Peripheral deafferentation-driven functional somatosensory map shifts are associated with local, not large-scale dendritic structural plasticity.

Long-term peripheral deafferentation induces representational map changes in the somatosensory cortex. It has been suggested that dendrites and axons structurally rearrange in such paradigms. However, the extent and process of this plasticity remains elusive. To more precisely quantify deafferentation-induced structural plasticity of excitatory cells we repeatedly imaged GFP-expressing L2/3 and L5 pyramidal dendrites in the mouse barrel cortex over months after the removal of a subset of the whisker follicles (FR), a procedure that completely and permanently removes whisker-sensory input. In the same mice we imaged whisker-evoked intrinsic optical signals (IOS) to assess functional cortical map changes. FR triggered the expansion of spared whisker IOS responses, whereas they remained unchanged over months in controls. The gross structure and orientation of apical dendrite tufts remained stable over a two-month period, both in controls and after deprivation. However, terminal branch tip dynamics were slightly reduced after FR, and the formation of new dendritic spines was increased in a cell-type and location-dependent manner. Together, our data suggest that peripheral nerve lesion-induced cortical map shifts do not depend on the large scale restructuring of dendritic arbors but are rather associated with local cell-type and position-dependent changes in dendritic synaptic connectivity.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

The RhoA-associated protein Citron-N controls dendritic spine maintenance by interacting with spine-associated Golgi compartments.

Dendritic spines are highly dynamic protuberances that are thought to be crucial for learning and memory. Although it is well known that actin filaments and membrane dynamics regulate spine plasticity, how these two events are linked locally is less clear. Here, we provide evidence that Citron-N (CIT-N), a binding partner of the small GTPase RhoA, is associated with the actin filaments and Golgi compartments of dendritic spines. We also show that CIT-N is required for recruiting F-actin and Golgi membranes at spines of in vitro-grown neurons. Studies in knockout mice show that this protein is essential for the maturation of dendritic spines. We suggest that CIT-N might function as a scaffold protein in spine organization through its ability to bind to Golgi membranes and by affecting actin remodelling.

Amyloid toxicity is independent of polypeptide sequence, length and chirality.

By using an amyloid sequence pattern, here we have identified putative six-residue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as A beta(1-42) do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length A beta(1-42) amyloid peptide or their amyloid stretch A beta(16-21) peptide preferentially localize in synapses, leading to the re-organization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.

Transmitting on actin: synaptic control of dendritic architecture.

Excitatory synaptic transmission in the central nervous system mainly takes place at dendritic spines, highly motile protrusions on the dendritic surface. Depending on the stimuli received, dendritic spines undergo rapid actin-based changes in their morphology. This plasticity appears to involve signaling through numerous proteins that control the organization of the actin cytoskeleton (actin regulators). At least in part, recruitment and activation of these depends on neurotransmitter receptors at the post-synapse, which directly link neurotransmission to changes in dendritic spine architecture. However, other, non-neurotransmitter-receptors present at dendritic spines also participate. It is likely that several receptor types can control the activity of a single actin-regulatory pathway and it is the complex integration of numerous signals that determines the overall architecture of a dendritic spine.

Localized recruitment and activation of RhoA underlies dendritic spine morphology in a glutamate receptor-dependent manner.

Actin is the major cytoskeletal source of dendritic spines, which are highly specialized protuberances on the neuronal surface where excitatory synaptic transmission occurs (Harris, K.M., and S.B. Kater. 1994. Annu. Rev. Neurosci. 17:341-371; Yuste, R., and D.W. Tank. 1996. Neuron. 16:701-716). Stimulation of excitatory synapses induces changes in spine shape via localized rearrangements of the actin cytoskeleton (Matus, A. 2000. Science. 290:754-758; Nagerl, U.V., N. Eberhorn, S.B. Cambridge, and T. Bonhoeffer. 2004. Neuron. 44:759-767). However, what remains elusive are the precise molecular mechanisms by which different neurotransmitter receptors forward information to the underlying actin cytoskeleton. We show that in cultured hippocampal neurons as well as in whole brain synaptosomal fractions, RhoA associates with glutamate receptors (GluRs) at the spine plasma membrane. Activation of ionotropic GluRs leads to the detachment of RhoA from these receptors and its recruitment to metabotropic GluRs. Concomitantly, this triggers a local reduction of RhoA activity, which, in turn, inactivates downstream kinase RhoA-specific kinase, resulting in restricted actin instability and dendritic spine collapse. These data provide a direct mechanistic link between neurotransmitter receptor activity and the changes in spine shape that are thought to play a crucial role in synaptic strength.

RhoA, Rac1, and cdc42 intracellular distribution shift during hippocampal neuron development.

Differences in cytoskeleton organization are key determinants of the architecture and dynamics of axons and dendrites. This is most clearly illustrated by the diverse pools of microtubule-associated proteins in axons and dendrites. Whether similar polarized organization occurs for actin regulatory proteins remains to be determined. To address this issue, we analyzed the intracellular distribution of the Rho GTPases, RhoA, Rac1, and cdc42 in hippocampal neurons in culture. We report that all three Rho members are evenly distributed during the time of axon and dendrite sprouting. This is not the case in mature neurons, as RhoA enriches in dendrites, Rac1 in axons, and Cdc42 is equally abundant in both domains. Polarized segregation of the actin regulatory machinery in mature neurons might play an important role in axonal and dendritic architectural plasticity.

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation.

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.