18 Cell Death in the Olfactory System

1. INTRODUCTION

The olfactory system is one of the most plastic regions in the brain, where neurons are continuously replaced and neuronal circuits are modulated throughout life. Although there is a constant replacement of receptor neurons in the adult olfactory epithelium, there is a perpetual integration of new neurons into the adult olfactory bulb. Hence a fine-tuned program to balance neuronal apoptosis and survival is necessary. This chapter summarizes current knowledge about life and death in the adult olfactory epithelium and olfactory bulb after illustrating normal olfactory anatomy. Finally, olfactory neuronal death is revisited in the context of neurodegenerative diseases.

2. ANATOMICAL ASPECTS

The neural system for smell is composed of the olfactory epithelium in the nose, the fila olfactoria forming the first cranial nerve (olfactory nerve), the olfactory bulb and tract, and depending cortical areas (Figure 18-1). The olfactory pathway is unique because smell is the only sense that reaches the phylogenetic old cortical regions without being relayed through the thalamus. The olfactory epithelium covers an area of approximately 2 to 5 cm2 in the dorsal posterior recess of the nasal cavity and contains receptor cells, basal cells, supporting cells, and glands (Figure 18-2). Odors are sensed by receptors on the endings of the short peripheral processes of bipolar receptor neurons. Their unmyelinated axons are bundled and surrounded by a Schwann cell, forming approximately 20 so-called fila olfactoria on each side, which together are considered as olfactory nerves. Olfactory receptor neurons (ORNs) project through the cribriform plate of the ethmoid bone

and terminate in the olfactory bulb (OB), a part of the telencephalon lying below the frontobasal cortex. Here, ORN axons form the first synapse of the olfactory pathway within specialized synaptic areas called glomeruli mainly on large mitral cells and on small tufted cells, the main output neurons of the OB. Sensory information is modulated in the OB by two types of inhibitory interneurons, granule cells and periglomerular cells, which receive synaptic input from other parts of the nervous system and to some extent also from ORNs (Figure 18-2). Neurites from mitral and tufted cells form the olfactory tract (second neuron), dividing into medial and lateral olfactory striae in front of the anterior perforated substance. Although the lateral stria projects to the amygdala and prepyriform area, where it connects to the third olfactory neuron, reaching cortical projection fields of the olfactory system in the entorhinal area and parahippocampal gyrus, fibers of the medial stria terminate in the septal area below the corpus callosum, from where the third neuron projects to the contralateral hemisphere via the anterior commissure. Other projections reach the habenular nucleus, reticular formation, and tegmental nuclei via the longitudinal striae, striae medullares, and medial forebrain bundle (Figure 18-1).

3. LIFE AND DEATH IN THE OLFACTORY SYSTEM

3.1. Olfactory epithelium

The olfactory epithelium was identified as an exceptional brain region with regenerative capacity and continuous turn-over of neuronal cells more than 30 years ago (Graziadei, 1973; Graziadei and Monti Graziadei, 1980). As holds true for large parts of the rest of the nervous system, the number of ORNs in the developing rat brain is fine-tuned predominantly by apoptosis

corpus callosum

medial olfactory stria

olfactory tract

olfactory bulb

olfactory epithelium

to amygdala

and prepyriform cortex

medial forebrain bundle

brainstem

cerebellum

(Figure 18-2), with a peak around E12 (Pellier and Astic, 1994) displaying typical morphological criteria under the electron microscope. However, unlike other parts of the nervous system, ORNs undergo both genesis and apoptosis throughout adult life. Initially, ORN apoptosis in intact olfactory epithelium was demonstrated by electron microscopy (Magrassi and Graziadei, 1995). Later, DNA fragmentation was documented histologically employing the terminal dUTP nick end labeling (TUNEL) technique in ORNs in the normal adult rat (Deckner et al., 1997) and mouse (Holcomb et al., 1995). Retroviral labeling of progenitor cells, the socalled globose basal cells in the olfactory epithelium (Suzuki and Takeda, 1991; Suzuki, 2004), revealed an ORN lifespan of approximately 1 month (Caggiano et al., 1994). However, some ORNs are believed to live more

than 3 months (Hinds et al., 1984; Mackay-Sim and Kittel, 1991x), depending on environmental input (see Farbman, 1990, for review). In aged mice, apoptosis is increased again when compared with normal or young animals with high expression levels of procaspase-3 and Bax (Robinson et al., 2002), suggesting increased fragility of the aged olfactory system.

The pathways underlying ORN apoptosis in vivo have mainly been studied by manipulating ORN death by lesion models such as naris occlusion, bulbectomy, and olfactory nerve transection. Although the latter two cause massive ORN apoptosis, sensory deprivation by naris occlusion leads to decreased ORN density and smaller size of the downstream regions in the olfactory bulb within 30 days (Farbman et al., 1988). Because the number of ORNs immunoreactive for olfactory marker

protein stays constant, reduced ORN numbers after naris occlusion are believed to be due to impaired neurogenesis (Cowan and Roskams, 2002). DNA laddering after olfactory nerve transaction or bulbectomy peaks between 24 and 48 hours post-lesion (Cowan and Roskams, 2002; Suzuki, 2004). Although surgical removal of the olfactory bulb, in contrast to axotomy, allows replaced ORNs to grow axons toward the former region of the olfactory bulb, they are deprived from their target and die chronically. This is reflected by the fact that all ORNs on the bulbectomized side appear to have a substantially shortened lifespan of less than 14 days (Schwob et al., 1992), possibly because of a lack of targetderived trophic support.

On the molecular level, mRNAs for Fas, Fas ligand, tumor necrosis factor receptor 1, and its ligand, tumor necrosis factor alpha (TNF-α), have been detected in the olfactory epithelium of adult rats (Farbman et al., 1999),

suggesting an important role of the so-called extrinsic apoptosis pathway for ORN apoptosis. Indeed, addition of Fas ligand or TNF-α to organotypic cultures of fetal E19 rat olfactory epithelium causes an increased apoptosis after 4 to 6 hours (Cowan and Roskams, 2002). There is more evidence, however, for the relevance of the mitochondria-dependent intrinsic apoptosis pathway because ORN death can be modulated by proand antiapoptotic members of the Bcl protein family. For example, over-expression of the antiapoptotic Bcl-2 protein in transgenic mice protects ORNs from apoptosis induced by bulbectomy for at least 5 days (Jourdan et al., 1998), a time point at which lesion-induced ORN loss is believed to be over. In line with that, knockout mice for the proapoptotic Bax protein are protected from bulbectomy-induced ORN loss (Robinson et al., 2003), even though neither expression of Bcl-2 nor Bax could be located in ORNs so far. Further support

for the involvement of the intrinsic apoptosis pathway in ORN loss comes from data by Cowan and co-workers demonstrating caspase-9 and -3 as major drivers for ORN apoptosis, both during development and in the mature brain (Cowan et al., 2001; Cowan and Roskams, 2004). Both caspases are present in mature ORNs, and their expression levels increase within hours upon apoptosis initiation by bulbectomy. Caspase-3 seems first to be activated at the synapse proceeding through the axon to the cell body, suggesting a timed activation pattern within the ORN population. Underscoring the importance for caspase-3 as executioner of ORN apoptosis also during development, caspase-3 knockout mice display an expanded ORN population (Cowan et al., 2001). On the other hand, not only caspase- 3 mRNA, but also mRNAs for caspase-1 and -2 have been detected in the olfactory epithelium of fetal and adult rats (Suzuki and Farbman, 2000). In the case of caspase-2, mRNA is not detectable between 3 and 5 days after bulbectomy but reappears within 21 days, suggesting that caspase-2 may be expressed in ORNs and contribute to the propagation of cell death. More evidence for caspase-dependent ORN apoptosis comes from studies employing caspase inhibitors, which not only blocked apoptosis in organotypic cultures of the olfactory epithelium after treatment with TNF-α, but also reduced apoptosis under control conditions (Suzuki and Farbman, 2000).

Finally, ORNs contain several caspase substrates that eventually result in DNA fragmentation. However, their significance for ORN remains unresolved. Cleaveage of alternative caspase targets, like amyloid precursor-like protein 2, which is presumed to mediate axonal outgrowth in ORNs, reflects the spatial caspase activation, further supporting the relevance of caspases for ORN loss in normal and lesioned olfactory epithelium (Cowan and Roskams, 2002).

3.2. Olfactory bulb

Besides the hippocampus, the olfactory bulb is the only region in the adult mammalian brain where the integration of newly generated neurons into pre-established neuronal circuits is uncontroversial (Altman, 1969; Gage, 2002; Hack et al., 2005; Gould, 2007). Neuronal progenitors destined for the olfactory bulb are generated in the subventricular zone (SVZ) between the lateral ventricle and the striatum from astrocytic neural stem cells (Doetsch et al., 1999; Garcia et al., 2004). Although evidence for its existence is lacking in humans (Sanai et al., 2004), in the rodent, newly produced postmitotic neuroblasts migrate tangentially along the so-called

rostral migratory stream (RMS) (Figure 18-2), where they are shielded against surrounding brain tissue by glial cells forming a tube-like structure (Lois et al., 1996; Anton et al., 2004). More than 30,000 neuroblasts exit the rodent SVZ each day (Alvarez-Buylla et al., 2001), but only a very limited number is integrated into the olfactory bulb after radial invasion from the migratory path (Lledo and Saghatelyan, 2005). Newborn olfactory neurons can only mature into two types of inhibitory interneurons. The vast majority differentiate into GABAergic granule cells (Figure 18-2), whereas only very few give rise to periglomerular cells expressing GABA and/or the dopamine-synthesizing enzyme tyrosine hydroxylase (Lledo and Saghatelyan, 2005). Both types of neurons only form local synapses and probably can directly or indirectly modulate the processing of sensory input by mitral and tufted cells, the olfactory bulb output neurons (Lledo et al., 2006). Surprisingly, only approximately 50% of the newly generated interneurons survive more than a month (Petreanu and AlvarezBuylla, 2002; Winner et al., 2002) before they die by apoptosis (Figure 18-2), as revealed by TUNEL labeling (Winner et al., 2002). This waste of resources poses the question regarding the functional relevance of adult neurogenesis. Although the answer still remains obscure, Lledo and co-workers (2006) offered a scheme of newborn neurons functioning at the cellular and network levels, either leaving existing neurons unchanged, changing existing networks, or just replacing apoptotic cells displaying adaptable or prespecified functions. Hence the hypothesis that bulbar neurogenesis mediates the adjustments of sensory processing in response to the constantly changing olfactory input appears feasible.

Be that as it may, a clear correlation between the survival of newborn bulbar granule cells and olfactory input has been demonstrated (Corotto et al., 1994; Rochefort et al., 2002; Miwa and Storm, 2005; Saghatelyan et al., 2005). On the molecular level, various factors have been shown to regulate neuronal survival in the olfactory bulb. For example, glutamate, the main excitatory amino acid in the brain, has been shown to enhance survival of SVZ-derived neurons in a dose-dependent manner, whereas phosphatase and tensin homolog, a lipid phosphatase with proapoptotic activity, has been identified as negative regulator (Brazel et al., 2005; Li et al., 2002). Moreover, loss or inactivation of the polysialylated isoforms of neural cell adhesion molecule (NCAM) leads to reduced survival of postnatally generated immature neurons in the olfactory bulb in response to neurotrophic factors due to an upregulation of the p75 neurotrophin death receptor (Gascon et al., 2007). In line with the increased death of olfactory bulb neurons in

the absence of NCAM, NCAM−/– mice display an approximately 30% reduction in olfactory bulb size and an approximately 10% reduction in overall brain size (Cremer et al., 1994; Tomasiewicz et al., 1993; Gheusi et al., 2000). Furthermore, extracellular signal-regulated kinase 1/2 (Erk1/2) as part of the mitogen-activated protein kinase (MAPK) pathway have been identified as promoters of granule cell survival in the olfactory bulb. Odor exposure of mice in vivo resulted in MAPK activation in granule cells within 10 minutes, with an increased survival of newly formed granule cells (Miwa and Storm, 2005). In contrast to ORNs, where expression of the antiapoptotic Bcl-2 and the proapoptotic Bax protein could not be demonstrated so far, increased Erk phosphorylation after odor exposure culminated in increased expression levels of the antiapoptotic Bcl-2 protein in the olfactory bulb (Miwa and Storm, 2005). Similarly, adult Baxdeficient mice exhibited markedly reduced apoptosis in the olfactory bulb, as revealed by TUNEL staining in comparison with wild-type mice (Kim et al., 2007). At the same time, neurons expressing active caspase- 3, which are physiologic in wild-type mice, could not be detected in Bax knockout mice. Interestingly, lack of apoptosis in the olfactory bulb of Bax-deficient mice was neither associated with increased size of the olfactory bulb nor changes in the number of proliferating neuroblasts in the SVZ, but rather caused an accumulation of ectopic neurons in the RMS as a result of an impairment of RMS glial tube formation (Kim et al., 2007). Conversely to the hypothesis suggesting a relevance of granule cell turn-over in the olfactory bulb for processing of the sensory information (Lledo et al., 2006), and in conflict with the correlation between the survival of newborn bulbar granule cells and olfactory input (Corotto et al., 1994; Rochefort et al., 2002; Miwa and Storm, 2005; Saghatelyan et al., 2005), adult and aged Bax knockout mice show normal olfactory behavior and odor discrimination (Kim et al., 2007). Together, these most recent data question the requirement of granule cell apoptosis in the olfactory bulb for odor discrimination or odor memory.

4. OLFACTION IN AGING AND NEURODEGENERATIVE

DISEASE

Impairment of smell perception with age in humans was discovered more than 20 years ago (Doty et al., 1984, Stevens and Cain, 1987) and was believed to be a matter of senescence. New data, however, indicate that a significant decline in odor discrimination ability can already be documented in the second half of the fourth decade in human life (Hawkes, 2006). Interestingly, the sensation of high hedonic or low-intensity odors is more severely

affected than the sensation of unpleasant ones (Hawkes, 2006). In rodents, this decline in olfactory discrimination ability correlates with a profound decrease in SVZ neurogenesis rather than increased apoptosis in the olfactory bulb. Still, decreased neurogenesis results in a reduction in the number of new neurons in the olfactory bulb affecting both periglomerular and granule interneurons (Enwere et al., 2004).

The discovery of hypor anosmia as common feature in idiopathic Parkinson’s disease (PD) and dementia of the Alzheimer type (AD) has boosted the interest in olfactory dysfunction in neurodegenerative diseases. In AD, typical histological abnormalities throughout the olfactory system are well documented (Yamagishi et al., 1994; Braak and Braak, 1998; Kovacs et al., 2001), and it has been suggested that AD could be diagnosed by biopsy of the nasal olfactory epithelium (Talamo et al., 1989). However, this procedure has not entered the clinic for several reasons, including doubts on specificity, histological limitations, and the fact that there is a constant replacement of ORNs that might be even accelerated in AD (Hawkes, 2006). Nevertheless, hyposmia/anosmia has been identified as risk factor for subsequent cognitive failure and AD, especially in combination with specific genetic markers (see Graves et al., 1999; Hawkes, 2006, for review).

Olfactory dysfunction in PD was first reported more than 30 years ago (Ansari and Johnson, 1975), and loss of smell was suggested as risk factor, as well as a screening tool for the development of PD in recent prospective studies (Sommer et al., 2004; Ross et al., 2006; Haehner et al., 2007). This is also of clinical importance with regard to differential diagnosis of PD because there are other neurodegenerative diseases associated with PD symptoms in which loss of smell is absent or much less severe (e.g., multisystem atrophy, essential tremor and others; for review see Hawkes, 2006). Pathological hallmarks of PD (Lewy neuritis and Lewy bodies) are first found in the brainstem, olfactory bulb, and associated anterior olfactory nucleus in the olfactory tract long before the disease becomes clinically apparent (Braak et al., 2003), with Lewy bodies being found in mitral cells of the olfactory bulb (Daniel and Hawkes, 1992) and dystrophic neurites being present in the olfactory epithelium (Crino et al., 1995). Although a detailed analysis of apoptosis in the olfactory bulb beyond anatomical description is still lacking, significant cell loss in the PD olfactory bulb is beyond doubt (Hawkes, 2006). From results in the rodent, it appears that this cell loss can at least partially and temporarily be compensated by increased neurogenesis and cell replacement in the olfactory bulb (Yamada et al., 2004), thereby slowing down symptom onset.