ACS Chemical Neuroscience

Dopamine-modified hyaluronic acid (DA-HA) has been initially developed as an efficient coating and adhesion material for industrial uses. However, the biological activity and safety of DA-HA in the brain have not been explored yet. Here, we report a series of evidence that DA-HA exhibits similar functionality as dopamine (DA), but with much lower toxicity arising from autoxidation. DA-HA shows very little autoxidation even after 48-hour incubation. This is profoundly different from DA and its derivatives including L-DOPA, which all induce severe neuronal death after pre-autoxidation, indicating that autoxidation is the cause of neuronal death. Furthermore, in vivo injection of DA-HA induces significantly lower toxicity compared to 6-OHDA, a well-known oxidized and toxic form of DA, and alleviates the apomorphine-induced rotational behavior in the 6-OHDA animal model of Parkinsons disease. Our study proposes that DA-HA with DA-like functionalities and minimal toxicity can be an effective therapeutic substitute for L-DOPA in Parkinsons disease.

SignificanceThe majority of drugs that target membrane-embedded protease {gamma}-secretase show unusual biphasic activation-inhibition dose-response in cells, model animals, and humans. Semagacestat and avagacestat are two biphasic-drugs that can facilitate cognitive decline in patients with Alzheimers disease. Initial mechanistic studies showed that the biphasic-drugs, and pathogenic mutations, can produce the same type of changes in {gamma}-secretase activity. ResultsDAPT, semagacestat LY-411,575, and avagacestat are four drugs that show different binding constants, and biphasic activation-inhibition dose-response curves, for amyloid-{beta}-40 products in SHSY-5 cells. Multiscale molecular dynamics studies showed that all four drugs bind to the most mobile parts in presenilin structure, at different ends of the 29 [A] long active site tunnel. Combined results from structure-activity studies, showed that the biphasic dose-response curves are a result of modulation of {gamma}-secretase activity by concurrent binding of multiple drug molecules at each end of the active site tunnel. The drugs activate {gamma}-secretase by forcing the active site tunnel to open, when the rate-limiting step is the tunnel opening, and formation of the enzyme-substrate complex. The drugs inhibit {gamma}-secretase as uncompetitive inhibitors, by binding next to the substrate to dynamic enzyme structures that regulate processive catalysis. The drugs can modulate the production of different amyloid-{beta} catalytic intermediates, by penetrating into the active site tunnel to different depth with different binding affinity. The drugs and pathogenic mutations affect the same dynamic processes in {gamma}-secretase structure. ConclusionsBiphasic-drugs like disease-causing mutations can reduce the catalytic capacity of {gamma}-secretase and facilitate pathogenic changes in amyloid metabolism.

Dopamine serves an important role in supporting both locomotor control and higher brain functions such as motivation and learning. Dopaminergic dysfunction is implicated in an equally multidimensional spectrum of neurological and neuropsychiatric diseases. Extracellular dopamine levels are known to be tightly controlled by presynaptic dopamine transporters (DAT), which is also a main target of psychostimulants. Still, detailed data on dopamine dynamics in space and time is needed to fully understand how dopamine signals are encoded and translated into cellular and behavioral responses, and to uncover the pathological effects of dopamine-related diseases. The recently developed genetically encoded fluorescent dopamine sensors enable unprecedented monitoring of dopamine dynamics and have changed the field of in vivo dopamine recording. However, the potential of these sensors to be used for in vitro and ex vivo assays remains unexplored. Here, we demonstrate a generalizable blueprint for making "sniffer" dopamine cells for multimodal detection of dopamine in vitro and ex vivo. We generated sniffer cell lines with inducible expression of six different dopamine sensors and performed a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we show how the sniffer cells can be applied to measure release of endogenous dopamine from cultured neurons and striatal slices, and for determining total dopamine content in striatal tissue. Furthermore, we use the sniffer cells to quantify DAT-mediated dopamine uptake, and AMPH-induced and constitutive dopamine efflux as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cells framework can readily be applied to other transmitter systems for which the list of genetically encoded fluorescent sensors is rapidly growing.

Presenilin homologues in the {gamma}-secretase complex play a pivotal role in substrate binding and processing, impacting {beta}-amyloid (A{beta}) peptide generation in Alzheimers disease. We conducted a molecular simulation study to determine substrate preferences between presenilin-1 (PS1) and presenilin-2 (PS2) {gamma}-secretase enzymes for amyloid precursor protein (APP) and Notch1 processing. Using homology modelling, we generated PS1- and PS2-{gamma}-secretase models bound to substrates in the A{beta}40 and A{beta}42 generation pathways and Notch1 S3 and S4 cleavage site substrates. Metadynamics simulations and binding free energy calculations were used to explore conformational ensembles and substrate preferences. PS2-{gamma}-secretase exhibited increased conformational flexibility and preferential binding energy for initiating the A{beta}42 pathway compared to PS1-{gamma}-secretase. Additionally, Notch1 exhibits a preference for binding to PS2-{gamma}-secretase over PS1-{gamma}-secretase. This study provides valuable insights into the conformational dynamics of {gamma}-secretase bound to different substrates within a cleavage pathway, improving our understanding of substrate processivity. The findings highlight the importance of considering both PS1- and PS2-{gamma}-secretase in structure-based drug design efforts, with implications for stabilizing or destabilizing specific states during APP processing.

Alzheimers disease (AD) remains the most prevalent neurodegenerative disease characterized by intracellular neurofibrillary tangles of Tau protein and extracellular senile plaques build on Amyloid-{beta} (A{beta}) peptides. The latter result from an abnormal processing of Amyloid Precursor Protein (APP) leading to its accumulation in plaques. Ex vivo analyses of AD patients brains show an abnormally elevated concentration of metals including Cu, Zn and Fe within these plaques. Altered Cu levels have also been reported in brain regions most affected in AD. These modifications are often accompanied by reduced neuronal Cu levels and by an increased pool of extracellular labile Cu, which in turn promotes reactive oxygen species (ROS) formation. To counteract this Cu dyshomeostasis and limit Cu-A{beta}-induced extracellular ROS generation, we designed and synthesized two Cu(II)-selective peptide shuttles based on kinetically optimized ATCUN sequences for fast Cu(II) extraction out of A{beta}: DapHH-R5W4NBD and HDapH-R5W4NBD. They were also equipped with a fluorophore that showed a very strong response to Cu(II)-binding and release. Interestingly, these two Cu(II) shuttles displayed a dual mode of action. They promptly retrieve Cu from extracellular A{beta}, stop the associated ROS formation, and hence protect both cell culture models and organotypic hippocampal slices (OHSCs) from Cu(A{beta})-induced neurotoxicity. Moreover, these shuttles import and redistribute bioavailable Cu inside cells with a sequence-dependent kinetics.

The cerebral accumulation of -Synuclein (-Syn) and amyloid {beta}-1-42 (A{beta}-42) proteins are known to play a crucial role in the pathology of neurocognitive disorders such as Parkinsons disease (PD). Currently, Levodopa (L-dopa) is the dopamine replacement therapy for treating bradykinetic symptoms visible in PD patients. Here, we use atomic force microscopy to evidence at nanometer length scales the effects of L-dopa on the morphology of -Syn and A{beta}-42 protein fibrils. L-dopa treatment reduces the length and diameter of both types of protein fibrils, with a stark reduction observed for A{beta}-42 both in physiological buffer and human spinal fluid. The insights gained on A{beta}-42 fibril disassembly from the nanoscale imaging experiments are substantiated using atomic-scale molecular dynamics simulations. Our results reveal the mechanism governing L-dopa-driven reversal of protein aggregation, which may be useful in drug design of small molecule drugs for potentially treating neurocognitive disorders and provide leads for designing chemical effector-mediated disassembly of protein architectures.

Serotonin 5-HT4 receptors (5-HT4Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT4Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT4R agonists have been suggested to have effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT4R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact of RS67333, a partial 5-HT4R agonist, on DA and ACh release dynamics in the striatum detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRABACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum and nucleus accumbens core, effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals by extending extracellular ACh lifetime, and correspondingly, RS67333 was found to inhibit striatal AChE enzymatic activity. By contrast, BIMU8, an alternative 5-HT4R ligand that did not inhibit striatal AChE, had no effect on evoked striatal ACh or DA release. These findings indicate that RS67333 modulates striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs, not through 5-HT4Rs but through AChE inhibition. These findings emphasize the caution due in attributing functions to 5-HT4Rs, but also highlight an alternative pharmacological profile of some purported 5-HT4R ligands as AChE inhibitors of potential utility for treating ACh/DA disorders.

Natural products are ligands and potential inhibitors of Alzheimers disease (AD) tau. Dihydromyricetin (DHM) is a CNS active natural product. Despite having signature polyphenolic character, DHM is ostensibly hydrophobic owing to intermolecular hydrogen bonds that shield hydrophilic phenols. Our research shows DHM becomes ionized at near-neutral pH allowing formulation of salts with transformed solubility. The MicroED co-crystal structure with trolamine reveals DHM salts as metastable solids with unlocked hydrogen bonding and a thermodynamic bent to solubilize in water. All salt formulations show better inhibitory activity against AD tau than the non-salt form, with efficacies correlating to enhanced solubilities. These results underscore the role of structural chemistry in guiding selection of solubilizing agents for chemical formulation. We propose DHM salts are appropriate formulations for research as dietary supplements to promote healthy aging by combating protein misfolding. Additionally, DHM is a suitable lead for medicinal chemistry and possible development of CNS pharmaceuticals.

-Synuclein is known to involve in the pathogenesis of Parkinsons diseases (PD) and related disorders. However, it is unclear how its aggregation causes neuronal degeneration and neuroinflammation. Due to intrinsic disorder nature, -synuclein produces a large number of structural ensembles and diverse aggregation intermediates. The post-translational modifications add a new layer of complexity to the aggregation mechanism. Recently, it has been demonstrated that glycation of -synuclein restricts into oligomeric intermediates and causes neuronal toxicity. However, the understanding of aggregation mechanism, dopaminergic neuronal death, and neuroinflammation by the glycated -synuclein is yet to be elucidated. The present study aims to address how glycated synuclein differs in oligomerization and neuroinflammation. The glycation of -synuclein perturbs the aggregation kinetics and prevents the fibrilization through the alteration of surface charges of N-terminal domain residues which prevents membrane binding and seed amplification mechanism. Mass spectrometry-based proteomics analysis of BV2 cells treated with glycated oligomers provides evidence of alteration of endocytic mechanism, mitochondrial dysfunction, and inflammatory cascade. Here, we show that -synuclein oligomers strongly bind to TLR2 and activate the TLR2 mediated signaling. However, glycated -synuclein oligomers impair the TLR2 binding and compromise TLR2 signaling. Interestingly, we also find that the glycated -synuclein oligomers favor NLRP3 inflammasome mediated neuroinflammation compared to non- glycated -synuclein oligomers. In conclusion, our findings suggest that microglia response towards -synuclein is conformation-specific and glycated oligomers can contribute to neurodegeneration differently.

Pittsburgh compound B (PiB) is a widely used Positron Emission Tomography (PET) tracer for detecting amyloid-{beta} (A{beta}) deposits in Alzheimers disease (AD). While PiB is assumed to bind selectively to A{beta}, emerging evidence suggests off-target interactions that may complicate PET signal interpretation. Here, we report that PiB can interact with acetylcholinesterase (AchE), a key enzyme in the cholinergic system. Similarity screening identified the AchE ligand thioflavin T (ThT) as the top structural analog of PiB. Docking studies and molecular dynamics simulations showed that PiB stably binds the peripheral anionic site (PAS) of AchE, with binding energies comparable to ThT and clinically relevant AchE inhibitors. In vitro fluorescence-based assays confirmed this interaction and suggest an involvement of the PAS. These findings indicate a stable off-target interaction between PiB and AChE with implications for interpreting PiB-PET signals in AD, particularly in regions with altered AchE expression or under AchE inhibitor therapy.

Alzheimers disease (AD) is defined by cognitive decline in conjunction with accumulation of aggregated amyloid {beta} (A{beta}) and tau, yet existing models of AD fail to provide a simple connection between A{beta} and tau. However, microtubules provide an intriguing nexus for pathological interactions between the two. Tau binds to microtubules and is critical to maintain their proper function. We demonstrate that A{beta} also binds to microtubules with affinity comparable that of tau itself. We hypothesize that displacement of tau by A{beta} leads to microtubule dysfunction and facilitates tau phosphorylation and aggregation. Importantly, in this model, aggregation of A{beta} is not the primary cause of toxicity, which allows many of the apparent contradictions between A{beta} pathology and cognition to be rationalized. This new model highlights the importance of both tau and A{beta} and enables novel therapeutic and intervention strategies to be considered.

Recent advances in the development of genetically encoded biosensors have resulted in a variety of different neurotransmitter sensors for the precise measurement of the dynamics of neurotransmitters, neuromodulators, peptides and hormones in real time. However, intensity-based measurements of fluorescent biosensors are limited by their dependence on the expression level of the sensor, the intensity of the excitation light, and photobleaching overtime. Here, we show that the FLIM of sDarken (a GPCR-based genetically encoded sensor for serotonin) decreases with increasing serotonin concentrations. Different members of the sDarken family, with different affinities for serotonin, show concentration-dependent changes in fluorescence lifetime according to their dynamic range. We believe that this feature of sDarken is a value-adding complement to intensity-based information and may lead to a better understanding of serotonin dynamics in health and disease.

Regulation and dysregulation of AMPA receptor (AMPAR) diffusional trapping at synapses are critical for synaptic efficacy and are implicated in various neurological and neuropsychiatric disorders. However, the limited availability of reliable tools to modulate this process hinders our ability to explore its role in both physiological conditions and disease, as well as in drug development. In this study, we designed and characterized a 21-amino-acid trans-activator of transcription (TAT)-fused peptide that mimics the binding region of the transmembrane AMPAR regulatory proteins (TARPs) C-tail to the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) N-lobe. Acute intrahippocampal infusion of this peptide enhanced perforant path-evoked synaptic transmission in the rat dentate gyrus (DG) in vivo, likely by strengthening the interaction between TARP and postsynaptic density protein 95 (PSD-95), a mechanism crucial for the synaptic anchoring of AMPARs. Additionally, the peptide also slowed AMPAR lateral diffusion and blocked KCl-induced AMPAR endocytosis, likely by enhancing the diffusional trapping of AMPARs at the synapse. Remarkably, a 7-day peptide infusion prevented social-isolation-induced forgetting of fear memory. Our findings suggest that targeting AMPAR diffusional trapping, particularly through disrupting the TARP-Arc N-lobe interaction, could hold promise as a therapeutic approach for neurological and neuropsychiatric disorders associated with impaired AMPAR synaptic retention.

Glaucoma and age-related macular degeneration (AMD) are two of the major causes of progressive vision loss and ultimately blindness worldwide. Both retinopathies share several pathological features with Alzheimers disease (AD) such as: impairment of neuronal function, astrocytosis, and activation of immune-competent microglia and Muller cells. It also has been shown that these conditions are characterized by the presence of an elevated concentration of amyloid beta (A{beta}). Under pathological conditions, A{beta}1-42 tends to aggregate, forming toxic soluble oligomers, considered to be the most harmful amyloid species. One strategy adopted to prevent cell damage caused by these oligomers is to impair their aggregation. Here we studied GAL-101, a small molecule designed to modify the aggregation of A{beta}1-42. To assess the role of GAL-101 in the aggregation of A{beta}1-42, in vitro electrophysiological measurements on retinal ganglion cells (RGCs) and retinal pigment epithelial (RPE) cells were performed to determine the polarization of the resting membrane potential. Cells treated only with A{beta}1-42 oligomers showed a strong depolarization of the resting membrane potential, which is believed to be the main reason for retinal cells malfunctioning in neurodegenerative diseases of the eye. Pre-incubation with GAL-101 stabilized the cell resting potential to around -50mV during exposure to A{beta}1-42, in both RGCs and RPE cells. GAL-101 was able to prevent changes in resting membrane potential and thus would be expected to prevent impairment of retinal cell function. These results are supportive of evaluating GAL-101 as a potential treatment of A{beta}-associated retinopathies like glaucoma and dry AMD.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder worldwide. One of its central features is the neurodegeneration that starts in the substantia nigra and progressively tends to involve other brain regions. -Synuclein (-syn) and its aggregation during pathogenesis have been drawn into the center of attention, where especially soluble oligomeric and fibrillar structures are thought to play a key role in cell-to-cell transmission and induction of toxic effects. Here, we report the development of all-D-enantiomeric peptide ligands that bind monomeric -syn with high affinity, thereby stabilizing the physiological intrinsically disordered structure and preventing initiation of aggregation, and more important, disassembling already existing aggregates. This "anti prionic" mode of action (MoA) has the advantage over other MoAs that it eliminates the particles responsible for disease propagation directly and independently of the immune system, thereby restoring the physiological monomer. Based on mirror image phage display on the D-enantiomeric full-length -syn target, we identified SVD-1 and SVD-1a by next generation sequencing, Thioflavin-T screens and rational design. The compounds were analyzed with regard to their anti-aggregation potential and both compounds showed aggregation delaying as well as seed capacity reducing effects in de novo and seeded environments, respectively. High affinity towards the monomeric -syn, in the low nano- to picomolar KD range was identified by surface plasmon resonance (SPR). SVD-1a reduced toxic effects as well as intracellular seeding capacity of -syn pre-fromed fibrils (PFF) in cell culture. SVD-1a disassembled -syn PFF into monomers as identified by atomic force microscopy (AFM), time dependent dynamic light scattering (DLS) and size exclusion chromatography (SEC) analysis. The present work provides promising results on the development of lead compounds with this anti-prionic mode of action for treatment of Parkinsons disease and other synucleinopathies.

Decreases in brain pH are associated with numerous neurological and neuropsychiatric conditions yet the molecular mechanisms linking decreased brain pH with these disorders are incomplete. The ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the brain and are inhibited by extracellular protons; however, the proton sensitivity of the delta-glutamate receptor subclass of iGluRs is unknown. Using whole-cell patch-clamp recordings of serotonin neurons in mouse brain slices and activating alpha 1-adrenergic receptors to induce delta 1 glutamate receptor (GluD1R) current, we demonstrated that GluD1R current is inhibited by physiological drops in extracellular pH. Unlike other iGluRs, protons inhibited GluD1R current via a voltage-independent decrease in unitary current. Moreover, mice lacking GluD1R showed impaired behavioral responses to inhalation of CO2. Taken together, this study continues to expand on the growing body of evidence positing GluD1R as functional ion channels and suggests that GluD1R facilitate pH sensing in vivo.

-Synuclein (-syn) fibrillar aggregates are the major component of Lewy bodies and Lewy neurites presenting as the pathology hallmark of Parkinsons disease (PD). Studies have shown that -syn is potential to form different conformational fibrils associated with different synucleinopathies, but whether the conformation of -syn fibrils changes in different phases of related diseases is to be explored. Here, we amplified -syn aggregates from the cerebrospinal fluid (CSF) of preclinical (pre-PD) and late-stage postmortem PD (post-PD) patients. Our results show that compared to the CSF of pre-PD, that of post-PD is markedly stronger in seeding in vitro -syn aggregation, and the amplified fibrils are more potent in inducing endogenous -syn aggregation in neurons. Cryo-electron microscopic structures further reveal that the difference between the pre-PD- and post-PD-derived fibrils lies on a minor polymorph which in the pre-PD fibrils is morphologically straight, while in the post-PD fibrils represents a single protofilament assembled by a distinctive conformation of -syn. Our work demonstrates structural and pathological differences between pre-PD and post-PD -syn aggregation and suggests potential alteration of -syn fibrils during the progression of PD clinical phases. Significance StatementIncreasing evidence support different conformational -syn fibrils in patients with different -synucleinopathies, but whether the conformation of -syn fibrils changes in different phases of related diseases is unknown. Here, we show that -syn fibrils amplified from the cerebrospinal fluid (CSF) of the late-stage postmortem PD (post-PD) patient are more potent in inducing endogenous -syn aggregation in neurons than that amplified from the preclinical (pre-PD) patient. Cryo-EM structures further reveal that the post-PD fibrils contain a novel conformation that is distinct from either the pre-PD fibrils or those previously reported. Our work suggests conformational evolution of -syn fibrils along with PD progression.

Apolipoproteins are involved in pathological conditions of Alzheimers disease (AD), truncated apolipoprotein fragments and {beta}-amyloid (A{beta}) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with A{beta}(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of A{beta}(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-A{beta}(M1-42) heteromers as being comprised of unstructured A{beta}(M1-42) and helical 4F. A uniform {approx}2-fold reduction in A{beta}42 15N/1H NMR signal intensities with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond scale atomistic molecular dynamics simulations showed that 4F interaction with A{beta}(M1-42) is electrostatically driven and induces unfolding of A{beta}(M1-42). Neurotoxicity profiling of A{beta}(M1-42) complexed with 4F confirms a significant reduction in cell-viability and neurite growth. The molecular architecture of heteromerization between 4F and A{beta}(M1-42) discovered in this study provides evidence towards our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Tonic glutamate signaling by ambient levels of extracellular glutamate has been implicated in development, brain injury, pathologies, and physiological activity. However, it has been difficult to assay extracellular glutamate changes with spatial and temporal resolution. Here, we utilize the rarely used ratiometric excitations properties of the fluorescence glutamate sensor iGluSnFr to enable the characterization of ambient glutamate levels in acute brain slices. This ratiometric imaging enables a spatial, temporal and calibratable assay of ambient glutamate and demonstrates regional differences in ambient glutamate and sensitivity to glutamate transporters and system Xc inhibition.

Transfer of autoantibodies targeting ionotropic N-methyl-D-aspartate receptors in autoimmune encephalitis patients into mice leads to typical disease signs. Long-term effects of the pathogenic antibodies consist of immunoglobulin G-induced crosslinking and receptor internalization. We focused on the direct and immediate impact of a specific pathogenic patient-derived monoclonal autoantibody (immunoglobulin G #003-102) on receptor function. We performed cell-attached recordings in cells transfected with the GluN1 and GluN2A subunit of the N-methyl-D-aspartate receptor. Immunoglobulin G #003-102 binds to the amino-terminal domain of the glycine-binding GluN1 subunit. It reduced simultaneous receptor openings significantly compared to controls at both low and high glutamate and glycine concentrations. Closer examination of our data in 50-second to 2-second intervals revealed, that Immunoglobulin G #003-102 rapidly decreases the number of open receptors. However, antigen-binding fragments of immunoglobulin G #003-102 did not reduce the receptor openings. In conclusion, patient-derived immunoglobulin G #003-102 inhibits N-methyl-D-aspartate receptors rapidly and directly before receptor internalization occurs and the entire immunoglobulin G is necessary for this acute inhibitory effect. This suggests an application of the antigen-binding fragment-like constructs of #003-102 as a potential new treatment strategy for shielding the pathogenic epitopes on the N-methyl-D-aspartate receptors.