Long-term health sequelae and quality of life at least 6 months after infection with SARS-CoV-2: design and rationale of the COVIDOM-study as part of the NAPKON population-based cohort platform (POP).

PURPOSE: Over the course of COVID-19 pandemic, evidence has accumulated that SARS-CoV-2 infections may affect multiple organs and have serious clinical sequelae, but on-site clinical examinations with non-hospitalized samples are rare. We, therefore, aimed to systematically assess the long-term health status of samples of hospitalized and non-hospitalized SARS-CoV-2 infected individuals from three regions in Germany. METHODS: The present paper describes the COVIDOM-study within the population-based cohort platform (POP) which has been established under the auspices of the NAPKON infrastructure (German National Pandemic Cohort Network) of the national Network University Medicine (NUM). Comprehensive health assessments among SARS-CoV-2 infected individuals are conducted at least 6 months after the acute infection at the study sites Kiel, Würzburg and Berlin. Potential participants were identified and contacted via the local public health authorities, irrespective of the severity of the initial infection. A harmonized examination protocol has been implemented, consisting of detailed assessments of medical history, physical examinations, and the collection of multiple biosamples (e.g., serum, plasma, saliva, urine) for future analyses. In addition, patient-reported perception of the impact of local pandemic-related measures and infection on quality-of-life are obtained. RESULTS: As of July 2021, in total 6813 individuals infected in 2020 have been invited into the COVIDOM-study. Of these, about 36% wished to participate and 1295 have already been examined at least once. CONCLUSION: NAPKON-POP COVIDOM-study complements other Long COVID studies assessing the long-term consequences of an infection with SARS-CoV-2 by providing detailed health data of population-based samples, including individuals with various degrees of disease severity. TRIAL REGISTRATION: Registered at the German registry for clinical studies (DRKS00023742).

Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis.

Cancer cells are hallmarked by high proliferation and imbalanced redox consumption and signaling. Various oncogenic pathways such as proliferation and evading cell death converge on redox-dependent signaling processes. Nrf2 is a key regulator in these redox-dependent events and operates in cytoprotection, drug metabolism and malignant progression in cancer cells. Here, we show that patients with primary malignant brain tumors (glioblastomas, WHO °IV gliomas, GBM) have a devastating outcome and overall reduced survival when Nrf2 levels are upregulated. Nrf2 overexpression or Keap1 knockdown in glioma cells accelerate proliferation and oncogenic transformation. Further, activation of the Nrf2-Keap1 signaling upregulates xCT (aka SLC7A11 or system Xc-) and amplifies glutamate secretion thereby impacting on the tumor microenvironment. Moreover, both fostered Nrf2 expression and conversely Keap1 inhibition promote resistance to ferroptosis. Altogether, the Nrf2-Keap1 pathway operates as a switch for malignancy in gliomas promoting cell proliferation and resistance to cell death processes such as ferroptosis. Our data demonstrate that the Nrf2-Keap1 pathway is critical for cancer cell growth and operates on xCT. Nrf2 presents the Achilles' heel of cancer cells and thus provides a valid therapeutic target for sensitizing cancer for chemotherapeutics.

ATF4 promotes angiogenesis and neuronal cell death and confers ferroptosis in a xCT-dependent manner.

Activating transcription factor 4 (ATF4) is a critical mediator of metabolic and oxidative homeostasis and cell survival. ATF4 is elevated in response to diverse microenvironmental stresses, including starvation, ER stress damages and exposure to toxic factors. Here we show that ATF4 expression fosters the malignancy of primary brain tumors (WHO grade III and IV gliomas) and increases proliferation and tumor angiogenesis. Hence, ATF4 expression promotes cell migration and anchorage-independent cell growth, whereas siRNA-mediated knockdown of ATF4 attenuates these features of malignancy in human gliomas. Further experiments revealed that ATF4-dependent tumor promoting effects are mediated by transcriptional targeting the glutamate antiporter xCT/SCL7A11 (also known as system Xc-). Thus, xCT is elevated as a consequence of ATF4 activation. We further found evidence that ATF4-induced proliferation can be attenuated by pharmacological or genetic xCT inhibition and ferroptosis inducers such as sorafenib, erastin and GPx4 inhibitor RSL3. Further, fostered xCT expression promotes cell survival and growth in ATF4 knockdown cells. Moreover, increased xCT levels ameliorate sorafenib and erastin-induced ferroptosis. Conversely, ATF4 knockdown renders cells susceptible for erastin, sorafenib and RSL3-induced ferroptosis. We further identified that ATF4 promotes tumor-mediated neuronal cell death which can be alleviated by xCT inhibition. Moreover, elevated ATF4 expression in gliomas promotes tumor angiogenesis. Noteworthy, ATF4-induced angiogenesis could be diminished by ferroptosis inducers erastin and by GPx4 inhibitor RSL3. Our data provide proof-of-principle evidence that ATF4 fosters proliferation and induces a toxic microenvironmental niche. Furthermore, ATF4 increases tumor angiogenesis and shapes the vascular architecture in a xCT-dependent manner. Thus, inhibition of ATF4 is a valid target for diminishing tumor growth and vasculature via sensitizing tumor cells for ferroptosis.

MIF-CD74 signaling impedes microglial M1 polarization and facilitates brain tumorigenesis.

Microglial cells in the brain tumor microenvironment are associated with enhanced glioma malignancy. They persist in an immunosuppressive M2 state at the peritumoral site and promote the growth of gliomas. Here, we investigated the underlying factors contributing to the abolished immune surveillance. We show that brain tumors escape pro-inflammatory M1 conversion of microglia via CD74 activation through the secretion of the cytokine macrophage migration inhibitory factor (MIF), which results in a M2 shift of microglial cells. Interruption of this glioma-microglial interaction through an antibody-neutralizing approach or small interfering RNA (siRNA)-mediated inhibition prolongs survival time in glioma-implanted mice by reinstating the microglial pro-inflammatory M1 function. We show that MIF-CD74 signaling inhibits interferon (IFN)-γ secretion in microglia through phosphorylation of microglial ERK1/2 (extracellular signal-regulated protein kinases 1 and 2). The inhibition of MIF signaling or its receptor CD74 promotes IFN-γ release and amplifies tumor death either through pharmacological inhibition or through siRNA-mediated knockdown. The reinstated IFN-γ secretion leads both to direct inhibition of glioma growth as well as inducing a M2 to M1 shift in glioma-associated microglia. Our data reveal that interference with the MIF signaling pathway represents a viable therapeutic option for the restoration of IFN-γ-driven immune surveillance.

Dissection of mitogenic and neurodegenerative actions of cystine and glutamate in malignant gliomas.

Malignant glioma represents one of the most aggressive and lethal human neoplasias. A hallmark of gliomas is their rapid proliferation and destruction of vital brain tissue, a process in which excessive glutamate release by glioma cells takes center stage. Pharmacologic antagonism with glutamate signaling through ionotropic glutamate receptors attenuates glioma progression in vivo, indicating that glutamate release by glioma cells is a prerequisite for rapid glioma growth. Glutamate has been suggested to promote glioma cell proliferation in an autocrine or paracrine manner, in particular by activation of the (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate (AMPA) subtype of glutamate receptors. Here, we dissect the effects of glutamate secretion on glioma progression. Glioma cells release glutamate through the amino-acid antiporter system X(c)(-), a process that is mechanistically linked with cystine incorporation. We show that disrupting glutamate secretion by interfering with the system X(c)(-) activity attenuates glioma cell proliferation solely cystine dependently, whereas glutamate itself does not augment glioma cell growth in vitro. Neither AMPA receptor agonism nor antagonism affects glioma growth in vitro. On a molecular level, AMPA insensitivity is concordant with a pronounced transcriptional downregulation of AMPA receptor subunits or overexpression of the fully edited GluR2 subunit, both of which block receptor activity. Strikingly, AMPA receptor inhibition in tumor-implanted brain slices resulted in markedly reduced tumor progression associated with alleviated neuronal cell death, suggesting that the ability of glutamate to promote glioma progression strictly requires the tumor microenvironment. Concerning a potential pharmacotherapy, targeting system X(c)(-) activity disrupts two major pathophysiological properties of glioma cells, that is, the induction of excitotoxic neuronal cell death and incorporation of cystine required for rapid proliferation.

Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation during development and after neurotrauma.

Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with lysophospholipase D activity that generates bioactive lysophosphatidic acid. Lysophosphatidic acid has been implicated in various neural cell functions such as neurite remodeling, demyelination, survival and inhibition of axon growth. Here, we report on the in vivo expression of autotaxin in the brain during development and following neurotrauma. We found that autotaxin is expressed in the proliferating subventricular and choroid plexus epithelium during embryonic development. After birth, autotaxin is mainly found in white matter areas in the central nervous system. In the adult brain, autotaxin is solely expressed in leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion. The present study revealed the cellular distribution of autotaxin in the developing and lesioned brain and implies a function of autotaxin in oligodendrocyte precursor cells and brain injuries.

Myelination in the hippocampus during development and following lesion.

Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion.

Cholecystokinin expression after hippocampal deafferentiation: molecular evidence revealed by differential display-reverse transcription-polymerase chain reaction.

The cortical information flow via the perforant path represents a major excitatory projection to the hippocampus. Lesioning this projection leads to massive degeneration and subsequently to reorganization in its termination zones as well as in primary non-affected subfields of the hippocampus. The molecular mechanisms and factors which are involved in the postlesional events are poorly defined. Using a differential display reverse transcription-polymerase chain reaction (DDRT-PCR) strategy, we located one band which occurred only in control hippocampus lanes and almost disappeared in the lanes of lesioned hippocampi. By sequencing, we identified the corresponding gene as cholecystokinin (CCK). Northern blot analysis confirmed a decreased transcription of CCK after lesion. In situ hybridization analysis was performed for localization and quantification of altered CCK transcription. We noted a significant downregulation of CCK transcription in the hippocampus (20%) and in the contralateral cortex (12%) 1-day after lesion (dal) and an increased signal in the ipsilateral cortex (10.5%). This pattern was altered, showing upregulation of CCK mRNA expression, reaching its highest level of 70% above control levels at 5 dal. In the hippocampus, the control level was reached again at 21 dal, whereas the cortex reached the control level at 10 dal. In comparison, the mRNA transcripts of the receptors CCK(A) and CCK(B) remained unchanged. Since CCK-containing neurons are involved in the modulation of pyramidal and granule cell excitability, our data indicate a time course correlation between CCK mRNA expression and postlesional axonal sprouting response in the hippocampus.

Molecular and functional analysis of hyperpolarization-activated pacemaker channels in the hippocampus after entorhinal cortex lesion.

Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

Molecules involved in reactive sprouting in the hippocampus.

Denervation of the hippocampus triggers reactive responses in neurons and glial cells in their affected strata in a temporally ordered fashion. Many of these responses have been studied extensively, focusing on the one hand on glial initiation and clearing responses during the degeneration phase and, on the other, on transneuronal reorganization and the newly adjusted physiological balance. We used the entorhinal cortex lesion (ECL) as a model system to study the cues that underlie the layer-specific sprouting response. This lesion destroys the perforant path, which is a massive excitatory projection to the dentate gyrus and hippocampus proper. In the deafferented zones of the hippocampus, sprouting of the remaining unlesioned fibers occurs, which replaces the lost afferences of the perforant path. We focus on candidate molecules which govern the layer-specific sprouting of the remaining axons and, in particular, on membrane-bound cues. The fact that layer-specific sprouting occurs even in the adult central nervous system (CNS) provides a valuable model for understanding the mechanisms of reactive neuronal growth and reorganization in the adult CNS. Isolation and analysis of the molecules involved in these mechanisms are important steps in understanding the potential and limitations of regeneration in the CNS.

Perforant path lesion induces up-regulation of stathmin messenger RNA, but not SCG10 messenger RNA, in the adult rat hippocampus.

In this study, we performed in situ hybridization analysis of the expression pattern of two growth-associated proteins, stathmin and SCG10, in the hippocampus after unilateral lesion of the perforant pathway, the main excitatory input from the entorhinal cortex to the hippocampus. Stathmin is one of the major neural-enriched cytosolic phosphoproteins and a potential target of cyclic-AMP-dependent kinases [Jin L. W. et al. (1996) Neurobiol. Aging 17, 331-341; Leighton I. A. et al. (1993) Molec. Cell Biochem. 127/128, 151-156]. Three days after the lesion, stathmin messenger RNA was up-regulated ipsilaterally in the hilus, in the granule cell layer of the dentate gyrus and in the pyramidal cell layer of the CA1 region. Simultaneously, the hilar region of the contralateral dentate gyrus showed an increased stathmin messenger RNA expression. This altered expression pattern was observed until 15 days after lesion. Stathmin messenger RNA expression returned to a normal level until 21 days after lesion in all regions analysed. SCG10, a membrane-bound neuronal growth-associated protein belonging to the SCG10/stathmin gene family, did not show any alteration of messenger RNA expression after perforant path lesion. The temporal changes of stathmin messenger RNA expression in the ipsilateral hippocampus correspond well to the process of reactive synaptogenesis. The enhanced messenger RNA expression in the hilar region of the contralateral dentate gyrus might suggest a role in neurite elongation, since this region is the origin of commissural fibres involved in the sprouting response in the deafferented hippocampus. The present study provides evidence that the induction of specific growth-associated proteins is differentially regulated in the hippocampus.

Entorhinal cortex lesion studied with the novel dye fluoro-jade.

We used the fluorescent dye Fluoro-Jade, capable of selectively staining degenerating neurons and their processes, in order to analyze degenerative effects of transecting the hippocampus from its main input, the entorhinal cortex in vivo and in organotypical hippocampal slice culture. Degenerating fibers stained with Fluoro-Jade were present as early as 1 day postlesion in the outer molecular layer of the dentate gyrus and could be detected up to 30 days postlesion. However, the intensity of the Fluoro-Jade staining in the outer molecular layer faded from postlesional day 20 onward. Punctate staining, various cells and neural processes became visible in this area suggesting that degenerating processes were phagocytosed by microglial cells or astrocytes. We conclude that Fluoro-Jade is an early and sensitive marker for studying degenerating neurites in the hippocampal system.

Outgrowth-promoting molecules in the adult hippocampus after perforant path lesion.

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.

Sema3C and netrin-1 differentially affect axon growth in the hippocampal formation.

The interaction between outgrowing neurons and their targets is a central element in the development of the afferent and efferent connections of the hippocampal system. This requires that axonal growth cones recognize specific guidance cues in the appropriate target area. At present, little is known about the mechanisms that determine the lamina-specific termination of hippocampal afferents. In order to understand the role of different guidance factors, we analyzed the effects of Sema3C and Netrin-1 on explants from the entorhinal cortex, dentate gyrus, cornu ammonis regions CA1 and CA3 and medial septum in a collagen coculture assay. Our observations suggest that both semaphorins and netrin play important roles in the neuron-target interactions in the hippocampal system. Sema3C is involved in the control of the ingrowth of the septohippocampal projection. We also show that netrin-1 is involved in attracting commissural neurons from dentate gyrus/hilus and CA3 to their target area in the contralateral hippocampus.

IG-molecule Kilon shows differential expression pattern from LAMP in the developing and adult rat hippocampus.

Cell recognition molecules of the immunoglobulin superfamily are involved in the formation, establishment, and plasticity of neural circuits in the central nervous system (CNS). We used a polymerase chain reaction-based approach to specifically amplify molecules with conserved sequence elements of immunoglobulin-like domains. This approach enabled us to isolate Kilon, a novel immunoglobulin that has been described by Funatsu et al. (J Biol Chem 1999;274: 8224-8230) from the hippocampus. The sequence of Kilon shows a high degree of homology to that of the chicken protein neurotractin, a molecule involved in neurite outgrowth and capable of interacting with LAMP. In situ hybridization analysis was performed to analyze the Kilon mRNA distribution in the developing and adult rat brain and to compare it to that of LAMP mRNA. Kilon mRNA was found to be specifically expressed in the dentate gyrus (DG) of the adult rat, whereas LAMP transcripts were present in all regions of the hippocampal formation. These results were corroborated by RT-PCR semiquantification of gene expression in microdissected tissue prepared from the DG and the CA1 region of the hippocampus. We also performed mRNA expression analysis of both genes following hippocampal deafferentation and seizure, but neither Kilon nor LAMP gene expression showed significant alterations after lesioning on the in situ hybridization level. Our results show that the expression patterns of Kilon and LAMP during development and in the mature hippocampus are clearly distinguishable from one another, which suggests different roles for these related molecules in the hippocampus.

A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting.

Neurons of layers II and III of the entorhinal cortex constitute the major afferent connection of the hippocampus. The molecular mechanisms that target the entorhinal axons to specific layers in the hippocampus are not known. EphA5, a member of the Eph receptor family, which has been shown to play critical roles in axon guidance, is expressed in the entorhinal cortex, the origin of the perforant pathway. In addition, ligands that interact with EphA5 are expressed in distinct hippocampal regions during development of the entorhino-hippocampal projection. Of these ligands, ephrin-A3 mRNA is localized both in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the cornu ammonis, whereas ephrin-A5 mRNA is only expressed in the pyramidal cell layer of the cornu ammonis. In the dentate gyrus, the ligand protein is not present in the termination zone of the entorhinal efferents (the outer molecular layer of the dentate gyrus) but is concentrated in the inner molecular layer into which entorhinal efferents do not grow. We used outgrowth and stripe assays to test the effects of ephrin-A3 and ephrin-A5 on the outgrowth behavior of entorhinal axons. This functional analysis revealed that entorhinal neurites were repelled by ephrin-A3 but not by ephrin-A5. These observations suggest that ephrin-A3 plays an important role in the layer-specific termination of the perforant pathway and that this ligand may interact with the EphA5 receptor to restrict entorhinal axon terminals in the outer molecular layer of the dentate gyrus.

Target- and maturation-specific membrane-associated molecules determine the ingrowth of entorhinal fibers into the hippocampus.

In this study the role of membrane-associated molecules involved in entorhinohippocampal pathfinding was examined. First outgrowth preferences of entorhinal neurites were analyzed on membrane carpets obtained from their proper target area, the hippocampus, and compared to preferences on control membranes from brain regions which do not receive afferent connections from the entorhinal cortex. On a substrate consisting of alternating lanes of hippocampal and control membranes, entorhinal neurites exhibited a strong tendency to grow on lanes of hippocampal membrane. These tissue-specific outgrowth preferences were maintained even on membrane preparations from adult brain tissue devoid of myelin. To determine the possible maturation dependence of these membranes, we examined guidance preferences of entorhinal neurites on hippocampal membranes of different developmental stages ranging from embryonic to postnatal and adult. Given a choice between alternating lanes of embryonic (E15-E16) and neonatal (P0-P1) hippocampal membranes, entorhinal neurites preferred to extend on neonatal membranes. No outgrowth preferences were observed on membranes obtained between E19 and P10. From P10 onward there was a reoccurrence of a preference for postnatal membrane lanes when neurites were presented with a choice between P15, P30, and adult membranes (>P60). This choice behavior of entorhinal neurites temporally correlates with the ingrowth of the perforant path into the hippocampus and with the stabilization of this brain area in vivo. Experiments in which postnatal and adult hippocampal membranes were heat inactivated or treated to remove molecules sensitive to phosphatidylinositol-specific phospholipase C demonstrated that entorhinal fiber preferences were controlled in this assay by attractive guidance cues and were independent of phosphatidylinositol-sensitive linked molecules. Moreover, entorhinal neurites displayed a positive discrimination for membrane-associated guidance cues of their target field, thus preferring to grow on membranes from the molecular layer of the dentate gyrus compared with CA3 or hilus membranes. Heat-inactivation experiments indicated that preferential growth of entorhinal axons is due to a specific attractivity of the molecular layer substrate. The data presented demonstrate that outgrowth of entorhinal fibers on hippocampal membranes is target and maturation dependent.

Semaphorin D acts as a repulsive factor for entorhinal and hippocampal neurons.

We analysed the effects of semaphorin D on axons from the developing rat entorhinal-hippocampal formation. Explants from superficial layers of the entorhinal cortex and of the hippocampus anlage were obtained from various developmental stages and co-cultured with cell aggregates expressing semaphorin D. Neurites extending from entorhinal explants that had been isolated from early embryonic stages (E16 and E17) were not affected by semaphorin D, but were repelled at later stages (E20 and E21). Axons from hippocampal neurons explanted at E21 were also repelled by semaphorin D. In situ hybridization studies revealed expression of the semaphorin D receptor neuropilin-1 in the entorhinal cortex from stage E17 to stage P7, and in the dentate gyrus and CA1-3 regions between E17 and adulthood. These data suggest that semaphorin D is involved in the formation of the perforant pathway and acts, via the neuropilin-1 receptor, as a repulsive signal that prevents entorhinal fibres from growing into the granular layer of the dentate gyrus. These data also suggest a role for semaphorin D in the development of intrahippocampal connections.

Myelin does not influence the choice behaviour of entorhinal axons but strongly inhibits their outgrowth length in vitro.

Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non-permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin-associated factors by a specific antibody (IN-1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin-free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation-dependent and is not influenced by myelin. The application of IN-1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin-associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin-associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.