Stefano Alberi

The Mental Retardation Protein PAK3 Contributes to Synapse Formation and Plasticity in Hippocampus

Mutations of the gene coding for PAK3 (p21-activated kinase 3) are associated with X-linked, nonsyndromic forms of mental retardation (MRX) in which the only distinctive clinical feature is the cognitive deficit. The mechanisms through which PAK3 mutation produces the mental handicap remain unclear, although an involvement in the mechanisms that regulate the formation or plasticity of synaptic networks has been proposed. Here we show, using a transient transfection approach, that antisense and small interfering RNA-mediated suppression of PAK3 or expression of a dominant-negative PAK3 carrying the human MRX30 mutation in rat hippocampal organotypic slice cultures results in the formation of abnormally elongated dendritic spines and filopodia-like protrusions and a decrease in mature spine synapses. Ultrastructural analysis of the changes induced by expression of PAK3 carrying the MRX30 mutation reveals that many elongated spines fail to express postsynaptic densities or contact presynaptic terminals. These defects are associated with a reduced spontaneous activity, altered expression of AMPA-type glutamate receptors, and defective long-term potentiation. Together, these data identify PAK3 as a key regulator of synapse formation and plasticity in the hippocampus and support interpretations that these defects might contribute to the cognitive deficits underlying this form of mental retardation.

Long-Term Rearrangements of Hippocampal Mossy Fiber Terminal Connectivity in the Adult Regulated by Experience

We investigated rearrangements of connectivity between hippocampal mossy fibers and CA3 pyramidal neurons. We found that mossy fibers establish 10-15 local terminal arborization complexes (LMT-Cs) in CA3, which exhibit major differences in size and divergence in adult mice. LMT-Cs exhibited two types of long-term rearrangements in connectivity in the adult: progressive expansion of LMT-C subsets along individual dendrites throughout life, and pronounced increases in LMT-C complexities in response to an enriched environment. In organotypic slice cultures, subsets of LMT-Cs also rearranged extensively and grew over weeks and months, altering the strength of preexisting connectivity, and establishing or dismantling connections with pyramidal neurons. Differences in LMT-C plasticity reflected properties of individual LMT-Cs, not mossy fibers. LMT-C maintenance and growth were regulated by spiking activity, mGluR2-sensitive transmitter release from LMTs, and PKC. Thus, subsets of terminal arborization complexes by mossy fibers rearrange their local connectivities in response to experience and age throughout life.

LTP, Memory and Structural Plasticity

Our current understanding of the mechanisms of information processing and storage in the brain, based on the concept proposed more than fifty years ago by D. Hebb, is that a key role is played by changes in synaptic efficacy induced by coincident pre- and postsynaptic activity. Decades of studies of the properties of long-term potentiation (LTP) have shown that this form of plasticity adequately fulfills these requirements and is likely to contribute to several models of learning and memory. Recent analyses of the molecular events implicated in LTP are consistent with the view that modifications of receptor properties or insertion of new receptors account for the potentiation of synaptic transmission. These experiments, however, have also uncovered an unexpected structural plasticity of synapses. Dendritic spines appear to be dynamic structures that can be formed, modified in their shape or eliminated under the influence of activity. Furthermore, recent studies suggest that LTP, in addition to changes in synaptic function, is also associated with mechanisms of synaptogenesis. We review here the evidence pointing to this activity-dependent remodeling and discuss the possible role of this structural plasticity for synaptic potentiation, learning and memory.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA‐type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1‐binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR‐mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21–GRIP1 binding is crucial for GluR2‐AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Glutamate receptor changes associated with transient anoxia/hypoglycaemia in hippocampal slice cultures.

Transient anoxia/hypoglycaemia in organotypic hippocampal slice cultures, a model of transient brain ischaemia, ultimately results in delayed cell death. Although the mechanisms underlying this delayed death remain unknown, an increase in excitatory drive has been postulated. We report here that transient anoxia/hypoglycaemia in rat hippocampal slice cultures resulted in a 70–80% enhancement of evoked, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolpropionic acid (AMPA) receptor‐mediated, excitatory responses lasting over 60 min. This effect was prevented by blockade of N‐methyl‐d‐aspartate (NMDA) receptors, did not involve changes of paired‐pulse facilitation ratio, but was associated with a 50% increase in amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs). Consistent with this, paired recordings revealed the appearance of AMPA receptor‐mediated EPSCs at previously silent synapses and occlusion by prior induction of long‐term potentiation (LTP). Transient anoxia/hypoglycaemia further resulted in a 63% potentiation of evoked NMDA receptor‐dependent synaptic responses, accounting for the 20% increase in ratio of AMPA to NMDA responses. No change in rectification properties of AMPA receptor‐mediated currents could be detected within the first hour following anoxia/hypoglycaemia‐induced potentiation. Western blot analyses of slice cultures exposed to either control conditions or a short anoxia/hypoglycaemia revealed a marked, 50–70% increase of GluR1, GluR2/3 and NR1 subunits 1 h, but not 15 min, after the anoxic/hypoglycaemic episode. This increase was blocked by an inhibitor of protein synthesis. Together these results indicate that a transient anoxia/hypoglycaemia is associated with a marked enhancement of excitatory transmission sharing similarities with the mechanisms underlying LTP, and is correlated with an increased synthesis of excitatory receptor subunits.

The endosomal protein NEEP21 regulates AMPA receptor-mediated synaptic transmission and plasticity in the hippocampus

The neuron-enriched endosomal protein 21 (NEEP21) has recently been implicated in the regulation of AMPA receptor (AMPAR) trafficking and proposed to participate in the control of synaptic strength. We tested here this possibility at CA3-CA1 synapses in hippocampal slice cultures using antisense-mediated down-regulation of NEEP21 expression or transfection of a fragment of the cytosolic domain of NEEP21. We found that NEEP21 suppression or expression of the dominant-negative fragment reduced spontaneous and evoked AMPAR-mediated synaptic currents without affecting presynaptic properties. The effect specifically resulted from a reduction of currents mediated by AMPA as opposed to NMDA receptors. Blockade of endocytosis, using a peptide interfering with dynamin, revealed a progressive increase of AMPAR responses due to receptor accumulation in control cells, but not following NEEP21 suppression or expression of the fragment. Also, the enhanced receptor cycling induced by bath application of NMDA resulted in a depression that was enhanced following interference with NEEP21 function. Finally, LTP induction, which involves expression of new synaptic receptors, was abolished in NEEP21-depleted cells or cells expressing the dominant-negative fragment. Together, we conclude that NEEP21 contributes to the regulation of synaptic transmission and plasticity in slice cultures by affecting the recycling and targeting of AMPA receptors to the synapse.

A point mutant of GAP-43 induces enhanced short-term and long-term hippocampal plasticity

The growth‐associated protein GAP‐43 (or neuromodulin or B‐50) plays a critical role during development in mechanisms of axonal growth and formation of synaptic networks. At later times, GAP‐43 has also been implicated in the regulation of synaptic transmission and properties of plasticity such as long‐term potentiation. In a molecular approach, we have analyzed transgenic mice overexpressing different mutated forms of GAP‐43 or deficient in GAP‐43 to investigate the role of the molecule in short‐term and long‐term plasticity. We report that overexpression of a mutated form of GAP‐43 that mimics constitutively phosphorylated GAP‐43 results in an enhancement of long‐term potentiation in CA1 hippocampal slices. This effect is specific, because LTP was affected neither in transgenic mice overexpressing mutated forms of non‐phosphorylatable GAP‐43 nor in GAP‐43 deficient mice. The increased LTP observed in transgenic mice expressing a constitutively phosphorylated GAP‐43 was associated with an increased paired‐pulse facilitation as well as an increased summation of responses during high frequency bursts. These results indicate that, while GAP‐43 is not necessary for LTP induction, its phosphorylation may regulate presynaptic properties, thereby affecting synaptic plasticity and the induction of LTP.

Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors.

The number of synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-d-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.

The Oxytocin‐induced Inward Current in Vagal Neurons of the Rat is Mediated by G Protein Activation but not by an Increase in the lntracellular Calcium Concentration

The neuropeptide oxytocin can depolarize parasympathetic preganglionic neurons in the dorsal motor nucleus of the vagus nerve of the rat by generating a sustained inward current, which is sodium‐dependent and tetrodotoxin‐insensitive. The second messenger activated by oxytocin receptor binding is, however, not yet known. In the present study, we attempted to characterize it by using the whole‐cell recording technique and brainstem slices. When loaded with GTP‐γ‐S, a non‐hydrolysable analogue of GTP, vagal neurons generated a persistent inward current in the absence of agonist and the oxytocin effect was suppressed, suggesting that the peptide‐evoked current was mediated by G‐protein activation. Loading vagal neurons with the calcium chelator 1,2–bis(2–aminophenoxy)ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA) suppressed a calcium‐dependent, slowly decaying potassium aftercurrent but did not affect the oxytocin response, suggesting that the latter was not mediated by an agonist‐induced increase in the intracellular calcium concentration. Protein kinase C (PKC) activation was probably not involved, since the peptide‐evoked current was not modified by loading neurons with the PKC inhibitor H7. Thus, the oxytocin‐evoked current in vagal neurons was probably not mediated by phospholipase C‐β (PLC‐β) activation. Loading neurons with 8–Br‐CAMP or with an adenylyl cyclase activator (forskolin) reduced the oxytocin‐evoked current by about half. SQ 22536, an adenylyl cyclase inhibitor, reduced this current by a similar amount. However, the peptide‐evoked current was unaffected by Rp‐CAMPS and Sp‐CAMPS, an inhibitor and an activator, respectively, of CAMP‐dependent protein kinase (PKA). We suggest that oxytocin activates two distinct signalling pathways in vagal neurons: one which is CAMP‐dependent, but PKA‐independent, and one, unidentified, which is PLC‐β‐and CAMP‐independent. Each pathway accounts for about half of the peptide effect and both appear to involve G‐protein activation.

Central nervous system functions of PAK protein family: from spine morphogenesis to mental retardation.

Several of the genes currently known to be associated, when mutated, with mental retardation, code for molecules directly involved in Rho guanosine triphosphatase (GTPase) signaling. These include PAK3, a member of the PAK protein kinase family, which are important effectors of small GTPases. In many systems, PAK kinases play crucial roles regulating complex mechanisms such as cell migration, differentiation, or survival. Their precise functions in the central nervous system remain, however, unclear. Although their activity does not seem to be required for normal brain development, several recent studies point to a possible involvement in more subtle mechanisms such as neurite outgrowth, spine morphogenesis or synapse formation, and plasticity. This article reviews this information in the light of the current knowledge available on the molecular characteristics of the different members of this family and discuss the mechanisms through which they might contribute to cognitive functions.