Faculty Bibliography

Neuroimaging research seeks to identify biomarkers to improve the diagnosis, prognosis, and treatment of attention-deficit/hyperactivity disorder (ADHD), although clinical translation of findings remains distant. Resting-state functional magnetic resonance imaging (R-fMRI) is increasingly being used to characterize functional connectivity in the brain. Despite mixed results to date and multiple methodological challenges, dominant hypotheses implicate hyperconnectivity across brain networks in patients with ADHD, which could be the target of pharmacological treatments. We describe the experience and results of the Clínica Universidad de Navarra (Spain) Metilfenidato (CUNMET) pilot study. CUNMET tested the feasibility of identifying R-fMRI markers of clinical response in children with ADHD undergoing naturalistical pharmacological treatments. We analyzed cross-sectional data from 56 patients with ADHD (18 treated with methylphenidate, 18 treated with lisdexamfetamine, and 20 treatment-naive patients). Standard preprocessing and statistical analyses with attention to control for head motion and correction for multiple comparisons were performed. The only results that survived correction were noted in contrasts of children who responded clinically to lisdexamfetamine after long-term treatment vs. treatment-naive patients. In these children, we observed stronger negative correlations (anticorrelations) across nodes in six brain networks, which is consistent with higher across-network functional segregation in patients treated with lisdexamfetamine, i.e., less inter-network interference than in treatment-naive patients. We also note the lessons learned, which could help those pursuing clinically relevant multidisciplinary research in ADHD en route to eventual personalized medicine. To advance reproducible open science, our report is accompanied with links providing access to our data and analytic scripts.

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although nonamplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the neocortex of wild-type C57BL/6J mice. While we observed weak but consistent nonamplified off-target double inverted open reading frame (DIO) expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed notable Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype, or promoter. We also evaluated whether Cre-independent expression had functional effects via designer receptors exclusively activated by designer drugs (DREADDs). The DREADD agonist C21 (compound 21) had no effect on contextual fear conditioning or c-Fos expression in DIO-hM3Dq-mCherry⁺ cells of C57BL/6J mice. Together, our results indicate that DIO constructs have off-target expression in wild-type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.Significance StatementAdeno-associated viruses (AAVs) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study, we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 (compound 21) had no detectable effect on double inverted open reading frame (DIO)-hM3Dq-mCherry-infected neurons in C57BL/6J mice. Given the widespread use of DIO recombinant AAVs by the neuroscience community, our data support careful consideration when using DIO constructs in control animals.

Protein kinase B (PKB/AKT) is a central kinase involved in many neurobiological processes. AKT is expressed in the brain as three isoforms, AKT1, AKT2, and AKT3. Previous studies suggest isoform-specific roles in neural function, but very few studies have examined AKT isoform expression at the cellular level. In this study, we use a combination of histology, immunostaining, and genetics to characterize cell-type-specific expression of AKT isoforms in human and mouse brains. In mice, we find that AKT1 is the most broadly expressed isoform, with expression in excitatory neurons and the sole detectable AKT isoform in gamma-aminobutyric acid ergic interneurons and microglia. By contrast, we find that AKT2 is the sole isoform expressed in astroglia and is not detected in other neural cell types. We find that AKT3 is expressed in excitatory neurons with AKT1 but shows greater expression levels in dendritic compartments than AKT1. We extend our analysis to human brain tissues and find similar results. Using genetic deletion approaches, we also find that the cellular determinants restricting AKT isoform expression to specific cell types remain intact under Akt deficiency conditions. Because AKT signaling is linked to numerous neurological disorders, a greater understanding of cell-specific isoform expression could improve treatment strategies involving AKT.

OBJECTIVES:Patients with severe or critical COVID-19 are at higher risk for developing acute kidney injury (AKI). However, whether AKI is diagnosed in all the patients and the correlation between the outcomes of COVID-19 are not well understood. PATIENTS AND METHODS:This cohort study was conducted from February 4, 2020 to April 16, 2020 in Wuhan, China. All consecutive inpatients with laboratory-confirmed COVID-19 were included in this study. AKI was defined according to the KDIGO 2012 criteria. The outcomes of patients with and without AKI and whether AKI was or was not recognized were compared. RESULTS:A total of 107 elderly patients were included in the final analysis. The median age was 70 (64-78) years, and 69 (64.5%) were men. Overall, 48 of 107 patients (44.9%) developed AKI during hospitalization. Meanwhile, 22 (45.8%) cases with AKI was not recognized (missed diagnosis) in this cohort. The Kaplan-Meier curves showed that survival was better in the non-AKI group than in the AKI group (log-rank, all P < 0.001); in the subgroups of the patients with AKI, the hospital survival rate decreased when AKI was not recognized. The survival of patients with recognized AKI was better than that of patients with unrecognized AKI (log-rank, all P < 0.001). According to the multivariate regression analysis, the independent risk factors for in-hospital mortality were AKI (recognized AKI vs non-AKI: HR = 2.413; 95% CI = 1.092-5.333; P = 0.030 and unrecognized AKI vs non-AKI: HR = 4.590; 95% CI = 2.070-10.175; P <0.001), C-reactive protein level (HR = 1.004; 95% CI = 1.000-1.008; P = 0.030), lactate level (HR = 1.236; 95% CI = 1.098-1.391; P < 0.001), and disease classification (critical vs severe: HR = 0.019; 95% CI = 1.347-26.396; P = 5.963). CONCLUSIONS:AKI is not an uncommon complication in elderly patients with COVID-19 who admitted to ICU. Extremely high rates of underdiagnosis and undertreatment of AKI have resulted in an elevated in-hospital mortality rate. Kidney protection is an important issue that cannot be ignored, and intensive care kidney specialists should take responsibility for leading the battle against AKI.

Flipping through social media feeds, viewing exhibitions in a museum, or walking through the botanical gardens, people consistently choose to engage with and disengage from visual content. Yet, in most laboratory settings, the visual stimuli, their presentation duration, and the task at hand are all controlled by the researcher. Such settings largely overlook the spontaneous nature of human visual experience, in which perception takes place independently from specific task constraints and its time course is determined by the observer as a self-governing agent. Currently, much remains unknown about how spontaneous perceptual experiences unfold in the brain. Are all perceptual categories extracted during spontaneous perception? Does spontaneous perception inherently involve volition? Is spontaneous perception segmented into discrete episodes? How do different neural networks interact over time during spontaneous perception? These questions are imperative to understand our conscious visual experience in daily life. In this article we propose a framework for spontaneous perception. We first define spontaneous perception as a task-free and self-paced experience. We propose that spontaneous perception is guided by four organizing principles that grant it temporal and spatial structures. These principles include coarse-to-fine processing, continuity and segmentation, agency and volition, and associative processing. We provide key suggestions illustrating how these principles may interact with one another in guiding the multifaceted experience of spontaneous perception. We point to testable predictions derived from this framework, including (but not limited to) the roles of the default-mode network and slow cortical potentials in underlying spontaneous perception. We conclude by suggesting several outstanding questions for future research, extending the relevance of this framework to consciousness and spontaneous brain activity. In conclusion, the spontaneous perception framework proposed herein integrates components in human perception and cognition, which have been traditionally studied in isolation, and opens the door to understand how visual perception unfolds in its most natural context.

Down syndrome (DS) is the primary genetic cause of intellectual disability (ID), which is due to the triplication of human chromosome 21 (HSA21). In addition to ID, HSA21 trisomy results in a number of neurological and physiological pathologies in individuals with DS, including progressive cognitive dysfunction and learning and memory deficits which worsen with age. Further exacerbating neurological dysfunction associated with DS is the concomitant basal forebrain cholinergic neuron (BFCN) degeneration and onset of Alzheimer's disease (AD) pathology in early mid-life. Recent single population RNA sequencing (RNA-seq) analysis in the Ts65Dn mouse model of DS, specifically the medial septal cholinergic neurons of the basal forebrain (BF), revealed the mitochondrial oxidative phosphorylation pathway was significantly impacted, with a large subset of genes within this pathway being downregulated. We further queried oxidative phosphorylation pathway dysregulation in Ts65Dn mice by examining genes and encoded proteins within brain regions comprising the basocortical system at the start of BFCN degeneration (6 months of age). In select Ts65Dn mice we demonstrate significant deficits in gene and/or encoded protein levels of Complex I-V of the mitochondrial oxidative phosphorylation pathway in the BF. In the frontal cortex (Fr Ctx) these complexes had concomitant alterations in select gene expression but not of the proteins queried from Complex I-V, suggesting that defects at this time point in the BF are more severe and occur prior to cortical dysfunction within the basocortical circuit. We propose dysregulation within mitochondrial oxidative phosphorylation complexes is an early marker of cognitive decline onset and specifically linked to BFCN degeneration that may propagate pathology throughout cortical memory and executive function circuits in DS and AD.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4⁺CD8⁺ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

Background: Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH) is a rare monogenic disorder caused by SLC34A3 pathogenic variants, characterized by renal phosphate wasting, hypophosphatemia, hypercalciuria (HC), elevated 1,25-dihydroxyvitamin D, nephrocalcinosis (NC), and urinary stone disease (USD). Previously we reported a high prevalence of kidney cysts in CYP24A1 deficiency. Thus, in the current study, we characterized cyst presence in HHRH, another monogenic cause of HC, NC, and USD.
Method(s): Medical records from Mayo Clinic and Rare Kidney Stone Consortium research results were queried for all patients with genetically confirmed HHRH diagnosis. Clinical characteristics and imaging data are summarized in table 1.
Result(s): Among 12 patients with SLC34A3 pathogenic variants (7 monoallelic, 5 biallelic), 42% (5/12) were males. Median age at clinical presentation was 17 yrs (range 8-46) and at genetic confirmation 42 yrs (range 9-66). None had a family history of cystic kidney disease. Kidney cysts (Figure 1) were present in 75% (9/12), among whom median age at first kidney imaging and first cyst detection was 41 yrs (range 9-64). Median number of cysts per patient was 3 (range 1-23). The number of cysts >=5 mm in size was above the 97.5th percentile of an age-and sex-matched control population in 6/9 (67%). At least 2 cysts >=5 mm in size were found in 100% of children.
Conclusion(s): We found a strong association between HHRH and kidney cysts. Similarities in the biochemical profiles of HHRH and CYP24A1 deficiency suggest elevated active vitamin D, and/or HC may be potential factors in cyst formation. Further studies are needed to evaluate the role of the SLC34A3 gene in cyst formation. (Figure Presented)

Neurofibrillary tangles (NFTs) are a major pathologic hallmark of Alzheimer's disease (AD). Several studies have shown that amyloid β oligomers (Aβo) and tau oligomers mediate their toxicity, in part, via binding to cellular prion protein (PrP^C) and that some anti-PrP antibodies can block this interaction. We have generated a novel monoclonal anti-PrP antibody (TW1) and assessed the efficacy of passive immunization with it in a mouse model of AD with extensive tau pathology: hTau/PS1 transgenic (Tg) mice. These mice were injected intraperitoneally once a week with TW1 starting at 5 months of age. Behavior was assessed at 8 months of age and brain tissue was subsequently harvested for analysis of treatment efficacy at 9 months. Mice treated with TW1 did not show any significant difference in sensorimotor testing including traverse beam, rotarod, and locomotor activity compared to controls. Significant cognitive benefits were observed with the novel object recognition test (ORT) in the immunized mice (two-tailed, t-test p = 0.0019). Immunized mice also showed cognitive benefits on the closed field symmetrical maze (day 1 two-tailed t-test p = 0.0001; day 2 two-tailed t-test p = 0.0015; day 3 two-tailed t-test p = 0.0002). Reduction of tau pathology was observed with PHF-1 immunohistochemistry in the piriform cortex by 60% (two-tailed t-test p = 0.01) and in the dentate gyrus by 50% (two-tailed t-test p = 0.02) in animals treated with TW1 compared to controls. There were no significant differences in astrogliosis or microgliosis observed between treated and control mice. As assessed by Western blots using PHF-1, the TW1 therapy reduced phosphorylated tau pathology (two-tailed t-test p = 0.03) and improved the ratio of pathological soluble tau to tubulin (PHF1/tubulin; two-tailed t-test p = 0.0006). Reduction of tau pathology also was observed using the CP13 antibody (two-tailed t-test p = 0.0007). These results indicate that passive immunization with the TW1 antibody can significantly decrease tau pathology as assessed by immunohistochemical and biochemical methods, resulting in improved cognitive function in a tau transgenic mouse model of AD.