Josef P. Kapfhammer

Disruption of CREB function in brain leads to neurodegeneration

Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem−/− background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.

Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice

Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

The ExDression Pattern of the Orphan Nuclear Receptor RORβ in the Developing and Adult Rat Nervous System Suggests a Role in the Processing of Sensory Information and in Circadian Rhythm

RORβ is an orphan nuclear receptor related to retinoid and thyroid hormone receptors and is exclusively expressed in the central nervous system (CNS). Here we present an in situ hybridization analysis of the distribution of RORβ mRNA in the developing and adult rat CNS. The receptor localizes to areas involved in the processing of sensory information. In the cerebral cortex, RORβ mRNA was exclusively detected in non‐pyramidal neurons of layer IV and, less so, layer V. The highest expression was found in primary sensory cortices. In the thalamus highest RORβ expression was found in the sensory relay nuclei projecting to the respective cortical areas. In contrast, sensory projection neurons in the periphery, for example retinal ganglion cells and neurons of the sensory ganglia showed only little RORβ expression. RORβ is also expressed in areas involved in the generation and maintenance of circadian rhythms ‐ the suprachiasmatic nucleus, the pineal gland and the retina. In the latter two tissues, RORβ mRNA abundance oscillates with circadian rhythmicity peaking during the hours of darkness. RORβ rnRNA could not be detected in striatum, hippocampus, cerebellum, the motor nuclei of the cranial nerves or the ventral part of the spinal cord. During development, RORβ is expressed in many areas as early as embryonic day (E) 15, anticipating the distribution pattern in the adult. Our data suggest that RORβ regulates genes whose products play essential roles in the context of sensory input integration as well as in the context of circadian timing system.

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol‐12‐myristate‐13‐acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2‐[1‐(3‐dimethylaminopropyl)indol‐3‐yl]‐3‐(indol‐3‐yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.

INCREASED EXPRESSION OF THE GROWTH-ASSOCIATED PROTEIN GAP-43 IN THE MYELIN-FREE RAT SPINAL CORD

In the normal central nervous system (CNS) the regional expression of the growth‐associated protein GAP43 is complementary to the pattern of myelination. This has led us to suspect that myelin‐associated neurite growth inhibitors might contribute to the suppression of GAP‐43 expression by suppressing sprouting and plastic changes of synaptic terminals in myelinated CNS areas. In order to study the relationship between myelination and GAP‐43 expression more directly, we experimentally prevented myelination of the lumbar spinal cord of rats through neonatal X‐irradiation. The GAP‐43 protein expression in myelin‐free spinal cords was analysed by immunohistochemistry and immunoblotting and compared to age‐matched normal spinal cords. We found that in the absence of myelination, GAP‐43 expression is strongly increased in the spinal cord of 4‐week‐old rats. GAP‐43 was most strongly expressed in descending fibre tracts, where expression in the normal spinal cord is very low. In grey matter the typical regional pattern of GAP‐43 expression did not develop; instead GAP‐43 expression was high in all regions of the spinal cord. The overall pattern of myelination and GAP‐43 expression in the myelin‐free cord resembled that of early postnatal stages. This indicates that the regional down‐regulation of GAP‐43 expression during normal postnatal development did not occur in the myelin‐free areas. Our results support the hypothesis that neurite growth inhibitors from oligodendrocytes and CNS myelin suppress sprouting and plastic changes of synaptic terminals in the normal CNS and are thereby involved in regulating the stability of neural connections.

Increased collateral sprouting of primary afferents in the myelin-free spinal cord

After partial lesions, uninjured nerve fibers have been shown to sprout and expand their connections within the CNS of adult mammals. The extent of this anatomical plasticity in adults is rather limited in comparison to embryonic or neonatal animals. Factors that might limit sprouting of nerve fibers and suppress anatomical plasticity in the CNS include myelin-associated neurite growth inhibitory molecules present in the CNS of adult mammals. To examine further the role of these neurite growth inhibitors, we have studied the ability of primary afferent fibers to sprout in the absence of myelin within a partially deafferented spinal cord. Myelination was suppressed in the lower thoracic and lumbar spinal cord of rats using neonatal x-irradiation. Dorsal roots of lumbar segments L2-L4 were cut in myelin-free and normal spinal cords of 8- or 15-d-old rats. Sprouting of primary afferents was measured after 20 d using thiamine monophosphatase (TMP) histochemistry. TMP is a specific marker enzyme for small-diameter primary afferents that terminate in the substantia gelatinosa (lamina II) of the spinal cord. When compared to the control groups, collateral sprouting of TMP-positive afferents was significantly enhanced in the myelin-free spinal cords: in animals deafferented at postnatal day P8, the average volume occupied by sprouting fibers in the upper dorsal horn was 0.103 mm3 +/- 0.008 (mean +/- SEM) in myelin-free spinal cords and 0.044 mm3 +/- 0.011 in control spinal cords. In spinal cords deafferented at P15, this difference was even larger, with 0.106 mm3 +/- 0.010 in the absence of myelin and 0.031 mm3 +/- 0.010 in controls. Our results indicate that myelin and its associated neurite growth inhibitors restrict collateral sprouting. These data provide further evidence that CNS myelin and its associated neurite growth inhibitors are involved in the regulation of anatomical plasticity in the normal CNS.

The Sushi Domains of GABAB Receptors Function as Axonal Targeting Signals

GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. Two receptor subtypes, GABAB(1a,2) and GABAB(1b,2), are formed by the assembly of GABAB1a and GABAB1b subunits with GABAB2 subunits. The GABAB1b subunit is a shorter isoform of the GABAB1a subunit lacking two N-terminal protein interaction motifs, the sushi domains. Selectively GABAB1a protein traffics into the axons of glutamatergic neurons, whereas both the GABAB1a and GABAB1b proteins traffic into the dendrites. The mechanism(s) and targeting signal(s) responsible for the selective trafficking of GABAB1a protein into axons are unknown. Here, we provide evidence that the sushi domains are axonal targeting signals that redirect GABAB1a protein from its default dendritic localization to axons. Specifically, we show that mutations in the sushi domains preventing protein interactions preclude axonal localization of GABAB1a. When fused to CD8α, the sushi domains polarize this uniformly distributed protein to axons. Likewise, when fused to mGluR1a the sushi domains redirect this somatodendritic protein to axons, showing that the sushi domains can override dendritic targeting information in a heterologous protein. Cell surface expression of the sushi domains is not required for axonal localization of GABAB1a. Altogether, our findings are consistent with the sushi domains functioning as axonal targeting signals by interacting with axonally bound proteins along intracellular sorting pathways. Our data provide a mechanistic explanation for the selective trafficking of GABAB(1a,2) receptors into axons while at the same time identifying a well defined axonal delivery module that can be used as an experimental tool.

Increased lesion-induced sprouting of corticospinal fibres in the myelin-free rat spinal cord.

Myelin contains potent inhibitors of neurite growth which have been implicated in the failure of long‐distance regeneration of nerve fibres within the CNS. These myelin‐associated neurite growth inhibitors may also be involved in the stabilization of neural connections by suppressing sprouting and fibre growth. After lesions of the CNS in neonatal animals, extensive rearrangements of the remaining fibre systems have been observed. In the rat, this plasticity of neuronal connections is severely restricted following the first few weeks of postnatal life, coincident with the progression of myelination of the nervous system. A well‐studied example of postnatal plasticity is the sprouting of one corticospinal tract (CST) into the denervated half of the spinal cord after unilateral motor cortex or pyramidal lesions. In the hamster and rat, significant CST sprouting is restricted to the first 10 postnatal days. Here we show that very extensive sprouting of corticospinal fibres occurs after deafferentations as late as P21 if myelination is prevented by neonatal X‐irradiation in the rat lumbar spinal cord. Sprouted fibres from the intact CST cross the midline and develop large terminal arbors in the denervated spinal cord, suggesting the establishment of synaptic connections. Our results suggest that myelin and its associated neurite growth inhibitors play an important role in the termination of neurite growth permissive periods during postnatal CNS development. Corticospinal sprouting subsequent to lesions early in life, i.e. in the absence of myelin‐associated neurite growth inhibitors may explain the frequent occurrence of mirror movements in patients with hemiplegic cerebral palsy.

Basic Fibroblast Growth Factor Modulates Density of Blood Vessels and Preserves Tight Junctions in Organotypic Cortical Cultures of Mice: A New In Vitro Model of the Blood–Brain Barrier

This study was performed to examine the maintenance of blood vessels in vitro in cortical organotypic slice cultures of mice with special emphasis on basic fibroblast growth factor (FGF-2), which is known to promote angiogenesis and to preserve the integrity of the blood–brain barrier. Slices of neonatal day 3 or 4 mouse brain were maintained for 3, 7, or 10 d in vitro (DIV) under standard culture conditions or in the presence of FGF-2. Immunohistochemistry for factor VIII-related antigen or laminin revealed a relative low number of blood vessels under standard conditions. In contrast, moderate FGF-2 concentrations increased the number of vessels: with 0.5 ng/ml FGF-2 it was 1.4-fold higher after DIV 3 or 1.5-fold after DIV 7 compared with controls; with 5 ng/ml it was almost doubled in both cases. With an excess of 50 ng/ml, FGF-2 vessels were reduced after DIV 3 or similar to controls after DIV 7. FGF receptor 1 was preferentially found on endothelial cells; its immunolabeling was reduced in the presence of the ligand. Cell death detected by an ethidium bromide analog or the apoptosis marker caspase-3 was barely detectable during the 10 d culture period. Immunolabeling of the tight junction proteins ZO-1 (zonula occludens protein 1), occludin, claudin-5, and claudin-3 revealed evidence for structural integrity of the blood–brain barrier in the presence of moderate FGF-2 concentrations. In conclusion, FGF-2 maintains blood vessels in vitro and preserves the composition of the tight junction. Hence, we propose FGF-2-treated organotypic cortical slices as a new tool for mechanistic studies of the blood–brain barrier.

Protein kinase C: its role in activity-dependent Purkinje cell dendritic development and plasticity.

The cerebellum is a central organ in the control of motor learning and performance. In this respect, the cellular plasticity model systems of multiple climbing fiber elimination and long-term depression have been intensively studied. The signalling pathways involved in these plastic changes are now well understood on a molecular level and protein kinase C (PKC) activity appears to be crucially involved in both processes. Furthermore, as shown in recent studies, Purkinje cell dendritic development also critically depends on the activity of PKC. Thereby, the Ca2+-dependent PKC subtypes, activated by synaptic inputs through metabotropic glutamate receptors, trigger functional changes as well as long-term anatomical maturation of the Purkinje cell dendritic tree during cerebellar development at different time levels. This review summarizes these findings and forwards the hypothesis of a link between the functional mechanisms underlying LTD and the differentiation of Purkinje cell dendrites.