Alexandra Kretz

Excess Bcl-XL increases the intrinsic growth potential of adult CNS neurons in vitro

The regenerative potential of adult mammalian CNS neurons is limited. Recent data suggest that inactivation of major growth inhibitors may not suffice to induce robust regeneration from mature neurons unless the intrinsic growth state is modulated. To investigate a possible role of Bcl-XL for axon regeneration in the adult mammalian CNS, Bcl-XL was adenovirally overexpressed in severed rat RGCs. Bcl-XL overexpression in mature axotomized RGCs in vivo increased both numbers [3.10-fold (+/-0.20)] and cumulative length [6.72-fold (+/-0.47)] of neurites regenerated from retinal explants, and this effect was further pronounced in the central retina where specific and dense axoplasmatic transduction occurs. Similarly, delayed Bcl-XL gene transfer to explanted retinae 12-13 days after lesion increased the numbers and length of emanating neurites by a factor of 5.22 (+/-0.41) and 8.29 (+/-0.69), respectively. In vivo, intraretinal sprouting of unmyelinated RGC axons into the nerve fiber layer was increased. However, fiber ingrowth into the optic nerve remained sparse, likely due to myelin inhibitors and scar components. Therefore, Bcl-XL overexpression may enhance, but may not be sufficient to, restitute functional regeneration in the adult CNS. As assessed by cell quantification analysis, Bcl-XL overexpression rescued a higher proportion of RGCs in vivo than in vitro. Therefore, Bcl-XL is capable to induce both neuronal survival and axon regeneration, but these two processes appear to be differentially modified by distinct pathways in vivo.

Optic nerve lesion increases cell proliferation and nestin expression in the adult mouse eye in vivo.

In the naïve adult rodent eye cell proliferation does not occur. The aim of this in vivo study was to evaluate if quiescent putative progenitor-like cells within the adult mouse eye can be activated by optic nerve (ON) injury. For a comprehensive analysis, three areas were assessed: the ON, the neural retina, and the ciliary body (CB). Two lesion types were performed, i.e. intraorbital ON transection, or ON crush lesion, in order to analyse possible differences in cellular response after injury. This mouse study shows, for the first time that ON lesion up-regulates cell proliferation and nestin expression in the mouse eye as compared to naïve controls. Numbers and distribution patterns of BrdU+ cells obtained were similar after both lesion types, suggesting analogous mechanisms of activation. Interestingly, a differential cell proliferative response was observed in the CB. After ON lesion, the absence of BrdU/TUNEL co-labelled cells confirmed that BrdU+ cells were indeed proliferating. Following ON lesion, in the retina approximately 18% of all BrdU+ cells were positive for the neural stem cell/progenitor cell (NSC/PC) marker nestin. The fraction of BrdU+/nestin+ cells in the CB was approximately 26%. Most of the BrdU+/nestin+ cells found in the neural retina were identified as reactive astrocytes and Müller cells. Since reactive glia cells can participate in adult neuro- and gliogenesis this may indicate a potential for regeneration after ON lesion in vivo.

Regulation of GDNF and its receptor components GFR-α1, -α2 and Ret during development and in the mature retino-collicular pathway

Abstract The development of the retino-tectal projection as part of the central visual pathway is accomplished around postnatal day (P) 10–14 in rodents, and trophic factors are important for topographic refinement of this projection. Emerging data indicate that GDNF may influence synaptic plasticity of this projection. To date, maturation-dependent kinetics of GDNF release and expression and biological function of single GDNF receptors along the retino-collicular pathway are ill-defined. Here, we examined mRNA and protein expression of GDNF and its multicomponent receptor complex in the retina and superior colliculus (SC) during postnatal development of the rat visual system, and after optic nerve (ON) injury by RT-PCR, immunoblotting and immunofluorescence. Stable mRNA transcription of GDNF and its receptors GFR-α1, -α2 and Ret was found in retina and SC throughout development into adulthood and after ON transection. Expression of GDNF protein increased during retinal development, declined in adulthood and was further reduced in injured retina. In the SC, GDNF peaked at P0, continuously declined with maturation, and was undetectable in the deafferentiated SC. GFR-α1 was abundant in retina and SC throughout, while GFR-α2 was not expressed. Since Ret was localized primarily to the vascular compartment, the receptor tyrosine kinase may play a minor role in neuronal GDNF signaling. In summary, we provide evidence for GDNF as survival and guidance factor during development of the retino-tectal projection with differential regulation in early and premature retina and SC. Postlesionally, midbrain targets do not induce GDNF, suggesting that retrograde GDNF is not essential for rescue of adult injured retinal ganglion cells (RGCs).

Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS.

Traditionally, depiction of isolated CNS fiber tracts is achieved by histological post mortem studies. As a tracer-dependent strategy, the calcium analog manganese has proved valuable for in vivo imaging of CNS trajectories, particularly in rats. However, adequate protocols in mice are still rare. To take advantage of the numerous genetic mouse mutants that are available to study axonal de- and regeneration processes, a MnCl2-based protocol for high-resolution contrast-enhanced MRI (MEMRI) of the visual pathway in mice acquired on a widely used clinical 3 Tesla scanner was established. Intravitreal application of MnCl2 significantly enhanced T1-weighted contrast and signal intensity along the retino-petal projection enabling its reconstruction in a 3D mode from a maximum intensity projection (MIP) calculated dataset. In response to crush injury of the optic nerve, axonal transport of MnCl2 was diminished and completely blocked proximal and distal to the lesion site, respectively. Conditions of Wallerian degeneration after acute optic nerve injury accelerated Mn2+-enhanced signal fading in axotomized projection areas between 12 and 24 h post-injury. In long-term regeneration studies 12 months after optic nerve injury, the MRI protocol proved highly sensitive and discriminated animals with rare spontaneous axonal regrowth from non-regenerating specimens. Also, structural MRI aspects shared high correlation with histological results in identical animals. Moreover, in a model of chronic neurodegeneration in p50/NF-κB-deficient mice, MnCl2-based neuron-axonal tracing supported by heat map imaging indicated neuropathy of the visual pathway due to atrophy of optic nerve fiber projections. Toxic effects of MnCl2 at MRI contrast-relevant dosages in repetitive administration protocols were ruled out by histological and optometric examinations. At higher dosages, photoreceptors, not retinal ganglion cells, turned out as most susceptible to the well-known toxicity of MnCl2. Our data accentuate in vivo MEMRI of the murine visual system as a highly specific and sensitive strategy to uncover axonal degeneration and restoration processes, even in a functional latent state. We expect MEMRI to be promising for future applications in longitudinal studies on development, aging, or regeneration of CNS projections in mouse models mimicking human CNS pathologies.

Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice

Although Harderian glands are especially large in rodents, many features of this retroocular gland, including its development and function, are not well established. Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes expressed in this gland. PP2A substrate specificity is determined by regulatory subunits with leucine 309 of the catalytic subunit playing a crucial role in the recruitment of regulatory subunits into the complex in vitro. Here we expressed an L309A mutant catalytic subunit in Harderian gland of transgenic mice. We found a delayed postnatal development and hypoplasia of the gland, causing enophthalmos. To determine why expression of the L309A mutant caused this phenotype, we determined the PP2A subunit composition. We found an altered subunit composition in the transgenic gland that was accompanied by pronounced changes of proteins regulating cell adhesion. Specifically, cadherin and beta-catenin were dramatically reduced and shifted to the cytosol. Furthermore, we found an inactivating phosphorylation of the cadherin-directed glycogen synthase kinase-3beta. In conclusion, the carboxy-terminal leucine L309 of the PP2A catalytic subunit determines PP2A heterotrimer composition in vivo. Moreover, our data demonstrate that PP2A subunit composition plays a crucial role in regulating cell adhesion and as a consequence in the development of the Harderian gland.

NF-κB controls axonal regeneration and degeneration through cell-specific balance of RelA and p50 in the adult CNS.

ABSTRACT NF-&kgr;B is dually involved in neurogenesis and brain pathology. Here, we addressed its role in adult axoneogenesis by generating mutations of RelA (p65) and p50 (also known as NFKB1) heterodimers of canonical NF-&kgr;B. In addition to RelA activation in astrocytes, optic nerve axonotmesis caused a hitherto unrecognized induction of RelA in growth-inhibitory oligodendrocytes. Intraretinally, RelA was induced in severed retinal ganglion cells and was also expressed in bystander Müller glia. Cell-type-specific deletion of transactivating RelA in neurons and/or macroglia stimulated axonal regeneration in a distinct and synergistic pattern. By contrast, deletion of the p50 suppressor subunit promoted spontaneous and post-injury Wallerian degeneration. Growth effects mediated by RelA deletion paralleled a downregulation of growth-inhibitory Cdh1 (officially known as FZR1) and upregulation of the endogenous Cdh1 suppressor EMI1 (officially known as FBXO5). Pro-degenerative loss of p50, however, stabilized retinal Cdh1. In vitro, RelA deletion elicited opposing pro-regenerative shifts in active nuclear and inactive cytoplasmic moieties of Cdh1 and Id2. The involvement of NF-&kgr;B and cell-cycle regulators such as Cdh1 in regenerative processes of non-replicative neurons suggests novel mechanisms by which molecular reprogramming might be executed to stimulate adult axoneogenesis and treat central nervous system (CNS) axonopathies.

A primary culture technique of adult retina for regeneration studies on adult CNS neurons.

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5–7 days. Mere preparation of a single retina should be completed within 20 min.

Morg1+/− heterozygous mice are protected from experimentally induced focal cerebral ischemia

Focal cerebral ischemia (stroke) and reperfusion injury leads to acute and chronic brain damage. The increase of the hypoxia-inducible transcription factor alpha (HIF-α), an important transcription factor for several genes, may attenuate ischemic brain injury. We recently identified a new WD-repeat protein designated Morg1 (MAPK organizer 1) that interacts with prolyl hydroxylase 3 (PHD3), an important enzyme involved in the regulation of HIF-1α and HIF-2α expression. While homozygous Morg1(-/-) mice are embryonically lethal, heterozygous Morg1(+/-) mice have a normal phenotype. Brain vasculature as well as systolic blood pressure in Morg1(+/-) mice were indistinguishable from wild-type (WT) animals. We show here that Morg1(+/-) mice were partially protected from cerebral ischemia/reperfusion injury in comparison to WT (Morg1(+/+)) animals using the middle cerebral artery occlusion model (MCAO). Morg1(+/-) mice compared with WT animals revealed a significantly reduced infarct volume as detected by Nissl and Map 2 staining despite a similar restriction of blood flow in both mice genotypes as measured by laser Doppler flowmetry. Immunohistochemistry revealed specific Morg1 expression in reactive astrocytes in the ipsilateral (ischemic) hemisphere in Morg1(+/-) and WT mice, especially in the penumbral regions. In the contralateral hemisphere, Morg1 was not detectable. Furthermore, Morg1 mRNA expression was significantly enhanced in the ischemic brain of WT, but not in ischemic brain tissue obtained from Morg1(+/-) animals. However, HIF-1α was expressed with the same intensity in Morg1(+/-) and WT mice with no difference between the ipsilateral and contralateral hemispheres. No positive staining for HIF-2α was found in ischemic (ipsilateral) and non-ischemic (contralateral) brain regions in Morg1(+/+) and Morg1(+/-) mice. Almost no PHD3 staining was found in the contralateral hemispheres of either WT or heterozygous Morg1(+/-) mice. Transcript expression for the HIF1α-dependent genes erythropoietin (Epo) and vascular endothelial growth factor 164 (VEGF 164) were significantly reduced in the ischemic brain from Morg1(+/-) mice. Positive staining for PHD3 in the ipsilateral hemisphere of WT mice was suggested to occur in astrocytes. A compensatory increase in Morg1 expression in astrocytes in the penumbra may negatively influence infarct volume. It appears that these effects are independent of the PHD3-HIF1α axis.

Dysfunctional NF-κB and brain myelin formation

In a recent issue of the European Journal of Human Genetics, Philippe et al1 reported on a duplication of the Xq28 locus and its supposed contribution to distorted brain myelination in patients carrying this mutation. They provided evidence that additional copies of the IKBKG gene (encoding NEMO, the regulatory subunit of the IKK complex) functionally impair NF-κB signaling, which in turn leads to the developmental brain abnormalities and mild mental retardation. MRI-based application of axial T2-weighted FSE and coronal FLAIR sequences to differentiate between CNS white and gray matter confirmed defective myelination in three out of five patients. The authors also presented convincing data that, as exemplified by isolated fibroblasts, NF-κB-dependent gene expression is indeed impaired in these patients carrying the IKBKG mutation.1 Accounting for the structural phenotype observed, they concluded that proper myelination requires NF-κB activation in CNS-intrinsic cell populations. This case study is highly valuable in providing evidence for a contribution of NF-κB to normal neurodevelopment. However, in line of our findings in transgenic mice, the proposal of a direct relationship between Xq28 duplication/NEMO hyperactivation in neuro-ectodermal cells and myelination deficits/microcephaly appears questionable. To realize neuro-ectodermal deletion of the transactivating NF-κB subunit RelA (RelACNSKO),2 we crossed mice carrying floxed relA alleles with transgenic mice expressing Cre recombinase under the control of the nestin promoter. Displaying almost complete removal of RelA protein from the CNS as verified by immunoblotting (see Supplementary Figure S1), this construct was used to analyze the requirement of RelA for proper myelin formation in the CNS using histological procedures, MR imaging, and behavioral tests. Histological and electron microscopic analyses of the optic nerve showed unimpaired oligodendrocyte densities (wild type, WT: 877.2±27.7 cells/mm2, RelACNSKO: 851.9±48.4 cells/mm2; P=0.43) and normal myelin sheath formation in RelACNSKO mice as assessed by g-ratios (WT: 0.71±0.02, RelACNSKO: 0.71±0.02; P=0.99). No abnormalities in brain structure were observed in T1- and T2-weighted sequences obtained in a 3 T magnetic field, and magnetization transfer (MT) sequences excluded reduced myelin contents in the cerebellar white matter of RelACNSKO mice (MT ratio WT: 35.8±2.3%, RelACNSKO: 34.1±2.9% P=0.7). Furthermore, tracer-based MR imaging (MEMRI) revealed normal active Mn2+ uptake into retinal ganglion cells and transport along the retino-tectal projection (signal-to-noise ratio in superior colliculi WT: 69.24±2.02, RelACNSKO: 70.52±5.77; P=0.86), indicating unimpaired nerve fiber vitality and function as compared to controls. Likewise, functional parameters of visual acuity and contrast sensitivity were indistinguishable between WT and RelACNSKO mice (published elsewhere). These findings are in line with previous reports showing that mice with inactivated upstream regulators of NF-κB (IκBα, IKK) in the neuro-glial compartment are indiscernible regarding overall neuro-anatomical and behavioral features3, 4 and, in particular, display normal myelination.5 Among the NF-κB family members (RelA, RelB, c-Rel, p105/50, p100/52), only deletion of the subunit p50, which lacks a transcriptional activator domain, results in a destructive neuronal phenotype as characterized by precocious aging, neuronal apoptosis and spontaneous demyelination in young adult mice.6 However, even in the case of p50 deletion, disturbances in congenital brain and myelin development have not been described to date. As p50 deficiency, as previously shown by others7, 8 and supported by our own data on NF-κB reporter mice (published elsewhere), results in enhanced rather than diminished NF-κB activity, such a phenotype does not offer a straightforward explanation for the neurodevelopmental deficits described under Xq28-dependent NF-κB inactivation. Nevertheless, RelA deficiency might lead to a compensatory replacement by other subunits, particularly by c-Rel. Indeed, switches in NF-κB dimer composition have been reported for cerebral ischemia.9 We are currently generating neuro-glia-specific RelA;c-Rel double-knockout mice to establish whether white matter abnormalities ensue. Undoubtedly, the highly informative character of the case study by Philippe et al1 highlights the clinical impact of NF-κB for proper neurodevelopment. Their data are supported by previous case studies on deregulated NF-κB signaling in patients carrying mutations in the TRAPPC9 gene, which encodes the NF-κB-inducing kinase (NIK)- and IκB kinase complex β (IKK-β)-binding protein.10 Although both factors are required for proper activation of NF-κB, NEMO and NIK-/IKK-β-binding protein might act via NF-κB-independent pathways in the context of myelination. Alternatively, a complex relationship between cerebral myelin formation and NF-κB-dependent gene expression in non-neuro-ectodermal cells might exist. One example of such an interaction is represented by the pathology of incontinentia pigmenti (IP), an X-linked dominant disorder with recurrent mutation in the NEMO gene, where about one-third of affected patients develop ocular and neurological deficits including mental retardation, microcephaly, and cerebellar ataxia.11 The fundamental neuropathology in IP, however, is not a neuro-ectodermal, but a mesenchymal dysfunction of cerebral blood vessels, leading to infantile microvascular ischemia and subsequent neuronal damage.12 Taken together, the currently available data from transgenic mice show discrepancies compared to the human phenotype described by Philippe and colleagues.1 Further research is necessary to clarify whether inhibition of upstream activators of NF-κB or of specific dimer compositions in (non-)neuro-ectodermal cells is of relevance in developmental white matter abnormalities.

In vivo imaging of optic nerve fiber integrity by contrast-enhanced MRI in mice.

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 µm)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-κB p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-κB mutations. In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.