Spatial and temporal distribution of laminins in permanent focal ischemic brain damage of the adult rat.

Laminins are extracellular matrix glycoproteins with multiple functions in the central nervous system, including maintenance of the blood-brain barrier. Because ischemic brain damage results in rapid degradation of extracellular matrix, we used immunocytochemistry on rat central nervous system after permanent focal ischemia to identify laminins involved in pathophysiology of stroke. At 24 hr after stroke, laminin-1 is transiently expressed by neurons inside the ischemic core, but from 2-3 days to 28 days it is expressed only in basement membrane structures. During the first 24 hr, alpha1, alpha5, beta1, and gamma1 laminins are transiently expressed in neurons within the ischemic core as an acute reaction of the brain to ischemia. Rapid induction of gamma1 laminin but no other laminin in reactive astrocytes surrounding the ischemic core is clear at 24 hr, and importantly, expression of gamma1 laminin in astrocytes surrounding the ischemic core intensifies during the first days and persists up to 28 days after stroke. At 2-3 days, gamma1 laminin immunoreactive barrier of reactive astrocytes is already fully formed, isolating the ischemic area from the healthy brain. Similar to gamma1 laminin, its KDI domain localizes in reactive astrocytes isolating the ischemic core. Results indicate that gamma1 laminin and its KDI domain are rapidly induced in glial cells after stroke and their expression persists, forming a molecular barrier between the healthy and the damaged brain. Thus, gamma1 laminin is involved in pathology of stroke and is likely to serve a protective function, considering its potent neuroprotective role after spinal cord injury and in neurodegenerative disorders.

Angeli's salt and spinal motor neuron injury.

UNLABELLED: Nitroxyl anion or its conjugate acid (NO-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied. RESULTS: Freshly prepared AS (5 or 10 micromol), but not papanonoate, caused a marked decrease in the rotarod performance 3-7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5 micromol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons. Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro.

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO-) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 microM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 microM, while inhibiting it at 1000 microM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro. Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced whenmeasured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [3H] dopamine uptake with concentrations over 200 microM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro, infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.

Selective overexpression of gamma1 laminin in astrocytes in amyotrophic lateral sclerosis indicates an involvement in ALS pathology.

Our earlier studies indicate that the KDI tripeptide of gamma1 laminin reverts paralysis and protects adult rat CNS from excitotoxicity of glutamate and from oxidative stress. Here we show that gamma1 laminin is selectively overexpressed in reactive astrocytes of the amyotrophic lateral sclerosis (ALS) spinal cord, with both gray and white matter astrocytes overexpressing gamma1 laminin. Intensely gamma1 laminin-positive, aggressive-looking reactive astrocytes of the lateral columns of both cervical and thoracic spinal cord surround the lateral ventral horns and roots and extend into the area of the lateral corticospinal tract. In the cervical ALS spinal cord, large numbers of strongly gamma1 laminin-immunoreactive astrocytes are also present in the dorsal columns of the ascending sensory pathways. No other laminin or any other ALS-associated protein localizes in this manner. This unique distribution of gamma1 laminin-immunoreactive astrocytes in the ALS white matter together with our recent results on the efficacy of the KDI domain as a neuronal protector strongly suggest that gamma1 laminin may be expressed by astrocytes of the ALS spinal cord as a protective measure intended to aid neuronal survival. Further comparative studies on ALS spinal cord tissues and those of the animal models of ALS are needed to clarify the specific role of gamma1 laminin and its KDI domain in ALS and its putative interactions with the additional ALS-associated factors, such as excitotoxicity, oxidative stress, and neurofilament accumulation. Most importantly, further studies are urgently needed to test the potential of the KDI tripeptide as a therapeutic treatment for ALS.

KDI tripeptide of gamma1 laminin protects rat dopaminergic neurons from 6-OHDA induced toxicity.

Our previous studies indicate that the KDI (Lys-Asp-Ile) tripeptide of gamma1 laminin protects central neurons from mechanical trauma and excitotoxicity. At least part of the neuroprotective effect of the KDI tripeptide may be mediated by its inhibitory function on ionotropic glutamate receptors. We studied the protective effect of the KDI tripeptide against 6-hydroxy-dopamine (6-OHDA) induced neurotoxicity in a rat experimental model of Parkinson's disease (PD). We found that a single unilateral injection of the KDI tripeptide into the substantia nigra before an injection of 6-OHDA protected the dopaminergic neurons from the neurotoxicity of 6-OHDA. Compared to rats treated with 6-OHDA alone, the KDI + 6-OHDA-treated substantia nigra was relatively intact with large numbers of dopaminergic neurons present at the injection side. In the rats treated with 6-OHDA alone, no dopaminergic neurons were detected, and the substantia nigra-area at the injection side was filled with blood-containing cavities. Quantification of the rescue effect of the KDI tripeptide indicated that, in animals receiving KDI before 6-OHDA, 33% of tyrosine hydroxylase-positive dopaminergic neurons of the substantia nigra were present as compared to the contralateral non-injected side. In animals receiving 6-OHDA alone, only 1.4% of the tyrosine hydroxylase expressing dopaminergic neurons could be verified. If this much protection were achieved in humans, it would be sufficient to diminish or greatly alleviate the clinical symptoms of PD. We propose that the KDI tripeptide or its derivatives might offer a neuroprotective biological alternative for treatment of PD.

The neuroprotective KDI domain of gamma 1-laminin is a universal and potent inhibitor of ionotropic glutamate receptors.

Previous work from this laboratory indicates that the KDI (Lys-Asp-Ile) domain of gamma 1-laminin promotes functional regeneration of adult rat spinal cord injuries and protects adult rat hippocampal neurons against massive neuronal death induced by intracerebral injection of the glutamate analogue kainic acid. In the present study, we used patch clamp recordings on cultured human embryonic neocortical neurons and HEK 293 cells expressing recombinant glutamate receptor subunits to study a putative interaction of the KDI with the glutamate system. We show that the KDI domain of gamma 1-laminin is a universal and potent inhibitor of AMPA, kainate, and NMDA subclasses of glutamate receptors, with a noncompetitive action on the AMPA receptor channel activity. Glutamate neurotoxicity plays a key role in both CNS trauma and neurodegenerative disorders, so this unexpected, novel function of the gamma 1-laminin-derived tripeptide may prove clinically valuable in treatment of CNS trauma and/or disease.

Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar.

We investigated the spatial and temporal expression of basement-membrane-forming and neurite-outgrowth-supporting matrix proteins after a unilateral dorsal root injury combined with a collagen I/laminin-1 graft and a stab wound lesion to the dorsal horn of the adult rat spinal cord. Ten days after injury, the gamma1 laminin was induced in the reactive glia. At this early stage, the glial cells failed to express type IV collagen and the alpha1 laminin. One month after injury, reactive astrocytes in the dorsal horn of the lesioned side expressed gamma1 laminin, type IV collagen, and the alpha1 laminin whereas astrocytes of the normal spinal cord or the uninjured contralateral dorsal horn were negative. Both astrocytes and neurons of the ipsilateral ventral horn were induced to express laminin-1 and gamma1 laminin. Astrocytes of the ipsilateral ventral horn also expressed type IV collagen. Simultaneously with the changes in expression of the extracellular matrix proteins, the expression pattern of basic fibroblast growth factor (FGF-2) was markedly altered after spinal cord injury. In normal and contralateral spinal cord, FGF-2 was expressed in nerve fibers, but its expression changed from neuronal into glial in the ipsilateral spinal cord within 1 month after injury. Four months after injury, expression of both type IV collagen and the alpha1 laminin had declined, but the astrocytes at the injury site continued expressing the gamma1 laminin. Cultured astrocytes were negative for type IV collagen, but several cytokines, including IL-1beta and TGFbeta1, induced expression of type IV collagen in the astrocytes. These factors also increased deposition of type IV collagen matrix in the glial cultures. These results indicate that type IV collagen and the alpha1 laminin are induced in reactive astrocytes after spinal cord injury in vivo. Induction of type IV collagen in astrocytes in vitro by cytokines indicates that blood-borne or local factors at the injury site may induce the spinal cord glial expression of type IV collagen in vivo. Simultaneous expression of laminin-1 and alpha1 laminin with type IV collagen is known to lead to production of basement membranes. This may hamper the neurite-outgrowth-promoting potential of the gamma1 laminin by initiating formation of the glial scar.

Differential distribution of laminins in Alzheimer disease and normal human brain tissue.

Immunocytochemistry, Western blotting, and RT-PCR were used to identify the isoforms of laminin expressed in the Alzheimer disease, but not in normal human brain tissue. We found that alpha 1 laminin was heavily over-expressed in Alzheimer disease frontal cortex, and localized in reactive astrocytes of the grey and white matter, and as punctate deposits in the senile placques of the Alzheimer brain tissue. Antibodies against the C-terminal neurite outgrowth domain of the gamma 1 laminin demonstrated expression of the gamma 1 laminin in GFAP-immunoreactive reactive astrocytes of the Alzheimer disease frontal cortex. The gamma 1 laminin was also heavily over-expressed in reactive astrocytes of both grey and white matter. Although antibodies against the C-terminal neurite outgrowth domain failed to localize gamma 1 laminin in senile plaques, antibodies against the N-terminal domains of the gamma 1 laminin demonstrated gamma 1 laminin as punctate deposits in the senile plaques. The present results indicate that enhanced and specialized expression patterns of alpha 1 and gamma 1 laminins distinctly associate these two laminins with the Alzheimer disease. The fact that domain specific antibodies localize both alpha1 and gamma 1 laminins in the senile plaques as punctate deposits and in astrocytes of both the gray and white matter indicate that these laminins and their specific domains may have distinct functions in the pathophysiology of the Alzheimer disease.

Is the soluble KDI domain of gamma1 laminin a regeneration factor for the mammalian central nervous system?

Regeneration of adult mammalian CNS is poor as a result of environmental factors that prevent axon growth. The major factors hampering regeneration of central axons include proteins released from the damaged myelin sheets of the injured neuronal pathways and formation of the glial scar. By using an experimental model of human CNS injury, we show that survival and neurite outgrowth of human central neurons are significantly enhanced by the soluble KDI domain of gamma1 laminin. Our results indicate that the KDI domain appears to neutralize both glia-derived inhibitory signals and inhibitory molecules released from the myelin of the adult human spinal cord. We propose that the KDI domain may enhance regeneration of injuries in the adult mammalian CNS.

Gamma 1 laminin and its biologically active KDI-domain may guide axons in the floor plate of human embryonic spinal cord.

Immunocytochemistry, in situ hybridization and Matrigel-embedded cultures were used to investigate the distribution of laminins during development of the human embryonic spinal cord (7-11 weeks). Our results indicate that alpha 1, beta 1, beta 3 and gamma 1 laminins localize as punctate deposits in the floor plate region in association with commissural fibers crossing the ventral midline. In addition, the neurite outgrowth domain of gamma 1 laminin accumulates heavily in the floor plate region, in the notochord and in GFAP-immunoreactive glial fibers of the embryonic spinal cord. In culture experiments, the biologically active KDI-domain of gamma 1 laminin selectively attracted directional outgrowth of neurites from explants of the dorsal spinal cord. The spatial and temporal colocalization of punctate deposits of laminins with nerve fibers crossing the ventral midline, and the guidance of neurites by the KDI-peptide domain, indicate that laminins, specifically the gamma 1 laminin, may be involved in guidance of axons during embryonic development of the human spinal cord.

Regeneration of adult rat spinal cord is promoted by the soluble KDI domain of gamma1 laminin.

Regeneration in the central nervous system (CNS) of adult mammals is hampered by formation of a glial scar and by proteins released from the myelin sheaths of injured neuronal pathways. Our recent data indicate that the KDI (Lys-Asp-Ile) domain of gamma1 laminin neutralizes both glial- and myelin-derived inhibitory signals and promotes survival and neurite outgrowth of cultured human spinal cord neurons. We show that after complete transection of the adult rat spinal cord, animals receiving onsite infusion of the KDI domain via osmotic mini-pumps recover and are able to sustain their body weights and walk with their hindlimbs. Animals treated with placebo suffer from irreversible hindlimb paralysis. Microscopic and molecular analyses of the spinal cords indicate that the KDI domain reduces tissue damage at the lesion site and enables neurite outgrowth through the injured area to effect functional recovery of the initially paralyzed animals. That the KDI domain enhances regeneration of acute spinal cord injuries in the adult rat suggests that it may be used to promote regeneration of spinal cord injuries in humans.

Cellular migration in the postnatal rat cerebellar cortex: confocal-infrared microscopy and the rapid Golgi method.

Confocal laser microscopy of DiI-labeled slices of postnatal rat cerebellum (postnatal Day 4-10; P4-10) was compared to infrared microscopy and the rapid Golgi method (P0-14) to investigate postnatal migration of granule neurons. Vertical migration of the granule neurons occurred already at birth (P0). Surprisingly, mossy fibers often reached the external granule cell layer and were in close contact with the external granule cells. These mossy fibers may play a role in initiating granule cell migration. At this age, cell bodies of the immature neurons were attached to the external basal lamina by a process and extended down toward the presumptive internal granule cell layer. At P14, some granule cells remained attached to the surface, although their cell bodies exhibited the typical morphology of mature granule neurons and were located deep in the internal granule cell layer. These cells extended their endfeet-like processes all the way to the surface of the brain. These results indicate that the vertical pathways of granule cell migration form early and persist throughout the period of granule cell migration. Confocal infrared microscopy of DiI-labeled sections and the rapid Golgi method also allowed demonstration of tangentially migrating neurons that made one or more turns on the way to the internal granule cell layer. The rapid Golgi method confirmed that many Bergmann glial processes end at the level of the tangentially migrating granule cells whereas others project to the surface. These observations show that migratory granule cells take several different routes to their final destination, which cannot be explained by so-called radial glial guidance. The only mode of migration in evidence is consistent with process elongation and translocation of the nucleus within the preformed processes.

Soluble KDI domain of gamma1 laminin protects adult hippocampus from excitotoxicity of kainic acid.

Recent data indicate that the soluble KDI domain of gamma1 laminin promotes survival and neurite outgrowth of human central neurons in vitro (Liebkind et al.[2003] J Neurosci Res 73:637-643), and seems to neutralize both glia- and myelin-derived signals that hamper regeneration in the central nervous system (CNS) of adult mammals. We show that damage of adult rat neocortical and hippocampal areas by a stereotaxic injection of kainic acid (KA) is prevented by a preceding injection of the soluble KDI domain. In the presence of the KDI domain, both neocortical and hippocampal areas show extensive gliosis but have viable neurons and glial cells, which are absent and the areas fully destroyed after injection of KA alone. This result indicates that the KDI domain of the gamma1 laminin protects the CNS against excitotoxic insults and promotes survival of both neurons and glial cells. The KDI domain may thus be a potential drug to prevent CNS damage induced by neurodegenerative disorders, mechanical injury, or ischemia.