Potential\ua0effects\ua0of\ua0Rac1\ua0GTPase\ua0signalling\ua0in\ua0the\ua0deregulation\ua0of\ua0Alzheimer's\ua0Disease\ua0relevant\ua0proteins\ua0

La malattia di Alzheimer (AD) rappresenta una patologia a carattere multifattoriale e la forma pi\uf9 diffusa di demenza. Nonostante sia stata descritta per la prima volta nel 1906, le cause della malattia sono ancora poco comprese. Negli ultimi anni diversi gruppi hanno descritto un possibile coinvolgimento delle small Rho GTPasi in AD (Bolognin et al., 2014). Tali proteine svolgono una molteplicit\ue0 di funzioni, tra cui una delle principali riguarda la regolazione delle dinamiche dell\u2019actina del citoscheletro (Hall e Lalli, 2010). Esse inoltre rivestono un ruolo importante nella morfologia delle spine dendritiche (Etienne\u2010Manneville e Hall, 2002), la cui alterazione \ue8 responsabile dei danni a livello sinaptico. Dal momento che la progressione dell\u2019AD \ue8 caratterizzata proprio da un\u2019estesa perdita di sinapsi, le alterazioni a livello del citoscheletro potrebbero essere la chiave di eventi patologici che contribuiscono alla sua insorgenza, e le Rho GTPasi potrebbero essere pertanto direttamente connesse alla malattia. Rac1, uno dei tre membri pi\uf9 studiati e meglio caratterizzati delle Rho GTPasi, \ue8 nota per avere un ruolo nella promozione della sopravvivenza neuronale (Le et al., 2005; Loucks et al., 2006). La stessa proteina inoltre si \ue8 vista essere deregolata in cervelli AD, suggerendo un suo possibile coinvolgimento nel processamento di beta amiloide (A\u3b2) dal suo precursore APP (Stankiewicz e Linseman, 2014). Partendo da queste premesse, lo scopo della tesi \ue8 stato quello di analizzare i pathways molecolari che connettono Rac1 e le proteine rilevanti della patologia AD, A\u3b2 e tau. Tutti gli esperimenti del lavoro di tesi sono stati eseguiti in vitro su neuroni corticali primari, andando a modulare le singole proteine. Il progetto \ue8 iniziato con il trattamento delle cellule attraverso somministrazione di A\u3b2 e induzione dell\u2019iperfosforilazione di tau. Entrambi gli approcci non hanno provocato effetti n\ue9 sulla localizzazione di Rac1 nelle cellule, n\ue9 sulla sua attivazione. Sono stati quindi effettuati alcuni esperimenti per manipolare l\u2019espressione e l\u2019attivit\ue0 di Rac1. Tale scopo \ue8 stato raggiunto attraverso l\u2019utilizzo di mutanti della proteina coniugati ad una sequenza TAT in grado di promuovere l\u2019internalizzazione della proteina stessa nelle cellule. Questo approccio ha dato come risultato una perturbazione nel metabolismo di A\u3b2 e promosso la traslocazione di SET, una proteina direttamente connessa all\u2019iperfosforilazione di tau. In particolare, solamente con l\u2019attivazione della proteina \ue8 stato possibile osservare un maggior livello di A\u3b2 o del suo precursore, mentre \ue8 bastata la sovra espressione di Rac1 a causare la traslocazione di SET dal nucleo alla membrana plasmatica. Considerati nel complesso, i dati illustrati descrivono un possibile pathway in cui Rac1 agisce a monte ed \ue8 in grado di influenzare la regolazione di proteine rilevanti in AD. Il coinvolgimento di Rac1 nel processamento di A\u3b2 non \ue8 un fatto completamente nuovo (Boo et al., 2008; Wang et al., 2009), tuttavia sembra che nessuno finora abbia approfondito quali siano i frammenti prodotti in seguito all\u2019attivazione di Rac1. Tale aspetto costituir\ue0 oggetto di futuri esperimenti. I dati riguardanti invece la traslocazione di SET dopo sovra espressione di Rac1 costituiscono un nuovo aspetto interessante che esperimenti futuri potranno meglio approfondire. Infatti, la diretta relazione tra Rac1 e SET \ue8 stata osservata solamente da due gruppi, ma non in cellule neuronali (ten Klooster et al., 2007; Switzer et al., 2011).Alzheimer\u2019s disease (AD) is a multifactorial pathology and the most common form of dementia in the elderly. Despite it was first described in 1906, the aetiology of AD is still poorly understood. Recently, different groups have highlighted the involvement of small Rho GTPases in AD (Bolognin et al., 2014), whose main function consists in the regulation of actin cytoskeleton dynamics (Hall and Lalli, 2010). Moreover, this family of protein plays an important role in dendritic spine morphology (Etienne\u2010Manneville and Hall, 2002), whose alterations are responsible for synaptic deficits. Since the progression of AD is characterized by a wide synaptic loss, alterations of cytoskeleton dynamics might be a key pathogenic event contributing to AD neuropathology, and Rho GTPases could be directly connected to the disease. Rac1, one of the three best characterized Rho GTPases, is known to act as neuronal survival promoter (Le et al., 2005; Loucks et al., 2006). Interestingly, it has been found that Rac1 is dysregulated in AD brain, suggesting a possible involvement in the processing of beta amyloid (A\u3b2) from its precursor APP (Stankiewicz and Linseman, 2014). Moving from these assumptions, the aim of this thesis was to investigate the molecular pathways connecting Rac1 and AD relevant proteins, A\u3b2 and tau. All the experiments were performed in vitro on primary cortical neurons, taking into account the modulation of the single proteins. The project has started with A\u3b2 administration and tau induced hyperphosphorylation, and both approaches did not result in any modification of Rac1 cellular distribution or activation. We proceeded then with the mis\u2010regulation of Rac1 activity, through the administration of mutant proteins fused with a TAT domain, which allows their internalization into the cells. This approach showed a perturbation of A\u3b2 metabolism, and elicited the translocation of SET, a protein directly connected to tau hyperphosphorylation. More in details, only Rac1 activation was able to enhance the levels of A\u3b2 or its precursor, while the over\u2010expression of the protein was sufficient to promote SET translocation from the nucleus to the plasma membrane. Taken together, the data describe a putative pathway in which Rac1 is up\u2010stream and it is able to affect the regulation of AD relevant proteins. The involvement of Rac1 in the processing of A\u3b2 is not completely new (Boo et al., 2008; Wang et al., 2009), but, to our knowledge, nobody has already investigated which fragments can be produced after Rac1 activation. On the other hand, the data on SET translocation after Rac1 over\u2010expression constitute a new insight and future experiments could be better clarify this direct connection between SET and Rac1 in AD context. Indeed, only two groups found the same pathway, but not in a neuronal population (ten Klooster et al., 2007; Switzer et al., 2011)

Potential effects of Rac1 GTPase signalling in the deregulation of Alzheimer's Disease relevant proteins

Alzheimer\u2019s disease (AD) is a multifactorial pathology and the most common form of dementia in the elderly. Despite it was first described in 1906, the aetiology of AD is still poorly understood. Recently, different groups have highlighted the involvement of small Rho GTPases in AD (Bolognin et al., 2014), whose main function consists in the regulation of actin cytoskeleton dynamics (Hall and Lalli, 2010). Moreover, this family of protein plays an important role in dendritic spine morphology (Etienne\u2010Manneville and Hall, 2002), whose alterations are responsible for synaptic deficits. Since the progression of AD is characterized by a wide synaptic loss, alterations of cytoskeleton dynamics might be a key pathogenic event contributing to AD neuropathology, and Rho GTPases could be directly connected to the disease. Rac1, one of the three best characterized Rho GTPases, is known to act as neuronal survival promoter (Le et al., 2005; Loucks et al., 2006). Interestingly, it has been found that Rac1 is dysregulated in AD brain, suggesting a possible involvement in the processing of beta amyloid (A\u3b2) from its precursor APP (Stankiewicz and Linseman, 2014). Moving from these assumptions, the aim of this thesis was to investigate the molecular pathways connecting Rac1 and AD relevant proteins, A\u3b2 and tau. All the experiments were performed in vitro on primary cortical neurons, taking into account the modulation of the single proteins. The project has started with A\u3b2 administration and tau induced hyperphosphorylation, and both approaches did not result in any modification of Rac1 cellular distribution or activation. We proceeded then with the mis\u2010regulation of Rac1 activity, through the administration of mutant proteins fused with a TAT domain, which allows their internalization into the cells. This approach showed a perturbation of A\u3b2 metabolism, and elicited the translocation of SET, a protein directly connected to tau hyperphosphorylation. More in details, only Rac1 activation was able to enhance the levels of A\u3b2 or its precursor, while the over\u2010expression of the protein was sufficient to promote SET translocation from the nucleus to the plasma membrane. Taken together, the data describe a putative pathway in which Rac1 is up\u2010stream and it is able to affect the regulation of AD relevant proteins. The involvement of Rac1 in the processing of A\u3b2 is not completely new (Boo et al., 2008; Wang et al., 2009), but, to our knowledge, nobody has already investigated which fragments can be produced after Rac1 activation. On the other hand, the data on SET translocation after Rac1 over\u2010expression constitute a new insight and future experiments could be better clarify this direct connection between SET and Rac1 in AD context. Indeed, only two groups found the same pathway, but not in a neuronal population (ten Klooster et al., 2007; Switzer et al., 2011)

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles.

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles with promising implications for the treatment of retinal diseases affecting the photoreceptor layer

Rac1 selective activation prevents the dendrite atrophy occurring after crush.

<p>Representative confocal maximum projections of YFPH mouse RGCs 15 days after crush and double injection of either vehicle or CA or DN mutants. A normal RGC is shown for comparison (A). After Rac1L61F37A and Y40C treatment (D and E respectively) the dendritic atrophy is prevented, whereas the same extent of degeneration was found in control (B) and after DN treatment (C). The plots relative to the Sholl analysis for the different treatments are shown in F–G: (F) the maximum number of intersections, the ramification index and the critical value were used for statistical evaluation (10 to 20 neurons per treatment), whereas (G) the N of intersections against the distance from the soma shows the morphological changes along the whole dendritic tree. Scale bar 20 µm. # p<0,01 versus the control, the L61F37A and the L61Y40C; *p<0,05 versus the indicated group (one way ANOVA followed by LSD post hoc test).</p

Quantification of axonal regeneration after optic nerve crush and Rac1 selective activation in Brainbow mice.

<p>Brainbow mouse optic nerves were crushed at day 0 and, after one or more injections of either Rac1 mutants, WT or vehicle, were dissected at 15 or 30 days post crush in order to investigate regeneration. A scheme of the different treatments is given in A. Nerves were studied by confocal microscopy and the results of the regeneration study are plotted in B, C and D. Only the double injection of L61F37A was able to increase the average number of axons crossing the crush site per 100 µm of nerve z-section (B) at 15 days post lesion. The number of regenerating axons is higher than control also after 2 and 5 injections of L61F37A at 30 dpl. The data at 15 days are confirmed also by the analysis of length distribution in the entire distal stump (C). The same analysis at 30 days (D) revealed that, despite after 2 and 5 L61F37A injections the total number of regenerating neurons are similar, the repetitive treatment resulted in longer axons. Since we found no differences between the various vehicle injection protocols of treatment at 15 and 30 days, we put together the data of the controls on the same column/curve (n = 6 and 8). Data are mean ±SEM. N is in brackets. ^#p<0.01, *p<0.05 by ANOVA (LSD post hoc test).</p

The role of rho-gtpases in the aetiology of Alzheimer's disease

AIMS: The progression of Alzheimer’s disease (AD) is characterized by a wide synaptic and dendritic loss whose mechanisms are still unknown. We aimed at studying the potential role of Rho-GTPases, key regulator of spine dynamics, as biomarkers for AD and interacting partners of tau and beta-amyloid peptide (Abeta). METHOD: Rac1 and Cdc42 levels were measured by ELISA in the plasma of AD patients (n=101), age and sex-matched healthy controls (n=102) as well as in frontal cortex of autoptic brain samples. The 3xTgAD mouse model was used to correlate the alteration of Rho-GTPases in the brain to the progression of the disease. To investigate the molecular mechanisms connecting Rho-GTPases to Abeta and tau, mouse primary cortical neurons were treated with Rho-GTPase mutant proteins, Rac1 and Cdc42, and immunocytochemistry and pull down assays were performed. RESULTS: Rac1 was significantly increased in plasma of AD patients and significantly decreased in AD brain homogenates compared to controls. The underlined involvement of Rac1 was confirmed by the decreased levels of Rac1 in the cortex of 7 month old 3xTgAD. Rac1 deregulation affected Aß metabolism and tau regulators. CONCLUSION: Our data proposes a putative pathway in which Rac1 is able to affect the regulation of AD relevant proteins. Moreover, we show that Rac1 is increased in AD plasma suggesting its novel role as a biomarker for AD and as a promising therapeutic target

Rac1 selective activation promotes axonal regeneration after optic nerve crush in Brainbow mice.

<p>In Brainbow mice the injection of the AAV-Cre-GFP around the day of crush triggers a genetic recombination that leads to YFP expression (white false color) only in surviving neurons. We injected either the Tat-Rac1 mutants, WT or vehicle on the day of crush (day 0) and on day 2 and studied regeneration 15 days post lesion. A scheme of the treatment is in A. Nerves were acquired and studied by confocal microscopy. Single examples of whole mounted nerves after mosaic merge reconstruction are given in B to E, and are relative to vehicle (B), Rac1WT (C), L61F37A mutant (D) and L61Y40C mutant (E) double injections. The crush sites of B, C, D and E are enlarged in F, G, H and I respectively. By comparison of the panels it is clear that after treatment with L61F37A a higher number of axons is able to cross the crush site and run distally (D and H). Scale bars 100 µm.</p