Interaction of amyloid-ß (Aß) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice.

Brain accumulation of the amyloid-ß protein (Aß) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aß oligomers (AßOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AßOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AßOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AßOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AßO binding to hippocampal neurons and prevented AßO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AßOs in mice. The results indicate that Nrx2α and NL1 are targets of AßOs and that prevention of this interaction reduces the deleterious impact of AßOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AßOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Rapid Mechanically Controlled Rewiring of Neuronal Circuits.

CNS injury may lead to permanent functional deficits because it is still not possible to regenerate axons over long distances and accurately reconnect them with an appropriate target. Using rat neurons, microtools, and nanotools, we show that new, functional neurites can be created and precisely positioned to directly (re)wire neuronal networks. We show that an adhesive contact made onto an axon or dendrite can be pulled to initiate a new neurite that can be mechanically guided to form new synapses at up to 0.8 mm distance in <1 h. Our findings challenge current understanding of the limits of neuronal growth and have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration. Significance statement: Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Using microtools and nanotools we have developed a new method to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved are ≥60 times faster than previously reported. Our findings have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration after trauma and in neurodegenerative diseases. It also opens the door for the direct wiring of robust brain-machine interfaces as well as for investigations of fundamental aspects of neuronal signal processing and neuronal function.

Netrin-1 promotes excitatory synaptogenesis between cortical neurons by initiating synapse assembly.

Netrin-1 is a secreted protein that directs long-range axon guidance during early stages of neural circuit formation and continues to be expressed in the mammalian forebrain during the postnatal period of peak synapse formation. Here we demonstrate a synaptogenic function of netrin-1 in rat and mouse cortical neurons and investigate the underlying mechanism. We report that netrin-1 and its receptor DCC are widely expressed by neurons in the developing mammalian cortex during synapse formation and are enriched at synapses in vivo. We detect DCC protein distributed along the axons and dendrites of cultured cortical neurons and provide evidence that newly translated netrin-1 is selectively transported to dendrites. Using gain and loss of function manipulations, we demonstrate that netrin-1 increases the number and strength of excitatory synapses made between developing cortical neurons. We show that netrin-1 increases the complexity of axon and dendrite arbors, thereby increasing the probability of contact. At sites of contact, netrin-1 promotes adhesion, while locally enriching and reorganizing the underlying actin cytoskeleton through Src family kinase signaling and m-Tor-dependent protein translation to locally cluster presynaptic and postsynaptic proteins. Finally, we demonstrate using whole-cell patch-clamp electrophysiology that netrin-1 increases the frequency and amplitude of mEPSCs recorded from cortical pyramidal neurons. These findings identify netrin-1 as a synapse-enriched protein that promotes synaptogenesis between mammalian cortical neurons.

Atomic force microscopy reveals important differences in axonal resistance to injury.

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.

Myosin is reversibly inhibited by S-nitrosylation.

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Reversing adhesion with light: a general method for functionalized bead release from cells.

Coated beads retain great importance in the study of cell adhesion and intracellular communication; we present a generally applicable method permitting spatiotemporal control of bead adhesion from cells. Herein we demonstrate in vitro release of a poly-d-lysine (PDL) layer from anionic polystyrene beads, allowing complete bead release from rat cortical neurons post-adhesion.

Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-ß peptide (Aß) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aß than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aß40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aß40 and blocking Aß-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aß40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aß, IQ and analogues of IQ on α3ß4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aß40 potently inhibit the function of α3ß4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3ß4 nAChRs by Aß40 reversible. These findings indicate that Aß40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Human apolipoprotein A-I binds amyloid-beta and prevents Abeta-induced neurotoxicity.

Aggregates of the amyloid-beta peptide (Abeta) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Abeta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Abeta(1-42), we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and Abeta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Abeta. Significantly, Abeta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Abeta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Abeta aggregation and Abeta-induced neuronal damage and that the Abeta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Abeta toxicity.

Amyloid-beta binds to the extracellular cysteine-rich domain of Frizzled and inhibits Wnt/beta-catenin signaling.

The amyloid-beta peptide (Abeta) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Abeta-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Abeta binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Abeta and are homologous to neuronal receptors putatively involved in Abeta interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Abeta with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Abeta and Fz. The results show that Abeta binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Abeta binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Abeta-binding site in Fz is homologous to cSP5 and that this is a relevant target for Abeta-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Abeta with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection.

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.

Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta.

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.

In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion.

Trypanosoma cruzi causing Chagas' disease needs to invade host cells to complete its life cycle. Macromolecules on host cell surfaces such as laminin, thrombospondin, heparan sulfate, and fibronectin are believed to be important in mediating parasite-host cell adhesions and in the invasion process of the host cell by the parasite. The SELEX technique (systematic evolution of ligands by exponential enrichment) was used to evolve nuclease-resistant RNA ligands (aptamer = to fit) that bind with affinities of 40-400 nm to parasite receptors for the host cell matrix molecules laminin, fibronectin, thrombospondin, and heparan sulfate. After eight consecutive rounds of in vitro selection four classes of RNA aptamers based on structural similarities were isolated and sequenced. All members of each class shared a common sequence motif and competed with the respective host cell matrix molecule that was used for displacement during the selection procedure. RNA pools following seven and eight selection rounds as well as individual aptamers sharing consensus motifs were active in inhibiting invasion of LLC-MK(2) monkey kidney cells by T. cruzi in vitro.