Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Individual and Familial Susceptibility to MPTP in a Common Marmoset Model for Parkinson's Disease.

INTRODUCTION: Insight into susceptibility mechanisms underlying Parkinson's disease (PD) would aid the understanding of disease etiology, enable target finding and benefit the development of more refined disease-modifying strategies. METHODS: We used intermittent low-dose MPTP (0.5 mg/kg/week) injections in marmosets and measured multiple behavioral and neurochemical parameters. Genetically diverse monkeys from different breeding families were selected to investigate inter- and intrafamily differences in susceptibility to MPTP treatment. RESULTS: We show that such differences exist in clinical signs, in particular nonmotor PD-related behaviors, and that they are accompanied by differences in neurotransmitter levels. In line with the contribution of a genetic component, different susceptibility phenotypes could be traced back through genealogy to individuals of the different families. CONCLUSION: Our findings show that low-dose MPTP treatment in marmosets represents a clinically relevant PD model, with a window of opportunity to examine the onset of the disease, allowing the detection of individual variability in disease susceptibility, which may be of relevance for the diagnosis and treatment of PD in humans.

Muscle contractile properties as an explanation of the higher mean power output in marmosets than humans during jumping.

The muscle mass-specific mean power output (PMMS,mean) during push-off in jumping in marmosets (Callithrix jacchus) is more than twice that in humans. In the present study it was tested whether this is attributable to differences in muscle contractile properties. In biopsies of marmoset m. vastus lateralis (VL) and m. gastrocnemius medialis (GM) (N=4), fibre-type distribution was assessed using fluorescent immunohistochemistry. In single fibres from four marmoset and nine human VL biopsies, the force-velocity characteristics were determined. Marmoset VL contained almost exclusively fast muscle fibres (>99.0%), of which 63% were type IIB and 37% were hybrid fibres, fibres containing multiple myosin heavy chains. GM contained 9% type I fibres, 44% type IIB and 47% hybrid muscle fibres. The proportions of fast muscle fibres in marmoset VL and GM were substantially larger than those reported in the corresponding human muscles. The curvature of the force-velocity relationships of marmoset type IIB and hybrid fibres was substantially flatter than that of human type I, IIA, IIX and hybrid fibres, resulting in substantially higher muscle fibre mass-specific peak power (PFMS,peak). Muscle mass-specific peak power output (PMMS,peak) values of marmoset whole VL and GM, estimated from their fibre-type distributions and force-velocity characteristics, were more than twice the estimates for the corresponding human muscles. As the relative difference in estimated PMMS,peak between marmosets and humans is similar to that of PMMS,mean during push-off in jumping, it is likely that the difference in in vivo mechanical output between humans and marmosets is attributable to differences in muscle contractile properties.

Mechanical output in jumps of marmosets (Callithrix jacchus).

In this study we determined the mechanical output of common marmosets (Callithrix jacchus) during jumping. Vertical ground reaction forces were measured in 18 animals while they jumped from an instrumented crossbar to a crossbar located 70 cm higher. From the vertical force time histories, we calculated the rate of change of mechanical energy of the centre of mass (dE/dt). The mean value of dE/dt during the push-off amounted to 51.8±6.2 W kg(-1) body mass, and the peak value to 116.4±17.6 W kg(-1) body mass. We used these values in combination with masses of leg muscles, determined in two specimens, to estimate mean and peak values of dE/dt of 430 and 970 W kg(-1) muscle, respectively. These values are higher than values reported in the literature for jumps of humans and bonobos, but smaller than those of jumps of bushbabies. Surprisingly, the mean value of dE/dt of 430 W kg(-1) muscle was close to the maximal power output of 516 W kg(-1) muscle reported in the literature for isokinetic contractions of rat medial gastrocnemius, one of the fastest mammalian muscles. Further study of the force-velocity relationship of muscle tissue of small primates is indicated.

New AAV9 engineered variants with enhanced neurotropism and reduced liver off-targeting in mice and marmosets.

Although adeno-associated virus 9 (AAV9) has been highly exploited as delivery platform for gene-based therapies, its efficacy is hampered by low efficiency in crossing the adult blood-brain barrier (BBB) and pronounced targeting to the liver upon intravenous delivery. We generated a new galactose binding-deficient AAV9 peptide display library and selected two new AAV9 engineered capsids with enhanced targeting in mouse and marmoset brains after intravenous delivery. Interestingly, the loss of galactose binding greatly reduced undesired targeting to peripheral organs, particularly the liver, while not compromising transduction of the brain vasculature. However, the galactose binding was necessary to efficiently infect non-endothelial brain cells. Thus, the combinatorial actions of the galactose-binding domain and the incorporated displayed peptide are crucial to enhance BBB crossing along with brain cell transduction. This study describes two novel capsids with high brain endothelial infectivity and extremely low liver targeting based on manipulating the AAV9 galactose-binding domain.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Preclinical Marmoset Model for Targeting Chronic Inflammation as a Strategy to Prevent Alzheimer's Disease.

Due to the aging population, modern society is facing an increasing prevalence of neurological diseases such as Alzheimer's disease (AD). AD is an age-related chronic neurodegenerative disorder for which no satisfying therapy exists. Understanding the mechanisms underlying the onset of AD is necessary to find targets for protective treatment. There is growing awareness of the essential role of the immune system in the early AD pathology. Amyloidopathy, the main feature of early-stage AD, has a deregulating effect on the immune function. This is reciprocal as the immune system also affects amyloidopathy. It seems that the inflammatory reaction shows a heterogeneous pattern depending on the stage of the disease and the variation between individuals, making not only the target but also the timing of treatment important. The lack of relevant translational animal models that faithfully reproduce clinical and pathogenic features of AD is a major cause of the delay in developing new disease-modifying therapies and their optimal timing of administration. This review describes the communication between amyloidopathy and inflammation and the possibility of using nonhuman primates as a relevant animal model for preclinical AD research.

Electrical stimulation of the nucleus basalis of meynert: a systematic review of preclinical and clinical data.

Deep brain stimulation (DBS) of the nucleus basalis of Meynert (NBM) has been clinically investigated in Alzheimer's disease (AD) and Lewy body dementia (LBD). However, the clinical effects are highly variable, which questions the suggested basic principles underlying these clinical trials. Therefore, preclinical and clinical data on the design of NBM stimulation experiments and its effects on behavioral and neurophysiological aspects are systematically reviewed here. Animal studies have shown that electrical stimulation of the NBM enhanced cognition, increased the release of acetylcholine, enhanced cerebral blood flow, released several neuroprotective factors, and facilitates plasticity of cortical and subcortical receptive fields. However, the translation of these outcomes to current clinical practice is hampered by the fact that mainly animals with an intact NBM were used, whereas most animals were stimulated unilaterally, with different stimulation paradigms for only restricted timeframes. Future animal research has to refine the NBM stimulation methods, using partially lesioned NBM nuclei, to better resemble the clinical situation in AD, and LBD. More preclinical data on the effect of stimulation of lesioned NBM should be present, before DBS of the NBM in human is explored further.

Ketamine treatment upon memory retrieval reduces fear memory in marmoset monkeys.

Emotionally arousing experiences are retained very well as seen in posttraumatic stress disorder (PTSD). Various lines of evidence indicate that reactivation of these memories renders them labile which offers a potential time-window for intervention. We tested in non-human primates whether ketamine, administered during fear memory reactivation, affected passive (inhibitory) avoidance learning. For the consolidation of contextual emotional memory, the unescapable foot-shock paradigm in a passive avoidance task with two compartments (dark vs illuminated) was used. After entering the dark compartment, marmoset monkeys received four random foot-shocks (1 mA, 4 s) within 15-min. This stressful exposure increased the saliva cortisol and heart rate and impaired REM-sleep (p<0.05). One week later the monkeys were re-exposed to the stressful situation for the reconsolidation of the fearful experience. During the re-exposure the monkeys were treated with ketamine (0.5 mg/kg) or saline. In week 3, the monkeys were placed in the experimental setting to test their memory for the fearful experience. In contrast to the vehicle-treated monkeys, who avoided the dark compartment, the ketamine-treated monkeys entered the dark compartment that was previously associated with the fearful experience (p<0.05). Post-mortem analysis of the hippocampus showed that ketamine-treated animals exhibited less doublecortin positive neurons and BrdU-labeled cells in the dentate gyrus. This study reveals that a single low dose of ketamine, administered upon fear retrieval in monkeys, reduce contextual fear memory and attenuate neurogenesis in the hippocampus. These are important findings for considering ketamine as a potential candidate to target traumatic memories in PTSD.

Involvement of the Red Nucleus in the Compensation of Parkinsonism may Explain why Primates can develop Stable Parkinson's Disease.

Neurological compensatory mechanisms help our brain to adjust to neurodegeneration as in Parkinson's disease. It is suggested that the compensation of the damaged striato-thalamo-cortical circuit is focused on the intact thalamo-rubro-cerebellar pathway as seen during presymptomatic Parkinson, paradoxical movement and sensorimotor rhythm (SMR). Indeed, the size of the red nucleus, connecting the cerebellum with the cerebral cortex, is larger in Parkinson's disease patients suggesting an increased activation of this brain area. Therefore, the red nucleus was examined in MPTP-induced parkinsonian marmoset monkeys during the presymptomatic stage and after SMR activation by neurofeedback training. We found a reverse significant correlation between the early expression of parkinsonian signs and the size of the parvocellular part of the red nucleus, which is predominantly present in human and non-human primates. In quadrupedal animals it consists mainly of the magnocellular part. Furthermore, SMR activation, that mitigated parkinsonian signs, further increased the size of the red nucleus in the marmoset monkey. This plasticity of the brain helps to compensate for dysfunctional movement control and can be a promising target for compensatory treatment with neurofeedback technology, vibrotactile stimulation or DBS in order to improve the quality of life for Parkinson's disease patients.

Efficacy of caffeine and modafinil in counteracting sleep deprivation in the marmoset monkey.

BACKGROUND AND PURPOSE: The effects of sleep deprivation are a burden in our 24-h society. The use of wake-promoting compounds could improve the performance in situations where sleep cannot be allowed. In this study, the efficacy of the wake-promoting compounds, modafinil and caffeine, in counteracting the effects of 24-h sleep deprivation in the marmoset monkey were tested. As caffeine is habitually used, the efficacy of both compounds after short- and long-term use was investigated. MATERIALS AND METHODS: After a normal active day, the animals were kept awake and received wake-promoting compounds during the whole night. Three times during the sleep-deprived night, putative fatigue was assessed with an activity test and the vigilance and ability to execute a task was assessed with a hand-eye coordination (HEC) task. RESULTS: Both compounds were able to counteract to some extent the decline in performance. Modafinil was able to keep the activity at baseline performance, but performance on the HEC task was not improved. Caffeine was able to keep performance in the HEC task at a level just below daytime level but was not able to keep activity at daytime levels during the last part of the night. Caffeine and modafinil administration for 2 weeks showed a comparable effect on activity as acute use. The performance on the HEC task was similar after chronic caffeine and improved after chronic modafinil. CONCLUSION: It is therefore concluded that modafinil and caffeine were both able to postpone or prevent the decline in vigilance and psychomotor performance and increase in fatigue induced by sleep deprivation.

Therapeutic effects of Delta9-THC and modafinil in a marmoset Parkinson model.

Current therapies for Parkinson's disease (PD) like l-dopa and dopamine (DA) agonists have declined efficacy after long term use. Therefore, research towards supplementary or alternative medication is needed. The implementation in PD can be expedited by application of compounds already used in the clinic. In this study the therapeutic effects of the psychoactive compounds Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and modafinil were tested in the 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-marmoset model for PD. The anti-parkinson effects of Delta(9)-THC (4 mg/kg) and modafinil (100 mg/kg) in parkinsonian marmosets were assessed with two behavioral rating scales covering parkinsonian symptoms and involuntary movements and two test systems assessing the locomotor activity and hand-eye coordination. Delta(9)-THC improved activity and hand-eye coordination, but induced compound-related side-effects. Modafinil improved activity and observed parkinsonian symptoms but not hand-eye coordination. It can be concluded that both compounds have therapeutic values and could supplement existing therapies for PD.

Subanesthetic Dose Ketamine in Posttraumatic Stress Disorder: A Role for Reconsolidation During Trauma-Focused Psychotherapy?

Despite efforts to develop more effective therapies, PTSD remains a difficult disorder to treat. Insight into the dynamic nature of memory formation and its required molecular machinery can provide an opportunity to target pathological memories for emotionally arousing events. As memories become labile upon retrieval, novel information can update the strength and course of these consolidated memories. Targeting the process of reconsolidation may offer a relevant approach to attenuate fearful and traumatic memories. Specific molecular mechanisms that are required for reconsolidation of arousing information include an intact functioning of the glutamatergic signaling pathways and, more specifically, the integrity of NMDA receptors. Ketamine, a noncompetitive NMDA-receptor antagonist, is receiving increasing interest for a variety of psychiatric indications. This compound can also be an interesting candidate for targeting emotional memories. We explore whether single intravenous infusion of a subanesthetic dose of ketamine can be considered as a viable augmentation strategy for trauma-focused psychotherapy in patients with PTSD. As a consequence, a systematic approach is needed to assess the pharmacodynamic effects of ketamine in relation to both psychotherapy and its pharmacokinetics prior to its application in patient populations. By using a "question-based drug development plan," we can explore such aspects for novel drugs, and we formulated five additional topics that need to be addressed concerning the psychotherapeutic approach and phase orientation of pharmacological assisted psychotherapy.

Author Correction: Transcription start site profiling of 15 anatomical regions of the Macaca mulatta central nervous system.

The authors regret that Luba M. Pardo was omitted in error from the author list of the original version of this Data Descriptor. This omission has now been corrected in the HTML and PDF versions. The authors also regret that Anemieke Rozemuller was omitted in error from the Acknowledgements of the original version of this Data Descriptor. This omission has now been corrected in the HTML and PDF versions.

Acceleration of Amyloidosis by Inflammation in the Amyloid-Beta Marmoset Monkey Model of Alzheimer's Disease.

BACKGROUND: The immune system is increasingly mentioned as a potential target for Alzheimer's disease (AD) treatment. OBJECTIVE: In the present pilot study, the effect of (neuro)inflammation on amyloidopathy was investigated in the marmoset monkey, which has potential as an AD animal model due to its natural cerebral amyloidosis similar to humans. METHODS: Six adult/aged marmosets (Callithrix jacchus) were intracranial injected with amyloid-beta (Aß) fibrils at three cortical locations in the right hemisphere. Additionally, in half of the monkeys, lipopolysaccharide (LPS) was co-injected with the Aß fibrils and injected in the other hemisphere without Aß fibrils. The other three monkeys received phosphate buffered saline instead of LPS, as a control for the inflammatory state. The effect of inflammation on amyloidopathy was also investigated in an additional monkey that suffered from chronic inflammatory wasting syndrome. Mirror histology sections were analyzed to assess amyloidopathy and immune reaction, and peripheral blood for AD biomarker expression. RESULTS: All LPS-injected monkeys showed an early AD immune blood cell expression profile on CD95 and CD45RA. Two out of three monkeys injected with Aß and LPS and the additional monkey, suffering from chronic inflammation, developed plaques. None of the controls, injected with Aß only, developed any plaques. CONCLUSION: This study shows the importance of immune modulation on the susceptibility for amyloidosis, a hallmark of AD, which offers new perspectives for disease modifying approaches in AD.

Sensorimotor rhythm neurofeedback as adjunct therapy for Parkinson's disease.

Neurofeedback may enhance compensatory brain mechanisms. EEG-based sensorimotor rhythm neurofeedback training was suggested to be beneficial in Parkinson's disease. In a placebo-controlled study in parkinsonian nonhuman primates we here show that sensorimotor rhythm neurofeedback training reduces MPTP-induced parkinsonian symptoms and both ON and OFF scores during classical L-DOPA treatment. Our findings encourage further development of sensorimotor rhythm neurofeedback training as adjunct therapy for Parkinson's disease which might help reduce L-DOPA-induced side effects.

Transcription start site profiling of 15 anatomical regions of the Macaca mulatta central nervous system.

Rhesus macaque was the second non-human primate whose genome has been fully sequenced and is one of the most used model organisms to study human biology and disease, thanks to the close evolutionary relationship between the two species. But compared to human, where several previously unknown RNAs have been uncovered, the macaque transcriptome is less studied. Publicly available RNA expression resources for macaque are limited, even for brain, which is highly relevant to study human cognitive abilities. In an effort to complement those resources, FANTOM5 profiled 15 distinct anatomical regions of the aged macaque central nervous system using Cap Analysis of Gene Expression, a high-resolution, annotation-independent technology that allows monitoring of transcription initiation events with high accuracy. We identified 25,869 CAGE peaks, representing bona fide promoters. For each peak we provide detailed annotation, expanding the landscape of 'known' macaque genes, and we show concrete examples on how to use the resulting data. We believe this data represents a useful resource to understand the central nervous system in macaque.

Behavioral effects of modafinil in marmoset monkeys.

RATIONALE: Modafinil is increasingly used in sleep disturbances in general and in neurodegenerative diseases and is recently being used in healthy people for attention control. However, the application of modafinil is possibly not only restricted to alertness enhancing effects. More insight in this compound may lead to new applications. Not all behavioral aspects have been studied sufficiently; therefore, more detailed investigations on modafinil's positive and aversive behavioral effects are addressed in this paper. OBJECTIVES: Determination of effects of modafinil in marmoset monkeys with observational methods and with behavioral tests measuring locomotor activity, hand-eye coordination, response to a threat situation and startle response. MATERIALS AND METHODS: Two hours after oral administration of modafinil in doses of 50, 100, 150, and 225 mg/kg, animals were observed and tested in the behavioral test systems. RESULTS: Locomotor activity was increased after 100 mg/kg modafinil in the Bungalow test and after 100, 150, and 225 mg/kg, as found in the movement parameters of the human threat test. Moreover, modafinil showed anxiolytic-like effects in the human threat test. No other side effects were observed, nor were the hand-eye coordination and startle response affected. CONCLUSIONS: Besides psychostimulation, modafinil has no aversive effects in the doses used in the domains measured. The potential anxiolytic-like effects of modafinil may create new possibilities for the therapeutic use of modafinil.

Stress adversely affects efficacy of physostigmine-scopolamine pretreatment against soman in guinea pigs.

During military operations, soldiers may be exposed to mixtures of chemicals and to physical, emotional and psychological stress factors, which all may influence efficacy of any treatment, including the nerve agent pretreatment regimen. The purpose of this study was therefore to investigate the influence of chronic intermittent, variable, unpredictable and uncontrollable stress conditions on the side effects and therapeutic efficacy of the combination of physostigmine (0.025 mg/kg/h) and scopolamine (0.018 mg/kg/h) as a pretreatment against 2 x LD50 soman intoxication in guinea pigs. Stress during pretreatment led to an increase of motor activity and an increase of power in the EEG delta2 frequency band. After chronic stress, exposure of pretreated animals to 2 x LD50 soman resulted in more severe intoxication symptoms, a more persistent effect on the startle response, and considerable more severe and persistent effects on the EEG power-spectrum, indicating irreversible brain damage.

Apocynin, a low molecular oral treatment for neurodegenerative disease.

Accumulating evidence suggests that inflammatory mediators secreted by activated resident or infiltrated innate immune cells have a significant impact on the pathogenesis of neurodegenerative diseases. This may imply that patients affected by a neurodegenerative disease may benefit from treatment with selective inhibitors of innate immune activity. Here we review the therapeutic potential of apocynin, an essentially nontoxic phenolic compound isolated from the medicinal plant Jatropha multifida. Apocynin is a selective inhibitor of the phagocyte NADPH oxidase Nox2 that can be applied orally and is remarkably effective at low dose.