Stephen M. Strittmatter

Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers

A pathological hallmark of Alzheimer’s disease is an accumulation of insoluble plaque containing the amyloid-β peptide of 40–42 amino acid residues. Prefibrillar, soluble oligomers of amyloid-β have been recognized to be early and key intermediates in Alzheimer’s-disease-related synaptic dysfunction. At nanomolar concentrations, soluble amyloid-β oligomers block hippocampal long-term potentiation, cause dendritic spine retraction from pyramidal cells and impair rodent spatial memory. Soluble amyloid-β oligomers have been prepared from chemical syntheses, transfected cell culture supernatants, transgenic mouse brain and human Alzheimer’s disease brain. Together, these data imply a high-affinity cell-surface receptor for soluble amyloid-β oligomers on neurons—one that is central to the pathophysiological process in Alzheimer’s disease. Here we identify the cellular prion protein (PrPC) as an amyloid-β-oligomer receptor by expression cloning. Amyloid-β oligomers bind with nanomolar affinity to PrPC, but the interaction does not require the infectious PrPSc conformation. Synaptic responsiveness in hippocampal slices from young adult PrP null mice is normal, but the amyloid-β oligomer blockade of long-term potentiation is absent. Anti-PrP antibodies prevent amyloid-β-oligomer binding to PrPC and rescue synaptic plasticity in hippocampal slices from oligomeric amyloid-β. Thus, PrPC is a mediator of amyloid-β-oligomer-induced synaptic dysfunction, and PrPC-specific pharmaceuticals may have therapeutic potential for Alzheimer’s disease.

Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein

Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.

Plexin-Neuropilin-1 Complexes Form Functional Semaphorin-3A Receptors

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. We find that the two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.

Nogo-66 receptor antagonist peptide promotes axonal regeneration.

Myelin-derived axon outgrowth inhibitors, such as Nogo, may account for the lack of axonal regeneration in the central nervous system (CNS) after trauma in adult mammals. A 66-residue domain of Nogo (Nogo-66) is expressed on the surface of oligodendrocytes and can inhibit axonal outgrowth through an axonal Nogo-66 receptor (NgR). The IN-1 monoclonal antibody recognizes Nogo-A and promotes corticospinal tract regeneration and locomotor recovery; however, the undefined nature of the IN-1 epitope in Nogo, the limited specificity of IN-1 for Nogo, and nonspecific anti-myelin effects have prevented a firm conclusion about the role of Nogo-66 or NgR. Here, we identify competitive antagonists of NgR derived from amino-terminal peptide fragments of Nogo-66. The Nogo-66(1–40) antagonist peptide (NEP1–40) blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro, demonstrating that NgR mediates a significant portion of axonal outgrowth inhibition by myelin. Intrathecal administration of NEP1–40 to rats with mid-thoracic spinal cord hemisection results in significant axon growth of the corticospinal tract, and improves functional recovery. Thus, Nogo-66 and NgR have central roles in limiting axonal regeneration after CNS injury, and NEP1-40 provides a potential therapeutic agent.

Rho kinase inhibition enhances axonal regeneration in the injured CNS

Myelin-associated inhibitors limit axonal regeneration in the injured brain and spinal cord. A common target of many neurite outgrowth inhibitors is the Rho family of small GTPases. Activation of Rho and a downstream effector of Rho, p160ROCK, inhibits neurite outgrowth. Here, we demonstrate that Rho is directly activated by the myelin-associated inhibitor Nogo-66. Using a binding assay to measure Rho activity, we detected increased levels of GTP Rho in PC12 and dorsal root ganglion (DRG) cell lysates after Nogo-66 stimulation. Rho activity levels were not affected by Amino–Nogo stimulation. Rho inactivation with C3 transferase promotes neurite outgrowth of chick DRG neurons in vitro, but with the delivery method used here, it fails to promote neurite outgrowth after corticospinal tract (CST) lesions in the adult rat. Inhibition of p160ROCK with Y-27632 also promotes neurite outgrowth on myelin-associated inhibitors in vitro. Furthermore, Y-27632 enhances sprouting of CST fibers in vivo and accelerates locomotor recovery after CST lesions in adult rats.

Axon Regeneration in Young Adult Mice Lacking Nogo-A/B

After injury, axons of the adult mammalian brain and spinal cord exhibit little regeneration. It has been suggested that axon growth inhibitors, such as myelin-derived Nogo, prevent CNS axon repair. To investigate this hypothesis, we analyzed mice with a nogo mutation that eliminates Nogo-A/B expression. These mice are viable and exhibit normal locomotion. Corticospinal tract tracing reveals no abnormality in uninjured nogo-A/B(-/-) mice. After spinal cord injury, corticospinal axons of young adult nogo-A/B(-/-) mice sprout extensively rostral to a transection. Numerous fibers regenerate into distal cord segments of nogo-A/B(-/-) mice. Recovery of locomotor function is improved in these mice. Thus, Nogo-A plays a role in restricting axonal sprouting in the young adult CNS after injury.

Rac1 Mediates Collapsin-1-Induced Growth Cone Collapse

Collapsin-1 or semaphorin III(D) inhibits axonal outgrowth by collapsing the lamellipodial and filopodial structures of the neuronal growth cones. Because growth cone collapse is associated with actin depolymerization, we considered whether small GTP-binding proteins of the rho subfamily might participate in collapsin-1 signal transduction. Recombinant rho, rac1, and cdc42 proteins were triturated into embryonic chick (DRG) neurons. Constitutively active rac1 increases the proportion of collapsed growth cones, and dominant negative rac1 inhibits collapsin-1-induced collapse of growth cones and collapsin-1 inhibition of neurite outgrowth. DRG neurons treated with dominant negative rac1 remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation, or collapsin-1 sensitivity. Whereas the addition of activated rho has no effect, the inhibition of rho withClostridium botulinum C3 transferase stimulates the outgrowth of DRG neurites. C3 transferase-treated growth cones exhibit little or no lamellipodial spreading and are minimally responsive to collapsin-1 and myelin. These data demonstrate a prominent role for rho and rac1 in modulating growth cone motility and indicate that rac1 may mediate collapsin-1 action.

Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.

Amyloid-beta (Aβ) oligomers are thought to trigger Alzheimers disease pathophysiology. Cellular prion protein (PrPC) selectively binds oligomeric Aβ and can mediate Alzheimers disease–related phenotypes. We examined the specificity, distribution and signaling of Aβ-PrPC complexes, seeking to understand how they might alter the function of NMDA receptors (NMDARs) in neurons. PrPC is enriched in postsynaptic densities, and Aβ-PrPC interaction leads to Fyn kinase activation. Soluble Aβ assemblies derived from the brains of individuals with Alzheimers disease interacted with PrPC to activate Fyn. Aβ engagement of PrPC-Fyn signaling yielded phosphorylation of the NR2B subunit of NMDARs, which was coupled to an initial increase and then a loss of surface NMDARs. Aβ-induced dendritic spine loss and lactate dehydrogenase release required both PrPC and Fyn, and human familial Alzheimers disease transgene–induced convulsive seizures did not occur in mice lacking PrPC. These results delineate an Aβ oligomer signal transduction pathway that requires PrPC and Fyn to alter synaptic function, with deleterious consequences in Alzheimers disease.

Nogo-66 Receptor Prevents Raphespinal and Rubrospinal Axon Regeneration and Limits Functional Recovery from Spinal Cord Injury

Axon regeneration after injury to the adult mammalian CNS is limited in part by three inhibitory proteins in CNS myelin: Nogo-A, MAG, and OMgp. All three of these proteins bind to a Nogo-66 receptor (NgR) to inhibit axonal outgrowth in vitro. To explore the necessity of NgR for responses to myelin inhibitors and for restriction of axonal growth in the adult CNS, we generated ngr(-/-) mice. Mice lacking NgR are viable but display hypoactivity and motor impairment. DRG neurons lacking NgR do not bind Nogo-66, and their growth cones are not collapsed by Nogo-66. Recovery of motor function after dorsal hemisection or complete transection of the spinal cord is improved in the ngr(-/-) mice. While corticospinal fibers do not regenerate in mice lacking NgR, regeneration of some raphespinal and rubrospinal fibers does occur. Thus, NgR is partially responsible for limiting the regeneration of certain fiber systems in the adult CNS.

Memory impairment in transgenic Alzheimer mice requires cellular prion protein.

Soluble oligomers of the amyloid-β (Aβ) peptide are thought to play a key role in the pathophysiology of Alzheimers disease (AD). Recently, we reported that synthetic Aβ oligomers bind to cellular prion protein (PrPC) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Aβ peptide. We hypothesized that PrPC is essential for the ability of brain-derived Aβ to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1ΔE9 into Prnp−/− mice to examine the necessity of PrPC for AD-related phenotypes. Neither APP expression nor Aβ level is altered by PrPC absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrPC expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1ΔE9 transgenic mice. The AD transgenic mice with intact PrPC expression exhibit deficits in spatial learning and memory. Mice lacking PrPC, but containing Aβ plaque derived from APPswe/PSen1ΔE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrPC expression dissociates Aβ accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrPC.