Genetic deletions of NCAM and PSA impair circadian function in the mouse.

The adult suprachiasmatic nuclei (SCN) express neural cell adhesion molecule (NCAM) that carries polysialic acid (PSA), a carbohydrate polymer which controls plastic cell-cell interactions in neural tissues. Expression of the three major isoforms of NCAM [180 (principal PSA carrier), 140, and 120 kDa) varies with developmental and physiological state. The requirements for NCAM and PSA in circadian timekeeping function were assessed among three transgenic mutant strains with differing expressions of PSA and NCAM. These included: NCAM(tm3Ciw) mice lacking only NCAM 180; NCAM(tm1Cwr) mice lacking NCAM 180, 140, and PSA; and NCAM(tm1Cgn) mice lacking all NCAM isoforms and PSA. Locomotor activity was stably entrained to a 12-h light/dark cycle (LD) in the NCAM(tm3Ciw) and NCAM(tm1Cwr) mutants, but not in NCAM(tm1Cgn) mutants, where the daily onset and offset of activity were irregular or lost. Under constant darkness (DD), the free-running rhythmicity of the NCAM(tm3Ciw) mutants expressing PSA was similar to wild-type controls, but was markedly disrupted in the majority of the NCAM(tm1Cwr) and NCAM(tm1Cgn) mutants lacking PSA. This analysis indicates that PSA and its NCAM carrier are necessary for stable free-running circadian rhythmicity under DD, and that NCAM, but not PSA, is needed for synchronization to LD. Notably, when NCAM 180 is deleted, NCAM 140 compensates as the PSA carrier, and may assume other SCN-related functions of the NCAM 180 isoform.

Phosphorylation of human tau protein by microtubule-associated kinases: GSK3beta and cdk5 are key participants.

Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the "normal" physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at "AD-like" sites. Results from this study indicate that PKA, CK1, GSK3beta, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3beta and cdk5 most readily contribute to the ATP-induced "AD-like" phosphorylation of tau.

Polysialylated neural cell adhesion molecule modulates photic signaling in the mouse suprachiasmatic nucleus.

Polysialic acid (PSA), a sialic acid polymer that regulates plasticity and cell-cell interactions in neural tissues, is expressed in the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). In vivo enzymatic removal of PSA from the mouse SCN significantly impaired both the photic induction of Fos protein in SCN cells and light-induced phase-resetting of the circadian locomotor activity rhythm. Genetic deletion of PSA and it's neural cell adhesion molecule (NCAM) carrier correspondingly attenuated light-induced circadian phase-shifting. Comparison of PSA levels between young and old mice revealed a large aging-related reduction in SCN PSA content that accompanies the diminished capacity for circadian photic response reported in old rodents. Collectively these data support the contention that PSA modulates photic signaling in the SCN, and that normal reductions in the cell surface molecule contribute to aging-related deficits in SCN circadian clock function.

Characterization of microtubule-associated proteins in teleosts.

Although microtubules are known to play an important role in many cellular processes, they have been virtually neglected in fish. In this report, microtubule-associated proteins (MAPs) in fish (teleost) were characterized using antibodies (Abs) directed against the mammalian MAPs tau, MAP1A and B, and MAP 2. Two different populations of tau-like proteins (TLPs) were found in fish brain using the anti-tau Abs Tau-1, Tau-2, tau5', and tau3'. The TLPs that were recognized by Tau-1, Tau-2, and tau5' were (1) heat-stable; (2) the same molecular weight as mammalian TLPs: 59-62 kDa; (3) not enriched in microtubules prepared from catfish brain; and (4) localized to the cell body of neurons in fish brains. While the TLPs recognized by tau3' Abs were (1) heat-stable; (2) lower molecular weight than mammalian TLPs: 32-55 vs. 50-65 kDa; (3) enriched in microtubule fractions prepared from catfish brain, and (4) localized to the axons of neurons. These results are consistent with two different populations of TLPs being present in fish brains. While MAP2 was found to be approximately the same molecular weight, 250 kDa, in zebrafish and goldfish as in mammals and to be distributed to dendrites in the fish brain, both MAP1A and MAP1B were found to be about 25% the mass of their mammalian homologs. These results suggest that MAPS in fish have some characteristics similar to their mammalian counterparts, but also possess some unique properties that require further study to elucidate their function.

Using Flp-recombinase to characterize expansion of Wnt1-expressing neural progenitors in the mouse.

Here we demonstrate how a Flp recombinase-based tagging system can be used to link temporally distinct developmental events in the mouse. By directly following Flp-mediated DNA rearrangements we have analyzed the adult expansion of embryonic neural progenitors which transiently express the signaling factor Wnt1. We report Wnt1 promoter activity in embryonic cells that give rise to aspects of the adult midbrain, cerebellum, spinal cord, and dorsal root ganglia. These findings show that cells transiently expressing Wnt1 play more than an inductive role during early brain regionalization, giving rise to distinct adult brain regions as well as neural crest derivatives. Moreover, these results reveal two new features of the Flp-FRT system: First, Flp(F70L) can effectively recombine target sites (FRTs) placed in an endogenous locus in a variety of tissues in vivo, despite previous in vitro evidence of thermolability; and second, Flp(F70L) action can be predictably and tightly regulated in the mouse embryo, making it suitable for fate mapping applications. A further advantage of the Flp-FRT system is that marked lineages can ultimately be combined with germline mutations and deficiencies currently being generated using the Cre-loxP recombination system-in this way it should be possible to analyze mutant gene activities directly for their effect on cell fate.

NCAM-180 knockout mice display increased lateral ventricle size and reduced prepulse inhibition of startle.

NCAM-180 knockout mice, which have documented deficits in neural migration, were used to determine whether developmental abnormalities could lead to morphological changes and alterations in sensory motor gating mechanisms. Measurement of the lateral ventricle showed that NCAM-180-/- mice had marked increases in both the left and right anterior horns of the lateral ventricle. Furthermore, these mice also displayed a reduction of prepulse inhibition that was differentially affected by the dopamine agonist apomorphine. These results are discussed in light of the known increase in lateral ventricle size and reduction in prepulse inhibition that are seen in schizophrenia.

Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function.

The suprachiasmatic nuclei (SCN) express the highly polysialylated form of the neural cell adhesion molecule (NCAM) that has been proposed to promote plasticity in the adult brain. To investigate a role for NCAM in SCN circadian clock function, we examined the daily locomotor rhythm of mice homozygous for a mutation, Ncamtm1Cwr, which results in deletion of the NCAM-180 isoform that in brain carries polysialic acid (PSA). Mutant mice entrained well to a 12 hr light/dark cycle but exhibited a significantly shortened free-running period and longer activity duration under constant darkness (DD) than did wild-type mice. By the third week of DD treatment, circadian rhythmicity in the mutant was abolished. Immunocytochemical analyses of the mutant SCN revealed an abnormal number and distribution of vasoactive intestinal polypeptide-producing neurons, suggesting a developmental effect of the mutant phenotype; however, a direct physiological effect of the mutation on clock function was indicated by the fact that removal of PSA from adult wild-type SCN by microinjection of endoneuraminidase shortened the free-running period to a similar extent as in the mutant. Together, these data indicate critical roles for NCAM and PSA in the development and physiology of the mammalian SCN circadian clock.

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform.

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli.

The role of polysialic acid in migration of olfactory bulb interneuron precursors in the subventricular zone.

Transplantation studies have been used to show that tangential migration of olfactory bulb interneuron precursors is retarded in NCAM-mutant mice, and that this defect reflects loss of NCAM polysialic acid (PSA). In contrast, radial migration of cells within the bulb did not require PSA. Reciprocal transplantations between wild-type and mutant mice have revealed that the mutation affects the in vivo migration environment in the subventricular zone, and not movement of individual cells. However, in vitro migration of the cells into a PSA-negative collagen matrix environment was also PSA dependent. The surprisingly similar results obtained in the in vivo and in vitro environments is consistent with the observation that migration of subventricular cells occurs as streams of closely apposed cells in which the PSA-positive cells appear to serve as their own migration substrate.

N-CAM mutation inhibits tangential neuronal migration and is phenocopied by enzymatic removal of polysialic acid.

The mutation of N-CAM in mice produces a phenotype dominated by an undersized olfactory bulb and accumulation of precursors in the subependymal layer. We demonstrate here that this defect can be duplicated by injection of an enzyme that specifically destroys the polysialic acid (PSA) moiety associated with N-CAM. Studies of BrdU-labeled and pyknotic cells suggest that this defect reflects a decrease in the rostral migration of olfactory precursors and not a change in the proliferation or rate of death of these cells. In addition to their ectopic location, these cells had fewer growth cone-like processes oriented along the migration route. In contrast to tangential movement, radial migration of granule cells in the olfactory bulb was not affected by loss of PSA. These results support the proposed role for PSA in cell translocation, discriminate between different mechanisms of cell migration, and provide insight as to the nature of the N-CAM mutant phenotype.

Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects in the central nervous system.

N-CAM is abundantly expressed in the nervous system in the form of numerous structural variants with characteristic distribution patterns and functional properties. N-CAM-180, the variant having the largest cytoplasmic domain, is expressed by all neurons. The N-CAM-180-specific exon 18 has been deleted to generate homozygous mice unable to express this N-CAM form. The most conspicuous mutant phenotype was in the olfactory bulb, where granule cells were both reduced in number and disorganized. In addition, precursors of these cells were found to be accumulated at their origin in the subependymal zone at the lateral ventricle. Analysis of the mutant in this region suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway. Subtle but distinct abnormalities also were observed in other regions of the brain.

Sequence analysis of the Escherichia coli dnaE gene.

We have determined the sequence of a 4,350-nucleotide region of the Escherichia coli chromosome that contains dnaE, the structural gene for the alpha subunit of DNA polymerase III holoenzyme. The dnaE gene appeared to be part of an operon containing at least three other genes: 5'-lpxB-ORF23-dnaE-ORF37-3' (ORF, open reading frame). The lpxB gene encodes lipid A disaccharide synthase, an enzyme essential for cell growth and division (M. Nishijima, C.E. Bulawa, and C.R.H. Raetz, J. Bacteriol. 145:113-121, 1981). The termination codons of lpxB and ORF23 overlapped the initiation codons of ORF23 and dnaE, respectively, suggesting translational coupling. No rho-independent transcription termination sequences were observed. A potential internal transcriptional promoter was found preceding dnaE. Deletion of the -35 region of this promoter abolished dnaE expression in plasmids lacking additional upstream sequences. From the deduced amino acid sequence, alpha had a molecular weight of 129,920 and an isoelectric point of 4.93 for the denatured protein. ORF23 encoded a more basic protein (pI 7.11) with a molecular weight of 23,228. In the accompanying paper (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987), the sequence of the upstream region that contains lpxA and lpxB is reported.

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein.

Transformation of fibroblasts by several retroviruses that produce transforming gene products associated with protein kinase activity results in the phosphorylation of a normal cellular protein with an Mr of 34,000 (the 34K protein). Evidence is presented here that, as extracted from chicken embryo fibroblasts, this protein exists in two forms that differ both in their elution from hydroxylapatite and in their native molecular weight. The form that eluted from hydroxylapatite at 210 to 295 mM potassium phosphate displayed a native molecular weight of 30,000 to 40,000, whereas the form that eluted at 320 to 440 mM displayed a native molecular weight of 60,000 to 70,000. The latter form copurified with a low-molecular-weight protein with an approximate Mr of 6,000 (6K). Both forms of 34K were completely separable from malate dehydrogenase activity. Phosphorylated 34K, isolated from Rous sarcoma virus-transformed cells, was also present in two forms; hence, in the cell neither form serves as a preferential substrate for pp60v-src. We found that the expression of 34K differed greatly in various avian tissues. In particular, it was present in the highest concentration in cultured fibroblasts and in very low concentration in brain tissue. Its expression in this tissue seems to be controlled at the level of transcription, since 34K mRNA in brain tissue was barely detectable. The expression of 6K was similar to that of 34K.