Susan Hockfield

A family of proteins implicated in axon guidance and outgrowth

Rapid progress in the identification and characterization of axon guidance molecules and their receptors has left the field poised to explore the intracellular mechanisms by which signals are transduced into growth cone responses. The TUC (TOAD/Ulip/CRMP) family of proteins has emerged as a strong candidate for a role in growth cone signaling. The TUC family members reach their highest expression levels in all neurons during their peak periods of axonal growth and are strongly down-regulated afterward. When axonal regrowth in the adult is triggered by axotomy, TUC-4 is reexpressed during the period of regrowth. Mutations in unc-33, a homologous nematode gene, lead to severe axon guidance errors in all neurons. Furthermore, the TUC family is required for the growth cone-collapsing activity of collapsin-1. An important role for the TUC family is also suggested by its high degree of interspecies amino acid sequence identity, with the rat TUC-2 protein showing 98% identity with its chick ortholog and 89% identity with its Xenopus ortholog. Information gained from the study of the TUC family will be of key importance in understanding how growth cones find their targets.

Brain-enriched Hyaluronan Binding (BEHAB)/Brevican Cleavage in a Glioma Cell Line Is Mediated by a Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) Family Member

Brain-enriched hyaluronan binding (BEHAB)/brevican is a brain-specific extracellular matrix protein containing a cleavage site between Glu395-Ser396, which bears remarkable homology to the “aggrecanase” site in the cartilage proteoglycan aggrecan. Expression of BEHAB/brevican is dramatically increased in human gliomas, notoriously invasive tumors. Recently, we showed that the rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms non-invasive tumors when grown as intracranial grafts, can form invasive tumors when transfected with a 5′ cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA. In marked contrast, the highly invasive CNS-1 glioma cell line expresses and cleaves BEHAB/brevican protein when grown as an intracranial graft. These results suggest that both synthesis and cleavage of BEHAB/brevican protein may play a role in the invasiveness of gliomas. We report here, using an antibody developed to the neoepitope created by BEHAB/brevican cleavage at the Glu395-Ser396 site, that the CNS-1 cells are able to cleave the protein in vitro. We characterized the CNS-1-derived cleavage activity by assaying its ability to cleave BEHAB/brevican proteoglycan, and determined that the enzyme is a constitutively expressed, secreted activity. Using a variety of protease inhibitors, reverse transcriptase-polymerase chain reaction, and specific antibodies, we determined that this activity is likely to be a member of the ADAMTS family of metalloproteinases, specifically ADAMTS4. These results suggest a novel function for ADAMTS family members in BEHAB/brevican cleavage and glioma and indicate that inhibition of ADAMTS in glioma may provide a novel therapeutic strategy.

Neuronal populations stained with the monoclonal antibody Cat-301 in the mammalian cerebral cortex and thalamus

The monoclonal antibody Cat-301 was used to examine neurons in the cerebral cortex and dorsal thalamus of several mammalian species, including Old World monkeys, cats, bush babies, guinea pigs, and rats. In each species, subpopulations of cortical and thalamic neurons are stained along the surfaces of their somata and proximal dendrites. Cat- 301-positive cortical neurons include specific groups of pyramidal cells (e.g., corticospinal but not corticobulbar or callosal neurons in the monkey sensory-motor areas) and certain GABA-immunoreactive nonpyramidal cells. In the thalamus, the relay neurons projecting to the cortex and not the intrinsic neurons are stained. The Cat-301- positive neurons are nonhomogeneously distributed in the cat and monkey cortex and thalamus. In the cortex, they are densely packed in 2 bands that in most areas include layers III and V, but that in primary sensory areas include layers IV and VI. Because the density of stained neurons, their distribution, and the intensity of their staining vary among cortical areas, the borders between neighboring areas can often be detected by the differences in Cat-301 staining. Broader, regional differences are also readily apparent, for areas in the parietal and occipital lobes contain large numbers of intensely stained cells, but most areas in the frontal and temporal lobes contain fewer, more lightly stained neurons. The same broad differences are seen within the thalamus: only those nuclei reciprocally connected with intensely stained cortical areas contain large numbers of Cat-301-positive neurons. Differences among species include variations in cell density and distribution when a given cortical area or thalamic nucleus is compared between cats and monkeys. Greater differences are seen among the other species. Immunoreactive neurons in the cerebral cortex are sparse and lightly stained in guinea pigs, are restricted to the hippocampal formation in rats, and are very rare and isolated in bush babies. Similarly, Cat-301-positive thalamic neurons are restricted to only one or 2 nuclei in the guinea pig and rat and are extremely rare in the bush baby. Cat-301 stains organized groups of neurons in the cat and monkey cortex and thalamus. In addition to the laminar organization of stained cells in all cortical areas (see above), the Cat-301- positive neurons of monkey areas 17 and 18 are grouped into radial arrays. In area 17, clusters of stained cells are present in layers above and below layer IVC. These clusters lie at the centers of ocular dominance columns, within patches stained for cytochrome oxidase (CO). Most of these cells are also GABA-immunoreactive.

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301.

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animals postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of integral of 680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.

Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHAB/brevican.

Hyaluronan (HA) plays an important role in tissue reorganization in response to injury. The mechanisms by which HA participates in these processes are likely to include HA-binding proteins. Previously, we reported the cloning and initial characterization of a central nervous system (CNS)-specific HA-binding protein, BEHAB (brain enriched hyaluronan binding), which was independently cloned in another laboratory and named brevican. BEHAB/brevican mRNA is expressed in the ventricular zone coincident with the initial proliferation and migration of glial cells and in surgical samples of human glioma, where glial-derived cells proliferate and migrate. To determine whether BEHAB/brevican is also expressed during the cellular proliferation and migration associated with CNS injury, we have examined BEHAB/brevican expression during reactive gliosis. BEHAB/brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol (GPI)-anchored, isoforms. The secreted, but not the GPI-anchored, isoform is up-regulated in response to a stab wound to the adult rat brain. The temporal regulation and spatial distribution of BEHAB/brevican expression parallel the gliotic response and the expression of the intermediate filament protein nestin. The up-regulation of BEHAB/brevican in response to CNS injury suggests a role for this extracellular matrix molecule in reactive gliosis. Glial process extension, a central element in the glial response to injury, may require the reexpression of both cytoskeletal and matrix elements that are normally expressed during the glial motility seen in the immature brain.

NEURONS PRODUCE A NEURONAL CELL SURFACE-ASSOCIATED CHONDROITIN SULFATE PROTEOGLYCAN

Monoclonal antibody Cat-315 recognizes a chondroitin sulfate proteoglycan (CSPG) expressed on the surface of subsets of neurons in many areas of the mammalian CNS (Lander et al., 1997). The cell type-specific expression exhibited by the Cat-315 CSPG and other perineuronal net CSPGs imparts a distinct molecular surface identity to a neuron (Celio and Blumcke, 1994; Lander et al., 1997). The cell type(s) producing these surface-associated proteins and yielding this cellular diversity has remained in question. The expression of the Cat-315 CSPG in primary rat cortical cultures has permitted an examination of the cellular source of the Cat-315 antigen, as well as a determination of its spatial relationship to the neuronal surface. Live-cell labeling of primary neuronal cultures demonstrates that the Cat-315 CSPG is on the extracellular surface of neurons. Furthermore, extraction experiments demonstrate that the Cat-315 CSPG lacks a transmembrane domain and that the entire molecule is extracellular and, therefore, can be considered a constituent of brain extracellular matrix. Several lines of evidence indicate that neurons with cell surface staining produce the Cat-315 CSPG. First, neurons with cell surface staining also show intracellular Cat-315 immunoreactivity. Second, β-xyloside or monensin, reagents that inhibit the synthesis and transport of CSPGs, increase intracellular Cat-315 immunoreactivity within neurons that express cell surface Cat-315 immunoreactivity. Third, double labeling with Cat-315 and a polyclonal antibody for the Golgi complex demonstrates a precise colocalization of the intracellular Cat-315 immunoreactivity with the Golgi. Together, these observations demonstrate that neurons contribute to the extracellular matrix of brain and that the Cat-315 CSPG is produced by the neurons that carry Cat-315 cell surface immunoreactivity.

Activity-dependent development of spinal cord motor neurons

Patterned neuronal activity in early postnatal life can regulate the acquisition of the mature morphological and electrophysiological properties of neurons. Many properties of motor neurons are developmentally regulated and may be influenced by epigenetic factors. The pattern of activation of motor neurons can regulate axon terminal morphology and synaptic efficacy at the neuromuscular junction. Motor neuron morphology and synaptic connections can also be modified by exposure to specific hormones in the early postnatal period. The acquisition of mature physiological and anatomical properties is paralleled by the acquisition of specific molecular properties. Recent experiments using molecular markers for motor neuron differentiation indicate that motor neurons undergo activity-dependent development during a circumscribed period in early postnatal life. Normal motor neuron differentiation requires a normal pattern of neuronal activity in early postnatal life. Differentiation also requires activation of the NMDA receptor over the same time period. The activity-dependent development of morphological, electrophysiological and molecular properties of motor neurons is similar to activity-dependent development in the vertebrate visual system. The neuromuscular system may provide an accessible system for characterizing the molecules subserving the translation of patterned neuronal activity into mature neuronal phenotype.

Large diameter primary afferent input is required for expression of the cat-301 proteoglycan on the surface of motor neurons

The expression of a cell surface proteoglycan, recognized by monoclonal antibody Cat-301, is regulated by neuronal activity in early life. Here we report that the expression of the Cat-301 proteoglycan on motor neurons depends on primary afferent input in the early postnatal period. Previously we showed that in two different systems, Y-cells in the cat lateral geniculate nucleus and motor neurons in the hamster spinal cord, the expression of the Cat-301 antigen requires neuronal activity during a circumscribed period in development. Disrupting the activity of Y-cells (by dark rearing or by monocular lid suture) or of motor neurons (by sciatic nerve crush or by spinal cord lesion) during the early postnatal period prevents Cat-301 expression. Disrupting neuronal activity in adults has no effect on Cat-301 expression. The onset of Cat-301 expression corresponds to the end of the period of activity-dependent development. In order to further dissect the components of the segmental reflex are required for the expression of Cat-301 on motor neurons, here we evaluated the effect of deafferentation by dorsal rhizotomy. In adult animals two weeks after deafferentation all sciatic motor neurons continue to express Cat-301. In contrast, in neonates two weeks after deafferentation the normal developmental expression of Cat-301 is reduced and less than 50% of sciatic motor neurons express Cat-301. We next selectively lesioned the small diameter afferents using the neurotoxin capsaicin. In contrast to rhizotomy, neonatal deletion of small diameter afferents has no effect on the development of Cat-301 expression on motor neurons. These results imply that input relayed by large diameter primary afferents (probably those conveying muscle and/or joint information) is required for normal maturation of motor neuronal properties during early life. They also provide further evidence for activity-dependent maturation of motor neurons.

Neuronal subsets express multiple high-molecular-weight cell-surface glycoconjugates defined by monoclonal antibodies Cat-301 and VC1.1.

Cat-301 and VC1.1 are monoclonal antibodies that recognize surface- associated molecules on subsets of mammalian CNS neurons. Earlier work demonstrated that Cat-301 recognizes a 680-kDa chondroitin sulfate proteoglycan (PG). VC1.1 has been shown to recognize 3 polypeptide bands on Western blot analysis; a major band at 95-105 kDa and additional bands at 145 kDa and 170 kDa. In the present report, we show that VC1.1 also reacts with a high-molecular-weight glycoconjugate. Immunoprecipitation experiments and biochemical characterizations indicate that Cat-301 and VC1.1 define at least 3 distinct high- molecular-weight antigens. The VC1.1 antigens react with antikeratan sulfate antibodies, while the Cat-301 antigens do not. By immunodepletion, we show that some VC1.1 antigens are Cat-301 positive, while others are Cat-301 negative. In addition, Cat-301-reactive proteoglycans are heterogeneous with respect to the presence or absence of VC1.1 epitopes. Double-label immunofluorescence studies with these 2 antibodies are consistent with the biochemical results and show that there are 3 classes of immunoreactive neurons in the cat CNS:Cat- 301+/VC1.1+, Cat-301-/VC1.1+, and Cat-301+/VC1.1-. These results indicate that structural microheterogeneity exists among Cat-301 and VC1.1 high-molecular-weight glycoconjugates. This heterogeneity may be a reflection of the diverse neuronal phenotypes that are recognized by Cat-301 and VC1.1 in the mammalian CNS.

An anatomical demonstration of projections to the medullary dorsal horn (trigeminal nucleus caudalis) from rostral trigeminal nuclei and the contralateral caudal medulla.

This study demonstrates that the medullary dorsal horn (MDH), the most caudal subdivision of the spinal trigeminal nucleus, receives input from neurons located in the trigeminal main sensory nucleus, the more rostral subdivisions of the spinal trigeminal nucleus, and the contralateral MDH. Using the retrograde transport of horseradish peroxidase (HRP), we show here that the MDH receives ipsilateral projections from rostral trigeminal nuclei but not from adjacent areas of the retricular formation. The rostral pole of spinal trigeminal nucleus oralis (nucleus oralis, pars beta) contains the highest density of MDH projection neurons. In addition, the MDH on one side receives projections from contralateral MDH neurons located in layers I, III, IV, V, VII and VIII but not from neurons in layers II and VI. We conclude that: (1) specific subdivisions of rostral trigeminal nuclei send projections to the MDH that could modulate the activity of MDH neurons; (2) projections from trigeminal nuclei to layers V and VI of the MDH, but not from adjacent areas of the reticular formation, provide further evidence that these deeper layers are related functionally to the MDH and trigeminal sensory processes; and (3) several populations of MDH neurons send axons across the midline into the contralateral MDH and may mediate contralateral inhibitory effects.