Dietmar Fischer

Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF

Intravitreal injections of exogenous CNTF stimulate axon regeneration of RGCs in vivo. Nevertheless, controversy exists over the ability of exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Here we demonstrate that CNTF potently stimulated axon outgrowth of mature RGCs in culture in a JAK/STAT3- and PI3K/AKT-signaling pathway-dependent fashion and stronger than oncomodulin. Additional cAMP elevation or inhibition of MAPK activity increased these effects. In vivo intravitreal injections of exogenous CNTF induced endogenous CNTF expression in astrocytes in a manner that depended on the MAPK/ERK-signaling pathway activation. Reduction of endogenous CNTF expression by MAPK/ERK pathway inhibitors or its absence in CNTF deficient mice markedly reduced the neurite growth-promoting effects of exogenous CNTF. These data demonstrate that CNTF is a potent axon growth-promoting factor for mature RGCs. However, exogenously applied CNTF stimulates RGCs in vivo partially indirectly via a mechanism that depends on astrocyte-derived CNTF.

Crystallins of the β/γ-superfamily mimic the effects of lens injury and promote axon regeneration

Adult retinal ganglion cells (RGCs) can survive axotomy and regrow lengthy axons when exposed to lens injury (LI). The neuroprotective and axon-growth-promoting effects of LI have been attributed to an infiltration of activated macrophages into the inner eye and recently also to astrocyte-derived CNTF. The present work reveals that certain purified lens proteins (crystallins) cause the effects of LI. Intravitreal injections of beta- or gamma-crystallins, but not of alpha-crystallin, strongly enhanced axon regeneration from retinal explants in culture, within peripheral nerve grafts or the crushed optic nerve. Deposition of the effective crystallins within the vitreous body was also associated with an influx of circulating macrophages and an activation of retinal astrocytes, Müller cells, and resident microglia. Furthermore beta-crystallin induced CNTF expression in retinal astrocytes and activation of CNTFs major downstream signaling pathway (JAK/STAT3) when intravitreally injected or added to the culture medium ex vivo. Consistently, in culture the addition of beta- and gamma-crystallins to the medium also increased axon regeneration from retinal explants. These results demonstrate that crystallins of the beta/gamma-superfamily are the lens-derived activators of cascades, which lead to axonal regeneration and suggest that their effects might be mediated by astrocyte-derived CNTF.

Neuroprotective and axon growth promoting effects of intraocular inflammation do not depend on oncomodulin or the presence of large numbers of activated macrophages

Retinal ganglion cells (RGCs) cannot regenerate their axons after injury and undergo apoptosis soon after an intraorbital injury of the optic nerve. However, RGCs reactivate their axonal growth program when inflammatory reactions occur in the eye, which enables them to survive axotomy and to regenerate lengthy axons into the lesioned optic nerve. Lens injury (LI) and zymosan injections can induce these beneficial processes and provoke also a strong accumulation of activated macrophages in the vitreous body. It has recently been suggested that macrophage-derived oncomodulin is the principal mediator of this phenomenon. We show here that oncomodulin is not significantly expressed in primary macrophages and that the intraocular levels of this protein do not increase after LI or zymosan treatment. Furthermore, greatly reducing the invasion of macrophages into the inner eye does not diminish the neuroprotective effects of LI, but rather increases axon regeneration into the optic nerve. Axon regeneration is correlated with the activation of retinal astrocytes and Müller cells. Our data suggest that intraocular inflammation mediates its main beneficial effects through factors other than oncomodulin and that the underlying mechanism might be independent of the presence of activated macrophages.

A dynein mutation attenuates motor neuron degeneration in SOD1G93A mice

Cu/Zn SOD1(G93A) transgenic mice develop phenotypical hallmarks of ALS and serve therefore as an established model to study the molecular mechanisms underlying this disease. Recent reports demonstrate that mutations in the motor protein dynein in Legs at odd angles (Loa) and Cramping (Cra1) mice lead to similar but milder phenotypes. Surprisingly, double transgenic mice (Loa/SOD1(G93A)) have been recently shown to attenuate rather than to accelerate the phenotypical expression of motor neuron degeneration. These results raise the question whether other functional relevant mutations in dynein cause a similar effect. To address this question, we have cross-bred SOD1(G93A) with Cra1/+ mice. These double transgenic mice show an attenuated decline of both motor activity and body weight and an increase of survival time compared to SOD1(G93A) mice. Thus, this study confirms that mechanisms associated with dynein such as retrograde axonal transport may play an important role in SOD1(G93A-) toxicity on motor neurons.

The first step into the brain: uptake of NIO-PBCA nanoparticles by endothelial cells in vitro and in vivo, and direct evidence for their blood-brain barrier permeation.

By using fluorescent polysorbateu200580 coated poly(n‐butylcyanoacrylate) (PBCA) nanoparticles in an inu2005vivo study, direct evidence was found for the presence of nanoparticles entering the brain and retina of rats. The nanoparticles, prepared with a miniemulsion process, were labeled inu2005situ with a fluorescent dye and coated with polysorbateu200580. After preparation the particle size, ζu2005potential, and the molecular weight distribution were determined. BMEC cells were used as an inu2005vitro model for the BBB. The cells showed significant uptake of the particles, but no transcytosis could be observed inu2005vitro. After applying the particles to the animals at two concentrations, cryosections of the brains and retinas were prepared. Regarding the sections of the rats that received the lower dose, co‐localization of the applied fluorescent particles and the stained endothelial cells could be detected in the brain and retina, indicating particle internalization in the endothelial cells. Applied at higher doses, the particles could be detected within the brain and retina with few co‐localized signals, suggesting passage through the blood–brain and blood–retina barriers.

PROTEIN PHOSPHATASE TYPE-2C ISOZYMES PRESENT IN VERTEBRATE RETINAE : PURIFICATION, CHARACTERIZATION, AND LOCALIZATION IN PHOTORECEPTORS

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11‐cis‐retinal and by dephosphorylation of the opsin moiety. Phosphatases type‐1 and type‐2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type‐2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Cα and PP2Cβ from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+‐sustained activity. Antiserum raised against a C‐terminal peptide of PP2Cβ specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age‐dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery. J. Neurosci. Res. 51:328–338, 1998.

A Method for Preparing Primary Retinal Cell Cultures for Evaluating the Neuroprotective and Neuritogenic Effect of Factors on Axotomized Mature CNS Neurons

Retinal ganglion cells (RGCs) are central nervous system neurons with a very limited ability for axon regeneration. This unit details a cell culture technique, which can be used to functionally screen factors/compounds for their neuritogenic and neuroprotective effects on RGCs. In this protocol, the retina is isolated, digested in a papain solution, and after trituration, the RGCs are cultured. The neuritogenic effect of applied factors/compounds on RGCs in the medium is functionally determined by measuring the average neurite length of βIII‐tubulin‐positive RGCs in culture after 3 days. This protocol takes 3 to 7 days to perform depending on the application to complete, and is suitable to reliably test pharmacological and genetic approaches for their axon growth‐promoting and neuroprotective potential on mature RGCs. Curr. Protoc. Neurosci. 53:3.22.1‐3.22.10.

Analysis of Cell Type-specific Expression of CK1ε in Various Tissues of Young Adult BALB/c Mice and in Mammary Tumors of SV40 T-Ag-transgenic Mice

Casein kinase 1 epsilon (CK1ε) is involved in various cellular processes, including cell growth, differentiation, and apoptosis, vesicle transport, and control of the circadian rhythm. Deregulation of CK1ε has been linked to neurodegenerative diseases and cancer. To better understand the cell type-specific functions of CK1ε, we determined its localization by immunhistochemistry in tissues of healthy, young adult BALB/c mice and in mammary tumors of SV40 T-antigen-transgenic mice. CK1ε expression was found to be highly regulated in normal tissues of endodermal, mesodermal, and ectodermal origin and in neoplastic tissue of mammary cancer. The data presented here give an overview of CK1ε reactivity in different organs under normal conditions and outline changes in its expression in mammary carcinomas. Our data suggest a cell/organ type-specific function of CK1ε and indicate that tumorigenic conversion of mammary glands in SV40 T-antigen-transgenic mice leads to downregulation of CK1ε. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

Background Casein kinase 1 delta (CK1δ) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1δ has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1δ in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. Methodology/Principal Findings Immunohistochemical staining of CK1δ was performed using three different antibodies against CK1δ. A high expression of CK1δ was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. Conclusions These results give an overview of the cell-type specific expression of CK1δ in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1δ, where CK1δ can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1δ in these tissues.

Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB

RelB is the key component of the alternative NF-kappaB (nuclear factor kappaB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)-Daxx (death domain-associated protein) complex to NF-kappaB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys(48) nor on Lys(63) conjugates, but might involve unconventional ubiquitin conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys(273/274) and Lys(305/308) significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.