Investigation on spatio-temporal dynamics of RhoGTPases and their role in neuronal growth cone and actin wave motility

Neurons are highly polarised cells that migrate elongating their axon to reach distant synaptic targets. In the developing nervous system they travel along highly conserved trajectories defined by the molecules present in the surrounding environment, the so-called guidance cues. They can exert the function either at short range by direct contact or at long range, secreted by surrounding and target cells to create gradients that can be sensed by migrating axons. During the PhD course I focused on investigating the spatio-temporal properties of neurons in response to chemical signals. I have studied in detail the morphology changes of Growth Cones (GC) upon local stimulation and the dynamics of signalling cascades regulating actin dynamics, with a particular attention on Rho-GTPases. Moreover I investigated the morphology, molecule composition of axonal Actin Waves (AWs), as well as the role of Rho-GTPases in their inception and movement kinetics. In these studies I adopted various techniques: from live-cell imaging of the actin dynamics in AWs to a combination of FRET imaging and optical manipulation to image the Rho-GTPases activation in GCs real time upon local chemical stimulus delivery. The cellular module designed to perceive the guidance stimuli is the Growth Cone (GC), a specialised structure at the tip of the growing axon divided into three regions. The central region contains organelles and has a structural function, the transition region is formed by acto-myosin contractile arcs and the peripheral region, formed by thin filopodia and veil-like lamellipodia structures, that sustain dynamic protrusion and retraction cycles and express on the surface all the receptors to sense the presence of guidance molecules gradients. The major component of these structures is actin, a molecule that polymerises to form filaments that can be arranged, with the cooperation of a wide variety of actin-binding molecules, into different architectures. Actin filaments are polarised structure with the \u201cbarbed\u201d end oriented towards the leading edge and a \u201cpointed\u201d end towards the central region. Filaments undergo continuous cycles of polymerisation at the barbed end and depolymerisation at the pointed end, creating two dynamic behaviours called treadmilling and retrograde flow. The relative prominence of one process over the other is regulated by external signals that are sensed by receptors and initiate different intracellular signalling cascades. These pathways involve a lot of diverse proteins at various levels, but almost all of them pass through a \u201cbottleneck\u201d step: the Rho family of Guanosine Tri-Phosphatases (Rho-GTPases). Rho-GTPases are molecular switches that cycle between activated, GTP-bound state and an inactivated, GDP-bound state. Their dynamics are modulated by upstream signals, and in turn they interact with downstream effectors to propagate the signal transduction to the actin cytoskeleton. A single Rho-GTPase can be regulated by many different molecules, called Guanosine Exchange Factors (GEFs), GTPase domain Activator Proteins (GAPs) and Guanine Nucleotide Dissociation Inhibitors (GDIs), and activate a wide range of cellular responses, depending on the cell type and the stimulus received. They are best known for their roles in the modulation of cytoskeleton rearrangements, cell motility and polarity and axon guidance. They exert their effect mainly by affecting actin dynamics, not only in the growth cone but also in the axon shaft. A particular behaviour of the polarising neuronal cells is the extrusion of GC-like structures that travel along the neurite shaft towards the tip and fuses with the GC to promote elongation. These structures are called Actin Waves (AWs): they have a mean velocity of 2-3 \ub5m/min and appear in a stochastic manner in all the growing neurites with a frequency of about 1-2 waves per hour. Their propagation is strongly dependent on the dynamic behaviour of the actin filaments, with the balance between barbed end polymerisation and pointed end de-polymerisation at its basis. Therefore all those proteins involved in the regulation of actin might have a prominent role in their structure and function, including the RhoGTPases. The main achievements and findings of my PhD are the following: 1. I combined successfully for the first time FRET imaging with optical tweezers to provide a strong tool to study dynamics of intracellular signalling molecules upon local delivery of chemical attractants and repellants. The versatility of the optical tweezers, that have the possibility to exert both contact stimulation and local gradient delivery, along with the precision and high spatio-temporal resolution of the FRET, allowed us to highlight fine spatio-temporal dynamics of Rho-GTPases in live cells. 2. Local repulsive stimulation by semaphorin-3A triggers local retraction of the side of the growth cone facing the stimulus, with distinct RhoGTPases spatio-temporal dynamics: a. I showed, in accordance to previous studies, that the stimulation triggers rapid activation of RhoA within 30 s in the central region of the growth cone, causing a delayed retraction (100-120 s from the stimulus application) that correlates with RhoA activation levels correlate with the induced morphological changes; b. I demonstrated that semaphorin-3A local delivery causes a decrease in Cdc42 activity within 60 s from the stimulation. Activity levels vary in a wave-like retrograde manner that proceeds almost in synchrony with the retraction. In few cases the stimulation induced the formation of active Cdc42 waves that propagate in a region away from the local stimulus and promote the spawning of new filopodia and lamellipodia, suggesting a role of Cdc42 in travelling actin waves; c. I showed that local stimulation with beads coated with semaphorin-3A induces the formation of active Cdc42 waves propagating from the GC edge to the central region with a mean period of 70 s. Same \u201ctravelling\u201d waves have been found in some cases of spontaneous retraction in the neuronal cell culture, but they oscillate with a longer period (110 s). These overall data show a more complex behaviour for Cdc42 than RhoA, and provide evidence for a higher degree of complexity in the Rho-GTPase signalling network. 3. Actin dynamics in neuronal actin waves are strongly dependent on Cdc42 and Rac1 activation dynamics. By means of immunofluorescence, STED nanoscopy and live cell imaging with inhibitors for different molecules, we showed that: a. In accordance with previous studies, actin waves are growth cone-like structures that generate at the proximal segment of neurites and then propagate along the shaft towards the growth cone. When it reaches its vicinity, the growth cone retracts and the two structure fuse together to form a new, bigger and more dynamic growth cone that elongates again; b. Myosin-IIB is localised at the rear of the propagating wave, suggesting a possible role of myosin in their dynamics. This role has been confirmed by further experiments in which myosin inhibition with 20 \ub5M blebbistatin highlighted the disruption of the GC-like morphology of actin waves and the disappearing of the GC retraction upon wave incoming at the neurite tip, along with an effect on AW frequency and velocity; c. Membrane tension has a role in maintenance of AW morphology and affects also AW initiation and propagation. Addition of 250 \ub5M of \u3b2-cyclodextrin disrupted the GC-like morphology and decreased the AW area of more than 50%. Moreover the treatment decreased the velocity and significantly the frequency of AW initiation, suggesting a major role of the membrane in AW dynamicity; d. Cdc42 and Rac1 have a strong impact on the initiation dynamics of the actin waves. The frequency of actin waves per hour is significantly reduced under 10 \ub5M of both Cdc42 (ML141) and Rac1 (EHT1864) inhibition: from 2-3 waves per hour to about 0,5 and 1 wave per hour, respectively. Moreover, addition of a high concentration (30\ub5M) of ML141 stopped the AW sprouting almost completely, demonstrating a prominent role of these Rho-GTPases in actin wave initiation at the initial segment of the neurite. e. Cdc42 and Rac1 have a role also in the propagation dynamics of actin waves. Inhibition of both GTPases resulted in a significant decrease in the velocity of actin waves, from a mean of 2,2 \ub5m/min to about 1,5 \ub5m/min and 1,2 \ub5m/min respectively. Moreover we observed a disruption of the GC-like morphology of AWs, as well as a reduction in the mean area of about 50%. These results provide new insights for a prominent role of Rho-GTPases in the overall dynamics of the actin cytoskeleton within the travelling waves, in perfect accordance with previously reported data

Pregabalin silences oxaliplatin-activated sensory neurons to relieve cold allodynia

Oxaliplatin is a platinum-based chemotherapeutic agent that causes cold and mechanical allodynia in up to 90% of patients. Silent Nav1.8-positive nociceptive cold sensors have been shown to be unmasked by oxaliplatin, and this event has been causally linked to the development of cold allodynia. We examined the effects of pregabalin on oxaliplatin-evoked unmasking of cold sensitive neurons using mice expressing GCaMP-3 in all sensory neurons. Intravenous injection of pregabalin significantly ameliorates cold allodynia, while decreasing the number of cold sensitive neurons by altering their excitability and temperature thresholds. The silenced neurons are predominantly medium/large mechano-cold sensitive neurons, corresponding to the 'silent' cold sensors activated during neuropathy. Deletion of α2δ1 subunits abolished the effects of pregabalin on both cold allodynia and the silencing of sensory neurons. Thus, these results define a novel, peripheral inhibitory effect of pregabalin on the excitability of 'silent' cold-sensing neurons in a model of oxaliplatin-dependent cold allodynia.Significance StatementPregabalin is an analgesic drug in the clinic, that is supposed to act by blocking neurotransmitter release. Here we show that silent nociceptors that are activated by chemotherapeutic insults like oxaliplatin are silenced by pregabalin, which blocks the associated pain. This mode of action suggests that peripheral acting pregabalin-like drugs could be very useful for pain during chemotherapy, as they would have no CNS side effects - a problem for many patients with pregabalin. This novel effect of pregabalin is mediated by its interaction with the α2δ1 calcium channel subunit, but how this works is not yet understood

Gate control of sensory neurotransmission in peripheral ganglia by proprioceptive sensory neurons

Melzak and Wall's gate control theory proposed that innocuous input into the dorsal horn of the spinal cord represses pain-inducing nociceptive input. Here we show that input from proprioceptive parvalbumin-expressing sensory neurons tonically represses nociceptor activation within dorsal root ganglia. Deletion of parvalbumin-positive sensory neurons leads to enhanced nociceptor activity measured with GCaMP3, increased input into wide dynamic range neurons of the spinal cord and increased acute and spontaneous pain behaviour, as well as potentiated innocuous sensation. Parvalbumin-positive sensory neurons express the enzymes and transporters necessary to produce vesicular GABA that is known to be released from depolarized somata. These observations support the view that gate control mechanisms occur peripherally within dorsal root ganglia

Actin waves do not boost neurite outgrowth in the early stages of neuron maturation

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed F\uf6rster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics

An improved method for growing neurons: Comparison with standard protocols

Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes

Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation

Local stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling

Patch method for culture of primary hippocampal neurons

Culture of primary neurons, and especially hippocampal neurons, is important for understanding cellular mechanisms in neurobiology. Actually, this is achieved by using culture dish or glass slide with surface coated proteins. Here, we proposed a patch method for culture of primary neurons on a monolayer of gelatin nanofibers electrospun and crosslinked on a honeycomb microframe of poly (ethylene glycol) diacrylate (PEGDA). This method allows us to minimize exogenous material contact of cells and largely increase the exposure area of cells to the culture medium. We found that neurons, and especially astrocytes, have a more in vivo like morphology comparing to that on culture dish or on glass slide. We also found that neurons were preferentially located in the suspended areas of the monolayer nanofibers. Finally, calcium imaging revealed that primary neurons have a higher degree of neural activity on the patch than on glass. These results suggest that crosslinked and monolayer gelatin nanofibers closely mimic the extracellular matrix structure and allow more effective culture of primary neurons than conventional methods, thus facilitating advanced studies of neural functions as well as cell-based assays

Tools for analysis and conditional deletion of subsets of sensory neurons

Background: somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: a floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests.Results: motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model.Conclusions: ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.<br/