PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522.

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 Aß oligomers. Expression of the PLCγ2-R522 variant resulted in increased stimulus-dependent PIP2 depletion and reduced basal PIP2 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.

The synthetic TRPML1 agonist ML-SA1 rescues Alzheimer-related alterations of the endosomal-autophagic-lysosomal system.

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.

Soluble mutant huntingtin drives early human pathogenesis in Huntington's disease.

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.

Complement receptor 1 is expressed on brain cells and in the human brain.

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.

Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

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.

Iatrogenic clozapine intoxication after hospital admission: A case-based rationale for an inpatient pharmacy clozapine monitoring service.

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.

Factors Associated With Postictal Agitation After Electroconvulsive Therapy.

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.

Nucleosome dynamics of human iPSC during neural differentiation.

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.

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library.

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 t1/2 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.

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

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.

Influence of Pressure on the Composition of Gaseous Reference Materials.

We have shown that the amount fraction of carbon dioxide in a nitrogen or synthetic air matrix stored in cylinders increases as the pressure of the gas mixture reduces, while the amount fraction of methane remains unchanged. Our measurements show the initial amount fraction of carbon dioxide to be lower than the gravimetric value after preparation, which we attribute to the adsorption of a proportion of the molecules to active sites on the internal surface of the cylinder and the valve. As the mixture is consumed, the pressure in the cylinder reduces and the amount fraction of the component is observed to increase. The effect is less pronounced in the presence of water vapor. More dramatic effects have been observed for hydrogen chloride. These findings have significant implications for the preparation of high accuracy gaseous reference materials with unprecedented uncertainties which underpin a broad range of requirements, in particular atmospheric monitoring of high impact greenhouse gases.

Use of a Gabapentin Protocol for the Management of Alcohol Withdrawal: A Preliminary Experience Expanding From the Consultation-Liaison Psychiatry Service.

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.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

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.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

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 GABAA 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.

Mirtazapine for Symptomatic Relief on a Psychiatric Consultation Service: A Case Series.

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.

Early transcriptional changes linked to naturally occurring Huntington's disease mutations in neural derivatives of human embryonic stem cells.

Huntington's disease (HD) is characterized by a late clinical onset despite ubiquitous expression of the mutant gene at all developmental stages. How mutant huntingtin impacts on signalling pathways in the pre-symptomatic period has remained essentially unexplored in humans due to a lack of appropriate models. Using multiple human embryonic stem cell lines derived from blastocysts diagnosed as carrying the mutant huntingtin gene by pre-implantation genetic diagnosis, we explored early developmental changes in gene expression using differential transcriptomics, combined with gain and loss of function strategies. We demonstrated a down-regulation of the HTT gene itself in HD neural cells and identified three genes, the expression of which differs significantly in HD cells when compared with wild-type controls, namely CHCHD2, TRIM4 and PKIB. Similar dysregulation had been observed previously for CHCDH2 and TRIM4 in blood cells from patients. CHCHD2 is involved in mitochondrial function and PKIB in protein kinase A-dependent pathway regulation, which suggests that these functions may be precociously impacted in HD.

A Case Series of 11 Patients With Subacute Serotonin Syndrome.

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.