Cost-Efficient Transcriptomic-Based Drug Screening.

Transcriptomics allows to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

Serotonin Degeneration and Amyloid-ß Deposition in Mild Cognitive Impairment: Relationship to Cognitive Deficits.

BACKGROUND: Neuropathological and neuroimaging studies have demonstrated degeneration of the serotonin system in Alzheimer's disease (AD). Neuroimaging studies have extended these observations to the preclinical stages of AD, mild cognitive impairment (MCI). Serotonin degeneration has been observed also in transgenic amyloid mouse models, prior to widespread cortical distribution of amyloid-ß (Aß). OBJECTIVE: The present study evaluated the regional distribution of the serotonin transporter (5-HTT) and of Aß in individuals with MCI and healthy older controls, as well as the contribution of 5-HTT and Aß to cognitive deficits. METHODS: Forty-nine MCI participants and 45 healthy older controls underwent positron emission tomography (PET) imaging of 5-HTT and Aß, structural magnetic resonance imaging and neuropsychological assessments. RESULTS: Lower cortical, striatal, and limbic 5-HTT and higher cortical Aß was observed in MCIs relative to healthy controls. Lower 5-HTT, mainly in limbic regions, was correlated with greater deficits in auditory-verbal and visual-spatial memory and semantic, not phonemic fluency. Higher cortical A ß was associated with greater deficits in auditory-verbal and visual-spatial memory and in semantic, not phonemic fluency. When modeling the association between cognition, gray matter volumes and Aß, inclusion of 5-HTT in limbic and in select cortical regions significantly improved model fit for auditory-verbal and visual-spatial memory and semantic, but not phonemic fluency. CONCLUSIONS: These results support the role of serotonin degeneration in the memory and semantic fluency deficits observed in MCI.

Chromatin accessibility profiling of targeted cell populations with laser capture microdissection coupled to ATAC-seq.

Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers.

P3a amplitude to trauma-related stimuli reduced after successful trauma-focused PTSD treatment.

An elevated P3a amplitude to trauma-related stimuli is strongly associated with posttraumatic stress disorder (PTSD), yet little is known about whether this response to trauma-related stimuli is affected by treatment that decreases PTSD symptoms. As an analysis of secondary outcome measures from a randomized controlled trial, we investigated the latency and amplitude changes of the P3a in responses in a three-condition oddball visual task that included trauma-related (combat scenes) and trauma-unrelated (threatening animals) distractors. Fifty-five U.S. veterans diagnosed with combat-related PTSD were randomized to receive either active or sham repetitive transcranial magnetic stimulation (rTMS). All received cognitive processing therapy, CPT+A, which requires a written account of the index trauma. They were tested before and 6 months after protocol completion. P3a amplitude and response time decreases were driven largely by the changes in the responses to the trauma-related stimuli, and this decrease correlated to the decrease in PTSD symptoms. The amplitude changes were greater in those who received rTMS + CPT than in those who received sham rTMS + CPT, suggesting that rTMS plays beneficial role in reducing arousal and threat bias, which may allow for more effective engagement in trauma-focused PTSD treatment.

Differences in electroencephalography oscillations between normal aging and mild cognitive impairment during semantic memory retrieval.

Semantic memory remains relatively stable with normal cognitive aging and declines in early stages of neurodegenerative disease. We measured electroencephalography (EEG) oscillatory correlates of semantic memory retrieval to examine the effects of normal and pathological aging. Twenty-nine cognitively healthy young adults (YA), 22 cognitively healthy aging adults (HA) and 20 patients with mild cognitive impairment (MCI) completed a semantic memory retrieval task with concurrent EEG recording in which they judged whether two words (features of objects) led to retrieval of an object (retrieval) or not (non-retrieval). Event-related power changes contrasting the two conditions (retrieval vs. non-retrieval) within theta, alpha, low-beta and high-beta EEG frequency bands were examined for normal aging (YA vs. HA) and pathological aging effects (HA vs. MCI). With no behavioural differences between the two normal age groups, we found later theta and alpha event-related power differences between conditions only in YA and a high-beta event-related power difference only in HA. For pathological aging effects, with reduced accuracy in MCI, we found different EEG patterns of early event-related beta power differences between conditions in MCI compared with HA and an event-related low-beta power difference only in HA. Beta oscillations were correlated with behavioural performance only in HA. We conclude that the aging brain relies on faster (beta) oscillations during the semantic memory task. With pathological aging, retrieval accuracy declines and pattern of beta oscillation changes. The findings provide insights about age-related neural mechanisms underlying semantic memory and have implications for early detection of pathological aging.

Corrigendum: Case report: Improving verbal retrieval deficits with high definition transcranial direct current stimulation targeting the pre-supplementary motor area in a patient with chronic traumatic brain injury.

[This corrects the article DOI: 10.3389/fneur.2021.678518.].

Molecular imaging of the association between serotonin degeneration and beta-amyloid deposition in mild cognitive impairment.

BACKGROUND: Degeneration of the serotonin system has been observed in Alzheimer's disease (AD) and in mild cognitive impairment (MCI). In transgenic amyloid mouse models, serotonin degeneration is detected prior to widespread cortical beta-amyloid (Aß) deposition, also suggesting that serotonin degeneration may be observed in preclinical AD. METHODS: The differences in the distribution of serotonin degeneration (reflected by the loss of the serotonin transporter, 5-HTT) relative to Aß deposition was measured with positron emission tomography in a group of individuals with MCI and a group of healthy older adults. A multi-modal partial least squares (mmPLS) algorithm was applied to identify the spatial covariance pattern between 5-HTT availability and Aß deposition. RESULTS: Forty-five individuals with MCI and 35 healthy older adults were studied, 22 and 27 of whom were included in the analyses who were "amyloid positive" and "amyloid negative", respectively. A pattern of lower cortical, subcortical and limbic 5-HTT availability and higher cortical Aß deposition distinguished the MCI from the healthy older control participants. Greater expression of this pattern was correlated with greater deficits in memory and executive function in the MCI group, not in the control group. CONCLUSION: A spatial covariance pattern of lower 5-HTT availability and Aß deposition was observed to a greater extent in an MCI group relative to a control group and was associated with cognitive impairment in the MCI group. The results support the application of mmPLS to understand the neurochemical changes associated with Aß deposition in the course of preclinical AD.

Transcranial Magnetic Stimulation and its Imaging Features in Patients With Depression, Post-traumatic Stress Disorder, and Traumatic Brain Injury.

Transcranial magnetic stimulation (TMS) is a type of noninvasive neurostimulation used increasingly often in clinical medicine. While most studies to date have focused on TMS's ability to treat major depressive disorder, it has shown promise in several other conditions including post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI). As different treatment protocols are often used across studies, the ability to predict patient outcomes and evaluate immediate and long-term changes using imaging becomes increasingly important. Several imaging features, such as thickness, connectedness, and baseline activity of a variety of cortical and subcortical areas, have been found to be correlated with a greater response to TMS therapy. Intrastimulation imaging can reveal in real time how TMS applied to superficial areas activates or inhibits activity in deeper brain regions. Functional imaging performed weeks to months after treatment can offer an understanding of how long-term effects on brain activity relate to clinical improvement. Further work should be done to expand our knowledge of imaging features relevant to TMS therapy and how they vary across patients with different neurological and psychiatric conditions.

High-definition transcranial direct current stimulation modulates theta response during a Go-NoGo task in traumatic brain injury.

OBJECTIVE: High Definition transcranial Direct Current Stimulation (HD-tDCS) has been shown to improve cognitive performance in individuals with chronic traumatic brain injury (TBI), although electrophysiological mechanisms remain unclear. METHODS: Veterans with TBI underwent active anodal (N = 15) vs sham (N = 10) HD-tDCS targeting the pre-supplementary motor area (pre-SMA). A Go-NoGo task was conducted simultaneously with electroencephalography (EEG) at baseline and after intervention completion. RESULTS: We found increased theta event-related spectral perturbation (ERSP) and inter-trial phase coherence (ITPC) during Go in the frontal midline electrodes overlying the pre-SMA after active HD-tDCS intervention, but not after sham. We also found increased theta phase coherence during Go between the frontal midline and left posterior regions after active HD-tDCS. A late increase in alpha-theta ERSP was found in the left central region after active HD-tDCS. Notably, lower baseline theta ERSP/ITPC in the frontal midline region predicted more post-intervention improvement in Go performance only in the active group. CONCLUSIONS: There are local and interregional oscillatory changes in response to HD-tDCS modulation in chronic TBI. SIGNIFICANCE: These findings may guide future research in utilizing EEG time-frequency metrics not only to measure interventional effects, but also in selecting candidates who may optimally respond to treatment.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Mature neutrophils and a NF-κB-to-IFN transition determine the unifying disease recovery dynamics in COVID-19.

Disease recovery dynamics are often difficult to assess, as patients display heterogeneous recovery courses. To model recovery dynamics, exemplified by severe COVID-19, we apply a computational scheme on longitudinally sampled blood transcriptomes, generating recovery states, which we then link to cellular and molecular mechanisms, presenting a framework for studying the kinetics of recovery compared with non-recovery over time and long-term effects of the disease. Specifically, a decrease in mature neutrophils is the strongest cellular effect during recovery, with direct implications on disease outcome. Furthermore, we present strong indications for global regulatory changes in gene programs, decoupled from cell compositional changes, including an early rise in T cell activation and differentiation, resulting in immune rebalancing between interferon and NF-κB activity and restoration of cell homeostasis. Overall, we present a clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues.

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

Regional amyloid correlates of cognitive performance in ageing and mild cognitive impairment.

Beta-amyloid deposition is one of the earliest pathological markers associated with Alzheimer's disease. Mild cognitive impairment in the setting of beta-amyloid deposition is considered to represent a preclinical manifestation of Alzheimer's disease. In vivo imaging studies are unique in their potential to advance our understanding of the role of beta-amyloid deposition in cognitive deficits in Alzheimer's disease and in mild cognitive impairment. Previous work has shown an association between global cortical measures of beta-amyloid deposition ('amyloid positivity') in mild cognitive impairment with greater cognitive deficits and greater risk of progression to Alzheimer's disease. The focus of the present study was to examine the relationship between the regional distribution of beta-amyloid deposition and specific cognitive deficits in people with mild cognitive impairment and cognitively normal elderly individuals. Forty-seven participants with multi-domain, amnestic mild cognitive impairment (43% female, aged 57-82 years) and 37 healthy, cognitively normal comparison subjects (42% female, aged 55-82 years) underwent clinical and neuropsychological assessments and high-resolution positron emission tomography with the radiotracer 11C-labelled Pittsburgh compound B to measure beta-amyloid deposition. Brain-behaviour partial least-squares analysis was conducted to identify spatial patterns of beta-amyloid deposition that correlated with the performance on neuropsychological assessments. Partial least-squares analysis identified a single significant (P < 0.001) latent variable which accounted for 80% of the covariance between demographic and cognitive measures and beta-amyloid deposition. Performance in immediate verbal recall (R = -0.46 ± 0.07, P < 0.001), delayed verbal recall (R = -0.39 ± 0.09, P < 0.001), immediate visual-spatial recall (R = -0.39 ± 0.08, P < 0.001), delayed visual-spatial recall (R = -0.45 ± 0.08, P < 0.001) and semantic fluency (R = -0.33 ± 0.11, P = 0.002) but not phonemic fluency (R = -0.05 ± 0.12, P < 0.705) negatively covaried with beta-amyloid deposition in the identified regions. Partial least-squares analysis of the same cognitive measures with grey matter volumes showed similar associations in overlapping brain regions. These findings suggest that the regional distribution of beta-amyloid deposition and grey matter volumetric decreases is associated with deficits in executive function and memory in mild cognitive impairment. Longitudinal analysis of these relationships may advance our understanding of the role of beta-amyloid deposition in relation to grey matter volumetric decreases in cognitive decline.

Auditory N2 Correlates of Treatment Response in Posttraumatic Stress Disorder.

Emotional processing and cognitive control are implicated as being dysfunctional in posttraumatic stress disorder (PTSD) and targeted in cognitive processing therapy (CPT), a trauma-focused treatment for PTSD. The N2 event-related potential has been interpreted in the context of emotional processing and cognitive control. In this analysis of secondary outcome measures from a randomized controlled trial, we investigated the latency and amplitude changes of the N2 in responses to task-relevant target tones and task-irrelevant distractor sounds (e.g., a trauma-related gunshot and a trauma-unrelated lion's roar) and the associations between these responses and PTSD symptom changes. United States military veterans (N = 60) diagnosed with combat-related PTSD were randomized to either active or sham repetitive transcranial magnetic stimulation (rTMS) and received a CPT intervention that included a written trauma account element (CPT+A). Participants were tested before and 6 months after protocol completion. Reduction in N2 amplitude to the gunshot stimulus was correlated with reductions in reexperiencing, |r| = .445, and hyperarousal measures, |r| = .364. In addition, in both groups, the latency of the N2 event-related potential to the distractors became longer with treatment and the N2 latency to the task-relevant stimulus became shorter, ηp 2  = .064, both of which are consistent with improved cognitive control. There were no between-group differences in N2 amplitude and latency. Normalized N2 latencies, reduced N2 amplitude to threatening distractors, and the correlation between N2 amplitude reduction and PTSD symptom reduction reflect improved cognitive control, consistent with the CPT+A objective of addressing patients' abilities to respond more appropriately to trauma triggers.

Positron emission tomography imaging of serotonin degeneration and beta-amyloid deposition in late-life depression evaluated with multi-modal partial least squares.

Depression in late-life is associated with increased risk of cognitive decline and development of all-cause dementia. The neurobiology of late-life depression (LLD) may involve both neurochemical and neurodegenerative mechanisms that are common to depression and dementia. Transgenic amyloid mouse models show evidence of early degeneration of monoamine systems. Informed by these preclinical data, the hypotheses were tested that a spatial covariance pattern of higher beta-amyloid (Aß) and lower serotonin transporter availability (5-HTT) in frontal, temporal, and parietal cortical regions would distinguish LLD patients from healthy controls and the expression of this pattern would be associated with greater depressive symptoms. Twenty un-medicated LLD patients who met DSM-V criteria for major depression and 20 healthy controls underwent PET imaging with radiotracers for Aß ([11C]-PiB) and 5-HTT ([11C]-DASB). A voxel-based multi-modal partial least squares (mmPLS) algorithm was applied to the parametric PET images to determine the spatial covariance pattern between the two radiotracers. A spatial covariance pattern was identified, including higher Aß in temporal, parietal and occipital cortices associated with lower 5-HTT in putamen, thalamus, amygdala, hippocampus and raphe nuclei (dorsal, medial and pontine), which distinguished LLD patients from controls. Greater expression of this pattern, reflected in summary 5-HTT/Aß mmPLS subject scores, was associated with higher levels of depressive symptoms. The mmPLS method is a powerful approach to evaluate the synaptic changes associated with AD pathology. This spatial covariance pattern should be evaluated further to determine whether it represents a biological marker of antidepressant treatment response and/or cognitive decline in LLD patients.

Baseline delayed verbal recall predicts response to high definition transcranial direct current stimulation targeting the superior medial frontal cortex.

Anodal high definition transcranial direct current stimulation (HD-tDCS) targeting the pre-supplementary motor area/dorsal anterior cingulate cortex (pre-SMA/dACC) has recently been shown to improve verbal retrieval deficits in veterans with chronic traumatic brain injury (TBI) (Motes et al., 2020), but predictors of treatment response are unclear. We hypothesized that baseline delayed verbal recall, a sensitive measure for post-TBI chronic cognitive decline, would predict therapeutic effects of HD-tDCS targeting the pre-SMA/dACC for verbal retrieval deficits. Standardized verbal retrieval measures were administered at baseline, immediately after and 8 weeks after treatment completion. We applied mixed generalized linear modeling as a post-hoc subgroup analysis to the verbal retrieval scores that showed significant improvement in Motes at el. (2020) to examine effects of active stimulation across the groups with baseline-intact delayed recall (N = 10) and baseline-impaired delayed recall (N = 8), compared to sham (N = 7). Individuals with impaired baseline delayed recall showed significant improvement (compared to baseline) in both category fluency and color-word inhibition/switch, while individuals with intact delayed recall showed significant improvement only in color-word inhibition/switch. Baseline delayed verbal recall may therefore be considered as a predictor for future electromodulation studies targeting frontal structures to treat TBI-related verbal deficits.

Case Report: Improving Verbal Retrieval Deficits With High Definition Transcranial Direct Current Stimulation Targeting the Pre-Supplementary Motor Area in a Patient With Chronic Traumatic Brain Injury.

We report a patient who has cognitive sequalae including verbal retrieval deficits after severe traumatic brain injury (TBI). The cortico-caudate-thalamic circuit involving the pre-Supplementary Motor Area (pre-SMA) has been proposed to underlie verbal retrieval functions. We hypothesized that High Definition-transcranial Direct Current Stimulation (HD-tDCS) targeting the pre-SMA would selectively modulate this circuit to remediate verbal retrieval deficits. After the patient underwent 10 sessions of 20 min of 1 mA HD-tDCS targeting the pre-SMA, we documented significant improvements for verbal fluency and naming, and for working memory and executive function tasks that involve the frontal lobes. The effects persisted for up to 14 weeks after completion of HD-tDCS treatment. We also demonstrated normalization of the event-related potentials suggesting modulation of the underlying neural circuit. Our study implicates that region-specific non-invasive brain stimulation, such as HD-tDCS, serves as a potential individualized therapeutic tool to treat cognitive deficits by inducing longer-lasting neuroplasticity even in the chronic phase of TBI.

Swarm Learning for decentralized and confidential clinical machine learning.

Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine1,2. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes3. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation4,5. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.

Molecular imaging of beta-amyloid deposition in late-life depression.

Late-life depression (LLD) is associated with an increased risk of all-cause dementia and may involve Alzheimer's disease pathology. Twenty-one LLD patients who met the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, criteria for a current major depressive episode and 21 healthy controls underwent clinical and neuropsychological assessments, magnetic resonance imaging to measure gray matter volumes, and high-resolution positron emission tomography to measure beta-amyloid (Aß) deposition. Clinical and neuropsychological assessments were repeated after 10-12 weeks of Citalopram or Sertraline treatment (LLD patients only). LLD patients did not differ from healthy controls in baseline neuropsychological function, although patients improved in both depressive symptoms and visual-spatial memory during treatment. Greater Aß in the left parietal cortex was observed in LLD patients compared with controls. Greater Aß was correlated with greater depressive symptoms and poorer visual-spatial memory, but not with improvement with treatment. The study of LLD patients with prospective measurements of mood and cognitive responses to antidepressant treatment is an opportunity to understand early neurobiological mechanisms underlying the association between depression and subsequent cognitive decline.