The actin binding protein drebrin helps to protect against the development of seizure-like events in the entorhinal cortex.

The actin binding protein drebrin plays a key role in dendritic spine formation and synaptic plasticity. Decreased drebrin protein levels have been observed in temporal lobe epilepsy, suggesting the involvement of drebrin in the disease. Here we investigated the effect of drebrin knockout on physiological and pathophysiological neuronal network activities in mice by inducing gamma oscillations, involved in higher cognitive functions, and by analyzing pathophysiological epileptiform activity. We found that loss of drebrin increased the emergence of spontaneous gamma oscillations suggesting an increase in neuronal excitability when drebrin is absent. Further analysis showed that although the kainate-induced hippocampal gamma oscillations were unchanged in drebrin deficient mice, seizure like events measured in the entorhinal cortex appeared earlier and more frequently. The results suggest that while drebrin is not essential for normal physiological network activity, it helps to protect against the formation of seizure like activities during pathological conditions. The data indicate that targeting drebrin function could potentially be a preventive or therapeutic strategy for epilepsy treatment.

ATM phosphorylation of the actin-binding protein drebrin controls oxidation stress-resistance in mammalian neurons and C. elegans.

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer's disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.

Defective actin dynamics in dendritic spines: cause or consequence of age-induced cognitive decline?

Ageing is a complex deteriorating process that coincides with changes in metabolism, replicative senescence, increased resistance to apoptosis, as well as progressive mitochondria dysfunction that lead to an increase production and accumulation of reactive oxygen species (ROS). Although controversy on the paradigm of the oxidative damage theory of ageing exists, persuasive studies in Caenorhabditis elegans and yeast have demonstrated that manipulation of ROS can modify the process of ageing and influences the damage of proteins, lipids and DNA. In neurons, ageing impacts on the intrinsic neuronal excitability, it decreases the size of neuronal soma and induces the loss of dendrites and dendritic spines. The actin cytoskeleton is an abundant and broadly expressed system that plays critical functions in many cellular processes ranging from cell motility to controlling cell shape and polarity. It is thus hardly surprising that the expression and the function of actin in neurons is crucial for the morphological changes that occur in the brain throughout life. We propose that alterations in actin filament dynamics in dendritic spines may be one of the key events contributing to the initial phases of ageing in the brain.

Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders.

PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN.

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.

The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity.

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.

p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor.

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.

PAK signalling in neuronal physiology.

Group I p21-activated kinases are a family of key effectors of Rac1 and Cdc42 and they regulate many aspects of cellular function, such as cytoskeleton dynamics, cell movement and cell migration, cell proliferation and differentiation, and gene expression. The three genes PAK1/2/3 are expressed in brain and recent evidence indicates their crucial roles in neuronal cell fate, in axonal guidance and neuronal polarisation, and in neuronal migration. Moreover they are implicated in neurodegenerative diseases and play an important role in synaptic plasticity, with PAK3 being specifically involved in mental retardation. The main goal of this review is to describe the molecular mechanisms that govern the different functions of group I PAK in neuronal signalling and to discuss the specific functions of each isoform.

The four mammalian splice variants encoded by the p21-activated kinase 3 gene have different biological properties.

The p21-activated kinases (PAK1), PAK2, and PAK3 are members of the PAK group I and share high sequence identity and common biochemical properties. PAK3 is specifically implicated in neuronal plasticity and also regulates cell cycle progression, neuronal migration, and apoptosis. Loss of function of PAK3 is responsible for X-linked non-syndromic mental retardation whereas gain of PAK3 function is associated with cancer. To understand the functional specificities of PAK3, we analyzed the structure of PAK3 gene products. We report here the characterization of a new alternatively spliced exon called c located upstream of the previously identified exon b. Exon b is detected in all tetrapods and not in fish, exon c is only present in mammals. Mammalian PAK3 genes encode four splice variants and the corresponding proteins were detected with specific antibodies in brain extracts. All PAK3 transcripts are specifically expressed in brain and in particular in neurons. The presence of the exons b and c renders the kinase constitutively active and decreases interaction with GTPases. The expression of the new splice variants in COS7 cells alters cell morphology and modifies the structure of focal adhesions. We propose that the appearance of new alternatively spliced exons during evolution and the resulting increase of complexity of PAK3 gene products may confer new functions to this kinase and contribute to its specific roles in neuronal signaling.

The p21-activated kinase 3 implicated in mental retardation regulates spine morphogenesis through a Cdc42-dependent pathway.

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.