Steven S. Carlson

A synaptic laminin-calcium channel interaction organizes active zones in motor nerve terminals.

Synapse formation requires the differentiation of a functional nerve terminal opposite a specialized postsynaptic membrane. Here, we show that laminin β2, a component of the synaptic cleft at the neuromuscular junction, binds directly to calcium channels that are required for neurotransmitter release from motor nerve terminals. This interaction leads to clustering of channels, which in turn recruit other presynaptic components. Perturbation of this interaction in vivo results in disassembly of neurotransmitter release sites, resembling defects previously observed in an autoimmune neuromuscular disorder, Lambert–Eaton myasthenic syndrome. These results identify an extracellular ligand of the voltage-gated calcium channel as well as a new laminin receptor. They also suggest a model for the development of nerve terminals, and provide clues to the pathogenesis of a synaptic disease.

The SV2 Protein of Synaptic Vesicles Is a Keratan Sulfate Proteoglycan

Abstract: We have determined that synaptic vesicles contain a vesicle‐specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an Mr of 100, 000, and the H form, with a lower mobility that migrates with an Mr of ∼250, 000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.

Identification and immunolocalization of a new class of proteoglycan (keratan sulfate) to the neuritic plaques of Alzheimer's disease

Previous studies have demonstrated three distinct classes of proteoglycans (PGs)/glycosaminoglycans (GAGs) localized to the characteristic lesions (i.e., neuritic plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles) of Alzheimers disease (AD). These include heparan sulfate (i.e., perlecan), dermatan sulfate (i.e., decorin), and chondroitin sulfate PGs/GAGs. In the present study, two different antibodies demonstrated the presence of a new class of PG (i.e., keratan sulfate) in the neuritic plaques of AD. Asynaptic vesicle keratan sulfate PG (known as SV2PG) was detected by the monoclonal antibodies, anti-SV2 and anti-SV4, which recognize the keratan sulfate core protein and GAG chains, of the SV2PG antigen, respectively. Both antibodies immunolocalized SV2PG primarily to synapses and to dystrophic neurites within neuritic plaques of AD and normal aged brain. The SV2PG was not immunolocalized to diffuse plaques, cerebrovascular amyloid deposits, or neurofibrillary tangles in AD or normal aged brain. SV2PG immunoreactivity in AD brain was similar in distribution to synaptophysin and showed apparent reduced immunoreactiviy+in AD cortex in comparison to age-matched controls. In conjunction with previous studies, these results now suggest that within the neuritic plaques of AD, there are at least four different classes of PGs present. Although heparan sulfate PGs are still the only class of PG immunolocalized to amyloid fibrils within the neuritic plaques of AD, the specific immunolocalization of keratan sulfate, dermatan sulfate, and chondroitin sulfate containing PGs to the periphery of plaques, suggests that these particular PGs/GAGs may also play distinct and important roles in neuritic plaque pathogenesis.

Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components

J. Neurochem. (2010) 115, 654–666.

Proteoglycans are present in the transverse tubule system of skeletal muscle

The transverse tubule system (T-tubule, T-system) of skeletal muscle is a membranous network that penetrates the interior of myofibers. The T-system is continuous with the sarcolemma and therefore provides a path for membrane excitation to reach internal myofibrils. In this study we demonstrate that T-tubules in elasmobranch fish, frog, and rat skeletal muscle contain a matrix of chondroitin sulfate proteoglycans. We used anti-T1, a mouse monoclonal antibody that recognizes a rare chondroitin sulfate epitope, for immunolocalization and biochemical studies. First, we find that T1 immunoreactivity colocalizes with a T-tubule marker, the dihydropyridine receptor alpha 2 subunit, in both frog and fish muscle. Secondly, the distribution of T1 immunoreactivity exactly matches the different distribution of T-tubules in rat and frog muscle. In rat muscle, two bands of T1 immunoreactivity are detected per sarcomere, a distribution that corresponds to the T-tubules located at the two A-I junctions of each sarcomere. In frog muscle, we detect one band of T1 immunoreactivity per sarcomere that corresponds to the one T-tubule per sarcomere located at the Z line. Lastly, we have isolated and biochemically characterized T1 antigenicity from fish skeletal muscle. Like extracellular matrix proteoglycans of cartilage, T1 antigenicity requires denaturing conditions to be solubilized. In fish muscle, two chondroitin sulfate proteoglycans bear T1: a heavily glycosylated proteoglycan with a molecular mass of about 1000 kDa, and a smaller proteoglycan that has a mobility on SDS-PAGE like a protein of molecular mass 280 kDa. We propose that proteoglycans function as structural components in the T-system. The proteoglycans may form a matrix, like the one formed by the cartilage proteoglycans they resemble, that can withstand the cytosolic osmotic pressures present in muscle cells and therefore may prevent the T-tubule from collapsing. We present a quantitative argument in support of this hypothesis.

Distribution of N-Acetylgalactosamine-Positive Perineuronal Nets in the Macaque Brain: Anatomy and Implications

Perineuronal nets (PNNs) are extracellular molecules that form around neurons near the end of critical periods during development. They surround neuronal cell bodies and proximal dendrites. PNNs inhibit the formation of new connections and may concentrate around rapidly firing inhibitory interneurons. Previous work characterized the important role of perineuronal nets in plasticity in the visual system, amygdala, and spinal cord of rats. In this study, we use immunohistochemistry to survey the distribution of perineuronal nets in representative areas of the primate brain. We also document changes in PNN prevalence in these areas in animals of different ages. We found that PNNs are most prevalent in the cerebellar nuclei, surrounding >90% of the neurons there. They are much less prevalent in cerebral cortex, surrounding less than 10% of neurons in every area that we examined. The incidence of perineuronal nets around parvalbumin-positive neurons (putative fast-spiking interneurons) varies considerably between different areas in the brain. Our survey indicates that the presence of PNNs may not have a simple relationship with neural plasticity and may serve multiple functions in the central nervous system.

Synaptic Vesicle Glycoproteins and Proteoglycans

Immunological markers specific for synaptic vesicles have been helpful in studying synapse regeneration (Glicksman and Sanes, 1983), development (Chun and Shatz, 1983), and tracing membrane traffic in the nerve terminal (von Wedel et al., 1981). Vesicle-specific antibodies have also identified a number of vesicle proteins. This chapter deals with four antigenic integral membrane proteins of the synaptic vesicle. These proteins, identified with monoclonal antibodies, are the best characterized vesicle membrane components. Three of these proteins are probably present in all vesicles of the regulated pathway of neuroendocrine cells. This includes both dense-core vesicles and small, clear synaptic vesicles. The cDNA for one of these proteins, p38, has been cloned and sequenced. The fourth protein is an integral membrane proteoglycan (SVPG) which has been found in some synaptic vesicles, but could have a wider distribution. Interestingly, this proteoglycan shares a unique antigenic site with a putative nerve terminal anchorage protein, TAP-1.

N-Acetylgalactosamine Positive Perineuronal Nets in the Saccade-Related-Part of the Cerebellar Fastigial Nucleus Do Not Maintain Saccade Gain

Perineuronal nets (PNNs) accumulate around neurons near the end of developmental critical periods. PNNs are structures of the extracellular matrix which surround synaptic contacts and contain chondroitin sulfate proteoglycans. Previous studies suggest that the chondroitin sulfate chains of PNNs inhibit synaptic plasticity and thereby help end critical periods. PNNs surround a high proportion of neurons in the cerebellar nuclei. These PNNs form during approximately the same time that movements achieve normal accuracy. It is possible that PNNs in the cerebellar nuclei inhibit plasticity to maintain the synaptic organization that produces those accurate movements. We tested whether or not PNNs in a saccade-related part of the cerebellar nuclei maintain accurate saccade size by digesting a part of them in an adult monkey performing a task that changes saccade size (long term saccade adaptation). We use the enzyme Chondroitinase ABC to digest the glycosaminoglycan side chains of proteoglycans present in the majority of PNNs. We show that this manipulation does not result in faster, larger, or more persistent adaptation. Our result indicates that intact perineuronal nets around saccade-related neurons in the cerebellar nuclei are not important for maintaining long-term saccade gain.