Christian K. Vorwerk

Traumatic Axonal Injury Results in Biphasic Calpain Activation and Retrograde Transport Impairment in Mice

Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intra-axonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.

An Experimental Basis for Implicating Excitotoxicity in Glaucomatous Optic Neuropathy

Most therapy for glaucoma is directed at the management of the intraocular pressure (IOP). Conventional wisdom holds that excessive pressure within the eye leads to the ganglion cell loss/optic nerve damage seen in this disease. Both glutamate and elevated IOP can selectively damage the retinal ganglion cells in the mammalian eye. We have identified an elevated level of glutamate in the vitreous humor of glaucoma patients (27 microM as compared to 11 microM in the control population). This concentration of glutamate suffices--on its own--to kill retinal ganglion cells. It is plausible that the IOP may represent an initial insult that precipitates the production of excessive glutamate. Therefore, even if glutamate elevation is an epiphenomenon associated with the course of the disease, it may contribute to ganglion cell loss in humans. Lowering the IOP may slow down glutamate production, but if nothing is done to block the toxic effects of glutamate as well, visual loss may result despite excellent IOP control. If interventions can be found to retard the production or toxic effects of glutamate, it may be possible to slow glaucomatous visual loss.

Different Activation of Mitogen-Activated Protein Kinase and Akt Signaling Is Associated with Aggressive Phenotype of Human Meningiomas

Purpose: Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. Experimental Design: Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. Results: Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. Conclusions: Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity

Purpose. Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. Methods. Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. Results. AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. Conclusion. AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

Nitrate therapy may retard glaucomatous optic neuropathy, perhaps through modulation of glutamate receptors

Nitrates have been a major part of the internists pharmacopoeia for more than 100 years, predominantly for the relief of anginal symptoms. The effects of nitroglycerin on the eye and specifically on intraocular pressure has been investigated with diverse results. However, nitroglycerin may also serve to protect retinal ganglion cells against glutamate mediated toxicity--a form of cell death that may be critical in glaucomatous blindness. Consequently, we therefore sought to evaluate whether nitroglycerin preparations, taken for non-ophthalmic reasons, had an effect on glaucomatous damage.

Co-expression of c-Jun and ATF-2 characterizes the surviving retinal ganglion cells which maintain axonal connections after partial optic nerve injury.

The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.

Effects of axonal injury on ganglion cell survival and glutamate homeostasis.

Axonal trauma leads to a series of pathologic events that can culminate in neuronal death. Optic nerve crush can be used to explore histologic and molecular changes in traumatic central nervous system malfunction. Although the precise mechanisms of retinal ganglion cell death after optic nerve crush have not been elucidated, glutamate antagonists can protect retinal ganglion cells after axotomy. We, therefore, evaluated the effect of optic nerve crush on levels of extracellular glutamate. Ganglion cell survival and extracellular glutamate levels were assessed from 1 to 28 days after optic nerve crush in Long-Evans rats. Optic nerve crush led to a rise in extracellular glutamate; this rise was blocked by treatment with memantine, riluzole, and nimodipine. Partial optic nerve crush leads to an increase in vitreal glutamate, perhaps through release of intracellular contents. This released glutamate can contribute to additional ganglion cell loss. Future work will help to additionally unravel the steps by which axotomy induces excitotoxic damage to ganglion cells, and perhaps indicate protective interventions.

Analysis of TP53 and PTEN in gliomatosis cerebri.

Gliomatosis cerebri (GC) is a rare glial neoplasm with extensive diffuse brain infiltration but relative preservation of the underlying architecture. Previous molecular studies, mostly analyzing biopsy samples, have suggested an astrocytic origin of GC, but a larger collective of autopsy tissue has not been investigated so far. Furthermore, whether the widespread neoplastic infiltration is based on a monoclonal process is still a matter of debate. In the present study, we screened paraffin-embedded brain tissue from different areas of 18 cases (8 autopsy cases and 10 biopsies) for alterations in the TP53 and PTEN genes. Nuclear accumulation of p53 protein was detected in 9 cases (50%). Somatic TP53 mutations occurred in two autopsy cases (11% of all cases). In the first case, a C→T transition in codon 273 (Arg→Cys) was detected in all tumor samples. In the second case, in tumor samples from one hemisphere, nuclear accumulation of p53 was caused by a G→A transition in codon 244 (Gly→Asp). In the present series, no mutations within the coding region of PTEN were found. Pten expression was observed in two autopsy cases (25%) and seven biopsy samples (70%). These data suggest that TP53 is affected in some cases, but other yet-unidentified genetic alterations might contribute to tumorigenesis in GC. Furthermore, although GC might be a monoclonal process, the presence of different tumor clones cannot be ruled out.

Saving the Nerve from Glaucoma: Memantine to Caspaces

Three recent discoveries regarding the pathophysiology of human glaucoma are that retinal ganglion cells die by apoptosis, that nitric oxide synthase levels are altered, and that glutamate is elevated in the vitreous. These findings provide encouraging new avenues for the development of neuroprotective strategies to alleviate ganglion cell loss and blindness that accompanies this disease. In this article, we discuss some of these data, as well as potential therapies that may arise from these findings.

bcl-2 Gene Therapy Exacerbates Excitotoxicity

The protooncogene bcl-2 can block neuronal death from both naturally occurring apoptosis and exogenous insults. bcl-2 is therefore a promising candidate for the prevention of excitotoxic neuronal death. Using an adeno-associated viral vector, we delivered the bcl-2 gene to the ganglion cell layer of the rat eye. We hypothesized that infection with bcl-2 would protect ganglion cells against excitotoxic cell death. However, retinal infection with bcl-2 increased ganglion cell susceptibility to both axonal injury and intravitreal NMDA. Our study--intended to explore the possibility of bcl-2 transduction as an in vivo therapeutic approach--revealed a deleterious effect of bcl-2 transduction.