Phosphoglycerate kinase is a central leverage point in Parkinson's disease-driven neuronal metabolic deficits.

Although certain drivers of familial Parkinson's disease (PD) compromise mitochondrial integrity, whether metabolic deficits underly other idiopathic or genetic origins of PD is unclear. Here, we demonstrate that phosphoglycerate kinase 1 (PGK1), a gene in the PARK12 susceptibility locus, is rate limiting in neuronal glycolysis and that modestly increasing PGK1 expression boosts neuronal adenosine 5'-triphosphate production kinetics that is sufficient to suppress PARK20-driven synaptic dysfunction. We found that this activity enhancement depends on the molecular chaperone PARK7/DJ-1, whose loss of function significantly disrupts axonal bioenergetics. In vivo, viral expression of PGK1 confers protection of striatal dopamine axons against metabolic lesions. These data support the notion that bioenergetic deficits may underpin PD-associated pathologies and point to improving neuronal adenosine 5'-triphosphate production kinetics as a promising path forward in PD therapeutics.

Amyloid fibril structures link CHCHD10 and CHCHD2 to neurodegeneration.

CHCHD10 is mutated in rare cases of FTD and ALS and aggregates in mouse models of disease. Here we show that the disordered N-terminal domain of CHCHD10 forms amyloid fibrils and report their cryoEM structure. Disease-associated mutations cannot be accommodated by the WT fibril structure, while sequence differences between CHCHD10 and CHCHD2 are tolerated, explaining the co-aggregation of the two proteins and linking CHCHD10 and CHCHD2 amyloid fibrils to neurodegeneration.

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Carbon dots as dual inhibitors of tau and amyloid-beta aggregation for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of senile dementia, presenting a significant challenge for the development of effective treatments. AD is characterized by extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Therefore, targeting both hallmarks through inhibition of amyloid beta (Aß) and tau aggregation presents a promising approach for drug development. Carbon dots (CD), with their high biocompatibility, minimal cytotoxicity, and blood-brain barrier (BBB) permeability, have emerged as promising drug nanocarriers. Congo red, an azo dye, has gathered significant attention for inhibiting amyloid-beta and tau aggregation. However, Congo red's inability to cross the BBB limits its potential to be used as a drug candidate for central nervous system (CNS) diseases. Furthermore, current studies only focus on using Congo red to target single disease hallmarks, without investigating dual inhibition capabilities. In this study, we synthesized Congo red-derived CD (CRCD) by using Congo red and citric acid as precursors, resulting in three variants, CRCD1, CRCD2 and CRCD3, based on different mass ratios of precursors. CRCD2 and CRCD3 exhibited sustained low cytotoxicity, and CRCD3 demonstrated the ability to traverse the BBB in a zebrafish model. Moreover, thioflavin T (ThT) aggregation assays and AFM imaging revealed CRCD as potent inhibitors against both tau and Aß aggregation. Notably, CRCD1 emerged as the most robust inhibitor, displaying IC50 values of 0.2 ± 0.1 and 2.1 ± 0.5 µg/mL against tau and Aß aggregation, respectively. Our findings underscore the dual inhibitory role of CRCD against tau and Aß aggregation, showcasing effective BBB penetration and positioning CRCD as potential nanodrugs and nanocarriers for the CNS. Hence, CRCD-based compounds represent a promising candidate in the realm of multi-functional AD therapeutics, offering an innovative formulation component for future developments in this area. STATEMENT OF SIGNIFICANCE: This article reports Congo red-derived carbon dots (CRCD) as dual inhibitors of tau and amyloid-beta (Aß) aggregation for the treatment of Alzheimer's disease (AD). The CRCD are biocompatible and show strong fluorescence, high stability, the ability to cross the blood-brain barrier, and the function of addressing two major pathological features of AD.

Phosphoglycerate kinase is a central leverage point in Parkinson's Disease driven neuronal metabolic deficits.

Phosphoglycerate kinase 1 (PGK1), the first ATP producing glycolytic enzyme, has emerged as a therapeutic target for Parkinson's Disease (PD), since a potential enhancer of its activity was reported to significantly lower PD risk. We carried out a suppressor screen of hypometabolic synaptic deficits and demonstrated that PGK1 is a rate limiting enzyme in nerve terminal ATP production. Increasing PGK1 expression in mid-brain dopamine neurons protected against hydroxy-dopamine driven striatal dopamine nerve terminal dysfunction in-vivo and modest changes in PGK1 activity dramatically suppressed hypometabolic synapse dysfunction in vitro. Furthermore, PGK1 is cross-regulated by PARK7 (DJ-1), a PD associated molecular chaperone, and synaptic deficits driven by PARK20 (Synaptojanin-1) can be reversed by increasing local synaptic PGK1 activity. These data indicate that nerve terminal bioenergetic deficits may underly a spectrum of PD susceptibilities and the identification of PGK1 as the limiting enzyme in axonal glycolysis provides a mechanistic underpinning for therapeutic protection.

Alpha synuclein modulates mitochondrial Ca2+ uptake from ER during cell stimulation and under stress conditions.

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson's disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress1. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction controls receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we identify key conserved residues and cancer-associated mutations that control CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells which can be modulated by disease-associated mutations or CTD phosphorylation. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Structure and DNA-bridging activity of the essential Rec114-Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Alpha Synuclein Modulates Mitochondrial Ca 2+ Uptake from ER During Cell Stimulation and Under Stress Conditions.

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson' disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress. 1 We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca 2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca 2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca 2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by 19F NMR and Cryo-EM.

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.

Structure and DNA bridging activity of the essential Rec114â€"Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo , these proteins form large immunostaining foci that are integrated with higher order chromosome structures. In vitro , they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C-terminus of Rec114 with the N-terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin-1C-Terminal Domain.

Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.1.

The Role of Membrane Affinity and Binding Modes in Alpha-Synuclein Regulation of Vesicle Release and Trafficking.

Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.

Proline-Rich Region II (PRR2) Plays an Important Role in Tau-Glycan Interaction: An NMR Study.

(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.

Molecular and functional interactions of alpha-synuclein with Rab3a.

Alpha-synuclein (a-Syn) is a presynaptic protein, the misfolding of which is associated with Parkinson's disease. Rab GTPases are small guanine nucleotide binding proteins that play key roles in vesicle trafficking and have been associated with a-Syn function and dysfunction. a-Syn is enriched on synaptic vesicles, where it has been reported to interact with GTP-bound Rab3a, a master regulator of synaptic vesicle trafficking. a-Syn is known to bind weakly to Rab8a in solution via a positively charged patch, but the physiological implications of such interactions have not been explored. Here, we investigate direct interactions between a-Syn and Rab3a in solution and on lipid membranes using NMR spectroscopy. We find that the C terminus of a-Syn interacts with Rab3a in a manner similar to its previously reported interaction with Rab8a. While weak in solution, we demonstrate that this interaction becomes stronger when the proteins are bound to a membrane surface. The Rab3a binding site for a-Syn is similar to the surface that contacts the Rab3a effector rabphilin-3A, which modulates the enzymatic activity of Rab3a. Accordingly, we show that a-Syn inhibits GTP hydrolysis by Rab3a and that inhibition is more potent on the membrane surface, suggesting that their interaction may be functionally relevant. Finally, we show that phosphorylation of a-Syn residue Ser 129, a modification associated with Parkinson's disease pathology, enhances its interactions with Rab3a and increases its ability to inhibit Rab3a GTP hydrolysis. These results represent the first observation of a functional role for synuclein-Rab interactions and for a-Syn Ser 129 phosphorylation.

Synaptic vesicle binding of α-synuclein is modulated by ß- and γ-synucleins.

α-synuclein, ß-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson's disease and related synucleinopathies. The functions of ß-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that ß-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of ß-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that ß-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein's physiological activity at the neuronal synapse.

Homogalacturonan from squash: Characterization and tau-binding pattern of a sulfated derivative.

A pectic polysaccharide (WAP) was isolated from squash and identified as a homogalacturonan with a molecular mass of 83.2 kDa by GPC, monosaccharide composition analysis, FT-IR and NMR spectra. Sulfation modification of WAP was carried out and a sulfated derivative (SWAP) was obtained with a substitution degree of 1.81. The NMR spectrum indicated that the sulfation modification mainly occurred at the C-2 and C-3 positions of galacturonan residues. The binding pattern of SWAP to tau K18 protein was observed in 2D 1H15N HSQC spectra of tau, which resembled the tau-heparin interaction, with R2 domain as the major binding region. These results suggest that SWAP has the potential to act as a heparin mimic to inhibit the transcellular spread of tau; thus natural polysaccharide from squash may be developed into therapies for AD and related tauopathies.

Altered succinylation of mitochondrial proteins, APP and tau in Alzheimer's disease.

Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer's disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aß accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.

Post-translational modifications within tau paired helical filament nucleating motifs perturb microtubule interactions and oligomer formation.

Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer's disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau-tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6∗ (paired helical filament [PHF] residues 275-280) and PHF6 (residues 306-311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer's disease-specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.