DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

Multi-method investigation of factors influencing amyloid onset and impairment in three cohorts.

Alzheimer's disease biomarkers are becoming increasingly important for characterizing the longitudinal course of disease, predicting the timing of clinical and cognitive symptoms, and for recruitment and treatment monitoring in clinical trials. In this work, we develop and evaluate three methods for modelling the longitudinal course of amyloid accumulation in three cohorts using amyloid PET imaging. We then use these novel approaches to investigate factors that influence the timing of amyloid onset and the timing from amyloid onset to impairment onset in the Alzheimer's disease continuum. Data were acquired from the Alzheimer's Disease Neuroimaging Initiative (ADNI), the Baltimore Longitudinal Study of Aging (BLSA) and the Wisconsin Registry for Alzheimer's Prevention (WRAP). Amyloid PET was used to assess global amyloid burden. Three methods were evaluated for modelling amyloid accumulation using 10-fold cross-validation and holdout validation where applicable. Estimated amyloid onset age was compared across all three modelling methods and cohorts. Cox regression and accelerated failure time models were used to investigate whether sex, apolipoprotein E genotype and e4 carriage were associated with amyloid onset age in all cohorts. Cox regression was used to investigate whether apolipoprotein E (e4 carriage and e3e3, e3e4, e4e4 genotypes), sex or age of amyloid onset were associated with the time from amyloid onset to impairment onset (global clinical dementia rating ≥1) in a subset of 595 ADNI participants that were not impaired before amyloid onset. Model prediction and estimated amyloid onset age were similar across all three amyloid modelling methods. Sex and apolipoprotein E e4 carriage were not associated with PET-measured amyloid accumulation rates. Apolipoprotein E genotype and e4 carriage, but not sex, were associated with amyloid onset age such that e4 carriers became amyloid positive at an earlier age compared to non-carriers, and greater e4 dosage was associated with an earlier amyloid onset age. In the ADNI, e4 carriage, being female and a later amyloid onset age were all associated with a shorter time from amyloid onset to impairment onset. The risk of impairment onset due to age of amyloid onset was non-linear and accelerated for amyloid onset age >65. These findings demonstrate the feasibility of modelling longitudinal amyloid accumulation to enable individualized estimates of amyloid onset age from amyloid PET imaging. These estimates provide a more direct way to investigate the role of amyloid and other factors that influence the timing of clinical impairment in Alzheimer's disease.

Support vector machine learning and diffusion-derived structural networks predict amyloid quantity and cognition in adults with Down's syndrome.

Down's syndrome results from trisomy of chromosome 21, a genetic change which also confers a probable 100% risk for the development of Alzheimer's disease neuropathology (amyloid plaque and neurofibrillary tangle formation) in later life. We aimed to assess the effectiveness of diffusion-weighted imaging and connectomic modelling for predicting brain amyloid plaque burden, baseline cognition and longitudinal cognitive change using support vector regression. Ninety-five participants with Down's syndrome successfully completed a full Pittsburgh Compound B (PiB) PET-MR protocol and memory assessment at two timepoints. Our findings indicate that graph theory metrics of node degree and strength based on the structural connectome are effective predictors of global amyloid deposition. We also show that connection density of the structural network at baseline is a promising predictor of current cognitive performance. Directionality of effects were mainly significant reductions in the white matter connectivity in relation to both PiB+ status and greater rate of cognitive decline. Taken together, these results demonstrate the integral role of the white matter during neuropathological progression and the utility of machine learning methodology for non-invasively evaluating Alzheimer's disease prognosis.

Neurofibrillary tau depositions emerge with subthreshold cerebral beta-amyloidosis in down syndrome.

INTRODUCTION: Adults with Down syndrome are genetically predisposed to develop Alzheimer's disease and accumulate beta-amyloid plaques (Aß) early in life. While Aß has been heavily studied in Down syndrome, its relationship with neurofibrillary tau is less understood. The aim of this study was to evaluate neurofibrillary tau deposition in individuals with Down syndrome with varying levels of Aß burden. METHODS: A total of 161 adults with Down syndrome (mean age = 39.2 (8.50) years) and 40 healthy, non-Down syndrome sibling controls (43.2 (12.6) years) underwent T1w-MRI, [C-11]PiB and [F-18]AV-1451 PET scans. PET images were converted to units of standardized uptake value ratios (SUVrs). Aß burden was calculated using the amyloid load metric (AßL); a measure of global Aß burden that improves quantification from SUVrs by suppressing the nonspecific binding signal component and computing the specific Aß signal from all Aß-carrying voxels from the image. Regional tau was assessed using control-standardized AV-1451 SUVr. Control-standardized SUVrs were compared across Down syndrome groups of Aß-negative (A-) (AßL < 13.3), subthreshold A+ (13.3 ≤ AßL < 20) and conventionally A+ (AßL ≥ 20) individuals. The subthreshold A + group was identified as having significantly higher Aß burden compared to the A- group, but not high enough to satisfy a conventional A + classification. RESULTS: A large-sized association that survived adjustment for chronological age, mental age (assessed using the Peabody Picture Vocabulary Test), and imaging site was observed between AßL and AV-1451 within each Braak region (p < .05). The A + group showed significantly higher AV-1451 retention across all Braak regions compared to the A- and subthreshold A + groups (p < .05). The subthreshold A + group showed significantly higher AV-1451 retention in Braak regions I-III compared to an age-matched sample from the A- group (p < .05). DISCUSSION: These results show that even the earliest detectable Aß accumulation in Down syndrome is accompanied by elevated tau in the early Braak stage regions. This early detection of tau can help characterize the tau accumulation phase during preclinical Alzheimer's disease progression in Down syndrome and suggests that there may be a relatively narrow window after Aß accumulation begins to prevent the downstream cascade of events that leads to Alzheimer's disease.

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. In vivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.

Measuring longitudinal cognition: Individual tests versus composites.

INTRODUCTION: Longitudinal cohort studies of cognitive aging must confront several sources of within-person variability in scores. In this article, we compare several neuropsychological measures in terms of longitudinal error variance and relationships with biomarker-assessed brain amyloidosis (Aß). METHODS: Analyses used data from the Wisconsin Registry for Alzheimer's Prevention. We quantified within-person longitudinal variability and age-related trajectories for several global and domain-specific composites and their constituent scores. For a subset with cerebrospinal fluid or amyloid positron emission tomography measures, we examined how Aß modified cognitive trajectories. RESULTS: Global and theoretically derived composites exhibited lower intraindividual variability and stronger age × Aß interactions than did empirically derived composites or raw scores from single tests. For example, the theoretical executive function outperformed other executive function scores on both metrics. DISCUSSION: These results reinforce the need for careful selection of cognitive outcomes in study design, and support the emerging consensus favoring composites over single-test measures.

Human brain imaging of nicotinic acetylcholine α4ß2* receptors using [18 F]Nifene: Selectivity, functional activity, toxicity, aging effects, gender effects, and extrathalamic pathways.

Nicotinic acetylcholinergic receptors (nAChR's) have been implicated in several brain disorders, including addiction, Parkinson's disease, Alzheimer's disease and schizophrenia. Here we report in vitro selectivity and functional properties, toxicity in rats, in vivo evaluation in humans, and comparison across species of [18 F]Nifene, a fast acting PET imaging agent for α4ß2* nAChRs. Nifene had subnanomolar affinities for hα2ß2 (0.34 nM), hα3ß2 (0.80 nM) and hα4ß2 (0.83 nM) nAChR but weaker (27-219 nM) for hß4 nAChR subtypes and 169 nM for hα7 nAChR. In functional assays, Nifene (100 µM) exhibited 14% agonist and >50% antagonist characteristics. In 14-day acute toxicity in rats, the maximum tolerated dose (MTD) and the no observed adverse effect level (NOAEL) were estimated to exceed 40 µg/kg/day (278 µg/m2 /day). In human PET studies, [18 F]Nifene (185 MBq; <0.10 µg) was well tolerated with no adverse effects. Distribution volume ratios (DVR) of [18 F]Nifene in white matter thalamic radiations were ∼1.6 (anterior) and ∼1.5 (superior longitudinal fasciculus). Habenula known to contain α3ß2 nAChR exhibited low levels of [18 F]Nifene binding while the red nucleus with α2ß2 nAChR had DVR ∼1.6-1.7. Females had higher [18 F]Nifene binding in all brain regions, with thalamus showing >15% than males. No significant aging effect was observed in [18 F]Nifene binding over 5 decades. In all species (mice, rats, monkeys, and humans) thalamus showed highest [18 F]Nifene binding with reference region ratios >2 compared to extrathalamic regions. Our findings suggest that [18 F]Nifene PET may be used to study α4ß2* nAChRs in various CNS disorders and for translational research.

Differential dopamine function in fibromyalgia.

Approximately 30 % of Americans suffer from chronic pain disorders, such as fibromyalgia (FM), which can cause debilitating pain. Many pain-killing drugs prescribed for chronic pain disorders are highly addictive, have limited clinical efficacy, and do not treat the cognitive symptoms reported by many patients. The neurobiological substrates of chronic pain are largely unknown, but evidence points to altered dopaminergic transmission in aberrant pain perception. We sought to characterize the dopamine (DA) system in individuals with FM. Positron emission tomography (PET) with [(18)F]fallypride (FAL) was used to assess changes in DA during a working memory challenge relative to a baseline task, and to test for associations between baseline D2/D3 availability and experimental pain measures. Twelve female subjects with FM and 11 female controls completed study procedures. Subjects received one FAL PET scan while performing a "2-back" task, and one while performing a "0-back" (attentional control, "baseline") task. FM subjects had lower baseline FAL binding potential (BP) in several cortical regions relative to controls, including anterior cingulate cortex. In FM subjects, self-reported spontaneous pain negatively correlated with FAL BP in the left orbitofrontal cortex and parahippocampal gyrus. Baseline BP was significantly negatively correlated with experimental pain sensitivity and tolerance in both FM and CON subjects, although spatial patterns of these associations differed between groups. The data suggest that abnormal DA function may be associated with differential processing of pain perception in FM. Further studies are needed to explore the functional significance of DA in nociception and cognitive processing in chronic pain.

Synthesis and evaluation of 2-(18)F-fluoro-5-iodo-3-[2-(S)-3,4-dehydropyrrolinylmethoxy]pyridine ((18)F-Niofene) as a potential imaging agent for nicotinic α4ß2 receptors.

Nicotinic α4ß2 acetylcholine receptors (nAChRs) have been implicated in various pathophysiologies including neurodegenerative diseases. Currently, 2-(18)F-A85380 (2-FA) and 5-(123)I-A85380 (5-IA) are used separately in human PET and SPECT studies respectively and require >4-6 hours of scanning. We have developed 2-fluoro-5-iodo-3-[2-(S)-3-dehydropyrrolinylmethoxy]pyridine (niofene) as a potential PET/SPECT imaging agent for nAChRs with an aim to have rapid binding kinetics similar to that of (18)F-nifene used in PET studies. Niofene exhibited a 10-fold better in vitro binding affinity in rat brain than that of nicotine. The relative binding of niofene was similar to that of niodene and twice as better as that of nifene. In vitro autoradiography in rat brain slices alongside niodene indicated selective binding of niofene to regions consistent with α4ß2 receptor distribution. Niofene, 10 nM, displaced >70% of (3)H-cytisine bound to α4ß2 receptors in rat brain slices. Radiolabeling of (18)F-niofene was achieved in 10-15% radiochemical yield in high specific activities >2 Ci/µmol and showed rapid in vivo kinetics similar to that of (18)F-nifene and (18)F-nifrolene. In vivo PET in rats showed rapid uptake in the brain and selective localization in receptor regions such as the thalamus (TH). Pseudoequilibrium with (18)F-niofene was achieved in 30-40 minutes, which is similar to that of (18)F-nifene. Further evaluation of (18)F-niofene as a potential PET imaging agent is underway. Future studies will be conducted to radiolabel niofene with iodine-123 for use in SPECT imaging.

Deficient import of acetyl-CoA into the ER lumen causes neurodegeneration and propensity to infections, inflammation, and cancer.

The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N(ε)-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.

The effects of lobeline on α4ß2* nicotinic acetylcholine receptor binding and uptake of [(18)F]nifene in rats.

UNLABELLED: Lobeline is a potential smoking cessation drug with affinity for the α4ß2 nicotinic acetylcholine receptor and may inhibit the blood-brain barrier (BBB) amine transporter. The goal of this work was to use PET imaging to evaluate the effects of lobeline on the kinetic properties of [(18)F]nifene in the rat brain. METHODS: Direct α4ß2* competition of lobeline with [(18)F]nifene was evaluated using imaging experiments with both displacing and blocking doses of lobeline (1mg/kg, i.v.) given between two injections of [(18)F]nifene separated by 50min. Inhibition of the BBB amine transporter was examined using a separate imaging protocol with three injections of [(18)F]nifene, first at baseline, then following (-)nicotine blocking, and finally following lobeline blocking. RESULTS: Rapid displacement of [(18)F]nifene was observed in the α4ß2*-rich thalamus following lobeline administration, suggesting direct competition of the drug at α4ß2* sites. Slight decreases in BBB transport of [(18)F]nifene were observed when the α4ß2* system was first saturated with (-)nicotine and then given lobeline. This perturbation may be due to inhibition of the BBB amine transporter by lobeline or reductions in blood flow. Significant cerebellar displacement of [(18)F]nifene was found following the administration of both lobeline and (-)nicotine, indicating detectable specific binding in the rat cerebellum. CONCLUSION: The competition of lobeline with [(18)F]nifene is largely dominated at the α4ß2* binding site and only small perturbations in BBB transport of [(18)F]nifene are seen at the 1mg/kg dose. Similar experiments could be used to study other drugs as therapeutic agents for smoking cessation with PET.

Fetal dose estimates for (18)F-fluoro-L-thymidine using a pregnant monkey model.

UNLABELLED: Estimating the radiation dose received by the fetus from nuclear medicine procedures is important because of the greater sensitivity of rapidly developing fetal tissues to ionizing radiation. (18)F-fluoro-L-thymidine (FLT) uptake is related to cellular proliferation and is currently used to monitor tumor progression and response to therapy. This study was undertaken to estimate-on the basis of biodistribution data obtained by PET/CT in pregnant rhesus monkeys-radiation absorbed dose to a human fetus administered (18)F-FLT. METHODS: Three pregnant rhesus macaques (gestational age, 113 +/- 8 d) were administered (18)F-FLT and imaged for 2 h on a PET/CT scanner. Time-activity curves for maternal and fetal organs were generated in anatomic regions of interest identified via CT. Doses were estimated using OLINDA/EXM and the 6-mo-pregnant human model. RESULTS: The extrapolated whole-body maternal dose obtained, 11.4 microGy/MBq, is similar to the previously reported adult female dose of 15.6 microGy/MBq. The estimated total-body dose to a human fetus is 24 microGy/MBq. Significant long-term (18)F-FLT accumulation in fetal liver resulted in a fetal liver dose of 53 microGy/MBq. CONCLUSION: The fetal dose estimate in a 6-mo-pregnant human using (18)F-FLT is slightly greater than that reported for (18)F-FDG. (18)F-FLT trapping in the fetal liver should be considered in the risk-benefit analysis of (18)F-FLT PET examination in pregnant patients.

Synthesis and evaluation of nicotine alpha4beta2 receptor radioligand, 5-(3'-18F-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine, in rodents and PET in nonhuman primate.

UNLABELLED: Nicotine alpha(4)beta(2) receptor subtypes are implicated in the study of Alzheimer's disease, schizophrenia, substance abuse, lung cancer, and other disorders. We report the development and evaluation of a putative antagonist, 5-(3'-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine (nifrolidine) as a PET agent for nicotine alpha(4)beta(2) receptors. METHODS: In vitro binding affinity of nifrolidine was measured in rat brain slices labeled with (125)I-iodoepibatidine or (125)I-bungaratoxin. Selectivity of binding was measured in the presence of cytisine. (18)F radiolabeling was performed by reacting the tosylate precursor with (18)F-fluoride followed by deprotection. In vitro autoradiographic studies in rat brain slices with 5-(3'-(18)F-fluoropropyl)-3-(2-(S)-pyrrolidinylmethoxy)pyridine ((18)F-nifrolidine) were read on a phosphor imager. Rats were injected with (18)F-nifrolidine (3.7 MBq each), and brain regions were counted at various times (2-120 min). Blocking studies were performed by subcutaneous injection of nicotine (10 mg/kg). A PET study of (18)F-nifrolidine (approximately 148 MBq) was performed on an anesthetized rhesus monkey using a high-resolution scanner. RESULTS: In vitro binding affinity of nifrolidine exhibited an inhibition constant of 2.89 nmol/L for the alpha(4)beta(2) sites. Radiosynthesis and high-performance liquid chromatography purifications yielded the product in approximately 20%-40% decay-corrected radiochemical yield to provide (18)F-nifrolidine specific activities of approximately 111-185 GBq/mumol. In vitro autoradiography in rat brain slices revealed selective binding of (18)F-nifrolidine to the anteroventral thalamic nucleus, ventral posteriomedial thalamus, dorsolateral geniculate, and, to a lesser extent, cortex and striata, which are known to contain alpha(4)beta(2) sites. This specific binding was completely abolished by 300 mumol/L nicotine. Ex vivo rat brain distribution studies indicated selective binding in the thalamus with a maximal thalamus-to-cerebellum ratio of approximately 3. The PET study revealed selective maximal uptake (0.01% injected dose/mL) in regions of the thalamus (anteroventral and anteromedial thalamus, ventrolateral thalamus) and extrathalamic regions such as cingulate gyrus, lateral geniculate, temporal cortex, and frontal cortex. CONCLUSION: Binding of (18)F-nifrolidine to alpha(4)beta(2) receptor-rich regions in rats and monkeys indicates promise as a PET agent. Additionally, the thalamus-to-cerebellum ratio approached a plateau of 1.7 in 120 min, indicating relatively faster kinetics compared with previously reported imaging agents.