From the beginning, induced pluripotent stem cell (iPSC) technology was touted as a path to improve our understanding of disease biology and enable drug discovery. Advances in iPSC culture, genome ...engineering, and differentiation protocols have rapidly expanded the use of iPSC-derived disease models from the specialized work of stem cell biology into the mainstream toolkit of cellular neuroscience. Here we provide guidance for using iPSC-derived neurons for disease modeling with a focus on enabling screening platforms amenable to therapeutic drug discovery. We also highlight the potential for incorporating three-dimensional systems that may create more translational in vitro models.
Stem cell biology has become part of the mainstream toolkit of cellular neuroscience. Engle et al. provide guidance on best practices for using iPSC-derived neurons for disease modeling with a focus on enabling screening platforms amenable to therapeutic drug discovery.
Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences ...critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes upregulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologie inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.
Abstract Brain-derived neurotrophic factor (BDNF) signaling is integral to a range of neural functions, including synaptic plasticity and exhibits activity-dependent regulation of expression. As ...altered BDNF signaling has been implicated in multiple psychiatric diseases, here we report a quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNAs encoding TrkB, total BDNF, and the four most abundant BDNF transcripts (I, IIc, IV, and VI) in postmortem tissue from matched tetrads of subjects with schizophrenia, bipolar disorder, or major depressive disorder (MDD) and healthy comparison subjects. In all three regions examined, dorsolateral prefrontal cortex (DLPFC), associative striatum and hippocampus, total BDNF mRNA levels did not differ in any disease state. In DLPFC, BDNF IIc was significantly lower in schizophrenia relative to healthy comparison subjects. In hippocampus, BDNF I, IIc, and VI were lower in subjects with both schizophrenia and bipolar disorder relative to comparison subjects. In striatum, TrkB mRNA was lower in bipolar disorder and MDD, while BDNF IIc was elevated in MDD, relative to comparison subjects. These data highlight potential alterations in BDNF signaling in the corticohippocampal circuit in schizophrenia, and within the striatum in mood disorders. Novel therapies aimed at improving BDNF–TrkB signaling may therefore have potential to impact on a range of psychiatric disorders.
3′,5′-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We ...mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26months) versus young (5months) Sprague–Dawley rats, with select experiments replicated in old (9months) versus young (2months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.
•Each PDE exhibits a unique expression pattern in brain that is highly conserved.•PDE11A, PDE8A, and PDE1C increase and PDE7A, PDE4D, and PDE1B decrease with age.•Age-related changes in PDEs may affect pGluR1, pCaMKII, TARPs and H3K27me3 signals.•Thus, aging is associated with changes in PDE expression and compartmentalization.•These results suggest the effects of certain PDE inhibitors may change with age.
Psychiatric disorders such as schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD) arise from complex interactions between genetic and environmental factors. Common genetic ...variants associated with multiple psychiatric disorders suggest that shared genetic architecture could contribute to divergent clinical syndromes. To evaluate shared transcriptional alterations across connected brain regions, Affymetrix microarrays were used to profile postmortem dorsolateral prefrontal cortex (DLPFC), hippocampus, and associative striatum from 19 well-matched tetrads of subjects with SCZ, BD, MDD, or unaffected controls. SCZ subjects showed a substantial burden of differentially expressed genes across all examined brain regions with the greatest effects in hippocampus, whereas BD and MDD showed less robust alterations. Pathway analysis of transcriptional profiles compared across diagnoses demonstrated commonly enriched pathways between all three disorders in hippocampus, significant overlap between SCZ and BD in DLPFC, but no significant overlap of enriched pathways between disorders in striatum. SCZ samples showed increased expression of transcripts associated with inflammation across all brain regions examined, which was not evident in BD or MDD, or in rat brain following chronic dosing with antipsychotic drugs. Several markers of inflammation were confirmed by RT-PCR in hippocampus, including S100A8/9, IL-6, MAFF, APOLD1, IFITM3, and BAG3. A cytokine ELISA panel showed significant increases in IL-2 and IL-12p70 protein content in hippocampal tissue collected from same SCZ subjects when compared to matched control subjects. These data suggest an overlapping subset of dysregulated pathways across psychiatric disorders; however, a widespread increase in inflammation appears to be a specific feature of the SCZ brain and is not likely to be attributable to chronic antipsychotic drug treatment.
Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in
(c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and ...Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type
in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT.
Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.
•Recent advances in the capability and affordability of digital technology allow for the intensive monitoring of psychological processes in daily life.•Ambulatory assessment is particularly suited to ...measure dynamic “real-world” processes and fluctuations in proximal risk.•We highlight key questions about proximal and distal risk that ambulatory methods allow researchers to evaluate.•These include between-subject questions to understand who is at risk, and within-subject questions to understand when risk is most acute.•We discuss how monitoring proximal risk with AA may be leveraged for translation into personalized, real-time interventions to reduce risk.
Rapid advances in the capability and affordability of digital technology have begun to allow for the intensive monitoring of psychological and physiological processes associated with affective disorders in daily life. This technology may enable researchers to overcome some limitations of traditional methods for studying risk in affective disorders, which often (implicitly) assume that risk factors are distal and static – that they do not change over time. In contrast, ambulatory assessment (AA) is particularly suited to measure dynamic “real-world” processes and to detect fluctuations in proximal risk for outcomes of interest.
We highlight key questions about proximal and distal risk for affective disorders that AA methods (with multilevel modeling, or fully-idiographic methods) allow researchers to evaluate.
Key questions include between-subject questions to understand who is at risk (e.g., are people with more affective instability at greater risk than others?) and within-subject questions to understand when risk is most acute among those who are at risk (e.g., does suicidal ideation increase when people show more sympathetic activation than usual?). We discuss practical study design and analytic strategy considerations for evaluating questions of risk in context, and the benefits and limitations of self-reported vs. passively-collected AA.
Measurements may only be as accurate as the observation period is representative of individuals’ usual life contexts. Active measurement techniques are limited by the ability and willingness to self-report.
We conclude by discussing how monitoring proximal risk with AA may be leveraged for translation into personalized, real-time interventions to reduce risk.
Discovery and development of new medicines requires the talent and passion of both academic and industrial scientists. Identifying the optimal set of circumstances for direct collaboration between ...academic and industry teams requires a mutual understanding of what each partner brings to the relationship, and an appreciation of the specialized capabilities and scope of work to be undertaken by each group. We provide our perspective on the who, what, where, why, and how for establishing therapeutic and translational research collaborations between academic and industry scientists.
Autism spectrum disorder (ASD) is a constellation of neurodevelopmental presentations with high heritability and both phenotypic and genetic heterogeneity. To date, mutations in hundreds of genes ...have been associated to varying degrees with increased ASD risk. A better understanding of the functions of these genes and whether they fit together in functional groups or impact similar neuronal circuits is needed to develop rational treatment strategies. We will review current areas of emphasis in ASD research, starting from human genetics and exploring how mouse models of human mutations have helped identify specific molecular pathways (protein synthesis and degradation, chromatin remodeling, intracellular signaling), which are linked to alterations in circuit function and cognitive/social behavior. We will conclude by discussing how we can leverage the findings on molecular and cellular alterations found in ASD to develop therapies for neurodevelopmental disorders.
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