The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell deathindependent role for Bax in facilitating mutant SOD1-mediated motor denervation.

In the dentate gyrus (DG) of the adult mouse hippocampus, a substantial number of new cells are generated daily, but only a subset of these survive and differentiate into mature neurons, whereas the majority undergo programmed cell death (PCD). However, neither the intracellular machinery required for adult stem cell-derived neuronal death nor the biological implications of the significant loss of these newly generated cells have been examined. Several markers for apoptosis failed to reveal cell death in Bax-deficient mice, and this, together with a progressive increase in neuron number in the DG of the Bax knock-out, indicates that Bax is critical for the PCD of adult-generated hippocampal neurons. Whereas the proliferation of neural progenitor cells was not altered in the Bax-knock-out, there was an accumulation of doublecortin, calretinin ϩ , and neuronal-specific nuclear protein ϩ postmitotic neurons, suggesting that Baxmediated PCD of adult-generated neurons takes place during an early phase of differentiation. The absence of PCD in the adult also influenced the migration and maturation of adult-generated DG neurons. These results suggest that PCD in the adult brain plays a significant role in the regulation of multiple aspects of adult neurogenesis.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspaseindependent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1G93A mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo-dendritic and axo-somatic synapses with an increase in C-type axo-somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1G93A mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.

The present study defined the time course of terminal proliferation (the growth of presynaptic processes) and reactive synaptogenesis in the dentate gyrus of the adult rat. Quantitative electron microscopic analyses were carried out in the dentate gyrus 2, 4, 6, 8, 10, 12, 14 days and 7 months after destruction of the ipsilateral entorhinal cortex and in the contralateral (control) dentate gyrus. At each survival interval, counts were made from photographic montages of 1) terminals (presynaptic processes with or without contacts with postsynaptic elements), 2) intact synapses, 3) degenerating synapses, 4) degeneration (degenerating presynaptic processes), and 5) multiple synapses (terminals making more than one synaptic contact). Terminal density was initially reduced to about 13% of control in the middle molecular layer at 2 and 4 days postlesion, and to about 26% of control in the outer. The density of terminals began to increase between 4 and 6 days postlesion, reaching a plateau by day 12. Synapse density was reduced to about 8% and 12% of cont,rol in the middle and outer molecular layer respectively. Synapse density increased about 5-fold between 8 and 12 days postlesion, but continued to increase in the period between 14 days and 7 months postlesion. At 2 days postlesion, the number of intact terminals that are lost corresponds to the number of degenerating presynaptic processes. This correspondence is not present at 4 days postlesion, however, suggesting a rapid removal of degenerating terminals. In contrast, even at 2 days postlesion, the number of intact synapses that are lost does not correspond to the number of degenerating synapses. Between 2 and 10 days postlesion, the number of postsynaptic specializations is about 60% of control, but recovers slightly by 12-14 days postlesion. Qualitative and quantitative evidence suggested a collapse of spines into configurations that resembled shaft synapses. There appeared to be a deformation of degenerating presynaptic processes resulting in the appearance of multiple synapse configurations prior to reinnervation. The combined results suggest that terminal proliferation precedes reactive synaptogenesis in the dentate gyrus by 2-4 days, that terminal proliferation is essentially complete by 12 days while reactive synaptogenesis continues, and that multiple synapses arise at least in part as a result of a deformation of degenerating presynaptic processes rather than as a consequence of the induction of additional contacts on existing presynaptic terminals.