Thymosin beta 4 and secretogranin II expression in the visual system of normal and sensory-deprived adult mice.

Summary Peptide research increasingly gained importance during the past few decades, and meanwhile comprises one of the most promising fields in drug discovery. However, research on peptides is very complex, because they may act at multiple sites and have variable effects on different cells. Knowledge of expression profiles of relevant peptides is a first step in unraveling possible pharmacologic interventions in the treatment of neurologic disorders. Many proteins involved in plasticity-dependent processes are also involved in neurodegenerative diseases, like Alzheimer s disease and Parkinson s disease. And only recently, it was suggested that recovery after neurologic damage involves reorganization of the neuronal circuitry, which enables non-damaged parts of the brain to acquire new functions. The role of plasticity in the treatment of stroke and Alzheimer s disease is already extensively reported. Research on visual system plasticity thus would not only be beneficial for the treatment of visual system disorders, but also for rehabilitation and recovery from other brain pathologies.In this thesis, we characterized the expression patterns of two peptides/peptide precursors in a visual system plasticity model in adult mice. We opted for the monocular enucleation model, because visual deprivation is frequently used to study brain plasticity, which is the reorganization of the brain in response to changes in environmental input. Although the mouse is a nocturnal animal, the gross organization of its visual system is similar to that of humans, which makes the mouse a suitable animal model to study visual system plasticity. The mouse gains more and more attention as a research animal in neurophysiologic research, because of its small size, short generation time, large litters, easy handling, and more importantly the relative easy manipulation by means of genetic engineering. As first peptide, we examined thymosin beta 4 (Tß4), a multifunctional peptide, widely distributed in the brain and other organs. However, research on the distribution of Tß4 at the cellular level in normal mice was limited so far. Therefore, we documented the detailed distribution of Tß4 throughout the entire mouse body. We applied two methods: in situ hybridization to allow an overall screening of the Tß4 mRNA distribution, and immunocytochemistry for a more detailed localization at the peptide level. We localized Tß4 in the brain, thymus, spleen, lung, kidney, liver, adrenal gland, stomach and intestine. Through a comparison with two macrophage markers, CD11b and CD68, it was shown that Tß4 is expressed by almost every type of tissue macrophage, including microglia in the brain. However, it appeared that Tß4 is only expressed by a subgroup of macrophages in the brain and some other tissues.The distribution of Tß4 in control mice provided a starting point to investigate the expression of Tß4 upon monocular enucleation. A first reaction of Tß4was observed in several direct retinal brain targets: more specificially in the medial preoptic area, the retrochiasmatic nucleus, the optic chiasm, the optic tract, the medial terminal nucleus, the dorsal lateral geniculate nucleus and the superficial layers of the superior colliculus (SC). A profound activation and proliferation of Tß4-immunopositive microglia was evident in these regions. Since Tß4 is only expressed by a subgroup of microglia, we also investigated the effect of enucleation on the entire microglial population with the CD11b macrophage marker, and found that the CD11b-immunoreactive microglial response was more pronounced than the response of the Tß4-immunoreactive microglia. Likewise, GFAP-immunocytochemistry revealed a prominent astrocyte activation in the different retinorecepient targets.We also analyzed the involvement of Tß4 in long-term brain plasticity in the deep layers of the SC, which are multimodal in nature. Large Tß4-immunoreactive neurons with long, extensively branching Tß4-immunoreactive neurites were observed after 2, 5 and 7 weeks of monocular enucleation. A number of varicosities were present on these neurons. Since these neurons were only located in the posteromedial deep layers of the SC, we hypothesized a role for Tß4 in growth and remodeling of collicular neuronal processes. The peptide may participate in the adaptation of the different sensory maps upon sensory deafferentation.Finally, the expression profile of a second peptide (precursor), secretogranin II (ScgII), was discussed. A marked decrease in ScgII mRNA levels was observed in the visual cortex after 1 day of monocular enucleation. The medial monocular driven regions were affected the most by the deprivation, especially in the supragranular layers. As from 1 week of deprivation, the infragranular layers were also profoundly affected. The ScgII mRNA levels began to recover after 5 weeks in the supragranular lateral visual cortex. Only in the 7 week animals, recovery was obvious, though this recovery was not completed after 7 weeks, except for the rostromedial cortex. By a comparison with the activity reporter gene zif268, clear differences became apparent, which indicates that ScgII cannot be appointed as an activity marker. A range of typical characteristics of neuromodulators could be applied to ScgII, which suggests a possible neuromodulatory role. In conclusion, we revealed the expression profiles of Tß4 and ScgII in normal and monocularly deprived adult mice, and thereby unraveled a small piece of the complicated puzzle of time-dependent responses of these peptides/peptide precursors upon visual deprivation. Our data provide a good starting point for future research to further elucidate the molecular and cellular mechanisms of Tß4 and ScgII functioning in health and disease.nrpages: 208status: publishe

Striking neuronal thymosin beta 4 expression in the deep layers of the mouse superior colliculus after monocular deprivation

Thymosin beta 4 (Tβ4), a small acidic peptide isolated originally from calf thymus, is recognized as a regulator of actin polymerization. It is widely distributed in the brain, in neurons as well as in microglia, and it has been implicated in lesion-induced neuroplasticity. In the present study, we quantitatively analyzed the immunocytochemical expression pattern of Tβ4 in the superior colliculus (SC) of the adult mouse following monocular enucleation. Immunocytochemistry revealed a remarkable neuronal profile in the SC of 2, 5 and 7-week enucleated animals. We investigated the morphology, number and layer-specific localization of these immunoreactive neurons. Neuronal Tβ4-immunoreactive cell bodies were only sparsely found in the deep layers of the SC of control mice and showed only few and short immunoreactive branches, whereas their number increased significantly with post-enucleation survival time, as well as the total extent of the neuritic tree and its degree of branching. Occasionally, some Tβ4-immunoreactive neurons were also detected in the periaqueductal gray. As a regulator of actin metabolism, Tβ4 may influence the remodeling of these collicular neuronal processes, possibly in the context of a re-alignment of the sensory and motor maps in the deep SC as a consequence of the visual deprivation paradigm.status: publishe

Monocular enucleation profoundly reduces secretogranin II expression in adult mouse

Secretogranin II (ScgII), a member of the chromogranin family, is almost exclusively found in large dense core vesicles of a wide variety of endocrine and neuronal tissues, being stored together with many different neurotransmitters, peptide hormones and neuropeptides. In the brain ScgII is almost completely processed to secretoneurin, a peptide involved in neurite outgrowth, neuroprotection and neuronal plasticity. Furthermore, correlations with neurotransmitter release and a variety of neurological diseases were reported. In this study we examined possible changes in ScgII mRNA expression in the visual system of adult mice after removal of one eye. Mice were monocularly deprived of vision and sacrificed 1 day or 1, 3, 5 and 7 weeks after enucleation. Starting 1 day after the deprivation, a marked decrease of ScgII was visible in the contralateral visual cortex. Recovery initiated in the lateral supragranular visual cortex after 5 weeks of enucleation, but was far from complete in the 7 week animals, especially in the monocularly driven medial cortex. By comparison with the immediate early gene zif268, it was proven that ScgII cannot be categorized as an activity marker, but more likely plays a role in visual system plasticity by modulating a range of neurotransmitters and neuropeptides.status: publishe

Thymosin beta 4 mRNA and peptide expression in phagocytic cells of different mouse tissues

Thymosin beta 4 (Tbeta4) is a peptide of 43 amino acids, mainly recognized as a regulator of actin polymerization by sequestering G-actin. Meanwhile, the peptide has been implicated in lymphocyte maturation, carcinogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. The peptide is also involved in lesion-induced neuroplasticity through microglia upregulation and it participates in the growth of neuronal processes. However, its precise cellular localization throughout the entire body of the mouse has not been documented. We therefore initiated a detailed investigation of the tissue distribution and cellular expression of the Tbeta4 peptide and its precursor mRNA by immunocytochemistry and in situ hybridization, respectively. In the brain, Tbeta4 was clearly present in neurons of the olfactory bulb, neocortex, hippocampus, striatum, amygdala, piriform cortex and cerebellum, and in microglia across the entire brain. We further localized Tbeta4 in cells, typically with many processes, inside thymus, spleen, lung, kidney, liver, adrenal gland, stomach and intestine. Remarkably, Tbeta4 was thus associated with microglia and macrophages, the differentiated phagocytic cells residing in every tissue. Motility and phagocytosis, two important activities of macrophages, depend on actin, which can explain the presence of Tbeta4 in these cells.status: publishe

Local cell proliferation upon enucleation in direct retinal brain targets in the visual system of the adult mouse

© the author(s), publisher and licensee Libertas Academica Ltd. In this study we used incorporation of the DNA synthesis marker 5-bromo-2'-deoxyuridine or BrdU to visualize cell proliferation in the visual system of the adult mouse as a response to monocular enucleation. We detected new BrdU-labeled cells in different subcortical retinal target regions and we established a specific time frame in which this cell proliferation occurred. By performing immunofluorescent double stainings for BrdU and different vascular (glucose transporter type 1, collagen type IV), glial (thymosin β4, glial fibrillary acidic protein) and neuronal (Neuronal Nuclei, doublecortin) markers, we identified these proliferating cells as activated microglia. Additional immunohistochemical stainings for thymosin β4 and glial fibrillary acidic protein also revealed reactive astrocytes in the different retinorecipient nuclei and allowed us to delineate a time frame for microglial and astroglial activation. A PCR array experiment further showed increased levels of cytokines, chemokines, growth factors and enzymes that play an important role in microglial-astroglial communication during the glial activation process in response to the deafferentation.status: publishe

Local cell proliferation upon enucleation in Direct Retinal Brain Targets in the Visual system of the Adult Mouse

In this study we used incorporation of the DNA synthesis marker 5-bromo-2′-deoxyuridine or BrdU to visualize cell proliferation in the visual system of the adult mouse as a response to monocular enucleation. We detected new BrdU-labeled cells in different subcortical retinal target regions and we established a specific time frame in which this cell proliferation occurred. By performing immunofluorescent double stainings for BrdU and different vascular (glucose transporter type 1, collagen type IV), glial (thymosin β 4 , glial fibrillary acidic protein) and neuronal (Neuronal Nuclei, doublecortin) markers, we identified these proliferating cells as activated microglia. Additional immunohistochemical stainings for thymosin β 4 and glial fibrillary acidic protein also revealed reactive astrocytes in the different retinorecipient nuclei and allowed us to delineate a time frame for microglial and astroglial activation. A PCR array experiment further showed increased levels of cytokines, chemokines, growth factors and enzymes that play an important role in microglial-astroglial communication during the glial activation process in response to the deafferentation

Cytoarchitecture of the mouse neocortex revealed by the low-molecular-weight neurofilament protein subunit

The expression patterns of the medium- and high-molecular-weight subunits of the neurofilament protein triplet have been extensively studied in several neuroanatomical studies. In the present study, we report the use of the low-molecular-weight neurofilament protein subunit (NF-L) as a reliable marker within the neurofilament protein family to reveal the regional architecture of mammalian neocortex. We document clearly its usefulness in anatomical parcellation studies and report unique expression patterns of NF-L throughout the mouse neocortex. NF-L was most abundant in the somatosensory cortex, the lateral secondary visual area, the granular insular cortex, and the motor cortex. Low NF-L staining intensity was observed in the agranular insular cortex, the prelimbic and infralimbic cortex, the anterior cingulate cortex, the visual rostromedial areas, the temporal association cortex, the ectorhinal cortex, and the lateral entorhinal cortex. NF-L immunoreactivity was present in the perikarya, dendrites, and proximal segment of axons primarily of pyramidal neurons, and was mainly located in layers II and III, and to a lesser extent in layers V and VI. Interestingly, Black-Gold myelin staining confirmed a close correlation between NF-L immunoreactivity and myelination patterns. The characteristic and distinctive distribution and laminar expression profiles of NF-L make it an excellent tool to assess accurately topographical boundaries among neocortical areas as illustrated herein in the adult mouse brain.status: publishe

AMIGO2 mRNA expression in hippocampal CA2 and CA3a

AMIGO2, or amphoterin-induced gene and ORF (open reading frame) 2, belongs to the leucine-rich repeats and immunoglobulin superfamilies. The protein is a downstream target of calcium-dependent survival signals and, therefore, promotes neuronal survival. Here, we describe the mRNA distribution pattern of AMIGO2 throughout the mouse brain with special emphasis on the hippocampus. In the Ammon's horn, a detailed comparison between the subregional mRNA expression patterns of AMIGO2 and Pcp4 (Purkinje cell protein 4)--a known molecular marker of hippocampal CA2 (Cornu Ammonis 2)--revealed a prominent AMIGO2 mRNA expression level in both the CA2 and the CA3a (Cornu Ammonis 3a) subregion of the dorsal and ventral hippocampus. Since this CA2/CA3a region is particularly resistant to neuronal injury and neurotoxicity [Stanfield and Cowan (Brain Res 309(2):299–307 1984); Sloviter (J Comp Neurol 280(2):183–196 1989); Leranth and Ribak (Exp Brain Res 85(1):129–136 1991); Young and Dragunow (Exp Neurol 133(2):125–137 1995); Ochiishi et al. (Neurosci 93(3):955–967 1999)], we suggest that the expression pattern of AMIGO2 indeed fits with its involvement in neuroprotection.status: publishe