PSA-NCAM in postnatally generated immature neurons of the olfactory bulb: a crucial role in regulating p75 expression and cell survival

In the mammalian brain, ongoing neurogenesis via the rostral migratory stream (RMS) maintains neuronal replacement in the olfactory bulb throughout life. Mechanisms that regulate the final number of new neurons in this system include proliferation, migration and apoptosis. Here we show that the polysialylated isoforms of the neural cell adhesion molecule (PSA-NCAM) act as a pro-survival molecule in immature newborn neurons. Confocal microscopic analysis revealed a threefold increase in TUNEL-positive cells in the subventricular zone (SVZ) and the RMS of transgenic animals lacking the gene encoding NCAM (NCAM(-/-)), as compared with wild types. The enhanced apoptotic cell death occurred specifically in the population of mCD24-positive newborn neurons, but not in GFAP-positive astrocytes. Using in vitro cultures of purified SVZ-derived neurons, we demonstrate that the loss or inactivation of PSA on NCAM, as well as the deletion of NCAM, lead to reduced survival in response to neurotrophins including BDNF and NGF. These changes in cell survival are accompanied by an upregulation of p75 neurotrophin receptor expression in vitro as well as in vivo. Furthermore, the negative effects of PSA-NCAM inactivation on cell survival could be prevented by the pharmacological blockade of the p75 receptor-signaling pathway. We propose that PSA-NCAM may promote survival by controlling the expression of the p75 receptor in developing neurons.     

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl₄. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1⁺ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1⁺ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl₄-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Slide-based cytometry and predictive medicine: the 8th Leipziger workshop and the 1st international workshop on slide-based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform, but for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows the specimen to be restained and subsequently reanalyzed. These separate measurements can be fused to one data file (merging), increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a typical feature of SBC, can be of integral importance for cytometric analysis. Predictive medicine is aimed at the detection of changes in the patient's state prior to the manifestation of deterioration or improvement. Such instances are concerned with multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients, or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Early anti-infectious or anti-shock therapy, as well as curative chemotherapy in combination with stem cell transplantation, may provide better survival chances for the patient as well as concomitant cost containment. Predictive medicine-guided, individualized, early reduction or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). With the 8th Leipziger Workshop and the 1st International Workshop on Slide-Based Cytometry, cytomics technologies moved to more practical applications in the clinics and the clinical laboratory. This development will be continued in 2004, at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.     

Halogenation of 4-hydroxy-3-methoxybenzyl thiourea TRPV1 agonists showed enhanced antagonism to capsaicin

Selected potent TRPV1 agonists (1-6) have been modified by 5- or 6-halogenation on the aromatic A-region to analyze their effects on potency and efficacy (agonism versus antagonism). The halogenation caused enhanced functional antagonism at TRPV1 compared to the corresponding prototype agonists. The analysis of SAR indicated that the antagonism was enhanced as the size of the halogen increased (I>Br>Cl) and when the 6-position was halogenated. Compounds 23c and 31b were found to be potent full antagonists with K(i) (as functional antagonist)=23.1 and 30.3 nM in rTRPV1/CHO system, respectively.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.     

The Polysialylated Neural Cell Adhesion Molecule Promotes Neurogenesis in vitro

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cells.     

Sharp wave/ripple network oscillations and learning-associated hippocampal maps

Sharp wave/ripple (SWR, 150–250 Hz) hippocampal events have long been postulated to be involved in memory consolidation. However, more recent work has investigated SWRs that occur during active waking behaviour: findings that suggest that SWRs may also play a role in cell assembly strengthening or spatial working memory. Do such theories of SWR function apply to animal learning? This review discusses how general theories linking SWRs to memory-related function may explain circuit mechanisms related to rodent spatial learning and to the associated stabilization of new cognitive maps.     

Characterization of the interaction of phorbol esters with the C1 domain of MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase) alpha/beta

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms alpha and beta with phorbol ester. The MRCK C1 domains bind [20-(3)H]phorbol 12,13-dibutyrate with K(d) values of 10 and 17 nm, respectively, reflecting 60-90-fold weaker affinity compared with the protein kinase C delta C1b domain. In contrast to binding by the C1b domain of PKCdelta, the binding by the C1 domains of MRCK alpha and beta was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCalpha and marked contrast to that of the C1b domain of PKCdelta. In intact cells, as in the binding assays, the MRCK C1 domains required 50-100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.