Leuco Ethyl Violet as Self-Activating Prodrug Photocatalyst for In Vivo Amyloid-Selective Oxygenation.

Aberrant aggregates of amyloid-ß (Aß) and tau protein (tau), called amyloid, are related to the etiology of Alzheimer disease (AD). Reducing amyloid levels in AD patients is a potentially effective approach to the treatment of AD. The selective degradation of amyloids via small molecule-catalyzed photooxygenation in vivo is a leading approach; however, moderate catalyst activity and the side effects of scalp injury are problematic in prior studies using AD model mice. Here, leuco ethyl violet (LEV) is identified as a highly active, amyloid-selective, and blood-brain barrier (BBB)-permeable photooxygenation catalyst that circumvents all of these problems. LEV is a redox-sensitive, self-activating prodrug catalyst; self-oxidation of LEV through a hydrogen atom transfer process under photoirradiation produces catalytically active ethyl violet (EV) in the presence of amyloid. LEV effectively oxygenates human Aß and tau, suggesting the feasibility for applications in humans. Furthermore, a concept of using a hydrogen atom as a caging group of a reactive catalyst functional in vivo is postulated. The minimal size of the hydrogen caging group is especially useful for catalyst delivery to the brain through BBB.

Reduced serotonergic transmission alters sensitivity to cost and reward via 5-HT1A and 5-HT1B receptors in monkeys.

Serotonin (5-HT) deficiency is a core biological pathology underlying depression and other psychiatric disorders whose key symptoms include decreased motivation. However, the exact role of 5-HT in motivation remains controversial and elusive. Here, we pharmacologically manipulated the 5-HT system in macaque monkeys and quantified the effects on motivation for goal-directed actions in terms of incentives and costs. Reversible inhibition of 5-HT synthesis increased errors and reaction times on goal-directed tasks, indicating reduced motivation. Analysis found incentive-dependent and cost-dependent components of this reduction. To identify the receptor subtypes that mediate cost and incentive, we systemically administered antagonists specific to 4 major 5-HT receptor subtypes: 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4. Positron emission tomography (PET) visualized the unique distribution of each subtype in limbic brain regions and determined the systemic dosage for antagonists that would achieve approximately 30% occupancy. Only blockade of 5-HT1A decreased motivation through changes in both expected cost and incentive; sensitivity to future workload and time delay to reward increased (cost) and reward value decreased (incentive). Blocking the 5-HT1B receptor also reduced motivation through decreased incentive, although it did not affect expected cost. These results suggest that 5-HT deficiency disrupts 2 processes, the subjective valuation of costs and rewards, via 5-HT1A and 5-HT1B receptors, thus leading to reduced motivation.

Distinct roles of monkey OFC-subcortical pathways in adaptive behavior.

To be the most successful, primates must adapt to changing environments and optimize their behavior by making the most beneficial choices. At the core of adaptive behavior is the orbitofrontal cortex (OFC) of the brain, which updates choice value through direct experience or knowledge-based inference. Here, we identify distinct neural circuitry underlying these two separate abilities. We designed two behavioral tasks in which macaque monkeys updated the values of certain items, either by directly experiencing changes in stimulus-reward associations, or by inferring the value of unexperienced items based on the task's rules. Chemogenetic silencing of bilateral OFC combined with mathematical model-fitting analysis revealed that monkey OFC is involved in updating item value based on both experience and inference. In vivo imaging of chemogenetic receptors by positron emission tomography allowed us to map projections from the OFC to the rostromedial caudate nucleus (rmCD) and the medial part of the mediodorsal thalamus (MDm). Chemogenetic silencing of the OFC-rmCD pathway impaired experience-based value updating, while silencing the OFC-MDm pathway impaired inference-based value updating. Our results thus demonstrate a dissociable contribution of distinct OFC projections to different behavioral strategies, and provide new insights into the neural basis of value-based adaptive decision-making in primates.

Photo-oxygenation of histidine residue inhibits α-synuclein aggregation.

Aggregation of α-synuclein (α-syn) into amyloid is the pathological hallmark of several neurodegenerative disorders, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. It is widely accepted that α-syn aggregation is associated with neurodegeneration, although the mechanisms are not yet fully understood. Therefore, the inhibition of α-syn aggregation is a potential therapeutic approach against these diseases. This study used the photocatalyst for α-syn photo-oxygenation, which selectively adds oxygen atoms to fibrils. Our findings demonstrate that photo-oxygenation using this photocatalyst successfully inhibits α-syn aggregation, particularly by reducing its seeding ability. Notably, we also discovered that photo-oxygenation of the histidine at the 50th residue in α-syn aggregates is responsible for the inhibitory effect. These findings indicate that photo-oxygenation of the histidine residue in α-syn is a potential therapeutic strategy for synucleinopathies.

Multimodal Imaging for Validation and Optimization of Ion Channel-Based Chemogenetics in Nonhuman Primates.

Chemogenetic tools provide an opportunity to manipulate neuronal activity and behavior selectively and repeatedly in nonhuman primates (NHPs) with minimal invasiveness. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are one example that is based on mutated muscarinic acetylcholine receptors. Another channel-based chemogenetic system available for neuronal modulation in NHPs uses pharmacologically selective actuator modules (PSAMs), which are selectively activated by pharmacologically selective effector molecules (PSEMs). To facilitate the use of the PSAM/PSEM system, the selection and dosage of PSEMs should be validated and optimized for NHPs. To this end, we used a multimodal imaging approach. We virally expressed excitatory PSAM (PSAM4-5HT3) in the striatum and the primary motor cortex (M1) of two male macaque monkeys, and visualized its location through positron emission tomography (PET) with the reporter ligand [18F]ASEM. Chemogenetic excitability of neurons triggered by two PSEMs (uPSEM817 and uPSEM792) was evaluated using [18F]fluorodeoxyglucose-PET imaging, with uPSEM817 being more efficient than uPSEM792. Pharmacological magnetic resonance imaging (phMRI) showed that increased brain activity in the PSAM4-expressing region began ∼13 min after uPSEM817 administration and continued for at least 60 min. Our multimodal imaging data provide valuable information regarding the manipulation of neuronal activity using the PSAM/PSEM system in NHPs, facilitating future applications.SIGNIFICANCE STATEMENT Like other chemogenetic tools, the ion channel-based system called pharmacologically selective actuator module/pharmacologically selective effector molecule (PSAM/PSEM) allows remote manipulation of neuronal activity and behavior in living animals. Nevertheless, its application in nonhuman primates (NHPs) is still limited. Here, we used multitracer positron emission tomography (PET) imaging and pharmacological magnetic resonance imaging (phMRI) to visualize an excitatory chemogenetic ion channel (PSAM4-5HT3) and validate its chemometric function in macaque monkeys. Our results provide the optimal agonist, dose, and timing for chemogenetic neuronal manipulation, facilitating the use of the PSAM/PSEM system and expanding the flexibility and reliability of circuit manipulation in NHPs in a variety of situations.

A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain.

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Quantitative Assays for Catalytic Photo-Oxygenation of Alzheimer Disease-Related Tau Proteins.

Catalytic photo-oxygenation of tau amyloid is a potential therapeutic approach to tauopathies, including Alzheimer disease (AD). However, tau is a complex target containing great molecular size and heterogeneous isoforms/proteoforms. Although catalytic photo-oxygenation has been confirmed when using catalyst 1 and recombinant tau pretreated with heparin, its effects on tau from human patients have not yet been clarified. In this study, focusing on the histidine residues being oxygenated, we have constructed two assay systems capable of quantitatively evaluating the catalytic activity when used on human patient tau: (1) fluorescence labeling at oxygenated histidine sites and (2) LC-MS/MS analysis of histidine-containing fragments. Using these assays, we identified 2 as a promising catalyst for oxygenation of human tau. In addition, our results suggest that aggregated tau induced by heparin is different from actual AD patient tau in developing effective photo-oxygenation catalysts.

Stable Neural Population Dynamics in the Regression Subspace for Continuous and Categorical Task Parameters in Monkeys.

Neural population dynamics provide a key computational framework for understanding information processing in the sensory, cognitive, and motor functions of the brain. They systematically depict complex neural population activity, dominated by strong temporal dynamics as trajectory geometry in a low-dimensional neural space. However, neural population dynamics are poorly related to the conventional analytical framework of single-neuron activity, the rate-coding regime that analyzes firing rate modulations using task parameters. To link the rate-coding and dynamic models, we developed a variant of state-space analysis in the regression subspace, which describes the temporal structures of neural modulations using continuous and categorical task parameters. In macaque monkeys, using two neural population datasets containing either of two standard task parameters, continuous and categorical, we revealed that neural modulation structures are reliably captured by these task parameters in the regression subspace as trajectory geometry in a lower dimension. Furthermore, we combined the classical optimal-stimulus response analysis (usually used in rate-coding analysis) with the dynamic model and found that the most prominent modulation dynamics in the lower dimension were derived from these optimal responses. Using those analyses, we successfully extracted geometries for both task parameters that formed a straight geometry, suggesting that their functional relevance is characterized as a unidimensional feature in their neural modulation dynamics. Collectively, our approach bridges neural modulation in the rate-coding model and the dynamic system, and provides researchers with a significant advantage in exploring the temporal structure of neural modulations for pre-existing datasets.

Attenuation of α-synuclein aggregation by catalytic photo-oxygenation.

We developed catalyst 11 to promote selective photo-oxygenation of α-synuclein amyloid and attenuate its aggregation. Catalyst 11 effectively oxygenated both small and large aggregates. The oxygenated α-synuclein exhibited lower seeding activity than intact α-synuclein. This study corroborates the feasibility of catalytic photo-oxygenation as an anti-synucleinopathy strategy.

INPP5D modulates TREM2 loss-of-function phenotypes in a ß-amyloidosis mouse model.

The genetic associations of TREM2 loss-of-function variants with Alzheimer disease (AD) indicate the protective roles of microglia in AD pathogenesis. Functional deficiencies of TREM2 disrupt microglial clustering around amyloid ß (Aß) plaques, impair their transcriptional response to Aß, and worsen neuritic dystrophy. However, the molecular mechanism underlying these phenotypes remains unclear. In this study, we investigated the pathological role of another AD risk gene, INPP5D, encoding a phosphoinositide PI(3,4,5)P3 phosphatase expressed in microglia. In a Tyrobp-deficient TREM2 loss-of-function mouse model, Inpp5d haplodeficiency restored the association of microglia with Aß plaques, partially restored plaque compaction, and astrogliosis, and reduced phosphorylated tau+ dystrophic neurites. Mechanistic analyses suggest that TREM2/TYROBP and INPP5D exert opposing effects on PI(3,4,5)P3 signaling pathways as well as on phosphoproteins involved in the actin assembly. Our results suggest that INPP5D acts downstream of TREM2/TYROBP to regulate the microglial barrier against Aß toxicity, thereby modulates Aß-dependent pathological conversion of tau.

Soluble form of the APP fragment, sAPPß, positively regulates tau secretion.

Extracellular tau has been highlighted in the pathogenesis of Alzheimer disease (AD), which is the most common neurodegenerative disease. Pathological analyses as well as model animal studies suggest that amyloid-ß peptide (Aß) deposition facilitates the spreading of tau aggregation pathology via extracellular tau. However, the precise mechanism of tau secretion remains unknown. Here, we show that the overexpression of amyloid precursor protein (APP) enhances the secretion of tau phosphorylated at threonine 181 in mouse neuroblastoma Neuro2a cells. Moreover, we found that soluble amyloid precursor protein ß (sAPPß), which is generated by ß-site APP cleaving enzyme 1 (BACE1), mediates tau secretion. Our results demonstrate that BACE1-mediated cleavage of APP plays pathological roles in AD pathogenesis by not only Aß production, but by the spreading of tau aggregation pathology via sAPPß in AD patients.

Chemogenetic attenuation of cortical seizures in nonhuman primates.

Epilepsy is a disorder in which abnormal neuronal hyperexcitation causes several types of seizures. Because pharmacological and surgical treatments occasionally interfere with normal brain function, a more focused and on-demand approach is desirable. Here we examined the efficacy of a chemogenetic tool-designer receptors exclusively activated by designer drugs (DREADDs)-for treating focal seizure in a nonhuman primate model. Acute infusion of the GABAA receptor antagonist bicuculline into the forelimb region of unilateral primary motor cortex caused paroxysmal discharges with twitching and stiffening of the contralateral arm, followed by recurrent cortical discharges with hemi- and whole-body clonic seizures in two male macaque monkeys. Expression of an inhibitory DREADD (hM4Di) throughout the seizure focus, and subsequent on-demand administration of a DREADD-selective agonist, rapidly suppressed the wide-spread seizures. These results demonstrate the efficacy of DREADDs for attenuating cortical seizure in a nonhuman primate model.

[Identification of novel regulators involved in AD pathogenesis using the CRISPR-Cas9 system].

The production of amyloid ß peptide (Aß) is an important process relating to the pathogenesis of Alzheimer disease (AD). It is widely known that the sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases lead to the production of Aß. However, the precise regulatory mechanism for Aß production remains unclear. We have established a CRISPR-Cas9 based screening system to identify the novel regulators of Aß production. Calcium and integrin-binding protein 1 (CIB1) was identified as a novel potential negative regulator of Aß production. The knockdown and knockout of Cib1 significantly increased Aß levels. In addition, immunoprecipitation showed that CIB1 interacts with the γ-secretase complex but did not alter its enzymatic activity. Moreover, Cib1 disruption specifically reduced the cell-surface localization of the γ-secretase complex. Finally, the single-cell RNA-seq analysis in the human brain demonstrated that early-stage AD patients have lower neuronal CIB1 mRNA levels compared to healthy controls. Taken together, we have shown that CIB1 controls the subcellular localization of γ-secretase, resulting in the regulation of Aß production, suggesting the involvement of CIB1 in the development of AD pathogenesis.

Promotion in the Clearance of Aggregated Aß In Vivo Using Amyloid Selective Photo-Oxygenation Technology.

Alzheimer's disease (AD) is characterized by the aggregation and deposition of 2 amyloid proteins: amyloid ß peptide (Aß) and tau protein. Immunotherapies using anti-Aß antibodies to promote the clearance of aggregated Aß have recently been highlighted as a promising disease-modifying approach against AD. However, immunotherapy has still some problems, such as low efficiency of delivery into the brain and high costs. We have developed the "amyloid selective photo-oxygenation technology" as a comparable to immunotherapy for amyloids. The photo-oxygenation can artificially attach the oxygen atoms to specific amino acids in amyloid proteins using photocatalyst and light irradiation. We revealed that in vivo photo-oxygenation for living AD model mice reduced the aggregated Aß in the brain. Moreover, we also showed that microglia were responsible for this promoted clearance of photo-oxygenated Aß from the brain. These results indicated that our photo-oxygenation technology has the potential as a disease-modifying therapy against AD to promote the degradation of amyloids, resulting in being comparable to immunotherapy. Here, we introduce our technology and its effects in vivo that we showed previously in Ozawa et al., Brain, 2021, as well as a further improvement towards non-invasive in vivo photo-oxygenation described in another publication Nagashima et al., Sci. Adv., 2021, as expanded discussion.

Photo-Oxygenation as a New Therapeutic Strategy for Neurodegenerative Proteinopathies by Enhancing the Clearance of Amyloid Proteins.

Alzheimer disease (AD) is associated with the aggregation of two amyloid proteins: tau and amyloid-ß (Aß). The results of immunotherapies have shown that enhancing the clearance and suppressing the aggregation of these two proteins are effective therapeutic strategies for AD. We have developed photocatalysts that attach oxygen atoms to Aß and tau aggregates via light irradiation. Photo-oxygenation of these amyloid aggregates reduced their neurotoxicity by suppressing their aggregation both in vitro and in vivo. Furthermore, photo-oxygenation enhanced the clearance of Aß in the brain and microglial cells. Here, we describe the effects of photo-oxygenation on tau and Aß aggregation, and the potential of photo-oxygenation as a therapeutic strategy for AD, acting via microglial clearance.

Chemogenetic Disconnection between the Orbitofrontal Cortex and the Rostromedial Caudate Nucleus Disrupts Motivational Control of Goal-Directed Action.

The orbitofrontal cortex (OFC) and its major downstream target within the basal ganglia-the rostromedial caudate nucleus (rmCD)-are involved in reward-value processing and goal-directed behavior. However, a causal contribution of the pathway linking these two structures to goal-directed behavior has not been established. Using the chemogenetic technology of designer receptors exclusively activated by designer drugs with a crossed inactivation design, we functionally and reversibly disrupted interactions between the OFC and rmCD in two male macaque monkeys. We injected an adeno-associated virus vector expressing an inhibitory designer receptor, hM4Di, into the OFC and contralateral rmCD, the expression of which was visualized in vivo by positron emission tomography and confirmed by postmortem immunohistochemistry. Functional disconnection of the OFC and rmCD resulted in a significant and reproducible loss of sensitivity to the cued reward value for goal-directed action. This decreased sensitivity was most prominent when monkeys had accumulated a certain amount of reward. These results provide causal evidence that the interaction between the OFC and the rmCD is needed for motivational control of action on the basis of the relative reward value and internal drive. This finding extends the current understanding of the physiological basis of psychiatric disorders in which goal-directed behavior is affected, such as obsessive-compulsive disorder.SIGNIFICANCE STATEMENT In daily life, we routinely adjust the speed and accuracy of our actions on the basis of the value of expected reward. Abnormalities in these kinds of motivational adjustments might be related to behaviors seen in psychiatric disorders such as obsessive-compulsive disorder. In the current study, we show that the connection from the orbitofrontal cortex to the rostromedial caudate nucleus is essential for motivational control of action in monkeys. This finding expands our knowledge about how the primate brain controls motivation and behavior and provides a particular insight into disorders like obsessive-compulsive disorder in which altered connectivity between the orbitofrontal cortex and the striatum has been implicated.

Chronic Behavioral Manipulation via Orally Delivered Chemogenetic Actuator in Macaques.

The chemogenetic technology referred to as designer receptors exclusively activated by designer drugs (DREADDs) offers reversible means to control neuronal activity for investigating its functional correlation with behavioral action. Deschloroclozapine (DCZ), a recently developed highly potent and selective DREADD actuator, displays a capacity to expand the utility of DREADDs for chronic manipulation without side effects in nonhuman primates, which has not yet been validated. Here we investigated the pharmacokinetics and behavioral effects of orally administered DCZ in female and male macaque monkeys. Pharmacokinetic analysis and PET occupancy examination demonstrated that oral administration of DCZ yielded slower and prolonged kinetics, and that its bioavailability was 10%-20% of that in the case of systemic injection. Oral DCZ (300-1000 µg/kg) induced significant working memory impairments for at least 4 h in monkeys with hM4Di expressed in the dorsolateral prefrontal cortex (Brodmann's area 46). Repeated daily oral doses of DCZ consistently caused similar impairments over two weeks without discernible desensitization. Our results indicate that orally delivered DCZ affords a less invasive strategy for chronic but reversible chemogenetic manipulation of neuronal activity in nonhuman primates, and this has potential for clinical application.SIGNIFICANCE STATEMENT The use of designer receptors exclusively activated by designer drugs (DREADDs) for chronic manipulation of neuronal activity for days to weeks may be feasible for investigating brain functions and behavior on a long time-scale, and thereby for developing therapeutics for brain disorders, such as epilepsy. Here we performed pharmacokinetics and in vivo occupancy study of orally administered deschloroclozapine to determine a dose range suitable for DREADDs studies. In monkeys expressing hM4Di in the prefrontal cortex, single and repeated daily doses significantly induced working-memory impairments for hours and over two weeks, respectively, without discernible desensitization. These results indicate that orally delivered deschloroclozapine produces long-term stable chemogenetic effects, and holds great promise for the translational use of DREADDs technology.

Chemogenetic sensory fMRI reveals behaviorally relevant bidirectional changes in primate somatosensory network.

Concurrent genetic neuromodulation and functional magnetic resonance imaging (fMRI) in primates has provided a valuable opportunity to assess the modified brain-wide operation in the resting state. However, its application to link the network operation with behavior still remains challenging. Here, we combined chemogenetic silencing of the primary somatosensory cortex (SI) with tactile fMRI and related behaviors in macaques. Focal chemogenetic silencing of functionally identified SI hand region impaired grasping behavior. The same silencing also attenuated hand stimulation-evoked fMRI signal at both the local silencing site and the anatomically and/or functionally connected downstream grasping network, suggesting altered network operation underlying the induced behavioral impairment. Furthermore, the hand region silencing unexpectedly disinhibited foot representation with accompanying behavioral hypersensitization. These results demonstrate that focal chemogenetic silencing with sensory fMRI in macaques unveils bidirectional network changes to generate multifaceted behavioral impairments, thereby opening a pivotal window toward elucidating the causal network operation underpinning higher brain functions in primates.

Chemogenetic activation of nigrostriatal dopamine neurons in freely moving common marmosets.

To interrogate particular neuronal pathways in nonhuman primates under natural and stress-free conditions, we applied designer receptors exclusively activated by designer drugs (DREADDs) technology to common marmosets. We injected adeno-associated virus vectors expressing the excitatory DREADD hM3Dq into the unilateral substantia nigra (SN) in four marmosets. Using multi-tracer positron emission tomography imaging, we detected DREADD expression in vivo, which was confirmed in nigrostriatal dopamine neurons by immunohistochemistry, as well as by assessed activation of the SN following agonist administration. The marmosets rotated in a contralateral direction relative to the activated side 30-90 min after consuming food containing the highly potent DREADD agonist deschloroclozapine (DCZ) but not on the following days without DCZ. These results indicate that non-invasive and reversible DREADD manipulation will extend the utility of marmosets as a primate model for linking neuronal activity and natural behavior in various contexts.

D1- and D2-like receptors differentially mediate the effects of dopaminergic transmission on cost-benefit evaluation and motivation in monkeys.

It has been widely accepted that dopamine (DA) plays a major role in motivation, yet the specific contribution of DA signaling at D1-like receptor (D1R) and D2-like receptor (D2R) to cost-benefit trade-off remains unclear. Here, by combining pharmacological manipulation of DA receptors (DARs) and positron emission tomography (PET) imaging, we assessed the relationship between the degree of D1R/D2R blockade and changes in benefit- and cost-based motivation for goal-directed behavior of macaque monkeys. We found that the degree of blockade of either D1R or D2R was associated with a reduction of the positive impact of reward amount and increasing delay discounting. Workload discounting was selectively increased by D2R antagonism. In addition, blocking both D1R and D2R had a synergistic effect on delay discounting but an antagonist effect on workload discounting. These results provide fundamental insight into the distinct mechanisms of DA action in the regulation of the benefit- and cost-based motivation, which have important implications for motivational alterations in both neurological and psychiatric disorders.