Global quantitative proteomic analysis of aged mouse hippocampus.

Understanding the molecular changes associated with the aged brain forms the basis for developing potential strategies for slowing cognitive decline associated with normal aging. Focusing on the hippocampus, a critical brain region involved in learning and memory, we employed tandem mass tag methodology to investigate global proteomic changes that occur in advanced-aged (20-month) versus young (3-month) C57BL/6 male mice. Our analysis revealed the upregulation of 236 proteins in the old hippocampal proteome, including those enriched within several age-related processes, such as the adaptive immune response and molecular metabolic pathways, whereas downregulated proteins (88 in total) are mainly involved in axonogenesis and growth cone-related processes. Categorizing proteins by cell-type enrichment in the brain identified a general upregulation of proteins preferentially expressed in microglia, astrocytes, and oligodendrocytes. In contrast, proteins with neuron-specific expression displayed an overall age-related downregulation. By integrating our proteomic with our previously published transcriptomic data, we discovered a mild but significant positive correlation between mRNA and protein expression changes in the aged hippocampus. Therefore, this proteomic data is a valuable additional resource for further understanding age-related molecular mechanisms.

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation.

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

SNAP-25 phosphorylation at Ser187 is not involved in Ca2+ or phorbolester-dependent potentiation of synaptic release.

SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch clamp recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca2+-independent or Ca2+-dependent replenishment. Furthermore, it did not affect the response to phorbol esters, which activate PKC. Expressing S187C in the context of the SNAP-25A isoform also did not affect synaptic transmission. Strikingly, the - potentially phosphomimetic - mutant S187E reduced spontaneous release and release probability, with the largest effect seen in the SNAP-25B isoform, showing that a negative charge in this position is detrimental for neurotransmission, in agreement with electrostatic fusion triggering. During the course of our experiments, we found that higher SNAP-25B expression levels led to decreased paired pulse potentiation, probably due to higher release probabilities. Under these conditions, the potentiation of evoked EPSCs by phorbol esters was followed by a persistent down-regulation, probably due to a ceiling effect. In conclusion, our results indicate that phosphorylation of Ser-187 in SNAP-25 is not involved in modulation of synaptic release by Ca2+ or phorbol esters.

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm.

Phosphorylation of syndapin I F-BAR domain at two helix-capping motifs regulates membrane tubulation.

Syndapin I (PACSIN 1) is a synaptically enriched membrane tubulating protein that plays important roles in activity-dependent bulk endocytosis and neuronal morphogenesis. While syndapin I is an in vitro phosphoprotein, it is not known to be phosphorylated in neurons. Here, we report the identification of two phosphorylation sites, S76 and T181, of syndapin I from nerve terminals. Both residues are located at the N-terminal helix-capping motifs (N-Cap) of different α-helices in the F-BAR domain, important for F-BAR homodimer curvature and dimer-dimer filament assembly, respectively. Phospho-mimetic mutations of these residues regulate lipid-binding and tubulation both in vitro and in cells. Neither phosphosite regulated syndapin I function in activity-dependent bulk endocytosis. Rather, T181 phosphorylation was developmentally regulated and inhibited syndapin I function in neuronal morphogenesis. This suggests a novel mechanism for phosphorylation control of an F-BAR function through the regulation of α-helix interactions and stability within the folded F-BAR domain.

A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

Multiomics of early epileptogenesis in mice reveals phosphorylation and dephosphorylation-directed growth and synaptic weakening.

To investigate the phosphorylation-based signaling and protein changes occurring early in epileptogenesis, the hippocampi of mice treated with pilocarpine were examined by quantitative mass spectrometry at 4 and 24 h post-status epilepticus at vast depth. Hundreds of posttranscriptional regulatory proteins were the major early targets of increased phosphorylation. At 24 h, many protein level changes were detected and the phosphoproteome continued to be perturbed. The major targets of decreased phosphorylation at 4 and 24 h were a subset of postsynaptic density scaffold proteins, ion channels, and neurotransmitter receptors. Many proteins targeted by dephosphorylation at 4 h also had decreased protein abundance at 24 h, indicating a phosphatase-mediated weakening of synapses. Increased translation was indicated by protein changes at 24 h. These observations, and many additional indicators within this multiomic resource, suggest that early epileptogenesis is characterized by signaling that stimulates both growth and a homeostatic response that weakens excitability.

Ketogenic diet modifies ribosomal protein dysregulation in KMT2D Kabuki syndrome.

BACKGROUND: Kabuki syndrome (KS) is a genetic disorder caused by DNA mutations in KMT2D, a lysine methyltransferase that methylates histones and other proteins, and therefore modifies chromatin structure and subsequent gene expression. Ketones, derived from the ketogenic diet, are histone deacetylase inhibitors that can 'open' chromatin and encourage gene expression. Preclinical studies have shown that the ketogenic diet rescues hippocampal memory neurogenesis in mice with KS via the epigenetic effects of ketones. METHODS: Single-cell RNA sequencing and mass spectrometry-based proteomics were used to explore molecular mechanisms of disease in individuals with KS (n = 4) versus controls (n = 4). FINDINGS: Pathway enrichment analysis indicated that loss of function mutations in KMT2D are associated with ribosomal protein dysregulation at an RNA and protein level in individuals with KS (FDR <0.05). Cellular proteomics also identified immune dysregulation and increased abundance of other lysine modification and histone binding proteins, representing a potential compensatory mechanism. A 12-year-old boy with KS, suffering from recurrent episodes of cognitive decline, exhibited improved cognitive function and neuropsychological assessment performance after 12 months on the ketogenic diet, with concomitant improvement in transcriptomic ribosomal protein dysregulation. INTERPRETATION: Our data reveals that lysine methyltransferase deficiency is associated with ribosomal protein dysfunction, with secondary immune dysregulation. Diet and the production of bioactive molecules such as ketone bodies serve as a significant environmental factor that can induce epigenetic changes and improve clinical outcomes. Integrating transcriptomic, proteomic, and clinical data can define mechanisms of disease and treatment effects in individuals with neurodevelopmental disorders. FUNDING: This study was supported by the Dale NHMRC Investigator Grant (APP1193648) (R.D), Petre Foundation (R.D), and The Sydney Children's Hospital Foundation/Kids Research Early and Mid-Career Researcher Grant (E.T).

Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.

Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.

Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity.

A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.

Kruppel-associated Box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1ß (HP1-ß) mobilization and DNA repair in heterochromatin.

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-ß chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-ß, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-ß from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-ß exchange from heterochromatin, promoting DNA repair.

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles.

Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.

Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis.

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

Autophosphorylation and ATM activation: additional sites add to the complexity.

The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser(367), Ser(1893), and Ser(1981)) and acetylation on Lys(3016). We now describe the identification of a new ATM phosphorylation site, Thr(P)(1885) and an additional autophosphorylation site, Ser(P)(2996), that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser(367) and Ser(2996), for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)(1981), ATM phosphorylated at Ser(367) and Ser(2996) localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser(367) and Ser(2996) was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis.

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked ß-N-acetylglucosaminyl-6-phosphate modification.

A novel post-translational modification of threonine, ß-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.

Fludarabine nucleoside induces major changes in the p53 interactome in human B-lymphoid cancer cell lines.

Triple combination FCR (fludarabine, cyclophosphamide and rituximab) is often used as front-line treatment for chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. Results from our laboratory indicate that 2-FaraAMP (fludarabine) has multiple mechanisms of cytotoxicity that include accumulation of isoforms and phosphorylated derivatives of p53, and induction of the unfolded protein response (UPR). Using protein pull-downs with Dynabeads coated with p53 antibody, we have found that 2-FaraA (fludarabine nucleoside) induces major changes in the p53 interactome in human Raji lymphoma and IM9 multiple myeloma cells. These changes are likely driven by DNA strand breaks induced by 2-FaraA that activate protein kinases such as ATM, ATR and Chk1.

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase.

Maintaining an appropriate balance between excitation and inhibition is critical for neuronal information processing. Cortical neurons can cell-autonomously adjust the inhibition they receive to individual levels of excitatory input, but the underlying mechanisms are unclear. We describe that Ste20-like kinase (SLK) mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feedforward circuit, but not in the feedback circuit. This effect is due to regulation of inhibition originating from parvalbumin-expressing interneurons, while inhibition via somatostatin-expressing interneurons is unaffected. Computational modeling shows that this mechanism promotes stable excitatory-inhibitory ratios across pyramidal cells and ensures robust and sparse coding. Patch-clamp RNA sequencing yields genes differentially regulated by SLK knockdown, as well as genes associated with excitation-inhibition balance participating in transsynaptic communication and cytoskeletal dynamics. These data identify a mechanism for cell-autonomous regulation of a specific inhibitory circuit that is critical to ensure that a majority of cortical pyramidal cells participate in information coding.

Differentiation of brain and retinal organoids from confluent cultures of pluripotent stem cells connected by nerve-like axonal projections of optic origin.

Advances in the study of neurological conditions have been possible because of pluripotent stem cell technologies and organoids. Studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids are transcriptionally comparable with organoids generated by other methods. Furthermore, these organoids showed spontaneous functional network activity and proteomic analysis confirmed organoids maturity. The generation of retinal and brain organoids in close proximity enabled their mutual isolation. Suspension culture of this complex organoid system demonstrated the formation of nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of neurological diseases of the eye and brain.