Laminin-α2 chain-like antigens in CNS dendritic spines

doi:10.1016/S0006-8993(97)00420-4

Brain Research

Volume 764, Issues 1–2, 1 August 1997, Pages 28-38

https://doi.org/10.1016/S0006-8993(97)00420-4 Get rights and content

Abstract

The laminin-α2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-α2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-α2 chain G-domain localized laminin-α2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-α2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-α2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-α2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-α2 chain and/or laminin-α2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-α2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

Introduction

Laminins are a family of trimeric proteins consisting of various combinations of a long chain (α chain) and two shorter chains (β and γ) and are characteristic components of basement membranes 3, 9. The laminin-α2 chain is expressed in a tissue-specific manner, mainly restricted to basement membrane of striated muscle, cardiac muscle, peripheral nerve, placenta and brain 2, 11, 24, 29, 43, 54. Mutations in the laminin-α2 gene result in greatly reduced mRNA and protein levels and have been linked to the dy and dy^2J murine muscular dystrophy 48, 55, 56and to a severe congenital human muscular dystrophy 18, 19, 51. Thus, laminins containing an α2 chain appear to be important for skeletal muscle organization and/or nerve–muscle interaction. Moreover, the α2 chain-containing laminin-2 (α2,β1,γ1) or merosin has also been implicated in neurite outgrowth and neuronal migration 4, 6, 10.

Neuronal laminin-like immunoreactivities and laminin transcripts as well as putative laminin receptors have been described in the adult CNS 14, 15, 16, 25, 28, 36, 44, 46, 50, 57. Recently, we have described a unique and distinctive laminin-α2 chain-like immunoreactivity associated with neuronal structures in hippocampus and other limbic brain regions, of rat, rabbit, pig and non-human primate [17]. In the present study, we have extended these initial observations by using immunoelectron microscopy and immunoblotting in adult, developing and lesioned animals to further characterize the nature of the neuronal laminin-α2-like proteins. Our results suggest a role for laminin-α2 chain-like proteins in synaptic function and CNS plasticity.

Section snippets

Animals and tissue preparation for immunohistochemistry

Male F344 and Sprague-Dawley rats (3 days to 6 months old) and adult 6-month-old New Zealand white rabbits were used. Some of the 6-month-old rats were subjected to unilateral electrolytic lesions of the entorhinal cortex (EC) according to a previously published protocol [23]and sacrificed 4, 12, 30 and 65 days later. All animals were overdosed with pentobarbital and perfused transcardially with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer. For

Laminin-α2 chain-like immunostaining in rat and rabbit hippocampus is associated with dendritic processes, primarily with spines

As reported previously [17], polyclonal antibodies to laminin-α2 chain (pAbI and pAbII) revealed capillary basement membrane labeling but also very distinctive punctuated labeling of the ml of DG and of CA1 of hippocampus in adult rats (Fig. 1A). Monoclonal antibodies (2G9 and 5H2) at the light microscopic level revealed similar labeling of the ml and additional labeling of CA3, but not CA1, of the rat (Fig. 2A) and rabbit hippocampus (Fig. 2D). In contrast to the distinct punctuated staining

Discussion

Recently, we have reported a unique and distinct neuronal laminin-α2 chain-like immunoreactivity in brains of various mammalian species [17]. In the present study, we have further characterized the antigens and report that the laminin-α2-like immunoreactivity is associated with dendrites and dendritic spines and corresponds to 80- and 140/160-kDa proteins on immunoblots of rat, rabbit and pig hippocampus.

Immunoblot analysis revealed that our polyclonal laminin-α2-chain antibody recognized

Acknowledgements

We would like to thank J. Kusiak (GRC, NIA, Baltimore, MD), L. Walker (Johns Hopkins University, Baltimore, MD), U. Bartsch, H. Hall and M. Schachner (Neurobiology, Zurich, Switzerland) for help and support. We also thank Dr. Y. Yamada (NIDR, NIH, Bethesda, MD) for the mouse laminin-α2 chain cDNA, and Dr. L. Burton (Genentech, South San Francisco, CA) for neurotrophin 4/5. M.T. was supported by the Aluminum Association.

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Research reportLaminin-α2 chain-like antigens in CNS dendritic spines

Abstract

Introduction

Section snippets

Animals and tissue preparation for immunohistochemistry

Laminin-α2 chain-like immunostaining in rat and rabbit hippocampus is associated with dendritic processes, primarily with spines

Discussion

Acknowledgements

Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats

J. Comp. Neurol.

Cloning and expression of laminin α2 chain (M-chain) in the mouse

Matrix Biol.

A new nomenclature for the laminins

Matrix Biol.

Relationship between neuronal migration and cell–substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive

J. Cell Biol.

A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering

Cell

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

EMBO J.

Merosin, a tissue-specific basement membrane protein, is a laminin-like protein

Proc. Natl. Acad. Sci. USA

Laminin variants: why, where, and when?

Kidney Int.

Merosin promotes cell attachement and neurite outgrowth and is a component of the neurite-promoting factor of RN22 Schwannoma cells

Exp. Cell Res.

Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits

Cell Regul.

Building synapses: agrin and dystroglycan stick together

Trends Neurosci.

Dystroglycan-α, a dystrophin-associated glycoprotein, is a functional agrin receptor

Cell

Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high-affinity to the major heparin binding domain of laminin

J. Biol. Chem.

Dystroglycan: brain localisation and chromosome mapping in the mouse

Human Mol. Genet.

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment

Neuron

Mutations in the laminin α2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy

Nature Genet.

Localization of merosin-negative congenital muscular dystrophy to chromosome 6q2 by homozygosity mapping

Human Mol. Genet.

Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix

Nature

Laminin immunohistochmistry in brain is dependent on the method of tissue fixation

Brain Res.

Laminin-like and laminin-binding protein-like immunoreactive astrocytes in rat hippocampus after transient ischemia

Ann. NY Acad. Sci.

Expression of the neural adhesion molecule L1 in the deafferented dentate gyrus

Neuroscience

Laminin α2 is a component of brain capillary basement membrane: reduced expression in dystrophic dy mice

Neuroscience

Localization of a laminin-binding protein in brain

Neuroscience

Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast epidermal growth factor, insulin and the insulin-like growth factors I and II

J. Neurosci.

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Nature

Differential laminin gene expression in dorsal root ganglion neurons and nonneuronal cells

Exp. Neurol.

Research report
Laminin-α2 chain-like antigens in CNS dendritic spines