Zu-Lin Chen

Neuronal Death in the Hippocampus Is Promoted by Plasmin-Catalyzed Degradation of Laminin

Excess excitatory amino acids can provoke neuronal death in the hippocampus, and the extracellular proteases tissue plasminogen activator (tPA) and plasmin (ogen) have been implicated in this death. To investigate substrates for plasmin that might influence neuronal degeneration, extracellular matrix (ECM) protein expression was examined. Laminin is expressed in the hippocampus and disappears after excitotoxin injection. Laminin disappearance precedes neuronal death, is spatially coincident with regions that exhibit neuronal loss, and is blocked by either tPA-deficiency or infusion of a plasmin inhibitor, both of which also block neuronal degeneration. Preventing neuron-laminin interaction by infusion of anti-laminin antibodies into tPA-deficient mice restores excitotoxic sensitivity to their hippocampal neurons. These results indicate that disruption of neuron-ECM interaction via tPA/plasmin catalyzed degradation of laminin sensitizes hippocampal neurons to cell death.

Laminin γ1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve

Laminins are heterotrimeric extracellular matrix proteins that regulate cell viability and function. Laminin-2, composed of α2, β1, and γ1 chains, is a major matrix component of the peripheral nervous system (PNS). To investigate the role of laminin in the PNS, we used the Cre–loxP system to disrupt the laminin γ1 gene in Schwann cells. These mice have dramatically reduced expression of laminin γ1 in Schwann cells, which results in a similar reduction in laminin α2 and β1 chains. These mice exhibit motor defects which lead to hind leg paralysis and tremor. During development, Schwann cells that lack laminin γ1 were present in peripheral nerves, and proliferated and underwent apoptosis similar to control mice. However, they were unable to differentiate and synthesize myelin proteins, and therefore unable to sort and myelinate axons. In mutant mice, after sciatic nerve crush, the axons showed impaired regeneration. These experiments demonstrate that laminin is an essential component for axon myelination and regeneration in the PNS.

Schwann Cell-Specific Ablation of Laminin γ1 Causes Apoptosis and Prevents Proliferation

To investigate the function of laminin in peripheral nerve development, we specifically disrupted the laminin γ1 gene in Schwann cells. Disruption of laminin γ1 gene expression resulted in depletion of all other laminin chains known to be expressed in Schwann cells. Schwann cells lacking laminin do not extend processes required for initiating axonal sorting and mediating axon-Schwann cell interaction. They fail to downregulate Oct-6 and arrest at the premyelinating stage. The impaired axon-Schwann cell interaction prevents phosphorylation of β-neuregulin-1 receptors and results in decreased cell proliferation. Postnatally, laminin-null Schwann cells exhibit reduced phosphatidylinositol 3 (PI3)-kinase activity and activation of caspase cascades, leading to apoptosis. Injection of a laminin peptide into mutant sciatic nerves partially restores PI3-kinase activity and reduces apoptotic signals. These results demonstrate the following: (1) that laminin initiates axonal sorting and mediates axon-Schwann cell interactions required for Schwann cell proliferation and differentiation, and (2) that laminin provides a PI3-kinase/Akt-mediated Schwann cell survival signal.

Regulation of Schwann cell function by the extracellular matrix.

Laminins and collagens are extracellular matrix proteins that play essential roles in peripheral nervous system development. Laminin signals regulate Schwann cell proliferation and survival as well as actin cytoskeleton dynamics, which are essential steps for radial sorting and myelination of peripheral axons by Schwann cells. Collagen and their receptors promote Schwann cell adhesion, spreading, and myelination as well as neurite outgrowth. In this article, we will review the recent advances in the studies of laminin and collagen function in Schwann cell development.

Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.

Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death.

Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

Laminins have dramatic and varied actions on neurons in vitro. However, their in vivo function in brain development is not clear. Here we show that knockout of laminin gamma1 in the cerebral cortex leads to defects in neuritogenesis and neuronal migration. In the mutant mice, cortical layer structures were disrupted, and axonal pathfinding was impaired. During development, loss of laminin expression impaired phosphorylation of FAK and paxillin, indicating defects in integrin signaling pathways. Moreover, both phosphorylation and protein levels of GSK-3beta were significantly decreased, but only phosphorylation of AKT was affected in the mutant cortex. Knockout of laminin gamma1 expression in vitro, dramatically inhibited neurite growth. These results indicate that laminin regulates neurite growth and neuronal migration via integrin signaling through the AKT/GSK-3beta pathway, and thus reveal a novel mechanism of laminin function in brain development.

Laminin is required for Schwann cell morphogenesis

Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, we demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting.

Exogenous tissue plasminogen activator enhances peripheral nerve regeneration and functional recovery after injury in mice.

Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in the nervous system. We show that exogenous tPA or tPA/plasminogen (plg) promotes axonal regeneration, remyelination, and functional recovery after sciatic nerve injury in the mouse. Local application of tPA or tPA/plg 7 days after sciatic nerve crush significantly increased the total number of axons and myelinated axons, which is accompanied by enhanced expression of neurofilament. Treatment with tPA or tPA/plg reduced the deposition of fibrin(ogen) after nerve injury. Moreover, tPA or tPA/plg increased the number of macrophages and induced MMP-9 expression at the injury site, coincident with reduced collagen scar formation and accelerated clearance of myelin and lipid debris after treatment. Consequently, tPA or tPA/plg treatment protected muscles from atrophy after nerve injury, indicating better functional recovery. These results suggest that administration of exogenous tPA or tPA/plg promotes axonal regeneration and remyelination through removal of fibrin deposition and activation of MMP-9-positive macrophages, which may be responsible for myelin debris clearance and preventing collagen scar formation. Therefore, tPA may be useful for treatment of peripheral nerve injury.

Activation of the factor XII-driven contact system in Alzheimer’s disease patient and mouse model plasma

Significance Alzheimer’s disease (AD) is a growing public health problem, in part because there are no effective therapies. Major roadblocks to the treatment of AD are the lack of adequate diagnostic tools and the absence of viable therapeutic targets. It is now clear that AD is characterized by inflammation. Our results indicate that AD patients and mouse models have increased activation of a set of proteins known as the contact activation system in their circulation. We also demonstrate that the AD-related peptide Aβ can initiate activation of this system in the circulation of animal models. Because the contact activation system contributes to inflammation, our results suggest new pathogenic mechanisms, diagnostic tests, and therapeutic targets for AD. Alzheimer’s disease (AD) is characterized by accumulation of the β-amyloid peptide (Aβ), which likely contributes to disease via multiple mechanisms. Increasing evidence implicates inflammation in AD, the origins of which are not completely understood. We investigated whether circulating Aβ could initiate inflammation in AD via the plasma contact activation system. This proteolytic cascade is triggered by the activation of the plasma protein factor XII (FXII) and leads to kallikrein-mediated cleavage of high molecular-weight kininogen (HK) and release of proinflammatory bradykinin. Aβ has been shown to promote FXII-dependent cleavage of HK in vitro. In addition, increased cleavage of HK has been found in the cerebrospinal fluid of patients with AD. Here, we show increased activation of FXII, kallikrein activity, and HK cleavage in AD patient plasma. Increased contact system activation is also observed in AD mouse model plasma and in plasma from wild-type mice i.v. injected with Aβ42. Our results demonstrate that Aβ42-mediated contact system activation can occur in the AD circulation and suggest new pathogenic mechanisms, diagnostic tests, and therapies for AD.

Disruption of laminin in the peripheral nervous system impedes nonmyelinating Schwann cell development and impairs nociceptive sensory function

The mechanisms controlling the differentiation of immature Schwann cells (SCs) into nonmyelinating SCs is not known. Laminins are extracellular matrix proteins critical for myelinating SC differentiation, but their roles in nonmyelinating SC development have not been established. Here, we show that the peripheral nerves of mutant mice with laminin‐deficient SCs do not form Remak bundles, which consist of a single nonmyelinating SC interacting with multiple unmyelinated axons. These mutant nerves show aberrant L1 and neural cell adhesion molecule (N‐CAM) expression pattern during development. The homophilic and heterophilic interactions of N‐CAM are also impaired in the mutant nerves. Other molecular markers for nonmyelinating SCs, including Egr‐1, glial fibrillary acidic protein, and AN2/NG2, are all absent in adult mutant nerves. Analysis of expression of SC lineage markers demonstrates that nonmyelinating SCs do not develop in mutant nerves. Additionally, mutant mice are insensitive to heat stimuli and show a decreased number of C‐fiber sensory neurons, indicating reduced nociceptive sensory function. These results show that laminin participates in nonmyelinating SC development and Remak bundle formation and suggest a possible role for laminin deficiency in peripheral sensory neuropathies.