The neurotrophic theory 3

Neurons are generated in excess during the development of the vertebrate nervous system. Approximately 50% of the neurons die shortly after their axons reach their target fields, i.e. the tissues they innervate, during a short period called "phase of naturally occurring cell death" (for review, see Davies, 1994a). This loss may be required to match the number of neurons to the size and requirements of their target fields. Neuronal death occurs by apoptosis, a descriptive name given to a genetically regulated process of cell death (Oppenheim, 1991). According to the neurotrophic hypothesis, target fields regulate the size of the neuronal populations that innervate them by producing limiting amounts of neurotrophic factors, which are essential for the survival of developing neurons. Because the supply of these factors is though to be limiting, only a proportion of the neurons are able to obtain enough trophic factor to survive. Thus, the level of trophic factor production in the target field directly influences the size of the innervating population of neurons (Davies, 1994). The neurotrophic theory was initially formulated on the basis of work on nerve growth factor (NGF), the first neurotrophic factor to be identified (Levi-Montalcini, 1987), and posteriorly generalized with the discovery of other neurotrophic factors such as BDNF (Barde et al, 1982).

In support of the neurotrophic hypothesis, certain populations of developing neurons are dependent on NGF for survival in vitro and in vivo. Administration of anti-NGF antibodies during their phase of tai'get field innervation increases neuronal death, whereas exogenous NGF enhances the number of surviving neurons that would otherwise die (Levi-Montalcini, 1987). Recent data obtained from mutant mice with targeted disruption of either the NGF gene or the NGF receptor tyrosine kinase gene have confirmed the dependency of these neurons on NGF for survival during development (Crowley e ta l, 1994; Smeyne et a l, 1994). Furthermore, NGF is synthesized in the tissues innervated by these neurons coinciding with the beginning of target field innervation (Davies e ta l, 1987; Korsching and Thoenen,

1988). The level of NGF expression in these tissues is found to be proportional to their final innervation density (Haiper and Davies, 1990).

It is currently believed that multiple neurotrophic factors may cooperate in regulating the survival of neurons during development and in some cases these factors are supplied using mechanisms apparently in contradiction with the neurotrophic theory. Thus, it has been shown that neurons can obtain neurotrophic factors via anterograde, autocrine, and paracrine routes (for reviews, see Oppenheim, 1991; Davies, 1996a), although the physiological relevance of these findings in the regulation of neuronal survival in vivo remains unclear.

2.2. APOPTOSIS

Apoptosis or programmed cell death is a genetically controlled mechanism used by cells to commit suicide in response to a variety of stimuli. It plays a major role in several tissues, including the nervous system, during development to ensure a proper matching between different interacting cell populations. The execution of this death program is often associated with characteristic morphological and biochemical changes that distinguishes it from necrosis; the nucleus and cytoplasm condense, and the dying cell fragments into membrane-bound apoptotic bodies that are rapidly phagocytosed by surrounding macrophages (Wyllie et al., 1980). The process of apoptosis is regulated through the expression of an increasingly discovered number of genes, conserved during evolution from nematodes and viruses to mammals. Some gene products are activators of apoptosis, whereas others are inhibitors (for review, see Davies, 1995).

A least fifteen genes encoding proteins that either inhibit or accelerate cell death compose the Bcl-2 family. The founder of the family, Bcl-2, is a proto­ oncogene that was originally identified at the breakpoint of translocations commonly occurring in human B cell follicular lymphoma (Bakhshi et ai, 1985; Tsujimoto et al., 1985; Cleary et al., 1986) and encodes a membrane-associated protein that enhances cell survival by preventing cell death (Vaux et al., 1988). The subgroup of vertebrate gene products that inhibit apoptosis is composed of: Bcl-2, B c 1 -x l,

Bcl-w, Mcl-l and Al, whereas the accelerators of cell death include: Bcl-xs, Bax, Bad, Bak and Nbk/Bikl (for review, see Rao and White, 1997). A striking feature of the Bcl-2 family members is their ability to homodimerize and heterodimerize

through the conserved regions, BHl, BH2 and BH3 (Bcl-2 homology regions 1, 2 and 3). The level of expression of pro-apoptotic and anti-apoptotic proteins and the interactions between them seem to regulate cell death (Rao and White, 1997). Furthermore, post-translational phosphorylation of some Bcl-2 family members is also involved in controlling apoptosis (for review, see Gajewski and Thompson,

1996).

Another family of proteins that regulates cell death is the interleukin-IB- converting enzyme (ICE) family of cysteine proteases, also termed caspases. They are involved in a cascade of proteolytic events and, upon activation during apoptosis, induce cellular disassembly as a result of cleavage of numerous cellular substrates (Fraser and Evan, 1996, Rao and White, 1997; Martins and Earnshaw,

1997).

Whereas the Bcl-2 family of proteins is in control of apoptosis, the ICE family of proteins is involved in the execution of apoptosis. It has been recently demonstrated that the Bcl-2 family of proteins regulate caspase activation by translocation of cytochrome C and a protease, AIF (apoptosis-inducing factor) from the mitochondria to the cytosol, functionally linking the two families (for reviews, see Golstein, 1997; Martins and Earnshaw, 1997).

Some signalling molecules, like neurotrophic factors, induce a survival response, whereas others, like the Fas ligand and TNF, promote apoptosis. A functional connection between the intracellular signalling pathway mediated by Fas or TNFR-I and the activation of the cell death machinary has been recently reported (Fraser and Evan, 1996). However, the intracellular events mediated by the neurotrophic factor receptors that lead to cell suiwival are mostly unknown.

The survival of neurons during the development of the vertebrate nervous system is mainly promoted by members of three groups of neurotrophic factors: the family of neurotrophins, the family of neuropoietic cytokines and the family of TGF-B-related proteins.

3. TH E F A M IL Y OF NEUROTROPHINS AND T H E IR RECEPTORS

The neurotrophins are the best characterized family of neurotrophic factors. The family comprises six highly homologous proteins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin- 4/5 (NT-4/5) and neurotrophin-6 (NT-6). These proteins regulate numerous events during the development of the vertebrate nervous system including neuroblast proliferation, neuronal survival and differentiation, axonal branching, and synaptic function (Davies, 1994b; Snider, 1994; Thoenen, 1995). It has been suggested that the NGF gene appealed more recently in evolution than BDNF and NT-3, probably as a result of gene duplication and diversification by mutations (Barde, 1994). Neurotrophins transduce their signal into the cell through binding to a receptor complex composed of two transmembrane proteins, a specific high-affinity receptor tyrosine kinase (Trk) and a common low affinity receptor (p75NTR) (for review, see

Snider, 1994).

3.1. MEMBERS OF THE NEUROTROPHIN FAMILY