Microtubules are essential for various cell processes  and are nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs) at various microtubule organizing centers (MTOCs), including centrosomes [2-6]. Recruitment of γ-TuRCs to different MTOCs at different times influences microtubule array formation, but how this is regulated remains an open question. It also remains unclear whether all γ-TuRCs within the same organism have the same composition and how any potential heterogeneity might influence γ-TuRC recruitment. MOZART1 (Mzt1) was recently identified as a γ-TuRC component [7, 8] and is conserved in nearly all eukaryotes [6, 9]. Mzt1 has so far been studied in cultured human cells, yeast, and plants; its absence leads to failures in γ-TuRC recruitment and cell division, resulting in cell death [7, 9-15]. Mzt1 is small (∼8.5 kDa), binds directly to core γ-TuRC components [9, 10, 14, 15], and appears to mediate the interaction between γ-TuRCs and proteins that tether γ-TuRCs to MTOCs [9, 15]. Here, we use Drosophila to investigate the function of Mzt1 in a multicellular animal for the first time. Surprisingly, we find that Drosophila Mzt1 is expressed only in the testes and is present in γ-TuRCs recruited to basal bodies, but not to mitochondria, in developing sperm cells. mzt1 mutants are viable but have defects in basal body positioning and γ-TuRC recruitment to centriole adjuncts; sperm formation is affected and mutants display a rapid age-dependent decline in sperm motility and male fertility. Our results reveal that tissue-specific and MTOC-specific γ-TuRC heterogeneity exist in Drosophila and highlight the complexity of γ-TuRC recruitment in a multicellular animal.
Literature context: Fisher Scientific Cat#A-11075; RRID:AB_2534119 Goat anti-Guinea Pig IgG (H+L)
Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.
Literature context: Fisher Scientific Cat# A-11075; RRID:AB_2534119 1:500
Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of the Drosophila visual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated by Drosophila Fezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building layered networks of neural connections.
Literature context: Fisher Scientific Cat# A-11075, RRID:AB_2534119 Goat anti-human IgG Alexa 488 T
Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.
Literature context: uor 568 conjugate, Cat# A-11075 RRID:AB_2534119, Thermo Fisher Scientific, USA)
Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.
Literature context: (A11075, RRID:AB_2534119, all from
Parvalbumin-expressing inhibitory neurons in the mammalian CNS are specialized for fast transmitter release at their output synapses. However, the Ca2+ sensor(s) used by identified inhibitory synapses, including the output synapses of parvalbumin-expressing inhibitory neurons, have only recently started to be addressed. Here, we investigated the roles of Syt1 and Syt2 at two types of fast-releasing inhibitory connections in the mammalian CNS: the medial nucleus of the trapezoid body to lateral superior olive glycinergic synapse, and the basket/stellate cell-Purkinje GABAergic synapse in the cerebellum. We used conditional and conventional knock-out (KO) mouse lines, with viral expression of Cre-recombinase and a light-activated ion channel for optical stimulation of the transduced fibers, to produce Syt1-Syt2 double KO synapses in vivo Surprisingly, we found that KO of Syt2 alone had only minor effects on evoked transmitter release, despite the clear presence of the protein in inhibitory nerve terminals revealed by immunohistochemistry. We show that Syt1 is weakly coexpressed at these inhibitory synapses and must be genetically inactivated together with Syt2 to achieve a significant reduction and desynchronization of fast release. Thus, our work identifies the functionally relevant Ca2+ sensor(s) at fast-releasing inhibitory synapses and shows that two major Syt isoforms can cooperate to mediate release at a given synaptic connection.SIGNIFICANCE STATEMENT During synaptic transmission, the influx of Ca2+ into the presynaptic nerve terminal activates a Ca2+ sensor for vesicle fusion, a crucial step in the activity-dependent release of neurotransmitter. Synaptotagmin (Syt) proteins, and especially Syt1 and Syt2, have been identified as the Ca2+ sensor at excitatory synapses, but the Ca2+ sensor(s) at inhibitory synapses in native brain tissue are not well known. We found that both Syt1 and Syt2 need to be genetically inactivated to cause a significant reduction of activity-evoked release at two types of fast inhibitory synapses in mouse brain. Thus, we identify Syt2 as a functionally important Ca2+ sensor at fast-releasing inhibitory synapses, and show that Syt1 and Syt2 can redundantly control transmitter release at specific brain synapses.
Literature context: # A-11075;RRID:AB_2534119 Goat anti
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.