Joshua A. Weiner

Identification of a Novel Protein Kinase A Anchoring Protein That Binds Both Type I and Type II Regulatory Subunits

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.

Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system

During central nervous system (CNS) development, programmed cell death (PCD) has been viewed traditionally as a fate reserved for differentiating neurons that are in the process of making synaptic connections. Recent studies in the embryonic cerebral cortex (Blaschke et al. [1996] Development 122:1165–1174), however, have shown that many neuroblasts in the proliferative ventricular zone undergo PCD as well and that this likely represents a novel form distinct from that found in regions of postmitotic neurons. To determine the commonality of this form of PCD throughout the CNS, the prevalence of dying cells identified by in situ end labeling plus (ISEL+; Blaschke et al. [1996]) was determined within populations of proliferating neuroblasts that were identified by rapid bromodeoxyuridine incorporation. Based on this approach, dying cells were observed to be a common feature of all proliferative neuroblast populations examined. In addition, when ISEL+ was combined with in situ hybridization for postmitotic neural gene‐1 (png‐1; Weiner and Chun [1997] J. Comp. Neurol. 381:130–142), which identifies newly postmitotic neurons, a positive correlation was found between the start of differentiation and the onset of PCD. These data indicate that PCD in neuroblast proliferative zones is a universal feature of nervous system development. Moreover, cell death represents a prominent cell fate that may be linked to mechanisms of differentiation. J. Comp. Neurol. 396:39–50, 1998.

Lysophosphatidic acid receptor gene vzg‐1/lpA1/edg‐2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain

The growth‐factor–like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G‐protein–coupled receptor for LPA, called ventricular zone gene‐1 (vzg‐1/lpA1/edg‐2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071–1083). Vzg‐1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg‐1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg‐1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg‐1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double‐label in situ hybridization studies demonstrated that vzg‐1–expressing cells co‐expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg‐1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg‐1 during cortical neurogenesis, these data suggest distinct pre‐ and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination. J. Comp. Neurol. 398:587–598, 1998.

Cell Adhesion Molecules in Synapse Formation

Neuronal transmission relies on signals transmitted through a vast array of excitatory and inhibitory neuronal synaptic connections. How do axons communicate with dendrites to build synapses, and what molecules regulate this interaction? There is a wealth of evidence suggesting that cell adhesion molecules (CAMs) provide much of the information required for synapse formation. This review highlights the molecular mechanisms used by CAMs to regulate presynaptic and postsynaptic differentiation.

Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate.

The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lpA1 (also referred to as vzg-1 or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lpB1, lpB2, and lpB3) were isolated, corresponding to human/rat Edg-1, rat H218/AGR16, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44-52% identical), the three lpB genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lpA1 and lpA2. Unlike lpA1 and lpA2, which contain multiple coding exons, all lpB members contained a single coding exon. Heterologous expression of individual lpB members in a hepatoma cell line (RH7777), followed by 35S-GTPgammaS incorporation assays demonstrated that each of the three LPB receptors conferred sphingosine-1-phosphate-dependent, but not lysophosphatidic acid-dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple sphingosine-1-phosphate receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family.

Combinatorial homophilic interaction between γ-protocadherin multimers greatly expands the molecular diversity of cell adhesion

The specificity of interactions between neurons is believed to be mediated by diverse cell adhesion molecules, including members of the cadherin superfamily. Whereas mechanisms of classical cadherin adhesion have been studied extensively, much less is known about the related protocadherins (Pcdhs), which together make up the majority of the superfamily. Here we use quantitative cell aggregation assays and biochemical analyses to characterize cis and trans interactions among the 22-member γ-Pcdh family, which have been shown to be critical for the control of synaptogenesis and neuronal survival. We show that γ-Pcdh isoforms engage in trans interactions that are strictly homophilic. In contrast to classical cadherins, γ-Pcdh interactions are only partially Ca2+-dependent, and their specificity is mediated through the second and third extracellular cadherin (EC) domains (EC2 and EC3), rather than through EC1. The γ-Pcdhs also interact both covalently and noncovalently in the cis-orientation to form multimers both in vitro and in vivo. In contrast to γ-Pcdh trans interactions, cis interactions are highly promiscuous, with no isoform specificity. We present data supporting a model in which γ-Pcdh cis-tetramers represent the unit of their adhesive trans interactions. Unrestricted tetramerization in cis, coupled with strictly homophilic interactions in trans, predicts that the 22 γ-Pcdhs could form 234,256 distinct adhesive interfaces. Given the demonstrated role of the γ-Pcdhs in synaptogenesis, our data have important implications for the molecular control of neuronal specificity.

Control of CNS Synapse Development by γ-Protocadherin-Mediated Astrocyte–Neuron Contact

Recent studies indicate that astrocytes, whose processes enwrap synaptic terminals, promote synapse formation both by releasing soluble factors and through contact-dependent mechanisms. Although astrocyte-secreted synaptogenic factors have been identified, the molecules underlying perisynaptic astroctye–neuron contacts are unknown. Here we show that the γ-protocadherins (γ-Pcdhs), a family of 22 neuronal adhesion molecules encoded by a single gene cluster, are also expressed by astrocytes and localize to their perisynaptic processes. Using cocultures in which either astrocytes or neurons are Pcdh-γ-null, we find that astrocyte–neuron γ-Pcdh contacts are critical for synaptogenesis in developing cultures. Synaptogenesis can eventually proceed among neurons cocultured with Pcdh-γ-null astrocytes, but only if these neurons themselves express the γ-Pcdhs. Consistent with this, restricted mutation of the Pcdh-γ cluster in astrocytes in vivo significantly delays both excitatory and inhibitory synapse formation. Together, these results identify the first known contact-dependent mechanism by which perisynaptic astrocyte processes promote synaptogenesis.

Molecular Control of Spinal Accessory Motor Neuron/Axon Development in the Mouse Spinal Cord

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Png‐1, a nervous system‐specific zinc finger gene, identifies regions containing postmitotic neurons during mammalian embryonic development

To identify genes associated with early postmitotic cortical neurons, gene fragments were examined for expression in postmitotic, but not proliferative, zones of the embryonic murine cortex. Through this approach, a novel member of the zinc finger gene family, containing 6 C2HC fingers, was isolated and named postmitotic neural gene‐1, or png‐1. Embryonic png‐1 expression was: 1) nervous system‐specific; 2) restricted to zones containing postmitotic neurons; and 3) detected in all developing neural structures examined. In the cortex, png‐1 expression was first observed on embryonic day 11, correlating temporally and spatially with the known generation of the first cortical neurons. Gradients of png‐1 expression throughout the embryonic central nervous system further correlated temporally and spatially with known gradients of neuron production. With development, expression remained restricted to postmitotic zones, including those containing newly‐postmitotic neurons. Png‐1 was also detected within two days of neural retinoic acid induction in P19 cells, and expression increased with further neuronal differentiation. These data implicate png‐1 as one of the earliest molecular markers for postmitotic neuronal regions and suggest a function as a panneural transcription factor associated with neuronal differentiation. J. Comp. Neurol. 130‐142, 1997.© 1997 Wiley‐Liss, Inc.

Neurobiology of receptor-mediated lysophospholipid signaling. From the first lysophospholipid receptor to roles in nervous system function and development

Abstract: Identification of the first lysophospholipid receptor, LPA1/Vzg‐1, cloned by way of neurobiological analyses on the embryonic cerebral cortex, has led to the realization and demonstration that there exist multiple, homologous LP receptors, including those encoded by a number of orphan receptor genes known as “Edg,” all of which are members of the G‐protein‐coupled receptor (GPCR) superfamily. These receptors interact with apparent high affinity for lysophosphatidic acid (LPA) or sphingosine‐1‐phosphate (S1P or SPP), and are referred to based upon their functional identity as lysophospholipid receptors: LPA and LPB receptors, respectively, with the expectation that additional subgroups will be identified (i.e., LPC, etc.). Here an update is provided on insights gained from analyses of these receptor genes as they relate to the nervous system, particularly the cerebral cortex, and myelinating cells (oligodendrocytes and Schwann cells).