Fazal Oozeer

Drebrin modulates F-actin networks and links them to other intracellular components, regulating crucial processes including neuritogenesis, synaptic plasticity, virus internalisation and cancer invasion. Using single-particle cryo-EM we characterise drebrin’s interaction with F-actin through two separate conserved actin binding domains (ABD1 and ABD2), revealing structural bases for its F-actin-modulating properties. We describe a multimodal interaction where drebrin’s ABD1 can adopt two conformations and a long flexible loop connecting to ABD2 allows the two ABDs to occupy multiple relative positions along F-actin. The flexible loop connecting the two ABDs also confers some propensity to loosely bundle F-actin. Drebrin’s ABDs bind across multiple actin protomers and their subdomains and modify the longitudinal inter-protomer interface, explaining its F-actin stabilising properties. Furthermore, we show drebrin’s binding site on F-actin is shared with other critical actin-binding and regulatory proteins, explaining their competitive displacement.

Neuronal activity must be regulated in a narrow permissive band for the proper operation of neural networks. Changes in synaptic connectivity and network activity—for example, during learning—might disturb this balance, eliciting compensatory mechanisms to maintain network function^1–3. In the neocortex, excitatory pyramidal cells and inhibitory interneurons exhibit robust forms of stabilizing plasticity. However, although neuronal plasticity has been thoroughly studied in pyramidal cells^4–8, little is known about how interneurons adapt to persistent changes in their activity. Here we describe a critical cellular process through which cortical parvalbumin-expressing (PV⁺) interneurons adapt to changes in their activity levels. We found that changes in the activity of individual PV⁺ interneurons drive bidirectional compensatory adjustments of the number and strength of inhibitory synapses received by these cells, specifically from other PV⁺ interneurons. High-throughput profiling of ribosome-associated mRNA revealed that increasing the activity of a PV⁺ interneuron leads to upregulation of two genes encoding multiple secreted neuropeptides: Vgf and Scg2. Functional experiments demonstrated that VGF is critically required for the activity-dependent scaling of inhibitory PV⁺ synapses onto PV⁺ interneurons. Our findings reveal an instructive role for neuropeptide-encoding genes in regulating synaptic connections among PV⁺ interneurons in the adult mouse neocortex.

Cortical neurons are specified during embryonic development but often only acquire their mature properties at relatively late stages of postnatal development. This delay in terminal differentiation is particularly prominent for fast-spiking parvalbumin-expressing (PV+) interneurons, which play critical roles in regulating the function of the cerebral cortex. We found that the maturation of PV+ interneurons is triggered by neuronal activity and mediated by the transcriptional cofactor peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). Developmental loss of PGC-1α prevents PV+ interneurons from acquiring unique structural, electrophysiological, synaptic, and metabolic features and disrupts their diversification into distinct subtypes. PGC-1α exerts its function as a master regulator of the differentiation of PV+ interneurons by directly controlling gene expression through a transcriptional complex that includes ERRγ and Mef2c. Our results uncover a molecular switch that translates neural activity into a specific transcriptional program promoting the maturation of PV+ interneurons at the appropriate developmental stage.

Neuronal activity is regulated in a narrow permissive band for the proper operation of neural networks. Changes in synaptic connectivity and network activity, for example, during learning, might disturb this balance, eliciting compensatory mechanisms to maintain network function. In the neocortex, excitatory pyramidal cells and inhibitory interneurons exhibit robust forms of stabilising plasticity. However, while neuronal plasticity has been thoroughly studied in pyramidal cells, little is known about how interneurons adapt to persistent changes in their activity. Here we uncover the critical cellular and molecular mechanisms through which cortical parvalbumin-expressing (PV+) interneurons adapt to changes in their activity levels. We found that changes in the activity of PV+ interneurons drive cell-autonomous, bi-directional compensatory adjustments of the number and strength of inhibitory synapses received by these cells, specifically from other PV+ interneurons. High-throughput profiling of ribosome-associated mRNA revealed that increasing the activity of PV+ interneurons leads to the cell-autonomous upregulation of two genes encoding multiple secreted neuropeptides, Vgf and Scg2 . Functional experiments demonstrated that VGF is critically required for the activity-dependent scaling of inhibitory PV+ synapses onto PV+ interneurons. Our findings reveal an instructive role for neuropeptide-encoding genes in regulating synaptic connections among PV+ interneurons in the adult mouse neocortex.

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV ⁺ ) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type–specific and synapse type–specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV ⁺ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.

Significance: Brain function requires appropriate numbers of different neuronal subtypes, but how these numbers are established remains poorly understood. Here, we identified a key role for the cerebral cortex in remotely controlling interneuron survival and thus establishing appropriate numbers of the two main types of interneurons in another brain region, the striatum. While cortical pyramidal cells directly control the survival of parvalbumin-expressing GABAergic neurons, the survival of cholinergic interneurons is indirectly controlled through the activity of the striatal medium spiny neurons. Our results demonstrate that the input-specific modulation of neuronal activity is universally required to control the survival of interneurons across very different brain circuits. This is important for the assembly of balanced neural networks.

Co‐ordinating the dynamic behaviour of actin filaments (F‐actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill‐defined. The drebrin/end‐binding protein 3 (EB3) pathway is a candidate pathway for linking F‐actin to microtubules in filopodia. Drebrin binds F‐actin and, simultaneously, the microtubule‐binding protein EB3 when bound to microtubule plus‐ends. We assessed the effect on neuritogenesis of gain‐ or loss‐of‐function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss‐of‐function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F‐actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over‐expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end‐binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over‐expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus‐ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F‐actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus‐ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F‐actin zippering in filopodia orchestrated by the drebrin/EB3 pathway. image

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones towards a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms. Impact statement: A novel genetic strategy for induction of reproducible neural tumors (or any other deleterious phenotype) in a single Drosophila stock (applicable to other organisms).