Loes H. Schrama

B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system.

The growth-associated protein B-50 (GAP-43) is a presynaptic protein. Its expression is largely restricted to the nervous system. B-50 is frequently used as a marker for sprouting, because it is located in growth cones, maximally expressed during nervous system development and re-induced in injured and regenerating neural tissues. The B-50 gene is highly conserved during evolution. The B-50 gene contains two promoters and three exons which specify functional domains of the protein. The first exon encoding the 1-10 sequence, harbors the palmitoylation site for attachment to the axolemma and the minimal domain for interaction with G0 protein. The second exon contains the GAP module, including the calmodulin binding and the protein kinase C phosphorylation domain which is shared by the family of IQ proteins. Downstream sequences of the second and non-coding sequences in the third exon encode species variability. The third exon also contains a conserved domain for phosphorylation by casein kinase II. Functional interference experiments using antisense oligonucleotides or antibodies, have shown inhibition of neurite outgrowth and neurotransmitter release. Overexpression of B-50 in cells or transgenic mice results in excessive sprouting. The various interactions, specified by the structural domains, are thought to underlie the role of B-50 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation. Apparently, B-50 null-mutant mice do not display gross phenotypic changes of the nervous system, although the B-50 deletion affects neuronal pathfinding and reduces postnatal survival. The experimental evidence suggests that neuronal morphology and communication are critically modulated by, but not absolutely dependent on, (enhanced) B-50 presence.

Long-lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment.

Behavioural experience (e.g. chronic stress, environmental enrichment) can have long‐lasting effects on cognitive functions. Because activity‐dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as hippocampus, we tested whether behaviour has also long‐lasting effects on synaptic plasticity by examining the induction of long‐term potentiation (LTP) and long‐term depression (LTD) in slices of hippocampal CA1 obtained from rats either 7–9u2003months after social defeat (behavioural stress) or 3–5u2003weeks after 5‐week exposure to environmental enrichment. Compared with age‐matched controls, defeated rats showed markedly reduced LTP. LTP was even completely impaired but LTD was enhanced in defeated and, subsequently, individually housed (during the 7–9‐month period after defeat) rats. However, increasing stimulus intensity during 100‐Hz stimulation resulted in significant LTP. This suggests that the threshold for LTP induction is still raised and that for LTD lowered several months after a short stressful experience. Both LTD and LTP were enhanced in environmentally enriched rats, 3–5u2003weeks after enrichment, as compared with age‐matched controls. Because enrichment reduced paired‐pulse facilitation, an increase in presynaptic release, facilitating both LTD and LTP induction, might contribute to enhanced synaptic changes. Consistently, enrichment reduced the number of 100‐Hz stimuli required for inducing LTP. But enrichment may also actually enhance the range of synaptic modification. Repeated LTP and LTD induction produced larger synaptic changes in enriched than in control rats. These data reveal that exposure to very different behavioural experiences can produce long‐lasting effects on the susceptibility to synaptic plasticity, involving pre‐ and postsynaptic processes.

Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat

To date, most research performed in the area of spinal cord injury focuses on treatments designed to either prevent spreading lesion (secondary injury) or to enhance outgrowth of long descending and ascending fiber tracts around or through the lesion. In the last decade, however, several authors have shown that it is possible to enhance locomotor function after spinal cord injury in both animals and patients using specific training paradigms. As a first step towards combining such training paradigms with pharmacotherapy, we evaluated recovery of function in adult rats sustaining a spinal cord contusion injury (MASCIS device, 12.5 mm at T8), either housed in an enriched environment or in standard cages (n = 15 in both groups). The animals in the enriched environment were stimulated to increase their locomotor activity by placing water and food on opposite sides of the cage. As extra stimuli, a running wheel and several other objects were added to the cage. We show that exposure to the enriched environment improves gross and fine locomotor recovery as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, the BBB subscale, the Gridwalk, and the Thoracolumbar height test. However, no group differences were found on our electrophysiological parameters nor on the amount of spared white matter. These data justify further studies on enriched housing and more controlled exercise training, with their use as potential additive to pharmacological intervention.

αCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation

Ca2+/calmodulin‐dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post‐synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C‐terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S‐transferase (GST)‐NR2A(1349‐1464) binds native CaMKII from solubilised hippocampal PSDs in ‘pull‐out’ and overlay experiments and this binding is competed by recombinant αCaMKII(1‐315). The longer GST‐NR2A(1244‐1464), although containing the CaMKII phosphosite Ser‐1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349‐1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.

Chronic imipramine treatment partially reverses the long-term changes of hippocampal synaptic plasticity in socially stressed rats.

In the present study, we investigated whether synaptic plasticity changes in the hippocampus of depressive-like socially stressed rats could be reversed by chronic antidepressant treatment. To that end, rats were either defeated and subsequently individually housed or subjected to control treatment followed by social housing. After a period of at least 3 months, rats were either treated chronically with imipramine (20 mg/kg per day, per os for at least 3 months) or the solvent solution (i.e. water). Then, long-term potentiation and depression were measured in the CA1 region of the hippocampus in vitro. Chronic imipramine treatment partially restored the attenuated induction of long-term potentiation and suppressed the facilitation of long-term depression-induction in socially stressed rats. The altered synaptic plasticity after social stress is discussed in relation to cognitive deficits and hippocampal changes that are observed in depressive patients.

Plasmid-mediated gene transfer in neurons using the biolistics technique.

Biolistics has been developed as a system for gene delivery into plant cells, but has recently been introduced for transfection into mammalian tissue, including few attempts in neural cells. Basically, in this system the plasmid DNA of interest is coated onto small particles, that are accelerated by a particular driving force. The combination of several so-called ballistic parameters and tissue parameters determine the transfection efficiency. The main advantage of the system is that it is, unlike other available transfection methods, a mechanical way to cross the plasma membrane and therefore less dependent on target cell characteristics. In terms of transfection efficiency, biolistics seems favorable above conventional techniques, like calcium phosphate precipitation and lipofection. Compared to viral techniques biolistics may be less efficient, but is quicker and easier to handle and seems to produce fewer complications for in vivo gene delivery. Therefore, although the technique is only in a developmental stage, preliminary results seem promising, and optimalization of the method may prove useful in scientific research and/or clinical use.

Identification of two promoter regions in the rat B-50/GAP-43 gene

To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5 flanking sequence was isolated. Sequence analysis of 1 kb 5 untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5 transcription start site is located between -465 and -440. Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.

B-50/GAP-43 potentiates cytoskeletal reorganization in raft domains

B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.

Expression of the pro-opiomelanocortin gene in dorsal root ganglia, spinal cord and sciatic nerve after sciatic nerve crush in the rat

Neuropeptides related to alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate nerve outgrowth following peripheral nerve injury and may play an important physiological role in peripheral nerve regeneration. The mechanism of action underlying the neurotrophic effect of pharmacologically administered alpha-MSH is unknown. Here we investigate the hypothesis that reexpression of the proopiomelanocortin (POMC) gene, the prohormone of alpha-MSH/adrenocorticotropic hormone (ACTH)-like peptides, is part of the endogenous repertoire of peripheral nerve responses following injury. The effect of sciatic nerve crush on the expression of POMC mRNA between 0.5 h and 14 days after crush was investigated using polymerase chain reaction (PCR) and Northern blot analysis. The presence of a POMC transcript in dorsal root ganglia (DRG), spinal cord and in the sciatic nerve at the crush site could be demonstrated in both control and lesioned animals by PCR using primers located in exon 1 and 3 of the POMC gene. Minute quantities of two POMC transcripts (1200 nt and 800 nt) could be detected by Northern blot analysis of total RNA prepared from DRG, spinal cord and the sciatic nerve of control animals and of animals subjected to nerve crush. POMC mRNA expression was, however, not increased following nerve crush. Probes specific for exons 1 and 2 or specific for exon 3 of the POMC gene were employed to demonstrate that the 800 nt transcript represents the truncated POMC mRNA previously shown to be present in extra-pituitary tissue. The larger 1200 nt transcript comigrates with the full length POMC mRNA expressed in the pituitary gland. The present results demonstrate the expression of small amounts of POMC mRNA in all compartments of the sciatic nerve. The absence of an induction of POMC expression in response to nerve crush suggests that the stimulating effect of exogenously applied alpha-MSH does not mimic a POMC derived neurotrophic peptide induced in the nerve following nerve injury.

Altered Protein Phosphorylation in Sciatic Nerve From Rats With Streptozocin-Induced Diabetes

The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with [32P]orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of 32P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with [32P]-γ-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.