Microtubule plus-end tracking proteins in neuronal development.

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TIPs) localize to and track with growing microtubule plus-ends in the cell. +TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our single-molecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170-EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5'-[alpha,beta-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.

Microtubule plus-end tracking proteins in differentiated mammalian cells

Development, polarization, structural integrity, and plasticity of neuronal cells critically depend on the microtubule network and its dynamic properties. SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that have been recently identified as regulators of microtubule dynamics. SLAINs are targeted to microtubule tips through an interaction with the core components of microtubule plus-end tracking protein network, End Binding family members. SLAINs promote persistent microtubule growth by recruiting the microtubule polymerase ch-TOG to microtubule plus-ends. Here, we show that SLAIN1/2 and ch-TOG-proteins are highly enriched in brain and are expressed throughout mouse brain development. Disruption of the SLAIN-ch-TOG complex in cultured primary rat hippocampal neurons by RNA interference-mediated knockdown and a dominant-negative approach perturbs microtubule growth by increasing catastrophe frequency and inhibits axon extension during neuronal development. Our study shows that proper control of microtubule dynamics is important for axon elongation in developing neurons.

The role of the drebrin/EB3/Cdk5 pathway in dendritic spine plasticity, implications for Alzheimer's disease.

Centralspindlin, a complex of the kinesin-6-family member MKLP1 and MgcRacGAP (also known as Kif23 and Racgap1, respectively), is required for cytokinesis and cell-cell junctions. During anaphase, Centralspindlin accumulates at overlapping central spindle microtubules and directs contractile ring formation by recruiting the GEF Ect2 to the cell equator to activate RhoA. We found that MgcRacGAP localized to the plus ends of equatorial astral microtubules during cytokinesis in Xenopus laevis embryos. How MgcRacGAP is stabilized at microtubule plus ends is unknown. We identified an SxIP motif in X. laevis MgcRacGAP that is conserved with other proteins that bind to EB1 (also known as Mapre1), a microtubule plus-end tracking protein. Mutation of the SxIP motif in MgcRacGAP resulted in loss of MgcRacGAP tracking with EB3 (also known as Mapre3) on growing microtubule plus ends, abnormal astral microtubule organization, redistribution of MgcRacGAP from the contractile ring to the polar cell cortex, and mislocalization of RhoA and its downstream targets, which together contributed to severe cytokinesis defects. Furthermore, mutation of the MgcRacGAP SxIP motif perturbed adherens junctions. We propose that the MgcRacGAP SxIP motif is functionally important both for its role in regulating adherens junction structure during interphase and for regulating Rho GTPase activity during cytokinesis.

O-GlcNAc glycosylation is a dynamic, inducible post-translational modification (PTM) essential for neuronal homeostasis and found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Intracellularly, O-GlcNAc modification is cycled by two enzymes in mammalian cells: O-GlcNAc transferase (OGT) appends O-GlcNAc to serine or threonine residues and O-GlcNAcase (OGA) removes O-GlcNAc. OGT modifies over 1000 different proteins, but the lack of a well-defined consensus sequence or substrate structural constraints has hampered efforts to predict sites a priori. Furthermore, the identification of O-GlcNAc modification sites has been obstructed by the difficulty of enriching and detecting O-GlcNAc using traditional biochemical methods. Here, we established and employed biological and chemical tools to illuminate the role of O-GlcNAc in neuronal function. In Chapter 2, we sought to determine the role of O-GlcNAc in learning, memory, and neurodegeneration. Deletion of the OGT gene causes early postnatal lethality in mice, complicating efforts to study O-GlcNAc glycosylation in mature neuronal function and dysfunction. We demonstrated that the loss of OGT in the forebrain of adult mice (OGT cKO) leads to progressive neurodegeneration, including neuronal death, neuroinflammation, hyperphosphorylated tau, amyloidogenic Aβ-peptides, and memory deficits. In the hippocampus, we showed that OGT ablation lead to the upregulation of neuroinflammatory genes and the downregulation of cholesterol biosynthetic genes. Additionally, a gene network analysis (WGCNA), qPCR, and immunohistochemistry (IHC) revealed that loss of O-GlcNAc perturbed cell cycle progression in the hippocampal neurons. In the hippocampus, we identified increased neuroinflammatory gene transcription in OGT cKO mice and both tau neurofibrillary tangle (NFT)-forming and amyloid-forming Alzheimer’s disease (AD) mouse models. However, only OGT cKO and NFT-forming mice displayed decreased synaptic gene expression, suggesting that NFT formation and OGT cKO compromise hippocampal synaptic transcription. These studies indicate that O-GlcNAcylation regulates pathways vital for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases. In order to understand the critical O-GlcNAc-mediated neuronal functions that underlie OGT cKO dysfunction, we next developed and utilized novel biological and chemical tools in order to identify key OGT interactors and substrates in the brain in Chapter 3. Due to the lack of a well-defined OGT substrate sequence and structural constraints, OGT is believed to obtain its substrate specificity through its interactome where specific interactors target OGT to specific substrates. In order to identify these interactors, we used CRISPR/Cas9 to generate a novel mouse with a minimally tagged OGT in order to identify the endogenous OGT brain interactome using tandem affinity purification and MS methods. The preliminary OGT brain interactome consisted of previously identified OGT interactors and substrates as well as novel interactors. The identified OGT interactors were enriched for ribosomal and cytoskeletal proteins in addition to axonal, dendritic, and neuronal cell body proteins, implicating OGT as a pivotal mediator of neuronal structure and function. In addition to the OGT interactome, we sought to uncover OGT’s substrates or the O-GlcNAcome. We developed an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labeling followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) installed a new MS-compatible linker designed for facile purification and release of O-GlcNAcylated proteins for downstream MS analysis. We validated the approach by identifying several established O-GlcNAc sites on the proteins α-crystallin and OGT as well as discovering new, previously unreported sites on both proteins. Notably, these novel sites on OGT lie in key functional domains of OGT, underscoring how this site identification method can reveal important biological insights into protein activity and regulation. Finally, in Chapters 4 and 5, we focus on the post-translational modification (PTM) code on a specific transcription factor (TF), CREB (cAMP response element binding protein). CREB regulates memory formation through its transcriptional control of neuronal metabolism, activity, differentiation, development, and survival. CREB phosphorylation at serine 133 has been previously shown to enhance CREB-mediated transcription while CREB glycosylation at serine 40 has been shown to decrease CREB-mediated transcription. However, the exact gene networks modulated by and potential interplay between CREB glycosylation and phosphorylation have not been explored. Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we showed that CREB O-GlcNAcylation is important for neuronal activity and excitability while phosphorylation at serine 133 regulated the expression of genes involved in neuronal differentiation. Using WGCNA, we demonstrated that CREB O-GlcNAcylation at serine 40 and phosphorylation at serine 133 mediate mutually exclusive gene networks. The glycosylation-deficient mutant enhanced neuronal activity- and excitotoxicity-related gene networks while the phosphorylation-deficient mutant perturbed neuronal differentiation and amino and fatty acid metabolism-related gene networks. Our work sheds light on the regulation of CREB through PTMs to modulate neuronal function and delineate the roles of O-GlcNAcylation and phosphorylation in modulating neuronal excitability and neuronal development and metabolism respectively. Altogether, these studies demonstrate that O-GlcNAc modification is a critical mediator of neuronal homeostasis and neurodegeneration.

Neurons form dense neural circuits by connecting to each other via synapses and exchange information through synaptic receptors to sustain brain activities. Excitatory postsynapses form and mature on spines composed predominantly of actin, while inhibitory synapses are formed directly on the shafts of dendrites where both actin and microtubules (MTs) are present. Thus, it is the accumulation of specific proteins that characterizes inhibitory synapses. In this study, we explored the mechanisms that enable efficient protein accumulation at inhibitory postsynapse. We found that some inhibitory synapses function to recruit the plus end of MTs. One of the synaptic organizers, Teneurin-2 (TEN2), tends to localize to such MT-rich synapses and recruits MTs to inhibitory postsynapses via interaction with MT plus-end tracking proteins EBs. This recruitment mechanism provides a platform for the exocytosis of GABAA receptors. These regulatory mechanisms could lead to a better understanding of the pathogenesis of disorders such as schizophrenia and autism, which are caused by excitatory/inhibitory (E/I) imbalances during synaptogenesis.

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.

A major goal in neurobiology is to study the mechanisms by which neurites navigate towards their targets to establish functional synapses. While a large amount of information is available on the extra cellular signals driving neuritogenesis and synaptogenesis, major gaps exist in the comprehension of the intracellular events. <br><br>Rho family GTPases regulate the dynamic of the actin cytoskeleton and plays a crucial role in regulating neuronal development. In particular, Rac has been implicated in axon elongation and guidance, in dendritic growth and in the regulation of dendritic spines formation and plasticity. There are three Rac genes in mammals: Rac1, Rac2 and Rac3, sharing about 90% protein identity, all participating in several functions, including actin dynamics and adhesion (Hall, 1998). Rac1 is ubiquitous, while Rac2 is specific to the hematopoietic lineage and Rac3 is developmentally regulated in the vertebrate brain (Corbetta et al., 2005), with the highest expression during neurite branching and synaptogenesis (Bolis et al., 2003). The co-expression of Rac1 and Rac3 in the vertebrate nervous system suggests that they are needed to solve individual developmental and maintenance issues in the nervous system. <br><br>The aim of my work is to analyse the function of Rac1 and Rac3 during nervous system development. I took advantage of both <i>in vitro</i> and <i>in vivo</i> approaches to answer this question. Comparison between wild-type and Rac3-/- hippocampal neurons showed no differences in neuritogenesis and synaptogenesis processes. On the other hand, Rac1 depletion by RNAi in both wild-type and Rac3-/- hippocampal neurons during early stage of neuronal development showed specific alteration of the dendritic development without affecting axonal outgrowth. Reduced Rac1 levels impaired growth cone dynamics and F-actin levels in both neurites and growth cones. These results show that endogenous Rac1 rather than Rac3 activity is essential for early dendritic development, but not required for the initial establishment of neuronal polarity and axonal development. Depletion of either Rac1 or Rac3 during later stages of <i>in vitro</i> neuronal development did not severe dendritic spines morphogenesis, while double depletion strongly impaired the formation and maturation of dendritic spines. All together these <i>in vitro</i> results reinforce the hypothesis that Rac1 and Rac3 are both important for neuronal development underlying a specific role for Rac3 in later events of neuronal development. <br><br>We next evaluated the effects of the depletion of Rac1 and Rac3 <i>in vivo</i>. I generated mice with conditional deletion of Rac1 in neurons (Rac1N), by crossing Rac1Flox/Flox mice (Walmsley et al., 2003) with transgenic Synapsin-Cre mice (Zhu et al., 2001) and double RaclN/Rac3-/- mice. <br><br>As Rac3-/-, also Rac1N developed normally and was fertile. Both Rac3-/- and Rac1N did not reveal major abnormalities in the brain cytoarchitecture, cell layering or alteration in the general organization of dendrites and synapses. Double RaclN/Rac3-/- mice died around 13 days after birth (P13) and showed strong neurological defects. Morphological analysis revealed evident alterations in the hippocampus. The abnormalities in the organization of the hippocampus may be correlated with the frequent spontaneous epileptic seizures developing after P7 in these mice. We found that double depletion of Rac1 and Rac3 induced an increase of WAVE1 (Ser310) and PAK1 (Thr423) phosphorylation, and reduced the total levels of RhoA. All together the data obtained in this thesis show for the first time that both Rac1 and Rac3 are important for neuronal development, while mutation of each gene leads to viable animals with no evident anatomical defects due to partial compensatory effects.

Obesity is a multifactorial disease in which environmental conditions and several genes play an important role in the development of this disease. Obesity is associated with neurodegenerative diseases (Alzheimer, Parkinson, and Huntington diseases) and with neurodevelopmental diseases (autism disorder, schizophrenia, and fragile X syndrome). Some of the environmental conditions that lead to obesity are physical activity, alcohol consumption, socioeconomic status, parent feeding behavior, and diet. Interestingly, some of these environmental conditions are shared with neurodegenerative and neurodevelopmental diseases. Obesity impairs neurodevelopment abilities as memory and fine-motor skills. Moreover, maternal obesity affects the cognitive function and mental health of the offspring. The common biological mechanisms involved in obesity and neurodegenerative/neurodevelopmental diseases are insulin resistance, pro-inflammatory cytokines, and oxidative damage, among others, leading to impaired brain development or cell death. Obesogenic environmental conditions are not the only factors that influence neurodegenerative and neurodevelopmental diseases. In fact, several genes implicated in the leptin–melanocortin pathway (LEP, LEPR, POMC, BDNF, MC4R, PCSK1, SIM1, BDNF, TrkB, etc.) are associated with obesity and neurodegenerative and neurodevelopmental diseases. Moreover, in the last decades, the discovery of new genes associated with obesity (FTO, NRXN3, NPC1, NEGR1, MTCH2, GNPDA2, among others) and with neurodegenerative or neurodevelopmental diseases (APOE, CD38, SIRT1, TNFα, PAI-1, TREM2, SYT4, FMR1, TET3, among others) had opened new pathways to comprehend the common mechanisms involved in these diseases. In conclusion, the obesogenic environmental conditions, the genes, and the interaction gene–environment would lead to a better understanding of the etiology of these diseases.

Modelling the Effects of Microtubule Plus End Tapering on EB1 Binding

Dopaminergic Neurons and Brain Reward Pathways: From Neurogenesis to Circuit Assembly

To begin the process of forming neural circuits, new neurons first establish their polarity and extend their axon. Axon extension is guided and regulated by highly coordinated cytoskeletal dynamics. Here we demonstrate that in hippocampal neurons, the actin-binding protein caldesmon accumulates in distal axons, and its N-terminal interaction with myosin II enhances axon extension. In cortical neural progenitor cells, caldesmon knockdown suppresses axon extension and neuronal polarity. These results indicate that caldesmon is an important regulator of axon development.

The CBL-CIPK Calcium Signaling Network: Unified Paradigm from 20 Years of Discoveries.

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.