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BACKGROUND: Two bronchoprovocation methods are widely used. Compared to the tidal breathing method, the dosimeter method delivers approximately half the dose and involves five deep inhalations. Both the lower dose and the bronchoprotective deep inhalations contribute to the lesser airway response of the dosimeter. OBJECTIVE: To determine the relative role of dose and deep inspiration in the difference between the two methods. METHODS: Subjects with asthma (n = 24) underwent three methacholine challenges: a dosimeter challenge, a 2-min tidal breathing challenge (twice the dose), and a modified 2-min tidal breathing challenge (twice the dose plus five deep inhalations). RESULTS: The dosimeter method produced a nonsignificantly lower response than the modified tidal breathing method (p = 0.14). Both deep inhalation methods produced significantly less response than did the standard tidal breathing method (p = 0.011). In the 12 subjects with the most mild airway hyperresponsiveness (AHR), the differences between the deep inhalation method and the tidal breathing method were greater (p = 0.007). By contrast, deep inhalations produced no effect in the 12 subjects with greater AHR; the two tidal breathing methods produced identical results, while the dosimeter produced less response than either (p = 0.033). Six current asthmatics with mild airway responsiveness (tidal breathing method) had negative dosimeter methacholine challenge results. CONCLUSIONS: In subjects with moderate airway responsiveness, the difference between the methods is due to the difference in dose, whereas in subjects with mild AHR, deep inhalations had a large effect overwhelming the dose effect and producing false-negative methacholine challenge results in 25% of the subjects. [ABSTRACT FROM AUTHOR]

The most common equine tapeworm, Anoplocephala perfoliata , has often been neglected amongst molecular investigations and has been faced with limited treatment options. However, the recent release of a transcriptome dataset has now provided opportunities for in-depth analysis of A. perfoliata protein expression. Here, global, and sub-proteomic approaches were utilized to provide a comprehensive characterization of the A. perfoliata soluble glutathione transferases (GST) (ApGST). Utilizing both bioinformatics and gel-based proteomics, GeLC and 2D-SDS PAGE, the A. perfoliata ‘ GST-ome’ was observed to be dominated with Mu class GST representatives. In addition, both Sigma and Omega class GSTs were identified, albeit to a lesser extent and absent from affinity chromatography approaches. Moreover, 51 ApGSTs were localized across somatic (47 GSTs), extracellular vesicles (EVs) (Whole: 1 GST, Surface: 2 GSTs) and EV depleted excretory secretory product (ESP) (9 GSTs) proteomes. In related helminths, GSTs have shown promise as novel anthelmintic or vaccine targets for improved helminth control. Thus, provides potential targets for understanding A. perfoliata novel infection mechanisms, host–parasite relationships and anthelmintic treatments.

Background: Serotonin syndrome is an acute, life-threatening illness characterized by mental status changes, neuromuscular symptoms, and autonomic instability. Some patients taking serotonergic antidepressants have been noted to have unexplained mental status changes and/or neuromuscular changes without autonomic instability raising the possibility of a more chronic or attenuated form of serotonin syndrome., Objective: Assessment of antidepressant blood levels to support the diagnosis of a subacute serotonin syndrome., Methods: At a tertiary psychiatric outpatient clinic, patients with unexplained mental status and/or neuromuscular changes without autonomic instability had antidepressant blood levels assessed., Results: Eleven patients were identified with signs and symptoms partially consistent with serotonin syndrome. Nine patients had cognitive changes, while four patients had motor changes, and three patients had psychosis. All patients had elevated blood levels of a single serotonergic antidepressant. Limited follow-up suggests that symptoms improve with reduction of antidepressant medication., Conclusions: These cases suggest that a more chronic, attenuated form of serotonin syndrome exists. Diagnostic criteria are proposed for a distinct clinical entity: subacute serotonin syndrome (SSS). Further research is required to validate these criteria. Clinicians should consider drawing antidepressant levels for patients with symptoms and signs suggestive of SSS-especially those at increased vulnerability for excessive serotonergic agonism. Given the high prevalence of antidepressant medication use, the awareness of SSS could lead to improved patient outcomes and public health., (Copyright © 2023 Academy of Consultation-Liaison Psychiatry. Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way., (© 2023. The Author(s).)

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5-mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2-ERK1/2 interactions., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The role of cyclin-dependent kinases (CDKs) that are ubiquitously expressed in the adult nervous system remains unclear. Cdk12 is enriched in terminally differentiated neurons where its conical role in the cell cycle progression is redundant. We find that in adult neurons Cdk12 acts a negative regulator of actin formation, mitochondrial dynamics and neuronal physiology. Cdk12 maintains the size of the axon at sites proximal to the cell body through the transcription of homeostatic enzymes in the 1-carbon by folate pathway which utilize the amino acid homocysteine. Loss of Cdk12 leads to elevated homocysteine and in turn leads to uncontrolled F-actin formation and axonal swelling. Actin remodeling further induces Drp1-dependent fission of mitochondria and the breakdown of axon-soma filtration barrier allowing soma restricted cargos to enter the axon. We demonstrate that Cdk12 is also an essential gene for long-term neuronal survival and loss of this gene causes age-dependent neurodegeneration. Hyperhomocysteinemia, actin changes, and mitochondrial fragmentation are associated with several neurodegenerative conditions such as Alzheimer's disease and we provide a candidate molecular pathway to link together such pathological events., (© 2023. Cell Death Differentiation Association (ADMC).)

Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner., (© 2023. The Author(s).)

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi-pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)-derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC-derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1-specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co-localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1, also known as MCOLN1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late-onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that induced pluripotent stem cell (iPSC)-derived human cortical neurons expressing APOE ε4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2 via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Neural stem cells (NSCs) are considered as candidates for cell replacement therapy in many neurological disorders. However, the propensity for their differentiation to proceed more glial rather than neuronal phenotypes in pathological conditions limits positive outcomes of reparative transplantation. Exogenous physical stimulation to favor the neuronal differentiation of NSCs without extra chemical side effect could alleviate the problem, providing a safe and highly efficient cell therapy to accelerate neurological recovery following neuronal injuries., Results: With 7-day physiological electric field (EF) stimulation at 100 mV/mm, we recorded the boosted neuronal differentiation of NSCs, comparing to the non-EF treated cells with 2.3-fold higher MAP2 positive cell ratio, 1.6-fold longer neuronal process and 2.4-fold higher cells ratio with neuronal spontaneous action potential. While with the classical medium induction, the neuronal spontaneous potential may only achieve after 21-day induction. Deficiency of either PI3Kγ or β-catenin abolished the above improvement, demonstrating the requirement of the PI3K/Akt/GSK-3β/β-catenin cascade activation in the physiological EF stimulation boosted neuronal differentiation of NSCs. When transplanted into the spinal cord injury (SCI) modelled mice, these EF pre-stimulated NSCs were recorded to develop twofold higher proportion of neurons, comparing to the non-EF treated NSCs. Along with the boosted neuronal differentiation following transplantation, we also recorded the improved neurogenesis in the impacted spinal cord and the significantly benefitted hind limp motor function repair of the SCI mice., Conclusions: In conclusion, we demonstrated physiological EF stimulation as an efficient method to boost the neuronal differentiation of NSCs via the PI3K/Akt/GSK-3β/β-catenin activation. Pre-treatment with the EF stimulation induction before NSCs transplantation would notably improve the therapeutic outcome for neurogenesis and neurofunction recovery of SCI., (© 2023. The Author(s).)

Background: Clozapine must be retitrated after 2 consecutive days or more of missed doses owing to the risk of severe hypotension, bradycardia, and cardiac arrest. However, other important adverse events such as somnolence, sialorrhea, or respiratory depression can occur without severe cardiovascular sequalae. These other unintended consequences are not well characterized in the literature. Three cases are reported, highlighting the concerns for continuing clozapine without retitration after periods of not taking the medication. Implications are discussed as well as how pharmacists can collaborate with other disciplines to mitigate safety risks associated with clozapine for hospitalized patients., Case Summaries: The first case highlights the importance of medication reconciliation and verifying adherence before clozapine continuation in the hospital. Waiting for collateral information and missing one dose are safer than unknowingly resuming clozapine. The second case suggests that it may be safer to consider patients with unexplained worsening psychiatric symptoms as nonadherent and even partially reduced clozapine doses after nonadherence may be unsafe. The final case demonstrates the importance assessing comedications (e.g., warfarin, phenytoin) that have available therapeutic drug monitoring to suggest nonadherence. Each case resulted in significant adverse events requiring transfer to a higher level of care or prolonged hospitalization., Practice Implications: Continuation of psychiatric medications when a patient is admitted to the hospital is important to prevent worsening of symptoms. However, assessment of clozapine adherence and confidence in that assessment is crucial to prevent clozapine intoxication, severe hypotension, and even death. Pharmacists are uniquely positioned to assess clozapine adherence and ensure patient safety. A hospital-based service was created at a 2000-bed academic medical center to improve transitions of care when patients are admitted with clozapine. The process was created in collaboration with the psychiatric consultation service. Through this process, pharmacists also complete appropriate hematologic monitoring and ongoing clinical monitoring for adverse events., (Copyright © 2022 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved., (© 2022 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The age at onset of motor symptoms in Huntington's disease (HD) is driven by HTT CAG repeat length but modified by other genes. In this study, we used exome sequencing of 683 patients with HD with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We discovered damaging coding variants in candidate modifier genes identified in previous genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier-onset HD. Nuclease activities of purified variants in vitro correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion similar to those observed with complete FAN1 knockout. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD., (© 2022. The Author(s).)

Objective: This study investigated the occurrence of postictal agitation (PA) in patients undergoing an acute series of electroconvulsive therapy (ECT) and further explored patient and treatment variables associated with PA., Methods: Charts were retrospectively searched for patients undergoing an acute series of ECT. Postictal agitation was identified by the administration of a sedative after ECT. Demographic, diagnostic, medication, and ECT variables that could also be associated with PA were collected and accounted for in statistical analysis., Results: In this population, 22 of 156 patients experienced PA. Associations that reached statistical significance included sex, weight, active substance use disorder, seizure duration (as observed by motor movements), and waking time. Only seizure duration and waking time maintained significance after multivariable analysis., Conclusions: These data identify clinical factors that could help predict PA. Patients with greater weight, male sex, or an active substance use disorder ought to be carefully monitored for PA, and staff in the recovery suite should be especially vigilant about such patients with longer seizures and waking times., Competing Interests: Conflicts of Interest: None. The authors have no conflicts of interest or financial disclosures to report., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Variants identified in genome-wide association studies have implicated immune pathways in the development of Alzheimer's disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant's role in macrophages and microglia of newly generated PLCG2-R522-expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc-receptor ligation or exposure to Aβ oligomers. Expression of the PLCγ2-R522 variant resulted in increased stimulus-dependent PIP 2 depletion and reduced basal PIP 2 levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD-related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

CAG repeat expansion in the HTT gene drives Huntington's disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion., Competing Interests: Declaration of interests A patent (application number 2105484.6) on the FAN1-MLH1 interaction and structural analogs for the treatment of disease has been filed by the University of Cambridge and UCL. The data presented in this patent are included in the main paper and supplemental information. G.B. is a co-founder and consultant for Adrestia Therapeutics. E.L.B. is the daughter of an advisor for Adrestia Therapeutics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In development, differentiation from a pluripotent state results in global epigenetic changes, although the extent to which this occurs in induced pluripotent stem cell-based neuronal models has not been extensively characterized. In the present study, induced pluripotent stem cell colonies (33Qn1 line) were differentiated and collected at four time-points, with DNA methylation assessed using the Illumina Infinium Human Methylation EPIC BeadChip array. Dynamic changes in DNA methylation occurring during differentiation were investigated using a data-driven trajectory inference method. We identified a large number of Bonferroni-significant loci that showed progressive alterations in DNA methylation during neuronal differentiation. A gene-gene interaction network analysis identified 60 densely connected genes that were influential in the differentiation of neurons, with STAT3 being the gene with the highest connectivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with several of the authors JI, EP, TK, AJ, JB, KL, at time of review., (Copyright © 2021 Imm, Pishva, Ali, Kerrigan, Jeffries, Burrage, Glaab, Cope, Jones, Allen and Lunnon.)

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

Primary care physicians (PCPs) are often asked to perform disability evaluations for patients with psychiatric disorders, which are now a leading cause of disability worldwide. After acknowledging the limitations of disability assessments for all conditions, this review aims to provide PCPs with practical knowledge to inform their assessments and interventions with a focus on patients with depression. After the disability definitions and programs in the United States are reviewed, a pragmatic approach to assessing function and discussing return to work is offered. Individualized assessment is key, and functional recovery rather than symptom relief should be prioritized. Finally, evidence-based interventions for enhancing the likelihood of return to work are considered. We believe the principles of functional assessment and recovery lend themselves to ready adaptation for use in other psychiatric conditions and chronic somatic syndromes, including chronic pain. The key principles of this approach are as follows: 1) a patient is not categorically disabled, but has specific limitations in specific contexts; 2) graded, work-oriented rehabilitation with tailored problem-solving strategies are essential; 3) involving a multidisciplinary team in coordinated care optimizes functional recovery; 4) return to work is an iterative process aimed at restoring meaningful function in a stepwise fashion; and 5) the relationship between symptoms and function is bidirectional. PCPs can use these principles to plan optimal recovery paths for psychiatrically ill patients presenting with a wide array of biopsychosocial realities., (Copyright © 2020. Published by Elsevier Inc.)

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely "high", "intermediate" and "low" firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology.

Good's buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca 2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca 2+ . Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function., Competing Interests: No competing interests were disclosed.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1 + neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Mirtazapine is generally well tolerated in medically ill patients with and without formal psychiatric comorbidity to target sleep, appetite, nausea, and pain. However, there is little data regarding mirtazapine's potential to prolong the corrected QT interval (QTc) in this population., Methods: From a retrospective cohort of patients hospitalized on a variety of medical units for whom a psychiatric consult recommended mirtazapine, electrocardiogram (ECG) data were extracted for ECGs obtained up to 3 days before and 6 days after the initial consult. Descriptive statistics were used to characterize the QTc changes and adverse cardiac outcomes, including incident ventricular tachycardia, torsades de pointes, and sudden cardiac death. Multiple linear regression models were completed to assess the effect of potential confounding variables on QTc changes., Results: Complete premirtazapine and postmirtazapine ECG data were available for 61 patients, and the average change in QTc was -0.31 ms (SD = 36.62 ms). No incidental adverse cardiac outcomes were found. QTc changes were not significantly affected by patient age and sex, initial and maximum mirtazapine dose, days between ECGs, number of concomitant QTc prolonging medications, Charlson comorbidity scores, and electrolyte abnormalities. Due to incomplete potassium, magnesium, and ionized calcium data, electrolytes were excluded from the final regression model., Discussion: Despite the limitations of this retrospective study, these data suggest that modest doses of mirtazapine may not significantly affect the QTc in medically ill patients. Retrospective cohorts are more feasibly analyzed, but prospective controlled trials could more systematically assess QTc changes with higher doses of mirtazapine in medical settings., Competing Interests: Disclosures: None., (© 2020 CPNP. The Mental Health Clinician is a publication of the College of Psychiatric and Neurologic Pharmacists.)

Late onset Alzheimer's disease is the most common form of dementia for which about 30 susceptibility loci have been reported. The aim of the current study is to identify novel genes associated with Alzheimer's disease using the largest up-to-date reference single nucleotide polymorphism (SNP) panel, the most accurate imputation software and a novel gene-based analysis approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 million genotypes from 17,008 Alzheimer's cases and 37,154 controls. In addition to earlier reported genes, we detected three novel gene-wide significant loci PPARGC1A (p = 2.2 × 10-6), RORA (p = 7.4 × 10-7) and ZNF423 (p = 2.1 × 10-6). PPARGC1A and RORA are involved in circadian rhythm; circadian disturbances are one of the earliest symptoms of Alzheimer's disease. PPARGC1A is additionally linked to energy metabolism and the generation of amyloid beta plaques. RORA is involved in a variety of functions apart from circadian rhythm, such as cholesterol metabolism and inflammation. The ZNF423 gene resides in an Alzheimer's disease-specific protein network and is likely involved with centrosomes and DNA damage repair., Competing Interests: The authors have declared that no competing interests exist.

In Huntington's disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K + currents, lengthened spontaneous Ca 2+ waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets.

Nucleosome positioning is important for neurodevelopment, and genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. To investigate changes in nucleosome positioning during neural differentiation, we generate genome-wide nucleosome maps from an undifferentiated human-induced pluripotent stem cell (hiPSC) line and after its differentiation to the neural progenitor cell (NPC) stage. We find that nearly 3% of nucleosomes are highly positioned in NPC, but significantly, there are eightfold fewer positioned nucleosomes in pluripotent cells, indicating increased positioning during cell differentiation. Positioned nucleosomes do not strongly correlate with active chromatin marks or gene transcription. Unexpectedly, we find a small population of nucleosomes that occupy similar positions in pluripotent and neural progenitor cells and are found at binding sites of the key gene regulators NRSF/REST and CTCF Remarkably, the presence of these nucleosomes appears to be independent of the associated regulatory complexes. Together, these results present a scenario in human cells, where positioned nucleosomes are sparse and dynamic, but may act to alter gene expression at a distance via the structural conformation at sites of chromatin regulation., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Benzodiazepines are the conventional mainstay to manage alcohol withdrawal; however, patients are subsequently at increased risk for poor sleep, cravings, and return to drinking. Research on alternative pharmacologic agents to facilitate safe alcohol withdrawal is scant. Gabapentin is one medication shown in small studies to reduce the need for benzodiazepines in the setting of alcohol withdrawal. The continuation of gabapentin after alcohol withdrawal appears to be safe during early sobriety and may aid in reducing alcohol-related cravings or returning to alcohol consumption. Use of a gabapentin-based, benzodiazepine-sparing protool began in early 2015 by the Mayo Clinic, Rochester, Consultation-Liaison Psychiatry Service., Objective: A retrospective chart review was conducted to detect any safety concerns with use of a gabapentin protocol for alcohol withdrawal syndrome., Methods: Secondary outcomes were derived by comparing a matched cohort of patients who received benzodiazepines for alcohol withdrawal syndrome., Results: Seventy-seven patients had their alcohol withdrawal managed via a gabapentin protocol during the study period. No patients required transfer to a higher level of care or had a documented withdrawal seizure. Length of stay between the gabapentin protocol group and benzodiazepine group were similar., Conclusion: This preliminary data has supported the frequent use of this protocol in the general internal medicine practice and formalization of an institutional order set of this protocol for mild to moderate alcohol withdrawal syndrome. Prospective studies are required to validate findings., (Copyright © 2018 Academy of Consultation-Liaison Psychiatry. Published by Elsevier Inc. All rights reserved.)

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t 1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation., Competing Interests: Conflict of interest statement: This work was supported by Zebra Biologics. P.S.D. and R.M.L. are affiliated with Zebra Biologics., (Copyright © 2018 the Author(s). Published by PNAS.)

Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABA A receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Producing induced pluripotent stem cells (iPSCs) from human tissue for use in personalized medicine strategies or therapeutic testing is at the forefront of medicine. Therefore, identifying a source of cells to reprogram that is easily accessible via a simple noninvasive procedure is of great clinical importance. Reprogramming these cells to iPSCs through nonintegrating methods for genetic manipulation is paramount for regenerative purposes. Here, we demonstrate reprogramming of oral mucosal lamina propria progenitor cells from patients undergoing routine dental treatment. Reprogramming was performed utilizing nonintegrating plasmids containing all 6 pluripotency genes (OCT4, SOX2, KLF4, NANOG, LIN28, and cMYC). Resulting iPSCs lacked genetic integration of the vector genes and had the ability to differentiate down mesoderm, ectoderm, and endoderm lineages, demonstrating pluripotency. In conclusion, oral mucosal lamina propria progenitor cells represent a source of cells that can be obtained with minimal invasion, as they can be taken concurrently with routine treatments. The resulting integration-free iPSCs therefore have great potential for use in personalized medicine strategies., (© International & American Associations for Dental Research 2016.)

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease.

Background: With a complex pharmacologic profile, mirtazapine may promote sleep, stimulate appetite, improve nausea, and reduce pain. Some practitioners working on the Mayo Clinic inpatient psychiatric consultation/liaison service have recommended mirtazapine in medically ill patients with or without formal psychiatric comorbidity to target these symptoms., Objective: To assess the success of this practice, we conducted a retrospective chart review covering a 4.5-year period., Methods: For patients recommended to start mirtazapine, global improvement in specific symptoms and suspected side effects were recorded., Results: During the study period, 528 medically ill patients started mirtazapine following a recommendation from the psychiatric consultation service. In total, 475 patients were provided mirtazapine to specifically target sleep, nausea, pain, or appetite. There was documented improvement in these symptoms for 37.7%, 37.0%, 36.4%, and 23.5% of the patients, respectively. These rates of improvement are conservative for the 229 patients without documented response, i.e., 48% of the patients who were given the medication for a somatic symptom were counted as having no improvement. Commonly documented adverse effects were daytime sedation (5.3%), worsening mental status (2.3%), and nightmares (1%)., Conclusions: Despite the limitations of this retrospective, qualitative study, these data confirm that mirtazapine is generally well tolerated and can provide at least short-term relief of certain symptoms in medically ill patients. Controlled trials are needed to assess these benefits more systematically, and it is not clear how long mirtazapine should be used for these symptoms., (Published by Elsevier Inc.)

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens., (Copyright © 2016 the American Physiological Society.)

There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington's disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1-expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation.

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

A scalable and cost-effective synthetic polymer substrate that supports robust expansion and subsequent multilineage differentiation of human pluripotent stem cells (hPSCs) with defined commercial media is presented. This substrate can be applied to common cultureware and used off-the-shelf after long-term storage. Expansion and differentiation of hPSCs are performed entirely on the polymeric surface, enabling the clinical potential of hPSC-derived cells to be realized., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bi-layered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular 'bystander signalling' both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development.