Lorenz Vogt

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture.

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Calsyntenin-1, a proteolytically processed postsynaptic membrane protein with a cytoplasmic calcium-binding domain.

In a screen for proteins released from synapse-forming spinal cord neurons, we found the proteolytically cleaved N-terminal fragment of a transmembrane protein localized in the postsynaptic membrane of both excitatory and inhibitory synapses. We termed this protein calsyntenin-1, because it binds synaptic Ca2+ with its cytoplasmic domain. By binding Ca2+, calsyntenin-1 may modulate Ca2+-mediated postsynaptic signals. Proteolytic cleavage of calsyntenin-1 in its extracellular moiety generates a transmembrane stump that is internalized and accumulated in the spine apparatus of spine synapses. Therefore, the synaptic Ca2+ modulation by calsyntenin-1 may be subject to regulation by extracellular proteolysis in the synaptic cleft. Thus, calsyntenin-1 may link extracellular proteolysis in the synaptic cleft and postsynaptic Ca2+ signaling.

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane

BACKGROUND Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.

Neuronal depolarization enhances the transcription of the neuronal serine protease inhibitor neuroserpin.

Neuroserpin is an axonally secreted neuronal serine protease inhibitor. Based on its inhibitory activity towards tissue plasminogen activator (tPA) and its predominant expression in the cerebral cortex, the hippocampus, and the amygdala, a role for neuroserpin in the regulation of neural plasticity has been suggested. We recently found that neuroserpin mRNA is increased in cultured hippocampal neurons upon depolarization with elevated extracellular KCl. Using luciferase reporter constructs containing segments of the promoter region of the neuroserpin gene, we identified a 200-bp segment near the transcription initiation site that is responsible for both the neuron-specific expression of the neuroserpin gene and the enhanced transcription resulting from depolarization. Nerve growth factor, which alone had no effect on the expression of neuroserpin mRNA in hippocampal neurons, had a marked potentiating effect when supplied in combination with elevated extracellular KCl. In contrast, the transcription factor zif/268 blocked neuroserpin transcription. These results implicate neuroserpin as an activity-regulated modulator of tPA activity at the synapse and provide further support for the occurrence of activity-regulated proteolytic processes at the synapse.

Chapter 9 Discrete clusters of axonin-1 and NgCAM at neuronal contact sites: Facts and speculations on the regulation of axonal fasciculation

Publisher Summary Recent investigations on the molecular interactions between the neuronal cell adhesion molecules axonin-1 and NgCAM have brought intriguing novel results suggesting that tetrameric complexes of the cell adhesion molecules axonin-1 and NgCAM could represent functional units of cellular recognition, capable of eliciting distinctive intracellular signals depending on their structural organisation. Based on a variety of independent experimental results, it is now established that axonin- 1 and NgCAM interact heterophilically in the plane of the axonal membrane. In neuritis without contact to other neurites, axonin-1 and NgCAM form heterodimeric complexes. Upon membrane-membrane contacts between neuritis expressing both axonin-I and NgCAM, higher molecular mass complexes, probably composed of two molecules of axonin-1 and two molecules of NgCAM, are formed. Concomitant with the formation of the presumptive tetrameric complexes during neurite fasciculation, a reduction of the axonin- 1 -associated tyrosine kinase fyn and an increase in the activity of the NgCAM-associated casein kinase II is observed. Based on the currently known structural features and binding site locations “unsaturated” complexes in which two axonin- l/NgCAM heterodimers of apposed membranes would be joined to a heterotetrarneric complex either by a homophilic axonin- 1 /axonin-1 or a homophilic NgCAM/NgCAM interaction are proposed. The possibility that differences in the concentrations of axonin-1 and NgCAM, as well as the factors regulating the binding affinities of axonin-1 and NgCAM might regulate the generation of structurally distinctive tetrameric complexes at membrane-membrane contact sites, raises speculations about a possible regulatory function of the axonin- l/NgCAM complexes in selection mechanisms of neuritis, such as preference determination for fasciculation or pathway choice at bifurcations.

Calcium binding proteins

Numerous low molecular weight albumins occurring in the white muscle of fish have been characterised according to biochemical properties283,284. While the physiological function of these proteins of approximately 11 000 molecular weight is not known, they manifest unusual characteristics in having amino-acid compositions of approximately 10 per cent phenylalanine and 20 per cent alanine content with little or no tryptophan, tyrosine, methionine, histidine, cysteine or arginine. In addition, they exhibit high calcium affinity. These two characteristics suggest that the carp-albumin protein may be analogous to the troponin-A protein of mammalian and avian muscle285, and may be involved, therefore, in mediation of the effect of calcium in muscle contraction286.