (i.e., reduced versus enhanced shedding, which result in similar phenotypic outcomes by reducing cell-surface TREM2) (6, 26).sTREM2 binds to diverse ligands, including phospholipids, apolipoproteins, DNA, and Aβ. Although the full physiological and pathological roles of these interactions remain to be revealed (11, 12, 27, 28), there is general agreement that the extracellular domain of TREM2 (sTREM2) binds to oligomeric forms of Aβ42. However, the observed apparent affinities vary over many orders-of-magnitude (7, 29–31). Most studies were conducted with dimeric Fc fusion proteins, tetrameric constructs, or biotinylated protein bound to the tetrameric streptavidin, which might artificially increase the avidity of the protein for oligomeric forms of Aβ peptides (7, 29–31). Moreover, the studies that report the highest affinities relied on biolayer interferometry or surface plasmon resonance, in which oligomeric protein constructs were immobilized on a surface and Aβ peptides were allowed to diffuse over the surface. Aβ oligomers were found to bind, but they either did not dissociate at all, or they dissociated slowly, leading to affinity estimates in the picomolar to nanomolar range (7, 30, 31). However, the extent of binding of Aβ to the surface did not saturate at concentrations that were orders-of-magnitude greater than the reported dissociation constants, suggesting that the slow off-rate was instead due to precipitation of insoluble Aβ on the bilayer surface (7). In another study, Aβ was fused to the dimeric protein glutathione S-transferase (29). Furthermore, there is inconsistency in the studies involving monomeric Aβ42, with some studies finding nanomolar to low micromolar dissociation constants for the interaction of monomeric Aβ42 and TREM2 ectodomain (29, 30), in contrast to two other studies that reported weak or no interaction (7, 31).To help elucidate the role of sTREM2 and its interaction with Aβ, we evaluated the binding of sTREM2, without any nonnative oligomerization domains added to the studied construct, to specific forms of Aβ40 and Aβ42. We used NMR to show that sTREM2 does not bind to monomeric Aβ, even at high micromolar concentrations. Next, we examined the binding of sTREM2 to fibrils, formed under well-defined conditions to provide a relatively homogenous structure, as assessed by solid-state NMR (32). Additionally, because oligomeric forms of Aβ are heterogeneous and kinetically labile, we opted to determine how sTREM2 affects the formation of intermediates in the fibrillization of Aβ and show that it has a profound effect on the secondary nucleation step of the process. We find that the R47H variant binds to Aβ40 and Aβ42 fibrils with a similar affinity and inhibits their fibrilization just as the WT sTREM2 does. Finally, we show that WT sTREM2, but not the mutant R47H, strongly enhances the uptake of Aβ fibrils in human neural and microglial cells.A second goal of this report was to define the structural underpinnings of the interaction between sTREM2 and Aβ fibrils. Although individual structures of sTREM2 and Aβ40 fibrils have been reported (8, 33), the structures of the complex are not available. The molecular surface of sTREM2 is particularly interesting with regards to its function (8, 29). The crystal structure of the ectodomain of TREM2 (TREM2_ECD) revealed an immunoglobulin fold motif with a highly asymmetric distribution of charged and hydrophobic residues. The surface of the hydrophobic and aromatic protrusion at the top of the structure (Fig. 1, red dotted area) has a highly positive electrostatic potential adjacent to it is a relatively flat surface of positively charged residues (Fig. 1, black dotted area, surface 1). Surface 1 appears suited for binding to acidic moieties (like in Protein Data Bank [PDB] ID code 6B8O) (8). R47 lies near the basic patch, consistent with the R47H mutation disrupting the conformation of the CD loop (8), which comprises a large portion of surface 1. Molecular dynamics simulations suggest that disease-promoting mutations disrupt the apolar character and electrostatic surface of this region of the protein (34). The R47H mutation is also known to disrupt sTREM2’s ability to bind to and signal in response to acidic phospholipids (29). Thus, the data indicate that this surface is important for binding or signaling in response to anionic lipids. In contrast, the determinants of binding to Aβ peptides are uncertain, with different studies coming to differing conclusions concerning the effect of AD mutants on binding or uptake of Aβ fibrils (7, 29–31). Recently, it was suggested that different surfaces might be involved in binding different TREM2 ligands (29). Indeed, sTREM2 has a second unusual, variegated electrostatic surface (surface 2 in Fig. 1), with an extended band of positively charged residues flanked by acidic patches near the top and bottom of the structure, which might interact with different binding partners. Here, we use integrative structural modeling guided by chemical cross-linking mass spectrometry (XL-MS) to map the structure of the fibrillar Aβ–sTREM2 complex, and how it is affected by the R47H substitution. The resulting model suggests that the patch of hydrophobic and basic residues on sTREM2 that contains R47 does not directly interact with Aβ40 fibrils. Instead, sTREM2 is predicted to interact with Aβ primarily via surface 2, while projecting surface 1 away from the amyloid fibrils, with implications for both cellular uptake and signaling.Open in a separate windowFig. 1.Crystal structure of sTREM2 (PDB ID code 5UD7) (8), showing electrostatic potential map of the ectodomain. The white, red, and blue colors in the map correspond to the neutral, acidic, and basic residues, respectively. The map was generated using CHIMERA v1.14 (69). The hydrophobic and aromatic protrusion in sTREM2 is highlighted with a red dashed curve (hydrophobic tip). The flat surface of basic residues adjacent to the hydrophobic tip is shown with black dashed curve (surface 1). Another patch of basic residues, opposite to surface 1, is highlighted with a yellow dashed curve (surface 2). Key residues in these three regions are indicated.     

Homocysteine fibrillar assemblies display cross-talk with Alzheimer’s disease β-amyloid polypeptide

High levels of homocysteine are reported as a risk factor for Alzheimer’s disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated β-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between β-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, currently affecting tens of millions of individuals globally and leading to immense social and financial impacts (1, 2). Despite the vast efforts to develop a pharmacological treatment for the disease, recent clinical trials had failed (3, 4). While it is very clear that amyloid formation is associated with AD (5, 6) and genetic variations in the gene coding for the β-amyloid polypeptide are associated with early onset of the disease (7), direct targeting of the aggregation of AD-associated pathological proteins and polypeptides has so far not resulted in clinical development of any disease-modifying treatment (8–10). Therefore, there is an essential need to understand the early biological changes that may induce the diverse pathologies observed in AD and other forms of dementia in order to identify new therapeutic targets (10).In the last decades, an enormous body of research had provided important information on the amyloid cascade, oxidative stress, and inflammatory response that are involved in AD (3, 11). Specifically, the notion of β-amyloid toxicity and extracellular plaque formation as the main cause of neuronal and synaptic loss has been extensively studied (12–14). Yet, the key to AD treatment and prevention remains elusive, and the initial steps leading to the formation and accumulation of these protein aggregates and the role of nonproteinaceous agents in this process are still not understood (10). Since only 1% of AD cases result from a familial mutation and the majority of patients are sporadic (2, 4), it is important to explore new directions to understand the biological mechanisms underlying these cases. Studies of metabolite profiling in brain regions and body fluids of AD and Parkinson’s disease (PD) patients show interference with specific metabolic pathways (15–19). Homocysteine (Hcy), a noncoded amino acid, was identified as a major risk factor for AD as high plasma concentrations were associated with the progression of the disease (20–22). Higher Hcy serum concentration was also correlated with behavioral and psychological symptoms of AD (23) and was associated with changes in motor function and cognitive decline in PD as well as with a more severe cognitive impairment in elderly adults (24–26). Furthermore, it was shown that significantly decreased hippocampal and cortical volume is associated with increased Hcy plasma concentration (27).Cystathionine β-synthase (CBS) deficiency, leading to excess of Hcy, results in the hyperhomocysteinemia inborn error of metabolism (IEM) disorder characterized by severe cognitive consequences (28–30). While elevated plasma Hcy is frequently reported as a strong and independent risk factor for the development of cognitive decline and dementia, the mechanism of its involvement is elusive (20, 31–34). Interestingly, the association between Hcy and AD-related pathological proteins was also demonstrated. Hcy-rich medium was shown to be cytotoxic to hippocampal and cortical neurons, resulting in increased β-amyloid–induced cell death (35–37). In addition, Hcy was found to bind β-amyloid_1-40, thereby stimulating β-sheet structure formation to facilitate its deposition. Indeed, induced Hcy accumulation in the brains of rats caused an elevation of β-amyloid deposition (38, 39). Furthermore, Hcy increased total tau and phosphorylated tau protein levels as well as the level of tau oligomers (40). Although the involvement of Hcy accumulation in AD pathology is evidential, no mechanistic insight has so far been suggested.We have previously demonstrated that small metabolites can self-assemble into amyloid-like structures with amyloidogenic characteristics (41–44). The presence of metabolite assemblies in IEM disorders (e.g., phenylalanine in Phenylketonuria) exemplifies their physiological importance in pathologically diversified diseases. Interestingly, recent studies demonstrated that metabolite assemblies could cross-seed the aggregation of proteins under physiological conditions, thereby suggesting a possible mechanism in which accumulated metabolites interfere with protein function and folding (45, 46). Assemblies of quinolinic acid, an endogenous neurometabolite that is involved in the pathology of PD, induce the aggregation of α-synuclein both in vitro and in cell culture (45). In addition, phenylalanine preformed fibrils were shown to initiate the aggregation of several proteins under physiological conditions (46). Metabolite accumulation, structure formation, and seeding of proteins may thus underlie the unknown role of metabolites in neurodegenerative pathologies (47). Specifically, seeding of amyloidogenic proteins by preformed fibrils may be part of the mechanisms underlying the stereotypical spreading of toxic aggregates in the brains of AD patients.We recently established an in vivo yeast model for IEM disorders by genetically modifying the yeast to reflect the mutations found in patients showing accumulation of the adenine nucleobase and its derivatives (48). Yeast model systems provide a powerful platform to elucidate the pathophysiology of diseases, as well as for the screening and development of disease-modifying therapeutics (49). This model was found to be a valid system, as supported by its robust sensitivity to adenine feeding and by the fact that adenine supramolecular structures could be detected. Furthermore, the addition of a generic fibrillation-modifying polyphenolic compound rescued the toxic effect without lowering the concentration of adenine, indicating the therapeutic potential of our model for the modulation of structure formation (48, 50).Here, aiming to explore the role of Hcy in AD, we demonstrate the formation of amyloid-like fibrils by Hcy in vitro and in vivo in a yeast model. Structural characterization of the Hcy fibrils and their cytotoxic effect were both studied. In addition, we revealed that polyphenolic inhibitors could rescue the toxic effect of Hcy assemblies and inhibit its structure formation. Remarkably, immunohistochemistry allowed the detection of Hcy fibrils in the brain of AD model mice as well as the apparent interplay between Hcy and β-amyloid. Finally, we demonstrated the cross-seeding of AD-related pathological protein by Hcy assemblies. Our work suggests a research direction for the association between metabolite accumulation and the initiation of neurodegenerative processes, thus offering a path for the development of therapeutic treatments that will target the key early stages of the disease.     

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are both associated with underlying Lewy body disease, which represents the second most common neurodegenerative disorder after Alzheimer’s disease (1, 2). The neuropathological hallmark of Lewy body disease is the intracellular aggregation of the protein α-synuclein (α-syn) into spherical cytoplasmic inclusions, termed Lewy bodies, but are also observed in neuronal processes as Lewy neurites (LNs) (3).α-Syn is thought to play a central role in the pathobiology of Lewy body disease. Single-point mutations and genetic modifications affecting α-syn expression—through duplications, triplications, or polymorphisms in its promoter—have been linked to both idiopathic and familial forms of Lewy body disease (4–6). Nevertheless, neuropathological studies utilizing pan–α-syn antibodies, recognizing both physiological and pathological forms of the protein, do not consistently report a relationship between the load of Lewy body pathology and clinical disease severity (2). To reconcile the apparent importance of α-syn in Lewy body disease with the difficulty relating Lewy body burdens in the brain to phenotypic severity, continued research has focused on the identification of particularly disease-relevant forms of α-syn. α-Syn undergoes various posttranslational modifications (PTMs)—including acetylation, nitration, ubiquitination, and glycosylation and phosphorylation at serine 129 (pS129)—increases from ∼4% under physiological conditions to 90% in Lewy body disease, suggesting it is associated with the disease state (7–9).Previous studies have reported that pS129 enhances intracellular aggregate formation in SH-SY5Y cells (10), and mediates cell death through activation of the unfolded protein response pathway (11). Furthermore, studies in rodent models have suggested that pS129 exacerbates the rate of pathological protein aggregation and deposition, with subsequent negative effects on neuronal functioning (12). However, these studies are counterbalanced by others reporting a potentially neuroprotective function of phosphorylation in animal models (13, 14) and cellular model systems (15). Additionally, studies have reported neutral findings regarding pS129 modification as neither enhancing nor diminishing cellular toxicity and α-syn aggregation (16, 17). Despite the uncertain pathogenic role of pS129 in Lewy body disease, antibodies against pS129 are widely used, based on the putative view that they label a species of α-syn that is particularly disease-relevant. These studies often employ pS129–α-syn as a marker of the abundance of protein inclusions to stage disease severity and evaluate the relationship between its abundance and important clinical or pathological variables, such as disease duration, phenotypic severity, or cell loss (18). Such studies typically identify that pS129 abundance throughout the brain correlates with disease severity (19–21), though it remains uncertain whether phosphorylation precedes protein aggregation or occurs secondarily to deposition of nonphosphorylated α-syn, and whether pS129 is a key driver of pathogenicity or simply a useful marker of a neurodegenerative process (22, 23). Therefore, although there is a substantial literature on pS129 in Lewy body disease, there is continued controversy regarding its potential contribution to disease states, with numerous studies reporting discordant findings. Despite contradictory findings regarding the disease-relevance of pS129, it is widely viewed as a particularly disease-associated modification, thus necessitating further research to address its importance for Lewy body disease.To address the key questions regarding the pathogenic relevance of pS129–α-syn, the present study aimed to undertake a comprehensive and multidisciplinary project to address this important and pressing question. The key aim of the study was to better understand the role of pS129 in the natural history of Lewy body disease, by determining when pS129 occurs in the development of α-syn aggregates and how it affects the aggregation-propensity and cytotoxicity of α-syn     

Amyloid-β peptide dimers undergo a random coil to β-sheet transition in the aqueous phase but not at the neuronal membrane

Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity of amyloid-β peptides in Alzheimer’s disease. To gain a detailed understanding of the mutual interference of amyloid-β oligomers and the neuronal membrane, we carried out microseconds of all-atom molecular dynamics (MD) simulations on the dimerization of amyloid-β (Aβ)42 in the aqueous phase and in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The dimerization in solution is characterized by a random coil to β-sheet transition that seems on pathway to amyloid aggregation, while the interactions with the neuronal membrane decrease the order of the Aβ42 dimer by attenuating its propensity to form a β-sheet structure. The main lipid interaction partners of Aβ42 are the surface-exposed sugar groups of the gangliosides GM1. As the neurotoxic activity of amyloid oligomers increases with oligomer order, these results suggest that GM1 is neuroprotective against Aβ-mediated toxicity.

In Alzheimer’s disease (AD), amyloid-β peptide (Aβ) aggregates into fibrils and subsequently accumulates as plaques within the neural tissue (1). An increasing number of studies suggest that the smaller soluble oligomers formed in the earlier stages of the aggregation process are the main cytotoxic species affecting the severity and progression of AD (2–4). Aβ dimers have been reported to be the smallest toxic oligomer that affects synaptic plasticity and impairs memory (5, 6). Therefore, a detailed characterization of Aβ dimerization is an essential step toward developing a better understanding of the aggregation process. However, its transient nature (resulting from its high aggregation tendency), its plasticity, and its equilibrium with both the monomer and higher-order oligomers all make the Aβ dimer extremely challenging to study experimentally. In fact, a large amount of the experimental studies performed on Aβ dimers employ some kind of cross-linking to stabilize them (7–9). On the other hand, covalently cross-linked Aβ dimers are certainly of biological relevance, as such species have been retrieved from the brains of AD patients and their neurotoxicity has been demonstrated (6, 10). Apart from this, recent technological developments, such as advanced single-molecule fluorescence spectroscopy and imaging, opened the way to characterize amyloid oligomers without the need to stabilize them by cross-linking (11, 12). Molecular dynamics (MD) simulations are also able to provide atomic insight into the temporal evolution of the dimer structure without the need of cross-linking (13, 14). Previous simulations of Aβ dimers were modeled in the aqueous phase only, and thus they lacked essential details from the cellular context. Consideration of the latter is particularly important if one wishes to reveal the mechanism of toxicity that has been shown to rely on direct contact with the lipid membrane of neurons by Aβ oligomers (15, 16).Many studies have been done to understand the consequences of Aβ–membrane interactions; however, it is extremely difficult to capture these transient interactions with experimental methods. This becomes possible with MD simulations and this problem is addressed in the current work. We use an aggregate of 24 μs of MD simulations to investigate the dimerization of the full-length Aβ42 peptide both in solution and in the presence of a model lipid bilayer including six lipid types to mimic the composition of a neuronal cell membrane (17–19): 38% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 24% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 5% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 20% cholesterol (CHOL), 9% sphingomyelin (SM), and 4% monosialotetrahexosylganglioside (GM1) (Fig. 1A). For modeling Aβ we employ Charmm36m, a force field adjusted for intrinsically disordered proteins (IDPs), to model their preference to adopt extended structures. When applied to monomeric Aβ, Charmm36m yields more than 80% of the structures in a random coil and extended state, and the remaining ones feature transient β-hairpins, which is in acceptable agreement with experimental data (20). Moreover, Charmm36m outperforms other force fields when it comes to modeling peptide aggregation (21, 22). To the best of our knowledge, this simulation study breaks ground on two fronts: 1) It exceeds the simulation time of previous studies modeling Aβ–membrane interactions by an order of magnitude, and 2) it studies the aggregation of Aβ on a bilayer containing more than three different lipid types. Lipid bilayers of a complexity comparable to the one modeled here have been thus far studied only at the coarse-grained level (23, 24). We also analyze the aggregation pathways by transition networks (25–27), which elucidate the similarities and differences between Aβ dimerization steps both in solution and at the neuronal membrane. We find that the neuronal membrane reduces the dynamics of membrane-bound Aβ42 while it also inhibits β-sheet formation. Here, the sugar groups of GM1 form hydrogen bonds with the peptide, thereby reducing the possibilities for other hydrogen bonds to otherwise form. In contrast, the dimerization in the aqueous phase is characterized by a random coil to β-sheet transition, leading to β-sheet structures similar to the ones found in Aβ fibrils.Open in a separate windowFig. 1.(A) A snapshot of the neuronal membrane containing 38% POPC, 24% POPE, 5% POPS (collectively shown as gray surface with their phosphorous atoms indicated by gray spheres), 20% CHOL (red sticks), 9% SM (green spheres), and 4% GM1 (yellow spheres). In the following, PC, PE, and PS are synonymously used for POPC, POPE, and POPS, respectively. (B and C) Radial distribution functions for (B) lipid pairings of identical type and (C) lipid–CHOL pairings. The P atoms of PC, PE, PS, and SM and the O atoms of CHOL and GM1 were used as reference atoms for the RDF calculations. The RDFs are averaged over both membrane leaflets. The x axis shows the distances between the respective atom pairs. Since CHOL resides deeper inside the membrane, it is possible that the O atom of CHOL and the reference atoms of the other lipids are above each other, explaining why not all of the RDFs approach zero for x=0. The colors of the functions refer to the lipids as indicated in the color key in B. Pairs with RDF >1 are considered to form clusters.     

The Parkinson’s disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release

Inositol-1,4,5-triphosphate (IP₃) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP₃, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson’s disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a variety of motor symptoms (including unbalanced gait, resting tremor, and bradykinesia) that are accompanied by psychosis and dementia at later stages of disease. The onset of motor symptoms is largely caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the corresponding depletion of dopamine innervation in the striatum. Although the cause of neuron loss is unknown, the hallmark pathological feature of PD is the presence of intraneuronal inclusions composed of misfolded and fibrillar α-synuclein (α-syn) in the neurites and soma, termed Lewy neurites and Lewy bodies, respectively (1). Protein-coding single-nucleotide polymorphisms (SNPs), duplications, and triplications in the gene encoding α-syn (SNCA) all cause early-onset, familial forms of PD and result in accelerated aggregation of α-syn protein into insoluble, fibrillar aggregates (1, 2). Recent evidence suggests that these aggregates can spread from cell to cell, leading to the propagation of pathology to neuroanatomically connected brain regions (3, 4). Therefore, therapeutic approaches that reduce the aggregation or spreading of pathological α-syn species represent potential disease-modifying therapies for PD.In addition to SNCA, mutations in several other genes have been identified that cause rare, familial forms of PD. These genes are primarily involved in the autophagic clearance of intracellular aggregates or damaged organelles, especially mitochondria (5–8). Genome-wide association studies (GWAS) have also identified common SNPs in several genes related to endolysosomal function, such as GBA and LRRK2, that are associated with increased risk of PD (8–11). GBA and LRRK2 mutations have been shown to affect lysosomal and mitochondrial phenotypes (12–14), which contribute to the accumulation of PD-like neuropathology in mouse models, primary neurons, and human iPSC-derived cells (15–18). These findings highlight the role of the lysosomal and mitochondria quality control pathways in PD and demonstrate that perturbations in these pathways are sufficient to increase α-syn aggregation. Despite this, the etiology of sporadic PD is not fully understood, and the specific genes and pathways that are tractable for therapeutic modulation remain elusive. Therefore, the discovery of new genes associated with sporadic PD may be critical for both understanding disease pathogenesis and identifying novel therapeutic approaches.Recently, Chang et al. conducted a GWAS analysis that identified 17 novel gene loci significantly associated with sporadic PD in a European population including more than 26,000 patients across three independent cohorts (19). The lead GWAS SNP in the novel 1q42 locus was rs4653767. This is an intronic SNP in the gene encoding inositol-1,4,5-triphosphate kinase B (ITPKB) and produces a thymine-to-cytosine nucleotide substitution that is protective against developing PD (odds ratio [OR] = 0.92, P = 2.4 × 10⁻¹⁰). A follow up meta-analysis study of 37,688 PD patients, which included the discovery cohort, and 18,618 proxy cases strengthened the GWAS finding at this locus (OR = 0.92, P = 1.4 × 10⁻¹⁵). Furthermore, this locus was still significant when the analysis was performed on only the new independent cases and proxy cases (OR = 0.92, P = 2.8 × 10⁻⁵) (20). The rs4653767-C allele is present in similar frequencies across populations (27% in non-Finnish European and 29% in East Asian populations) and was found to have the same direction of effect (OR = 0.87, P = 0.016) in a targeted replication study of the European PD loci in an East Asian cohort (21). ITPKB is also highly expressed in several brain regions related to PD, including the SNpc, striatum, and cerebral cortex (22).ITPKB is one of three ubiquitously expressed kinases known to phosphorylate inositol-1,4,5-triphosphate (IP₃), an intracellular messenger produced from phosphatidylinositol-4,5-bisphosphate by phospholipase C (23, 24). IP₃ binds to IP₃ receptors (IP₃Rs) in the endoplasmic reticulum (ER) to stimulate the release of calcium ions from the ER into the cytosol to mediate various downstream signaling pathways. IP₃ kinases (ITPKA, ITPKB, and ITPKC) add a fourth phosphate group to IP₃ producing inositol-1,3,4,5-tetrakisphosphate (IP₄), which has no activity on IP₃Rs. Thus, IP₃ kinases negatively regulate IP₃-mediated calcium release from the ER. While the role of this pathway in peripheral cell types under normal physiological conditions is well understood (25, 26), whether ITPKB is involved in the pathogenesis of PD is unknown. Here, we investigate whether the modulation of ITPKB expression or kinase activity impacts the accumulation of α-syn pathology in cellular models of PD.     

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.     

Biomaterials for Drugs Nose–Brain Transport: A New Therapeutic Approach for Neurological Diseases

In the last years, neurological diseases have resulted in a global health issue, representing the first cause of disability worldwide. Current therapeutic approaches against neurological disorders include oral, topical, or intravenous administration of drugs and more invasive techniques such as surgery and brain implants. Unfortunately, at present, there are no fully effective treatments against neurodegenerative diseases, because they are not associated with a regeneration of the neural tissue but rather act on slowing the neurodegenerative process. The main limitation of central nervous system therapeutics is related to their delivery to the nervous system in therapeutic quantities due to the presence of the blood–brain barrier. In this regard, recently, the intranasal route has emerged as a promising administration site for central nervous system therapeutics since it provides a direct connection to the central nervous system, avoiding the passage through the blood–brain barrier, consequently increasing drug cerebral bioavailability. This review provides an overview of the nose-to-brain route: first, we summarize the anatomy of this route, focusing on the neural mechanisms responsible for the delivery of central nervous system therapeutics to the brain, and then we discuss the recent advances made on the design of intranasal drug delivery systems of central nervous system therapeutics to the brain, focusing in particular on stimuli-responsive hydrogels.     

Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2–encoded Nsp1

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2–encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

Proteostasis refers to a cellular state in which protein synthesis, folding, and degradation are maintained at a homeostatic state, such that an intact proteome is preserved (1). Cellular capacity to preserve proteostasis declines with age, which is assumed to contribute to the pathogenesis of age-related diseases (2). Proteostasis failure manifested as formation of aberrant protein aggregates is a defining feature of neurodegenerative diseases (3). The root cause of the proteostasis failure and protein aggregation is still enigmatic. Problems of proteostasis can begin with nascent peptide chains (NPCs) still associated with translating ribosomes, necessitating the deployment of ribosome-associated quality control (RQC) to handle faulty translation (4). During translation elongation, ribosome slowdown and stalling can occur for various reasons. Some are functional and serve to facilitate cellular dynamics. Others are detrimental and can be triggered by damaged mRNAs, mRNA secondary structures, insufficient supply of aminoacyl-tRNAs, or environmental stress (5, 6). Ribosome slowdown and stalling can result in ribosome collision (7), which is sensed by cells as a proxy for aberrant translation and triggers RQC (4, 8–11). Key factors involved in the process are the ubiquitin ligase ZNF598 and the 40S subunit protein Rack1, which recognize the distinct 40S–40S interface of collided ribosomes and promote ubiquitination of specific 40S proteins (12, 13), and the ASC complex that disassembles the leading collided ribosome (14, 15). This then triggers a series of downstream events, including ribosome subunit splitting and recycling by ABCE1 (16), CAT-tailing modification of NPCs still attached to the 60S subunit (17), and degradation of stalled NPCs. The importance of this ribosome-mediated QC process is highlighted by the findings that RQC factors regulating translation elongation and termination are critical for neuronal function and integrity (18–20), and that inefficient RQC results in translation stalling and accumulation of faulty translation products that perturb proteostasis and contribute to Alzheimer’s disease (AD) (21), Parkinson’s disease (PD) (22), and amyotrophic lateral sclerosis (ALS) (23).Translational control is also crucial in the battle between viruses and their hosts during infection. Due to the lack of their own translational apparatus, viruses rely on the protein synthesis machinery of the host cells to complete their life cycle (24). Viral replication requires sustained high levels of active ribosomes to carry out rapid translation of their mRNAs. To achieve this, viruses must manage to neutralize host innate immune defenses and stress responses, which often target the ribosomes and the translation machinery (25). Gaining control of the ribosomes is therefore crucial in the arms race between viruses and their host cells. Of the three stages of translation—initiation, elongation, and termination—much effort has been directed toward control of initiation, the rate-limiting step of translation (24, 25). However, rapid translation of viral RNAs during replication, and certain features of the viral RNA, such as complex secondary structures throughout the genome, may engender translational stress beyond the initiation step that necessitates the deployment of additional regulatory mechanisms.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic that has led to a dramatic loss of human life and disruption of social and economic activities worldwide (26). COVID-19 is characterized by a wide spectrum of symptoms, from fever and cough to multiorgan failures (27). The coronavirus family includes SARS-CoV (28), SARS-CoV-2 (26), and Middle-East respiratory syndrome coronavirus (MERS-CoV) (29). SARS-CoV-2 encodes the spike (S), envelope (E), membrane (M), nucleocapsid (N), nonstructural (NSP1-16), and accessory (ORF3a, -3b, -6, -7a, -7b, -8, -9b, -9c, -10, and -14) proteins (30). NSP1 is one of the first SARS-CoV proteins synthesized upon cell entry and deemed a major virulence factor (31). Structural, biochemical, and cell culture studies have implicated NSP1 in inhibiting the translation initiation of host genes through blocking the mRNA entry channel of the 40S ribosome (32–35), and by promoting mRNA degradation (32, 36, 37). NSP1 has also been implicated in viral evasion of host innate immune response (38–40), although the underlying mechanism is unclear. Till now functional studies on NSP1 are performed in vitro or in cell culture. The in vivo effects of Nsp1 remain untested. Recent proteomics studies of COVID-19 autopsies revealed diverse up- and down-regulation of the proteome with limited overlap across tissues (41), emphasizing the need to study tissue-specific effects of viral proteins including Nsp1.Although rodent and primate models have been developed, their general use for studying the function of individual SARS-CoV-2 viral proteins has been limited by low availability and requirement for special biocontainment facilities. Drosophila offers an alternative model system. In addition to its small size, short life cycle, low maintenance cost, and powerful genetics, Drosophila shares conserved innate immune responses and other processes commonly hijacked by pathogens (42). Here we describe our systematic investigation into the in vivo effects of SARS-CoV-2–encoded viral proteins in Drosophila neuromuscular tissues under normal and disease conditions. This led to an unexpected finding of amelioration of neuromuscular degeneration by Nsp1 in multiple neurodegenerative disease models and new insights into the biochemical function of Nsp1 in manipulating the host translation machinery.