Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes

Astrocyte scar formation aids central nervous system axon regeneration

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration

Characterization of a Novel Rat Model of Penetrating Traumatic Brain Injury

A penetrating traumatic brain injury (pTBI) occurs when an object impacts the head with sufficient force to penetrate the skin, scull and meninges and inflict injury directly to the brain parenchyma. This type of injury has been notoriously difficult to model in small laboratory animals, such as rats or mice. To this end, we have established a novel, non-fatal, model for penetrating brain injury, based on a modified air-rifle that accelerates a pellet, which in turn, impacts a small probe that then causes the injury to the experimental animal’s brain. In the present study, we have focused on the acute phase and characterized the tissue destruction, including the increasing cavity formation, white matter degeneration, hemorrhage, edema and gliosis. We also used a battery of behavioral models to examine the neurological outcome, with the most noteworthy finding being impairment of reference memory function. In conclusion, we have described a number of events taking place after pTBI in our model. We expect this model will prove useful in our efforts to unravel the biological events underlying injury and regeneration after pTBI and possibly serve as a useful animal model in development of novel therapeutic and diagnostic approaches

Neuroprotective effects of N-acetylcysteine amide on experimental focal penetrating brain injury in rats

We examined the effects of N-acetylcysteine amide (NACA) in the secondary inflammatory response following a novel method of focal penetrating traumatic brain injury (TBI) in rats. N-acetylcysteine (NAC) has limited but well-documented neuroprotective effects after experimental central nervous system ischemia and TBI, but its bioavailability is very low. We tested NACA, a modified form of NAC with higher membrane and blood-brain barrier permeability. Focal penetrating TBI was produced in male Sprague-Dawley rats randomly selected for NACA treatment (n = 5) and no treatment (n = 5). In addition, four animals were submitted to sham surgery. After 2 hours or 24 hours the brains were removed, fresh frozen, cut in 14 mu m coronal sections and subjected to immunohistochemistry, immunofluorescence, Fluoro-Jade and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. All treated animals were given 300 mg/kg NACA intraperitoneally (IP) 2 minutes post trauma. The 24 hour survival group was given an additional bolus of 300 mg/kg IF after 4 hours. NACA treatment decreased neuronal degeneration by Fluoro-Jade at 24 hours with a mean change of 35.0% (

Differential gene expression changes for complement C1q and C3 after injuries to dorsal and ventral nerve roots

C1q is an initiating protein in the classical complement cascade and is a key element in the inflammatory response to injuries in the nervous system. Interestingly, it has been shown to be expressed by immature neurons and is localized to synapses. Mice that are deficient to C1q or the downstream complement factor C3 show severe defects in elimination of synapses during development (Stevens et al., 2007). This can lead to nonappropriate connections, increased excitatory connectivity and epileptiform activity. Recent in vitro studies indicate that C1q can directly promote neuronal survival (Benoit and Tenner, 2011). In this study we have examined expression changes after injuries to dorsal and ventral roots in 18 adult rats using Affymetrix Rat Gene ST 1.0 arrays.The data suggest that the cute response in genes for complement factors C1q and C3 is different after different nerve root lesions. The ventral root replantation and nerve injuries are followed by a regenerative response while dorsal root transection and ventral root avulsion are examples of non-regenerative conditions

Differential acute gene expression changes after 5 types of traumatic injury in spinal cord and the brain

Although a general poor outcome of lesions in mammalian central nervous system there are some interesting regional differences in the response to traumatic injury. There are indications that the inflammatory pattern and the duration of traumatic defects in the Blood Brain Barrier are dissimilar in the brain and the spinal cord. In this study we have examined the acute gene expression response in the adult rat after two types of traumatic brain injury (TBI) and two types of lesions affecting the spinal cord. The TBI models were an exposure to blast overpressure (200 kPa), a sagittal acceleration injury and a cortical penetration injury. The spinal injuries were lumbar ventral root avulsion at the border between the CNS and PNS. Ventral root avulsion is not followed by spontaneous regrowth. The second spinal injury was replantation of avulsed spinal ventral roots, enabling significant and useful regrowth of motor axons. In this study we have analyzed the acute response to these 5 types of injury with gene arrays combined with cluster analysis of gene ontology search terms. 3 adult Sprague-Dawley rats for each of the 5 models were used. 24 h after the injury, the animals were anesthetized and the inferior vena cava was cut open. The hippocampus and the cortex were used for analysis of the 3 TBI models and the ipsilateral ventral quadrat of the affected spinal cord segment was used for the spinal injuries. RNA samples were analysed was then hybridized to Affymetrix Rat Gene ST 1.0 array. The data show significant differences between rats subjected to ventral replantation compared to avulsion only. Whereas, the number of genes related to cell death is similar in the two models after 24 hours, we observed a significantly larger number of genes related to neurite growth and development in the rats treated with ventral root replantation. In addition, an acute inflammatory response was observed after avulsion, while effects on genes related to synaptic transmission were much more pronounced after replantation than after avulsion without replantation. Blast overpressure induced limited shifts in gene expression in the hippocampus. The most interesting findings were a down regulation of genes involved in neurogenesis and synaptic transmission. Acceleration and penetration injuries resulted in changes in the expression in a large number of gene families including cell death, inflammation and neurotransmitters in the hippocampus and the cortex. We, conclude that cluster analysis of gene ontology search terms analysis may facilitate the comparison of the acute response in different types of injury

Differential acute gene expression changes after 5 types of traumatic injury in spinal cord and the brain

Although a general poor outcome of lesions in mammalian central nervous system there are some interesting regional differences in the response to traumatic injury. There are indications that the inflammatory pattern and the duration of traumatic defects in the Blood Brain Barrier are dissimilar in the brain and the spinal cord. In this study we have examined the acute gene expression response in the adult rat after two types of traumatic brain injury (TBI) and two types of lesions affecting the spinal cord. The TBI models were an exposure to blast overpressure (200 kPa), a sagittal acceleration injury and a cortical penetration injury. The spinal injuries were lumbar ventral root avulsion at the border between the CNS and PNS. Ventral root avulsion is not followed by spontaneous regrowth. The second spinal injury was replantation of avulsed spinal ventral roots, enabling significant and useful regrowth of motor axons. In this study we have analyzed the acute response to these 5 types of injury with gene arrays combined with cluster analysis of gene ontology search terms. 3 adult Sprague-Dawley rats for each of the 5 models were used. 24 h after the injury, the animals were anesthetized and the inferior vena cava was cut open. The hippocampus and the cortex were used for analysis of the 3 TBI models and the ipsilateral ventral quadrat of the affected spinal cord segment was used for the spinal injuries. RNA samples were analysed was then hybridized to Affymetrix Rat Gene ST 1.0 array. The data show significant differences between rats subjected to ventral replantation compared to avulsion only. Whereas, the number of genes related to cell death is similar in the two models after 24 hours, we observed a significantly larger number of genes related to neurite growth and development in the rats treated with ventral root replantation. In addition, an acute inflammatory response was observed after avulsion, while effects on genes related to synaptic transmission were much more pronounced after replantation than after avulsion without replantation. Blast overpressure induced limited shifts in gene expression in the hippocampus. The most interesting findings were a down regulation of genes involved in neurogenesis and synaptic transmission. Acceleration and penetration injuries resulted in changes in the expression in a large number of gene families including cell death, inflammation and neurotransmitters in the hippocampus and the cortex. We, conclude that cluster analysis of gene ontology search terms analysis may facilitate the comparison of the acute response in different types of injury

Differential gene expression changes for complement C1q and C3 after injuries to dorsal and ventral nerve roots

C1q is an initiating protein in the classical complement cascade and is a key element in the inflammatory response to injuries in the nervous system. Interestingly, it has been shown to be expressed by immature neurons and is localized to synapses. Mice that are deficient to C1q or the downstream complement factor C3 show severe defects in elimination of synapses during development (Stevens et al., 2007). This can lead to nonappropriate connections, increased excitatory connectivity and epileptiform activity. Recent in vitro studies indicate that C1q can directly promote neuronal survival (Benoit and Tenner, 2011). In this study we have examined expression changes after injuries to dorsal and ventral roots in 18 adult rats using Affymetrix Rat Gene ST 1.0 arrays.The data suggest that the cute response in genes for complement factors C1q and C3 is different after different nerve root lesions. The ventral root replantation and nerve injuries are followed by a regenerative response while dorsal root transection and ventral root avulsion are examples of non-regenerative conditions