Apoptotic Neuronal Cell Death

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In the last few years, histopathological studies of postmortem tissue of patients, as well as data from experimental animal and cell culture models, have revealed the presence of neuronal apoptosis in multiple sclerosis (MS), an autoimmune CNS disease that has long been thought to be primarily characterized by inflammation and demyelination. In human autopsy brain tissue, a limited number of apoptotic neurons could be detected in chronic active and chronic inactive lesions from patients with MS (Peterson et al., 2001). In contrast, acute active lesions did not show neurons that fulfilled the morphological or intracellular criteria of apoptotic neuronal cell death. As also known from studies of "classical" neurodegenerative diseases such as Alzheimer's disease, it is difficult to detect apoptotic neurons in human brain tissue because of the rapid time kinetics of the apop-tosis-related intracellular signaling cascades (for review, see Mattson, 2000b). In addition, experiments that involve manipulation of apoptotic signaling pathways to investigate their functional relevance are not possible in patients. For these reasons, much of the present knowledge about apop-totic neuronal cell death and its mechanisms in MS comes from studies in animal models of experimental autoimmune encephalomyelitis (EAE).

By the use of different agents and modes of immunization as well as different animal strains, EAE models mimicking many aspects of the human disease have been established (Wekerle et al., 1994; Storch et al., 1998; Weissert et al., 1998; Kornek et al., 2000). EAE induced by active immunization of female brown Norway (BN) rats with the extracellular domain of recombinant rat myelin oligodendrocyte glycoprotein 1-125 (rrMOG) especially reflects the neurodegenerative aspect of MS (Meyer et al., 2001; Diem et al., 2003b). In this model, it has been demonstrated that EAE lesions often affect the ON with concomitant decrease of electrophysiological function as revealed by recordings of visual evoked potentials, the electrical cortical response to a visual stimulus. Simultaneous measurements of electroretinograms in response to pattern stimulation, which specifically correspond to RGC function, gave evidence for severe impairment of the neurons that give rise to the axons of the ON as well (Meyer et al., 2001). Immunohistochemical analysis of RGC somata showed that these neurons undergo lesion-induced apoptosis even before the onset of clinically manifest EAE (Hobom et al., 2004). RGCs positive for active caspase-3, an important member of the downstream caspase cascade, as well as those showing DNA degradation, were detected during induction and manifestation of MOG-EAE (Fig. 2 A-D). The importance of the animal's individual immunogenetic background for the kinetics and severity of neurodegeneration during EAE was emphasized by a comparative study of caspase activation in two different EAE models. Whereas neuronal caspase-3 expression in the spinal cord of Lewis rats in the interleukin-12 EAE model did not increase until the third relapse, active neuronal caspase-3 in ABH mice immunized with spinal cord homogenate was already detectable during the acute stage of the disease (Ahmed et al., 2002).

Comparing neuronal apoptosis during rrMOG-induced optic neuritis in rats with the loss of neurons in a noninflammatory, purely neurodegenerative model based on mechanical lesion of the retinocollicular fiber tract, similarities in kinetics and extent can be observed. Surgical axotomy of the ON in the rat is a frequently used model to investigate secondary neuronal cell loss in degenerative processes of the mammalian CNS because of its good surgical accessibility and well-known kinetics of cell death (Villegas-Perez et al., 1988; Bahr and Bonhoeffer, 1994; Bahr and Wizenmann, 1996; Klocker et al., 1997, 1998; Diem et al., 2001; 2003a).

Figure 2 Cell death of retinal ganglion cells (RGCs) during autoimmune optic neuritis is accompanied by DNA-degradation and caspase-3 activation. (A, B) Double staining of a retinal section from a rat with optic nerve affection during experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. RGCs were identified by retrograde labeling with the fluorescent dye Fluorogold (FG) (A). FG staining co-localizes with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling indicating DNA fragmentation (B). (C, D) Detection of active caspase-3 (D) by immunohistochemistry in RGCs identified by FG labeling (C). Scale bars: (A, B) 100 |m; (C, D) 70 |m. (Reprinted from Meyer et al., 2001.)

Figure 2 Cell death of retinal ganglion cells (RGCs) during autoimmune optic neuritis is accompanied by DNA-degradation and caspase-3 activation. (A, B) Double staining of a retinal section from a rat with optic nerve affection during experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. RGCs were identified by retrograde labeling with the fluorescent dye Fluorogold (FG) (A). FG staining co-localizes with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling indicating DNA fragmentation (B). (C, D) Detection of active caspase-3 (D) by immunohistochemistry in RGCs identified by FG labeling (C). Scale bars: (A, B) 100 |m; (C, D) 70 |m. (Reprinted from Meyer et al., 2001.)

It has been demonstrated that transection of the ON induces a delayed death of 80% to 90% of RGCs within 2 weeks, starting on or about day 4, reaching a maximum on day 7, and decreasing on or about day 14 after axotomy (Eschweiler and Bahr, 1993; Mansour-Robaey et al., 1994). In the rat model of rrMOG-induced optic neuritis, neuronal apoptosis occurred as acute and severe as after complete surgical transection of the ON: A first significant reduction of RGC density was seen at day 5 after immunization. Between day 7 after immunization and the day of disease onset, the highest rate of RGC apoptosis was observed. After this acute phase, neuronal cell death kinetics slowed down on or about day 8 of MOG-EAE (Hobom et al., 2004). At this time, rats had lost 73% of their RGCs compared to cell counts of healthy animals. Whereas these data raised the hypothesis of axonal pathology or dysfunction being responsible for the induction of apoptotic neuronal cell death in EAE as a secondary, retrograde event, the concept of primary neuronal apoptosis independent from axonal damage was suggested by in vitro studies on cortical neuron cultures. In this model, cerebrospinal fluid (CSF) from patients with primary progressive MS and active disease course or CSF obtained during acute relapse of relapsing-remitting MS induced apoptotic cell death of rat cortical neurons (Alcazar et al., 1998; Cid et al., 2003). Although the underlying mechanisms could not be identified, neurons were rescued from CSF-induced apopto-sis by application of caspase-3 inhibitors in vitro.

Investigating the relationship between neuronal cell loss during EAE and neurological function, it has been shown that similar to the classical neurodegenerative disorders, a linear correlation between these two parameters could not be observed. Postmortem studies in brains from parkinsonian patients revealed that the loss of nigrostriatal dopamine neurons must be greater than 75% in order for the disease to become clinically evident (Lloyd, 1977). For amyotrophic lateral sclerosis (ALS), it has been estimated that 50% to 80% of target neurons must be degenerated before clinical disability develops (Bradley, 1987). Accordingly, in the rat optic neuritis model induced by immunization with rrMOG, visual function was not severely altered until RGC loss had reached a threshold of 50% to 60%; below that compensatory mechanisms maintained functional integrity (Meyer et al., 2001; Hobom et al., 2004). Figure 3 shows the relationship between RGC density in this model and visual acuity values determined by electroretinogram recordings. In support of these data, up to 82% of axonal loss was found in MS-like lesions from individuals with no reported neurological symptoms during their lifetime (Mews et al., 1998). After autoimmune optic neuritis, functional compensation could occur at a cortical level based on topographical changes of the primary visual cortex, as has been shown for balancing visual field defects (Safran and Landis, 1996). Local molecular mechanisms on different levels of the optic pathway that involve NMDA receptor-mediated activity (Binns and Salt,

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Figure 3 Correlation between retinal ganglion cell (RGC) density and visual function in rats with acute experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG). (A) Normal cell density of RGCs in a healthy control rat. (B) The numbers of remaining RGCs are significantly reduced in a rat with acute optic neuritis during MOG-EAE. (C) The decrease of RGC density in rats with optic neuritis correlates with a reduction of visual acuity determined by measurements of electroretinograms. Filled circles indicate visual acuities and RGC densities of rats with histopathologically proven optic neuritis, whereas filled triangles represent those of healthy controls. Visual acuity values and RGC counts of MOG-immunized rats without optic neuritis are given as open squares. Note that RGC densities in animals suffering from optic neuritis must be markedly decreased to lead to a reduction of visual acuity. (Reprinted from Meyer et al., 2001.)

1998) or substance P expression (Tu et al., 2000) may contribute to neuronal plasticity in EAE or MS. Nitric oxide (NO) has also been postulated to act as a retrograde signal that can mediate multiple aspects of synaptic plasticity in the visual system (Cogen and Cohen-Cory, 2000).

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