Mol Neurobiol DOI 10.1007/s12035-014-8637-5

The Potential Role of Rho GTPases in Alzheimer's Disease Pathogenesis Silvia Bolognin & Erika Lorenzetto & Giovanni Diana & Mario Buffelli

Received: 25 October 2013 / Accepted: 2 January 2014 # Springer Science+Business Media New York 2014

Abstract Alzheimer's disease (AD) is characterized by a wide loss of synapses and dendritic spines. Despite extensive efforts, the molecular mechanisms driving this detrimental alteration have not yet been determined. Among the factors potentially mediating this loss of neuronal connectivity, the contribution of Rho GTPases is of particular interest. This family of proteins is classically considered a key regulator of actin cytoskeleton remodeling and dendritic spine maintenance, but new insights into the complex dynamics of its regulation have recently determined how its signaling cascade is still largely unknown, both in physiological and pathological conditions. Here, we review the growing evidence supporting the potential involvement of Rho GTPases in spine loss, which is a unanimously recognized hallmark of early AD pathogenesis. We also discuss some new insights into Rho GTPase signaling framework that might explain several controversial results that have been published. The study of the connection between AD and Rho GTPases represents a quite unchartered avenue that holds therapeutic potential. Keywords Beta-amyloid . Cytoskeleton . Rho-GTPases S. Bolognin (*) : E. Lorenzetto : M. Buffelli Department of Neurological and Movement Sciences, Section of Physiology, University of Verona, Strada le Grazie 8, 37134 Verona, Italy e-mail: [email protected] G. Diana Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy M. Buffelli Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona, Italy M. Buffelli National Institute of Neuroscience, Verona, Italy

Introduction Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by dramatic synaptic degeneration and memory impairment. Despite extensive efforts, the molecular triggers leading to synaptic dysfunction are still obscure. For at least two decades, insoluble β-amyloid peptide (Aβ) deposits were considered the only responsible cause for the wide array of alterations observed in AD brain, ultimately leading to neuronal death (amyloid cascade hypothesis [1, 2]). This hypothesis has changed over the years and the most recent version states that Aβ-derived diffusible ligands (ADDLs) are the true neurotoxic species [3–5]. Small soluble oligomers confer synaptic dysfunction and neurodegeneration [6–8], whereas insoluble Aβ deposits from senile plaques (SP) might function as a reservoir of bioactive oligomers. However, although a central role for Aβ in AD pathogenesis is likely to be indisputable, considerable evidence indicates that Aβ production is not the sole culprit of AD pathogenesis [9–12]. Most of the genetic mutations causing the familial form of the disease are connected with an increased Aβ processing [13], but some unexpected exceptions have been reported. For instance, a pathogenic mutation of presenilin 1 (PS1) nearly abolishes γ-secretase activity toward amyloid precursor protein (APP), inactivating Aβ generation [14]. In the context of the puzzling role of Aβ in the disease, several papers have highlighted the physiological role of the peptide in normal synapse functions [15–17]. Further compounding the issue, AD “characteristic” features (SP, tangles) also occur in noncognitively impaired individuals [18, 19]. These findings clearly suggest that several molecular mechanisms underlying neuronal degeneration in AD have not been completely determined as yet. This highlights gaps in what we thought was our understanding of the nature of the pathological process. Here, after briefly describing the synaptic alterations observed in AD, we will focus on Rho GTPases, one of the most

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important families of proteins orchestrating spine dynamics. We critically review the existing data specifically connecting Rho GTPase deregulation to neurotoxic mechanisms relevant to AD etiology. We also highlight novel conceptual frameworks that might be of help in the study of this connection. Lastly, we analyze whether Rho GTPase modulation might realistically represent a potential therapeutic approach.

Synaptic Dysfunction and Synapse Loss in AD In earlier studies, a feature that has consistently been observed in AD patients, compared to age-matched controls, is a substantial synapse loss with a dramatic decrease of presynaptic boutons [20, 21]. Indeed, the progression of the pathology is accompanied by a wide dendritic spine loss that well correlates with cognitive decline in patients [22]. Several authors have shown how this feature appears even more marked than what expected based on neuronal death. This indicates that synaptic dysfunction is not just a consequence of cell death, but it has a central role in AD pathogenesis [23–25]. The size of the remaining synapses is also altered, most likely for a compensatory response [26]. Furthermore, the expression of several synaptic proteins is decreased [27], synaptophysin in particular [28]. Thus, synapse loss currently constitutes the major counterpart to cognitive impairment in AD [27]. As mentioned earlier, the factors driving to these alterations are still obscure. Aβ oligomers have been proposed as good candidates, as they bind to neuron surfaces in small punctate clusters of synaptic terminals [29] and their burden correlates with the extent of synapse loss in AD patients [30]. Besides, oligomers reduced spine density when administered to neuronal cultures [7] and organotypic hippocampal slice cultures [31]. In accord with these morphological changes, many studies showed that the administration of high concentration of soluble Aβ oligomers inhibited long-term potentiation (LTP) and facilitated long-term depression (LTD) [31–43], two forms of synaptic plasticity that are believed to represent the cellular bases of the memory process. Also in transgenic mouse models, increased Aβ oligomer production altered synaptic transmission strength and plasticity, decreasing the number of surface receptors at the synapses [31, 41, 44]. Although these studies are very convincing, the molecular link between Aβ oligomers and the occurrence of synaptic dysfunction still remains elusive [45], and these harmful assemblies might not be the only causative factors. Recently, also the soluble form of tau protein has shown synaptic toxicity in vivo and in vitro [46, 47], supporting the potential contributing role of tau delocalization, from axon to somatodendritic compartment, in spine loss [48]. For a more detailed characterization of the synaptic alterations correlated to both Aβ and tau, we refer to excellent recently published reviews [45, 49–52].

Rho GTPases: Key Regulators of Spine Dynamics Synaptic transmission and plasticity of neuronal circuits rely on dendritic spines, the postsynaptic receptive structure of a synapse [53]. Spine morpho-dynamics and synaptic functions are closely related: in response to extracellular stimuli spine area and volume change to regulate synaptic efficacy. For instance, spine volume increases in LTP and decreases in LTD, realizing the modifications at the basis of learning and memory [54, 55]. Thus, it is not surprising that cognitive dysfunctions have been correlated to spine abnormalities [56]. These morpho-functional changes rely on the dynamics of the F-actin-rich cytoskeleton present in spines and thus on the related intracellular signaling pathways [57], in which the Rho GTPases play a pivotal role (Fig. 1). Rho GTPases connect signals from the postsynaptic receptors to changes in actin binding proteins, leading to the remodeling of dendritic spine shape and density [23, 58–63]. Rho GTPases encompass Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and Ras homologous member A (RhoA) and act as molecular switches, oscillating between a GTP-bound active and a GDPbound inactive form. Three types of regulatory proteins modulate Rho GTPases activation cycling: (i) guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP [64]; (ii) GTPase-activating proteins (GAPs) catalyze the opposite reaction, inactivating these proteins; and (iii) guanosine nucleotide dissociation inhibitors (GDIs) maintain GTPases in their inactive state, sequestering them in the cytosol. Rac1 and Cdc42 activation leads to spine formation, growth, and stabilization, while RhoA activation results in spine pruning [62, 65]. Indeed, the signaling pathway appears to be extremely complicated: the spatiotemporal activation of GTPases in dendritic spines is tightly regulated by the association with specific GEFs and GAPs to postsynaptic receptors, such as N-methyl-D-aspartate (NMDA), or scaffolding proteins like postsynaptic density protein 95 (PSD-95) or synapse-associated protein 97 (SAP-97). The association of many GEFs with different receptors allows GTPases to sense environmental cues like synaptic activity and extracellular ligands such as neurotransmitters and trophic factors (brainderived neurotrophic factor, neuregulin-1) [58, 64, 66]. A close interaction between the different Rho GTPases has been demonstrated more than a decade ago, but recently, it has become clear that they also exert complementary functions to regulate actin dynamics and to direct complex neuronal functions [67, 68]. As an example, for RhoA to be active, there is a requirement for Rac1 inactivation [69]. For this purpose, cell often includes specific GEFs and GAPs in the same complexes. For instance, the partition-defective protein (PAR) polarity complex that regulates spine morphogenesis is characterized by the association of three components: PAR3,

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Fig. 1 Rho GTPase signaling regulates dendritic spine morphogenesis. a 3D reconstruction of some dendrites: few spines are enlarged in the right panel. b Scheme of Rho GTPase signaling in excitatory synapses: GEFs (red) and GAPs (black) turn on and off, respectively, a specific Rho GTPase (violet), which in turn activates a pool of downstream effectors (yellow). The different pathways terminate on F-actin binding proteins, leading to changes in spine morphology. c Cartoon of a synapse showing

the structural organization of the signaling: GEFs and GAPs are connected to receptors, scaffolding proteins, and cell adhesion molecules of the PSD, allowing the connection of Rho GTPases signaling to extracellular stimuli and realizing the compartmentalization of different pathways. Downstream signaling targets the F-actin binding proteins, realizing the remodeling of spine cytoskeleton. Here, only cofilin and Arp2/3 are shown, but many other actin-binding proteins are present in the spine

PAR6, and atypical protein kinase C γ (aPKCγ). To stimulate spine development, PAR3 binds Tiam1 that activates Rac1 and, at the same time, PAR6 and aPKC bind the p190 RhoGAP that in turn maintains RhoA-inactivated [70, 71]. An example of how Rho GTPases signaling are spatiotemporally regulated is given by Lfc, a GEF that is normally associated to the dendrite shaft and translocates to spines only after a specific stimulus such as NMDA receptor activation [72]. Many studies showed that GTP-bound Rac1 and Cdc42 stimulate F-actin polymerization through their common main downstream effector p21-activated kinase (PAK) which, acting on the LIM kinase (LIMK)-cofilin axes, stimulates actin polymerization [73–75]. Other important Rac1 and Cdc42 downstream effectors are N-WASP and WASP, respectively. These are F-actin binding proteins positively influencing spine morphogenesis and PSD-95 clustering [76, 77]. Rac1

downstream signaling targets also the WAVE (Wiskott– Aldrich syndrome protein family member 1) complex to control actin clustering and polymerization [78]. N-WASP and WAVE are important for spine maintenance and maturation [79, 80] as they target the Arp2/3 complex, an F-actin binding protein responsible for filament branching at the spine head [81, 82]. Concerning RhoA, its constitutive active form was shown to decrease spine density, antagonizing the effect of Rac1 [83]. Following interaction with NMDA receptors, the GTP-bound RhoA recruits the Rho-associated protein kinase (ROCK)/ profilin complex to the PSD, leading to actin stabilization [84]. In addition, Rho GTPases play a role in synapse function and plasticity (LTP and LTD) [85–87]. In particular, this class of proteins is highly expressed in the hippocampus and

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cerebellum [88, 89], and the activation of NMDA receptors induced traslocation and activation of Rac1 in cultured hippocampal slice [89]. The Rac1-specific inhibitor NSC23766 blocked the induction of LTP [90], while the ROCK-specific inhibitor Y-27632 increased the magnitude of LTP [91]. The spine plasticity induced at single dendritic spine by twophoton glutamate uncaging activated rapidly RhoA and Cdc42 [85]. In addition, the pharmacological manipulation of Rho GTPases in vivo was associated with changes in synaptic plasticity, learning, and memory [92–94].

Rho GTPases Alteration in AD As a key regulator of actin dynamics and consequently of spine stability, defective Rho GTPase signaling might have a critical role in the pathological processes driving to synaptic degeneration. A wealth of studies has shown that in AD, the mentioned actin cytoskeleton stability is impaired [23, 95]. Actin depolymerization has been shown to cause synaptic and glutamate receptor loss [96]. AD brain sections displayed actin rod-like inclusions [97], implying imbalance of actin dynamics, especially in the area surrounding the SP. One can argue that these histological features are also observed in other neurodegenerative disorders [97] and in cell cultures exposed to different kinds of stimuli [98–100]. It is likely that the mechanisms underlying the formation of cofilin-actin rods are multiple, but this does not rule out that the occurrence of this assembly can still be connected to the critical causes of AD, even though several steps downstream. Indeed, Aβ has been shown to play a role in the imbalance of actin dynamics [101]. Thus, alteration of cytoskeleton properties, potentially driven by Rho GTPase deregulation, might be a key pathogenic event contributing to synaptic deficits in AD. It is quite surprising that there are relatively few studies trying to connect Rho GTPase signaling to loss of synaptic plasticity in AD (Table 1). Here, we will analyze in detail the findings correlating the three major members of the family to AD-like pathogenic mechanisms. Rac1 Evidence suggesting an interesting link between Rac1 and AD has been collected both in autoptic brain samples as well as in in vitro studies based primarily on the effect of Aβ exogenous administration. Unfortunately, the two approaches failed to give a clear-cut view of Rac1 deregulation in the pathology. Rac1 was upregulated in AD brain compared to agematched controls and, strikingly, this upregulation occurred in selected hippocampal neurons affected by cytoskeletal abnormalities [e.g., neurofibrillary tangles (NFTs)] [102]. The presence of this inclusion also in other apparently normal

neurons in the AD brain (but not in controls) led the authors to speculate that this increase might be an early event in the disease process. Notably, Rac1 has recently been connected to NFTs formation as a constitutively active splice variant, Rac1b, co-localized with cytoskeletal markers in cholinergic neurons in AD brain [103]. Rac1b accumulation increased with the severity of cognitive impairment and correlated with the decreased expression of genes involved in lipid metabolism and cell cycle. The appearance of Rac1 in these neurons was similar to the cytoskeletal changes described with an antibody (AT8) against two important sites of phosphorylation of tau. This suggests that Rac1b participates to the induction of cytoskeletal alterations related to NTF formation. Moreover, in mild and severe AD cases, Rac1b-positive neurons in the cortex showed Aβ pathology. Increased Rac1 immunoreactivity has been also shown in the cortex of Tg2576 [104], a model expressing APP Swedish mutation. On the other hand, a more recent study on AD brains showed that the levels of active Rac1 and PAK1 decreased in the frontal and in the occipital lobes in comparison with age-matched controls [105]. These changes were linearly correlated with the decrease of PSD-95 in the frontal lobe, which is related to synaptic impairment. In the occipital lobe, the authors did not find the same correlation, despite the fact that Rac1 and PAK decreased much more in the occipital lobe than in the frontal lobe of AD brains. This study interestingly suggests a reduced Rac1 signaling in AD brains, but it lacks of an immunohistochemical investigation that might have clarified the precise localization of these alterations. In vitro studies on primary neurons demonstrated a role for Rac1 in the processing of Aβ from its precursor APP. Rac1specific inhibitor NSC23766 has been shown to decrease APP protein levels in a concentration-dependent manner by modulating its transcriptional activity in primary hippocampal neurons [106]. Rac1 is present in the raft domain of neuronal membranes of dendrites/cell bodies [107] and this localization sustains Rac1 potential involvement in APP processing [108]. Boo et al. [109] showed in COS-7 cells that treatment with Rac1-dominant negative form attenuated γ-secretase activity resulting in decreased production of the APP intracellular domain and accumulation of the C-terminal fragments. Rac1 inhibition was also found to dramatically alter its interaction with PS1 [109]. It is conceivable that the translocation of γsecretase to lipid rafts increases the cleavage of certain substrates including APP C-terminal fraction while limiting the cleavage of other substrates. Despite these studies, the exact relation between Rac1 and APP in neuronal cells remains poorly understood, as most of the studies have been conducted in clonal cells expressing genes related to the familial form of AD. Moreover, studies directly connecting Rac1 to Aβ are contradictory when Aβ is exogenously administered to neuronal cells. 10 μM fibrillar Aβ1–42 induced actin

Mol Neurobiol Table 1 Rho GTPase and related protein dysfunction in AD Protein

Outcome

Model

Racl

↑ ↑ ↑ ↑ ↑ ↓ ←→ ↑

AD AD hippocampus Tg2576 Hippocampal cultures Cortical cultures SH-SY5Y, Tg2576 AD hippocampus and cortex SH-SY5Y neuroblastoma cell, Tg2576 AD hippocampus Tg2526 cortex and hippocampus

RhoA

↓ ←→

SN4741 AD hippocampus Hippocampal and cortical cultures Cortical cultures AD hippocampus and cortex SN4741 AD cortex, hippocampal cultures SH-SY5Y, cortex Tg2576 Hippocampal and cortical cultures SN4741 AD hippocampus AD cortex AD hippocampus Hippocampal cultures 3x-Tg AD hippocampus

Drebrin Cofilin

←→ ↑ ↑ ←→ ←→ ←→ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↑ mild AD, ↓ severe AD Abnormal translocation ↓ ↑

Cofilin EphB2

↑ ↓

Tg19959 AD and hAPP hippocampus

Cdc42

LIMK1 pCRMP-2 Tiaml Tiaml Kalirin Kalirin PAK1, PAK3 PAK kinase PAK PAKl-3 PAKl, PAK3

AD hippocampus, neuronal cultures, Tg2576 Hippocampal cultures Organotypic slice cultures

polymerization associated with the increased activity of Rac1/Cdc42 in primary hippocampal cultures [110]. Coherently, a recent study identified Rac1 activation as a potential required pathway for Aβ oligomers (1.25 μM) to induce neuronal death in primary neuronal cultures [111]. On the other hand, other groups described the opposite effect: incubation of SH-SY5Y cells with 0.5 and 1 μM Aβ1–40 induced a twofold reduction of Rac1 activation [112]. In the same study, Rac1 was largely inactivated in mouse Tg2576 brains starting from 6 months of age, when cognitive impairment is already manifest but amyloid deposition is not. Notably, at 6 months, Aβ accumulated in lipid raft fractions in the Tg2576 mouse brain [113], and this accumulation, according to the authors, may be due to Rac1 alteration.

Peptide form

Technique

References

Notes

IF IF IF IF, IP IP IP WB IP

[85] [84] [86] [91] [92] [93] [105] [93]

Rac1b

WB WB

[105] [105]

IP IF IP IP WB IP IF WB IP IP RT-PCR WB WB WB, IF IF

[92] [84] [91] [101] [105] [92] [132] [93] [91] [92] [133] [134] [97] [97] [135] [136]

250 nM oligomeric Aβ

WB, IF

[96]

Oligomeric Aβ 1 μM oligomeric Aβ

WB Live-cell imaging WB WB

[97] [81]

F-actin binding proteins

[137] [138]

Receptor

10 μM fibrillar Aβ 1.25 μM oligomeric Aβ 0.5–1 μM Aβ 1–40 0.5–1 μM Aβ 1–40

5 μM oligomeric Aβ 10 μM fibrillar Aβ 10 μM fibrillar Aβ 1–40 5 μM oligomeric Aβ 20 μM fibrillar Aβ 0.5–1 μM Aβ 1–40 10 μM fibrillar Aβ 1.25 μM oligomeric Aβ

oligomeric Aβ

GEF Effectors

Explanations for the discrepancies in the mentioned studies can be countless. Differences in the protocols of Aβ preparation can partly account for them. Nevertheless, as pointed out by Benilova et al. [114], the exact meaning of toxic Aβ specie is still very puzzling today and is in danger of becoming an easy way to justify inconsistencies in literature. However, the lack of a common experimental protocol of Aβ preparation renders comparisons between studies and interpretation very cumbersome. Another potential explanation would be that, considering Rho GTPase subtle and tight regulation in the time-scale frame, a kinetic of activation should be monitored rather than a single time point following several hours from the Aβ treatment. This would allow a clear perception of the dynamic of this activation.

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It is not the purpose of this review to give an exhaustive description of the data available for Rac1 direct interactors, but we want to highlight the role of its primary downstream effector, PAK. Interestingly, activation of Rac-PAK signaling in synapses has been recognized as a part of the system stabilizing the cytoskeletal changes necessary to consolidate LTP [87]. Indeed, Aβ oligomers have been suggested to interfere with Rac1 signaling inducing loss of actin polymerization through the interaction with PAK. Downstream both to Rac1 and Cdc42, this kinase plays a key role in cytoskeletal remodeling, acting through LIMK on actin/cofilin dynamics. Treatment of hippocampal neurons with Aβ1–42 oligomers resulted in PAK aberrant regulation, together with loss of Factin and decrease of PSD-95 indicating synaptic impairment, that were prevented by treatment with wild-type PAK [115]. The inhibition of PAK was sufficient to induce cognitive impairment [116, 117]. Several studies showed that active PAK levels increased in early stages of AD and decreased during mild and late stage of the disease [103, 115, 116]. Beside, a loss of an actin regulatory protein called Drebrin was observed [116]. This aberrantly regulated PAK colocalized with Rac1 in intracellular inclusions. In AD brains, active PAK forming a complex with Rac1/Cdc42 was found to localize to the plasma membrane to a greater extent compared to age-matched control brains [115]. Interestingly, the authors proposed that TIAM1 and Rac1 mediated PAK aberrant activation and translocation. Moreover, using the Src/Fyn kinase inhibitor PP2 they hypothesized that Fyn might be involved in this alteration of dendritic spine dynamic (Fig. 2). According to this hypothesis, Fyn might be upstream to Tiam1/Rac1/ Pak1. The attention on the role of this kinase has recently grown as Fyn plays a role in Aβ-induced synaptic neurotoxicity [6, 118–120]. Cdc42 Cdc42 upregulation was shown in AD brains compared to age-matched controls [102] but, as for Rac1, the in vitro studies failed to provide a common overview. One study showed that Cdc42 activity increased in a time- and dosedependent manner after 10 μM fibrillar Aβ1–42 treatment in hippocampal neurons [110], suggesting that Cdc42 deregulation participates in the cytoskeletal alterations observed in AD. Beside, Cdc42 was recruited at the plasma membrane following Aβ1–42 treatment, especially along the neurites and in the soma [110]. In contrast, another group showed in rat primary cortical neurons that 24 h Aβ1–40 (10 μM) exposure did not change Cdc42 expression and activation, although apoptotic death occurred within the same time frame [121]. The discrepancy might be related to the different peptides or to the different neuronal population. We can argue that Aβ1–40 sequence is considered less toxic compared to Aβ1– 42 [122] and this can explain the unchanged Cdc42 level.

Notably, the high concentration range used in both studies, which is unlikely to occur in vivo, suggests that Cdc42 activity might not be so affected by Aβ in the fibrillar form. On the other hand, treatment with Aβ oligomers resulted in a different outcome: Cdc42 expression was found to be decreased 24h after oligomeric Aβ25–35 treatment in PC12 cells [123]. This finding can match with a parallel decrease of the Cdc42 downstream effector PAK observed following oligomeric Aβ1–42 treatment [115, 116]. Again, the use of a different Aβ fragment and of a nonprimary cell culture renders difficult to conclusively interpret the available evidence. Another aspect that requires further analysis is the rod-like inclusion formation, which in neurons are due to an actin dynamics imbalance as consequence of stress stimuli or neurodegeneration. In organotypic slice cultures after oligomeric Aβ treatment cofilin was activated (dephosphorylated) and cofilin-actin based rods were visible in neurons 81. Although this might be a protective response to protect cell and spare the ATP normally consumed in actin dynamics [124], ultimately rod formation leads to synaptic impairment together with the blocking of axonal transports. Cdc42 could be a regulator of cofilin activity downstream of Aβ oligomers. Accordingly, in vitro hippocampal neurons stimulated with Aβ oligomers and transfected with the dominant negative Cdc42 resulted in less rod formation compared to control neurons [99]. RhoA The RhoA/ROCK pathway was found to be involved in several neurodegenerative diseases but its role in the pathogenesis of AD seems to be of particular interest as some of the proposed therapeutical strategies, such as nonsteroidal antiinflammatory drugs (NSAIDs) and statins that act directly or indirectly through the Rho/ROCK signaling pathway (Fig. 3). Huesa et al. [125] and Petratos et al. [112] investigated the expression, activity and localization of Rho GTPases together with some downstream effectors in AD patients and in Tg2576 mouse model, finding a prominent RhoA mislocalization. In Tg2576 mice, RhoA expression decreased at synapses and increased in dystrophic neurites [125], and around SP [112]. In AD human brains, RhoA decreased in the neuropil and increased in neurons, where it co-localized with hyperphosphorylated tau aggregates. This feature has been similarly observed in other tauopathies like Pick's disease [125]. On the contrary total RhoA was decreased in the hippocampus of human AD brains [125]. The authors observed no major changes for Rac1, Cdc42 or PAK in the same samples. Among the in vitro studies, Chacon and coworkers [126] found that after oligomeric Aβ1–42 treatment RhoA was activated within 2h in PC12 cells. This effect was abolished by treatment with nerve growth factor. In agreement, inhibition of RhoA by C3 transferase interrupted Aβ signaling in

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Fig. 2 Potential Aβ1–42-induced transduction pathways involving Rac1. Manterola et al. [111] has recently proposed that exogenously administered Aβ1–42 oligomers might induce neuronal death through the activation of a PI3K, PDK, novel (n) PKC pathway, ultimately activating Rac1. Aβ1-42 oligomers activated PI3K, involved in neural survival pathways, which in turn activated PDK1. PDK1 can phosphorylate several intracellular signaling molecules as some PKC family members. PKC is one of the most important serine–threonine kinase families involved in intracellular signal transduction. As PI3K and PDK1, nPKC

is involved in several pathways, which control cell survival and apoptosis. In the paper, inhibition of PKC δ and γ activities prevented Rac1 activation and apoptosis induced by Aβ1–42. The effects of Rac1 activation have not been fully elucidated as yet, but actin remodeling and NAPDH oxidase complex activation have been hypothesized. According to Ma et al. [115], Aβ1–42 oligomer treatment might also activate Tiam1, involved in regulating dendritic spine stability. Interestingly, Tiam1 might be tyrosine-phosphorylated and activated by Fyn

primary hippocampal neurons, protecting them from death [126]. The same effect was achieved in a neuroblastoma cell line and the authors suggested a mechanism in which Aβ1–42 was cleared by a Dynamin and RhoA-dependent endocytosis [127]. In contrast to these two studies, others showed that when cortical cultures were exposed to 10 μM Aβ1–40 for 24h, no changes in RhoA expression and activation were detected despite the neurodegeneration and apoptotic death occurring within the same time frame [121]. Zhou and coworkers [128] showed that RhoA/ROCK pathway is involved in Aβ1–42 overproduction both in vitro on neuroblastoma cells transfected with a mutated APP (Swedish mutation) as well as in vivo in a murine model of AD (PDAPP mouse). In particular, they observed in these mice a decrease of cortical Aβ1–42 after intraventricular injection with the ROCK inhibitor Y-27632 [128]. The authors hypothesized that RhoA/ROCK pathway might have an effect on γsecretase cleavage specificity as Y-27632 treatment altered the ratio between Aβ1–42 and Aβ1–38 production. Also the treatment with a Rac1 inhibitor (EHT 1864) determined a decreased APP processing by γ-secretase in vitro, and a reduction of the physiological level of Aβ 40 and 42 peptides in vivo in guinea pigs [129]. It is worth noticing that among the three small GTPases, only Rac1 was found in lipid rafts of adult rat brains, where the γ-secretase is located, while RhoA

and Cdc42 were found in the cytosolic fraction [130]. Thus Rac1 might directly influence the cleavage processing of APP, whereas RhoA/ROCK1 might act indirectly. Another interesting point concerns the ability of some NSAIDs to lower Aβ levels [131]. Although a direct effect of certain NSAIDs on γ-secretase activity was demonstrated [132, 133], many NSAIDs inhibit RhoA or directly ROCK [134]. For instance, Zhou and coworkers [128] showed that a subset of NSAIDs reduceed Aβ1–42 production and gave a dose-dependent inhibition of RhoA in vitro. Very recently, Fasudil, an NSAID with a potent inhibitory effect on ROCK, was found to have a neuroprotective effect in a rat model of AD [135]. Also for statins, evidence indicates that the antiamyloidogenic effect involves ROCK inhibition. First, statins impair isoprenylation, which is mandatory for Rho GTPases activation and recruiting to the plasma membrane [136]. Second, they activate the alpha secretase-type ectodomain shedding, generating the soluble APP ectodomain (sAPPα) [137]. DN-ROCK1 transfection led to enhanced sAPPalpha shedding in vitro, and the same effect was obtained with an inhibitor of isoprenylation [137]. Furthermore, a CA-ROCK1 blocked the statin-dependent shedding of sAPPα. This study pointed the attention on isoprenylation as the main target of statins, and on ROCK1 as a modulator of the APP

Mol Neurobiol Fig. 3 RhoA/ROCK1-dependent modulation of APP processing. Calorie restriction, statins, and NSAIDs are able to inhibit RhoA and ROCK1 by different mechanisms. Calorie restriction upregulates the transcription factor SIRT1 which in turn regulates the activity of other prosurvival transcription factors and downregulates ROCK1. Statins prevents the RhoA activation by inhibiting the isoprenylation, which is necessary for RhoGTPase activation. NSAIDs can inhibit directly either RhoA or ROCK1. APP can be processed sequentially by β- and γsecretases, leading to Aβ peptide formation in the so-called amyloidogenic APP processing, or by alpha and gamma secretases, leading to a nonamyloidogenic APP cleavage. ROCK1 is able to inhibit the second process. However, the intermediates and the precise mechanism connecting ROCK1 to its effect on APP processing are still unclear

nonamyloidogenic processing. A similar mechanism was found during the investigation of the relationship between calorie restriction and a decreased probability to develop AD [138]. Among the mechanisms proposed, the upregulation of silent information regulator of transcription 1 (SIRT1), a NAD(+)-dependent histone deacetilase, seem to be the protective key element against neurodegeneration [139], improving the nonamyloidogenetic α-secretase activity. SIRT1 regulates the activity of other transcription factors, such as NFkB, p53, and FOXO, that are critically involved in transcription of survival genes [140–142]. It has been shown that the mechanism by which SIRT1 improves the nonamyloidogenic pathway involved a downregulation of ROCK1, one of the

two ROCK isoforms. Rock1 was responsible for the inhibition of the α-secretase-dependent nonamyloidogenic processing of APP both in primary neurons [143, 144] as well as in mice and primates [143, 145]. However, this mechanism is not strictly pertaining to AD, but generally applicable to aging and neuronal degeneration. Very recently, it was also demonstrated that the neurotoxic Aβ25-35 peptide was able to suppress SIRT1 in vitro on PC12 cells and that resveratrol, a grapes-derived compound which is thought to be protective against AD, is able to rescue this suppression downregulating ROCK1 [146]. These studies point the attention on ROCK1 inhibition as a putative therapeutic strategy for AD. However, still unclear is the link between ROCK1 and APP or the α-secretase.

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Alternative Neurotoxic Mechanisms Involving Rho GTPases In the context of a potential connection between Rho GTPases and AD, we have to mention that Rho GTPases are involved in additional biological processes other than spine plasticity as inflammatory signal transduction cascade and neuronal survival [147, 148]. For instance, Rac1 is essential for the assembly and activation of NADPH oxidase, which is responsible for the production of reactive oxygen species (ROS). Dysfunctional mitochondria increase the production of harmful radicals causing oxidative stress in AD but also normal aged brain. Although during the last 20 years, the literature devoted to AD has primarily focused on Aβ and, to a lesser extent, on tau, several authors pointed to oxidative stress as a primary etiological factor [149]. From this perspective, the alteration of APP processing within AD vulnerable neurons would be a compensatory mechanism to counteract increased oxidative stress [149]. Stress-induced activation of Rac1 has been reported to cause oxidative DNA damage and neurodegeneration in rat dopaminergic cells [150]. Concerning neuronal death, to the best of our knowledge, the only paper trying to correlate in neurons AD and Rho GTPase signaling has been published this year. Aβ has been hypothesized to transduce cell death signals through PI3K/PKD/nPKC cascade as specific upstream regulatory pathway for Rac1 [111] (Fig. 2). Thus, activation of Rac1 would be required for Aβ toxicity as shown by the abolishment of apoptosis following Rac1 inhibitor NCS23766 administration in organotypic cultures co-treated with Aβ 100 nM for 4 days. The identification of Rac1 as a putative therapeutic target for AD is of high interest, but further studies are needed to characterize the receptors potentially participating upstream of this signal transduction pathway. Another study showed that the binding of transforming growth factor α 2 (TGFα2) to APP induced neuronal cell death by an intracellular signaling pathway mediated by a heterotrimeric G protein G0, Rac1, or Cdc42, ASK1, JNK, the NADPH oxidase and caspases [151]. In the familial forms of AD, the mutated APP could trigger itself the neuronal death without TGFα binding. Recently, it was hypothesized that the apoptotic signals coming from PS1 and APP could merge at a point between Rac1 and/or Cdc42 and apoptosis signal-regulated kinase 1 (ASK1) via the modifier of cell adhesion as a common downstream effector [152]. However, in these two studies, the authors did not specify which GTPase is involved in this signaling pathway, whether Rac1, Cdc42, or both. Further complicating the analysis, it is really difficult to ascertain which pathway might peculiarly pertain to AD mechanisms or which is just a consequence of the disease or, more broadly, even a consequence of the aging itself. Since there is no linear chain of events in AD, some of the observed alterations might not be pathogenic but protective.

Therapeutic Potential The involvement of Rho GTPases in dendritic spine and axon regulation suggests that their modulation might be tested to counteract the loss of neuronal connectivity in AD. The first attempt in this direction was the use of the Rho GTPase activator cytotoxic necrotizing factor 1 (CNF1). CNF1, intracerebroventricularly injected, reduced locomotor hyperactivity in 4-month-old TgCRND8 mice and reversed the cognitive impairment compared to wild-type animals (Fig. 4). In this study, TgCRND8 mice showed a selective deficit in reversal learning. CNF1 treatment corrected this derangement of behavioral plasticity while leaving unchanged reference/long-term memory during the initial place learning [153]. In another study, the administration of CNF1 to aged apoE4 mice, an animal model associated with a high risk of sporadic AD, improved spatial and emotional memory deficits and decreased the level of Aβ in the hippocampus [154]. This suggests that the modulation of Rho GTPases might be a tool to produce reversal effects on cognition through dendritic spine remodeling. Nevertheless, it is worth noting that CNF1 has been shown to enhance learning and memory in several conditions [92, 155, 156]. In addition, the molecule has been tested also in animal models of intellectual disability with a similar outcome. For instance, CNF1 administration markedly improved the behavioral phenotype in a Rett syndrome mouse model [157]. Therefore, the positive effect on cognition is most likely not directly correlated with the reversal of specific AD-like mechanisms. We can argue that CNF1 is a massive activator of all Rho GTPases, which contrasts with the fact that some of the members of the family exert antagonistic effects. Thus, a selective modulation of a single Rho GTPase member should be preferred rather than a global manipulation, which might produce artifacts and mask a specific contribution of the different members. Another interesting element to add to the discussion is the relation between Rho GTPases and statins, well-known antihypercholesterolemic drugs that have been shown to decrease the risk of developing AD [158]. One of the mechanisms of action of these molecules pertains to the inhibition of protein isoprenylation [136]. Interestingly enough, this is a typical posttranslational modification necessary for Rho GTPase recruitment to the cell membrane and subsequent activation by GEFs [159]. A study has addressed this relation: the novel statin pitavastatin decreased the amount of tau expression and phosphorylation and of membrane-bound Rac1, RhoA, and Cdc42 in primary neurons and in a cell line overexpressing tau [160]. This finding suggests that inhibition of small Rho GTPase maturation by low to moderate doses of pitavastatin may participate in the reduction of tau levels. In this paper, the authors focused the attention on RhoA, speculating that the decrease in phosphorylated tau by pitavastatin was caused by glycogen synthase kinase 3β (GSK3β) inactivation through RhoA. We

Mol Neurobiol

Fig. 4 Effects of the Rho GTPase-activating cytotoxic necrotizing factor 1 (CNF1) in a mouse model of Alzheimer's disease. The administration of CNF1 in the lateral cerebral ventricle of TgCRND8 mice reduced locomotor hyperactivity and improved behavioral plasticity. Top left: the molecule was injected in the lateral ventricle (10 fmol/kg, single injection) 10 days before the beginning of the behavioral assessment. Bottom left: Structure of CNF1 and its mechanism of action at a cellular level, a receptor binding domain, b membrane translocation domain, c catalytic domain. H1 and H2 hydrophobic helixes; R membrane receptor. Right:

Effects on spatial learning in 4-month-old TgCRND8: representative paths of swimming in the 8° trial of reversal learning of a water maze task (top) and representative paths of swimming in the spatial probe following the reversal learning (bottom). The mice had been previously trained in a place learning task with the platform in a fixed position (5 days, 4 trials/day, intertrial time=60 min). After moving the platform to a different position, TgCRND8 mice showed a selective deficit in reversal learning (2 days, 4 trials/day, intertrial time=60 min), which was corrected by the treatment (from Ref. [153])

cannot exclude that statins might have a more general impact on Rho GTPase signaling, considering also that pitavastatin decreased the membrane-bound fraction of many other small GTPases other than RhoA: Rac1, Cdc42 as well as two members of the Rab family of small G-proteins (Rab5 and Rab6). Lastly, it is also worth mentioning that Rac1-dependent pathways affecting survival and regeneration might be associated as downstream to ciliary neurotrophic factor (CNTF). CNTF, a member of the interleukin-6 cytokine family, has been shown to enhance neurogenesis in the hippocampus [161] and to promote self-renewal and maintenance of neuronal precursor cells in vitro [162]. Recently, a 11-mer peptide corresponding to the active region of CNTF rescued neurodegeneration and cognitive deficit in a rat model of sporadic AD [163]. In the frame of identifying strategies to rescue synaptic and behavioral dysfunction, Rho GTPase modulation can be used as a tool to enhance synaptogenesis and neuronal plasticity increasing the resistance of vulnerable neurons, thereby delaying the onset of the clinical manifestation of the pathology.

Boosting and improving neuronal plastic properties may indeed protect the memory network.

Future Directions The complexity of AD makes it extremely difficult to identify the molecular players relevant to its etiology. Progress in the understanding of the mechanisms underling the pathology has been substantial over the past decades, but this has failed to translate into successful therapeutic treatment. This has to do with the strong multifactorial character of this age-related disorder. Therefore, growing awareness on the fact that overlapping mechanisms may underpin dendrites withdrawal and synaptic dysfunction culminating in neuronal loss. In turn, we cannot rule out that different etiological factors may act through common pathways. The key roles of Rho GTPases in the regulation of actin dynamics on spines may itself be a sufficient rationale to

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hypothesize their involvement in neurodegenerative conditions. The studies discussed here clearly encourage further investigations, provided the analysis of the Rho GTPase regulation in AD-like models, and took into account recent insight into the framework of their signaling. As pointed out in the excellent review [164], Rho GTPase modification needs to be studied with tools allowing to appreciate their subtle spatiotemporal modification following a stimulus. Their ability to regulate different biological functions implies that they are also tightly regulated according to the cell compartment. For these reasons new imaging technologies need to be implemented to truly represent Rho GTPase spatiotemporal resolution. This newer approach, compared to the classical immunoprecipitation method, based on fluorescent biosensors, showed that Rho GTPases are regulated on a micrometer length scale and subminute time scales [85, 165–167]. From this perspective, even the use of immunofluorescent antibodies directed against the GTP bound fraction of the different Rho isoforms may be insufficient to clearly detect their focal recruitment at the membrane. To overcome the flaws of the classical tools, a variety of cutting edge biosensors have been designed, based on fluorescence resonance energy transfer technology [164, 167, 168]. The use of these probes has highlighted a much finer regulation of Rho GTPase signaling characterized by a close crosstalk between isoforms and a strict subcellular compartmentalization. RhoA activation, for example, seemed to diffuse from the stimulated spines and spread over about 5 μM along the dendrites [85]. Another key aspect to consider is that each single member of the family can activate several effectors acting through different and complementary routes [169]. This makes the identification of a potential pathway relevant to AD very challenging as the cascade of effects, which might be observed, are countless. One potentially fruitful approach to dissect the single Rho GTPase contribution is the use of mutant peptides other than simply the inactive form or the constitutive active forms. Early in vitro experiments have characterized several effector domain mutants that separate Rac1's role in lamellipodia formation from its role in activation of the Jun kinase (JNK) cascade and nuclear signaling [170]. In these mutants, an L61 mutation that tonically activates the protein is coupled to a second mutation in the effector domain (F37A or Y40C) that blocks the interaction with some downstream effectors. The Y40C mutation abolishes the binding of several CRIB-containing proteins (e.g., PAK) and, when combined to an activating Rac1 mutation, blocks the stimulation of the JNK pathways, without affecting membrane ruffling and lamellipodia formation. Conversely, F37A still binds PAK and activates the JNK pathway [170]. These data suggest that binding to the tyrosine residue 40 is required for the activation of JNK (e.g., PAK and other CRIBcontaining proteins), whereas effectors dependent on phenylalanine residue 37 are candidate mediators for Rac1-

dependent regulation of lamellipodia formation. We tested these peptides in a model of neuronal degeneration and found that the F37A or Y40C mutants improved survival and prevented dendrite degeneration, but only the one harboring the F37 mutation also improved the axonal regeneration [171]. These findings suggest that the selective activation of distinct Rac1-dependent pathways might indeed represent a therapeutic strategy to counteract neuronal degeneration. It follows that the speculation on a therapeutically relevance of Rho GTPases for AD is hampered by the availability of smart targeted agents able to proper modulate single Rho GTPase and, even more importantly, a specific signaling pathway.

Conclusions The descriptive connections between Rho GTPases and AD outlined in this review underline the importance of further investigating the role of the Rho GTPase family. Data published so far are interesting in many ways and have highlighted an interesting connection between Rho-GTPase signaling and Aβ, but we think there is much more to be discovered. The key question which is still lacking a definite answer is whether the synaptic dysfunction characterizing AD is caused by the pathological activation of a single triggering pathway or whether this dysfunction is triggered by several concomitant events ultimately leading to neuronal death. Most likely, given the multifactorial nature of AD, a certain degree of crosstalk should be expected from the wide spectrum of alterations characterizing the disease. In this perspective, we hypothesize that Rho GTPases might be crucial mediators of these detrimental modifications. Thus, regardless which is the primum movens of the disease, one logical way to proceed would be to find a way to counteract spine loss through a modulation of plasticity. As synapse degeneration is an established early event in AD, boosting the machinery regulating actin to render neurons less vulnerable and to protect spines from collapse. Acknowledgments This work was supported by funding of the University of Verona, “Fondazione Cariverona” project Verona Nanomedicine Initiative.

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