Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells.

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly.

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Direct activation of KCC2 arrests benzodiazepine refractory status epilepticus and limits the subsequent neuronal injury in mice.

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.

Phosphorylation on Ser-359 of the α2 subunit in GABA type A receptors down-regulates their density at inhibitory synapses.

GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in α4, ß, and γ subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the α2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the α2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic α2-containing GABAAR clusters and caused an absence of α2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the α2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.

Inhibitory Synapse Formation at the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype-the α2-containing receptor-is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.

Identification of a Core Amino Acid Motif within the α Subunit of GABAARs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures.

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), ß(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments.

"If you're gonna help me, help me": Barriers to housing among unsheltered homeless adults.

Despite a legally-mandated right to shelter and extensive outreach efforts, an estimated 3,675 homeless individuals were living on the streets of New York City in 2018. Through interviews with 43 unsheltered homeless individuals in the borough of Manhattan (age range 21-74 years), this qualitative study examined barriers they face in accessing housing and other services as well as experiences surviving on the street. Through thematic analysis of the interview data, the most common barriers found were obtaining required identification documents, lack of accessibility of shelters amid complex healthcare needs, waiting as part of the process, and exclusion of pets from shelters and housing options. Themes capturing survival on the street included sleeping safe, avoiding shelters, and meeting daily needs. Virtually all barriers street homeless New Yorkers face stem from bureaucratic policies that, however well-intentioned, do not address their diverse needs. Thus, long delays and poor communication, combined with crowded, unsafe shelters, lead to frustration and alienation. While homelessness is ultimately the result of a severe and chronic shortage of affordable housing, creating accessible, safe, pet-friendly shelter and safe haven options and instituting a smoother, more transparent process for moving from the streets could substantially reduce street homelessness.

Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin.

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2-1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

A Pharmacogenetic 'Restriction-of-Function' Approach Reveals Evidence for Anxiolytic-Like Actions Mediated by α5-Containing GABAA Receptors in Mice.

Benzodiazepines have been widely used for their anxiolytic actions. However, the contribution of GABAA receptor subtypes to anxiolysis is still controversial. Studies with mutant mice harboring diazepam-insensitive α-subunits α1, α2, α3, or α5 have revealed that α2-containing GABAA receptors (α2-GABAARs) are required for diazepam-induced anxiolysis, with no evidence for an involvement of any other α-subunit, whereas TP003, described as a selective modulator of α3-containing GABAA receptors, was shown to be anxiolytic. Here, we describe a novel, systematic approach to evaluate the role of positive allosteric modulation of each of the four diazepam-sensitive α-subtypes in anxiety-related behavioral paradigms. By combining H to R point mutations in three out of the four diazepam-sensitive α-subunits in mice with a 129X1/SvJ background, diazepam becomes a subtype-specific modulator of the remaining non-mutated α-subtype. Modulation of α5-GABAARs, but not of α2-GABAARs, increased the time in the light side of the light-dark box as well as open-arm exploration in the elevated plus maze. In contrast, modulation of α3-GABAARs decreased open-arm exploration, whereas modulation of α2-GABAARs increased time in the center in the open-field test. Modulation of any single α-subtype had no effect on stress-induced hyperthermia. Our results provide evidence that modulation of α5-GABAARs elicits anxiolytic-like actions, whereas our data do not provide evidence for an anxiolytic-like action of α3-GABAARs. Thus, α5-GABAARs may be suitable targets for novel anxiolytic drugs.

Aggression and increased glutamate in the mPFC during withdrawal from intermittent alcohol in outbred mice.

RATIONALE: Disrupted social behavior, including occasional aggressive outbursts, is characteristic of withdrawal from long-term alcohol (EtOH) use. Heavy EtOH use and exaggerated responses during withdrawal may be treated using glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonists. OBJECTIVES: The current experiments explore aggression and medial prefrontal cortex (mPFC) glutamate as consequences of withdrawal from intermittent access to EtOH and changes in aggression and mPFC glutamate caused by NMDAR antagonists memantine and ketamine. METHODS: Swiss male mice underwent withdrawal following 1-8 weeks of intermittent access to 20 % EtOH. Aggressive and nonaggressive behaviors with a conspecific were measured 6-8 h into EtOH withdrawal after memantine or ketamine (0-30 mg/kg, i.p.) administration. In separate mice, extracellular mPFC glutamate after memantine was measured during withdrawal using in vivo microdialysis. RESULTS: At 6-8 h withdrawal from EtOH, mice exhibited more convulsions and aggression and decreased social contact compared to age-matched water controls. Memantine, but not ketamine, increased withdrawal aggression at the 5-mg/kg dose in mice with a history of 8 weeks of EtOH but not 1 or 4 weeks of EtOH or in water drinkers. Tonic mPFC glutamate was higher during withdrawal after 8 weeks of EtOH compared to 1 week of EtOH or 8 weeks of water. Five milligrams per kilogram of memantine increased glutamate in 8-week EtOH mice, but also in 1-week EtOH and water drinkers. CONCLUSIONS: These studies reveal aggressive behavior as a novel symptom of EtOH withdrawal in outbred mice and confirm a role of NMDARs during withdrawal aggression and for disrupted social behavior.