Phosphatidylinositol-3,4,5-trisphosphate interacts with alpha-synuclein and initiates its aggregation and formation of Parkinson's disease-related fibril polymorphism.

Lipid interaction with α-synuclein (αSyn) has been long implicated in the pathogenesis of Parkinson's disease (PD). However, it has not been fully determined which lipids are involved in the initiation of αSyn aggregation in PD. Here exploiting genetic understanding associating the loss-of-function mutation in Synaptojanin 1 (SYNJ1), a phosphoinositide phosphatase, with familial PD and analysis of postmortem PD brains, we identified a novel lipid molecule involved in the toxic conversion of αSyn and its relation to PD. We first established a SYNJ1 knockout cell model and found SYNJ1 depletion increases the accumulation of pathological αSyn. Lipidomic analysis revealed SYNJ1 depletion elevates the level of its substrate phosphatidylinositol-3,4,5-trisphosphate (PIP3). We then employed Caenorhabditis elegans model to examine the effect of SYNJ1 defect on the neurotoxicity of αSyn. Mutations in SYNJ1 accelerated the accumulation of αSyn aggregation and induced locomotory defects in the nematodes. These results indicate that functional loss of SYNJ1 promotes the pathological aggregation of αSyn via the dysregulation of its substrate PIP3, leading to the aggravation of αSyn-mediated neurodegeneration. Treatment of cultured cell line and primary neurons with PIP3 itself or with PIP3 phosphatase inhibitor resulted in intracellular formation of αSyn inclusions. Indeed, in vitro protein-lipid overlay assay validated that phosphoinositides, especially PIP3, strongly interact with αSyn. Furthermore, the aggregation assay revealed that PIP3 not only accelerates the fibrillation of αSyn, but also induces the formation of fibrils sharing conformational and biochemical characteristics similar to the fibrils amplified from the brains of PD patients. Notably, the immunohistochemical and lipidomic analyses on postmortem brain of patients with sporadic PD showed increased PIP3 level and its colocalization with αSyn. Taken together, PIP3 dysregulation promotes the pathological aggregation of αSyn and increases the risk of developing PD, and PIP3 represents a potent target for intervention in PD.

An autopsy case of progressive supranuclear palsy treated with monoclonal antibody against tau.

We report an autopsy case of progressive supranuclear palsy (PSP-Richardson syndrome). The individual had been enrolled in a phase 2 trial and received a monoclonal tau antibody (tilavonemab, ABBV-8E12); he died of intrahepatic cholangiocarcinoma and gastrointestinal bleeding during the clinical trial. Neuropathological examination demonstrated neuronal loss, gliosis, and widespread deposits of phosphorylated tau in the neurofibrillary tangles, tufted astrocytes, coiled bodies, and threads, which mainly occurred in the inferior olive nucleus, dentate nucleus of the cerebellum, substantia nigra, midbrain tegmentum, subthalamic nuclei, globus pallidus, putamen, and precentral gyrus, confirming typical PSP pathology. Phosphorylated tau was also found to accumulate in Betz cells, Purkinje cells, and pencil fibers in the basal ganglia. In conclusion, no additional changes or pathological modifications, which were expected from immunotherapy targeting tau, were visible in the present case.

Extracellular transportation of α-synuclein by HLA class II molecules.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive accumulation of α-synuclein aggregates in form of Lewy bodies. Genome-wide association studies have revealed that human leukocyte antigen (HLA) class II is a PD-associated gene, although the mechanisms linking HLA class II and PD remain elusive. Here, we identified a novel function of HLA class II in the transport of intracellular α-synuclein to the outside of cells. HLA class II molecules and α-synuclein formed complexes and moved to the cell surface at various degrees among HLA-DR alleles. HLA-DR with a DRB5∗01:01 allele, a putative PD-risk allele, substantially translocated normal and conformationally abnormal α-synuclein to the cell surface and extracellular vesicles. α-Synuclein/HLA class II complexes were found in A2058 melanoma cells, which express intrinsic α-synuclein and HLA-DR with DRB5∗01:01. Our findings will expand our knowledge of unconventional HLA class II function from autoimmune diseases to neurodegenerative disorders, shedding light on the association between the GWAS-prioritized PD-risk gene HLA-DR and α-synuclein.

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis.

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of ß2-microglobulin (ß2m). Although high serum ß2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on ß2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

Development of HANABI, an ultrasonication-forced amyloid fibril inducer.

Amyloid fibrils involved in amyloidoses are crystal-like aggregates, which are formed by breaking supersaturation of denatured proteins. Ultrasonication is an efficient method of agitation for breaking supersaturation and thus inducing amyloid fibrils. By combining an ultrasonicator and a microplate reader, we developed the HANABI (HANdai Amyloid Burst Inducer) system that enables high-throughput analysis of amyloid fibril formation. Among high-throughput approaches of amyloid fibril assays, the HANABI system has advantages in accelerating and detecting spontaneous amyloid fibril formation. HANABI is also powerful for amplifying a tiny amount of preformed amyloid fibrils by seeding. Thus, HANABI will contribute to creating therapeutic strategies against amyloidoses by identifying their biomarkers.

Strong acids induce amyloid fibril formation of ß2-microglobulin via an anion-binding mechanism.

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of ß2-microglobulin (ß2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of ß2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.

Half-Time Heat Map Reveals Ultrasonic Effects on Morphology and Kinetics of Amyloidogenic Aggregation Reaction.

Ultrasonication has been recently adopted in amyloid-fibril assays because of its ability to accelerate fibril formation, being promising in the early stage diagnosis of amyloidoses in clinical applications. Although applications of this technique are expanding in the field of protein science, its effects on the aggregation reactions of amyloidogenic proteins are poorly understood. In this study, we comprehensively investigated the morphology and structure of resultant aggregates, kinetics of fibril formation, and seed-detection sensitivity under ultrasonication using ß2-microglobulin and compared these characteristics under shaking, which has been traditionally adopted in amyloid-fibril assays. To discuss the ultrasonic effects on the amyloid-fibril formation, we propose the half-time heat map, which describes the phase diagram of the aggregation reaction of amyloidogenic proteins. The experimental results show that ultrasonication greatly promotes fibril formation, especially in dilute monomer solutions, induces short-dispersed fibrils, and is capable of detecting ultra-trace-concentration seeds with a detection limit of 10 fM. Furthermore, we indicate that ultrasonication highly alters the energy landscape of an aggregation reaction due to the effect of ultrasonic cavitation. These insights contribute not only to our understanding of the effects of agitation on amyloidogenic aggregation reactions but also to their effective application in the clinical diagnosis of amyloidoses.

Polyphenol-solubility alters amyloid fibril formation of α-synuclein.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol-7─O─glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α-synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.

Polyphosphates induce amyloid fibril formation of α-synuclein in concentration-dependent distinct manners.

Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-µM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.

Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation.

Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using ß2-microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation.

Breakdown of supersaturation barrier links protein folding to amyloid formation.

The thermodynamic hypothesis of protein folding, known as the "Anfinsen's dogma" states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen's dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer's and Parkinson's diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen's intramolecular folding universe and the intermolecular misfolding universe.

Go-sha-jinki-Gan Alleviates Inflammation in Neurological Disorders via p38-TNF Signaling in the Central Nervous System.

Go-sha-jinki-Gan (GJG) is a traditional Japanese herbal medicine. In clinical practice, GJG is effective against neuropathic pain and hypersensitivity induced by chemotherapy or diabetes. In our previous study using a chronic constriction injury mouse model, we showed that GJG inhibited microglia activation by suppressing the expression of tumor necrosis factor-α (TNF-α) and p38 mitogen-activated protein kinase (p38 MAPK) in the peripheral nervous system. To investigate whether GJG can suppress inflammation in the central nervous system (CNS) in the context of neurological disorders, we examined the effect of GJG on the activation of resident glial cells and on p38-TNF signaling in two mouse models of neurological disorders: the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. GJG administration relieved the severity of clinical EAE symptoms and MPTP-induced inflammation by decreasing the number of microglia and the production of TNF-α in the spinal cord of EAE mice and the substantia nigra of MPTP-treated mice. Accordingly, GJG suppressed the phosphorylation of p38 in glial cells of these two mouse models. We conclude that GJG attenuates inflammation of the CNS by suppressing glial cell activation, followed by a decrease in the production of TNF-α via p38-TNF signaling.

Characteristic Features of FUS Inclusions in Spinal Motor Neurons of Sporadic Amyotrophic Lateral Sclerosis.

Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.

Ultrasonication-based rapid amplification of α-synuclein aggregates in cerebrospinal fluid.

α-Synuclein aggregates, a key hallmark of the pathogenesis of Parkinson's disease, can be amplified by using their seeding activity, and the evaluation of the seeding activity of cerebrospinal fluid (CSF) is reportedly useful for diagnosis. However, conventional shaking-based assays are time-consuming procedures, and the clinical significance of the diversity of seeding activity among patients remains to be clarified. Previously, we reported a high-throughput ultrasonication-induced amyloid fibrillation assay. Here, we adapted this assay to amplify and detect α-synuclein aggregates from CSF, and investigated the correlation between seeding activity and clinical indicators. We confirmed that this assay could detect α-synuclein aggregates prepared in vitro and also aggregates released from cultured cells. The seeding activity of CSF correlated with the levels of α-synuclein oligomers measured by an enzyme-linked immunosorbent assay. Moreover, the seeding activity of CSF from patients with Parkinson's disease was higher than that of control patients. Notably, the lag time of patients with Parkinson's disease was significantly correlated with the MIBG heart-to-mediastinum ratio. These findings showed that our ultrasonication-based assay can rapidly amplify misfolded α-synuclein and can evaluate the seeding activity of CSF.

[Analysis of PEG-J associated complications in 14 adult patients treated with levodopa-carbidopa intestinal gel].

We analyzed 14 patients in our hospital, who underwent levodopa-carbidopa intestinal gel (LCIG) treatment through a percutaneous endoscopic gastrojejunostomy (PEG-J). The PEG-J related complications were observed in 10 patients (71.4%). Detailed complications are as followings: J-tube related complications such as kinking (3 cases, 21.4%), pump malfunctions (3 cases, 21.4%), skin troubles in the gastrostoma (7 cases, 50.0%), duodenal perforation, peritonitis, and ulcers (2 cases, 14.3%). These results indicated that the sufficient care for PEG-J associated complications are important in LCIG treatment.

Altered Tau Isoform Ratio Caused by Loss of FUS and SFPQ Function Leads to FTLD-like Phenotypes.

Fused in sarcoma (FUS) and splicing factor, proline- and glutamine-rich (SFPQ) are RNA binding proteins that regulate RNA metabolism. We found that alternative splicing of the Mapt gene at exon 10, which generates 4-repeat tau (4R-T) and 3-repeat tau (3R-T), is regulated by interactions between FUS and SFPQ in the nuclei of neurons. Hippocampus-specific FUS- or SFPQ-knockdown mice exhibit frontotemporal lobar degeneration (FTLD)-like behaviors, reduced adult neurogenesis, accumulation of phosphorylated tau, and hippocampal atrophy with neuronal loss through an increased 4R-T/3R-T ratio. Normalization of this increased ratio by 4R-T-specific silencing results in recovery of the normal phenotype. These findings suggest a biological link among FUS/SFPQ, tau isoform alteration, and phenotypic expression, which may function in the early pathomechanism of FTLD.

Pioglitazone suppresses neuronal and muscular degeneration caused by polyglutamine-expanded androgen receptors.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. Mutant AR has been postulated to alter the expression of genes important for mitochondrial function and induce mitochondrial dysfunction. Here, we show that the expression levels of peroxisome proliferator-activated receptor-γ (PPARγ), a key regulator of mitochondrial biogenesis, were decreased in mouse and cellular models of SBMA. Treatment with pioglitazone (PG), an activator of PPARγ, improved the viability of the cellular model of SBMA. The oral administration of PG also improved the behavioral and histopathological phenotypes of the transgenic mice. Furthermore, immunohistochemical and biochemical analyses demonstrated that the administration of PG suppressed oxidative stress, nuclear factor-κB (NFκB) signal activation and inflammation both in the spinal cords and skeletal muscles of the SBMA mice. These findings suggest that PG is a promising candidate for the treatment of SBMA.

Amyotrophic lateral sclerosis: an update on recent genetic insights.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting both upper and lower motor neurons. The prognosis for ALS is extremely poor, but there is a limited course of treatment with only one approved medication. A most striking recent discovery is that TDP-43 is identified as a key molecule that is associated with both sporadic and familial forms of ALS. TDP-43 is not only a pathological hallmark, but also a genetic cause for ALS. Subsequently, a number of ALS-causative genes have been found. Above all, the RNA-binding protein, such as FUS, TAF15, EWSR1 and hnRNPA1, have structural and functional similarities to TDP-43, and physiological functions of some molecules, including VCP, UBQLN2, OPTN, FIG4 and SQSTM1, are involved in a protein degradation system. These discoveries provide valuable insight into the pathogenesis of ALS, and open doors for developing an effective disease-modifying therapy.

Disruption of axonal transport in motor neuron diseases.

Motor neurons typically have very long axons, and fine-tuning axonal transport is crucial for their survival. The obstruction of axonal transport is gaining attention as a cause of neuronal dysfunction in a variety of neurodegenerative motor neuron diseases. Depletions in dynein and dynactin-1, motor molecules regulating axonal trafficking, disrupt axonal transport in flies, and mutations in their genes cause motor neuron degeneration in humans and rodents. Axonal transport defects are among the early molecular events leading to neurodegeneration in mouse models of amyotrophic lateral sclerosis (ALS). Gene expression profiles indicate that dynactin-1 mRNA is downregulated in degenerating spinal motor neurons of autopsied patients with sporadic ALS. Dynactin-1 mRNA is also reduced in the affected neurons of a mouse model of spinal and bulbar muscular atrophy, a motor neuron disease caused by triplet CAG repeat expansion in the gene encoding the androgen receptor. Pathogenic androgen receptor proteins also inhibit kinesin-1 microtubule-binding activity and disrupt anterograde axonal transport by activating c-Jun N-terminal kinase. Disruption of axonal transport also underlies the pathogenesis of spinal muscular atrophy and hereditary spastic paraplegias. These observations suggest that the impairment of axonal transport is a key event in the pathological processes of motor neuron degeneration and an important target of therapy development for motor neuron diseases.