We have shown that in embryonic motoneurons γ-synuclein is uniformly distributed through the cytoplasm of cell bodies and axons. However, with the exception of a few neurons in the facial nucleus, the cytoplasm of cell bodies of motoneurons of the adult cranial somato- and branchiomotor nuclei is γ-synuclein negative, whereas their axons and synaptic boutons in neuromuscular junctions are intensively stained with anti-γ-synuclein antibody. Previously published in situ hybridization data demonstrated high levels of γ-synuclein
mRNA in cranial motor nuclei of adult brain (7
). Therefore, in motoneurons during postnatal development, γ-synuclein undergoes a compartmentalization shift, which might reflect a functional shift. Our results contradict recently published data (32
) which demonstrated the presence of γ-synuclein in cell bodies of motoneurons in brain stem motor nuclei of adult rats. This might reflect a difference between species; however, a more plausible explanation is the difference in the antibodies used in the two studies. In the present study, a highly specific antibody generated against mouse γ-synuclein C-terminal peptide was used, whereas Li et al. (32
) used an antibody generated against human recombinant γ-synuclein whose specificity has been checked with recombinant human synucleins but not with samples from null mutant animals.
It is not clear why some neurons of the adult facial nucleus do not follow the common rule and continue to accumulate γ-synuclein in the cytoplasm of their cell bodies. In this aspect they resemble peripheral sensory neurons, in which no developmental changes of intracellular compartmentalization of γ-synuclein take place and which have equally high levels of γ-synuclein in their cell bodies and processes during embryogenesis and postnatally. We found that both neural crest-derived DRG and placode-derived trigeminal sensory neurons have this unchanging pattern of γ-synuclein intracellular compartmentalization. Taken together, our expression studies demonstrated that between neurons expressing the highest levels of γ-synuclein throughout development, two subpopulations could be specified. The first includes peripheral sensory neurons and some motoneurons of the facial nucleus, which localize γ-synuclein in their cell bodies and axons at all developmental stages. Most other motoneurons of the brain stem nuclei comprise the second group, which is characterized by the developmental shift of γ-synuclein compartmentalization.
The high levels of expression suggest that γ-synuclein should have an important role in the development and function of sensory and motoneurons. Consequently, the loss of this protein could affect the morphology and/or physiology of animal sensory and motor systems. To check this, we produced mutant mice with complete inactivation of the γ-synuclein
gene. Similarly to previously reported α-synuclein
null mutants (1
), these mice showed no obvious phenotypical changes. Detailed studies of sensory and motoneurons in vivo and in vitro failed to detect any difference between γ-synuclein
null mutant and wild-type mice. The number of neurons is not changed in either of the subpopulations described above, suggesting that proliferation, migration, differentiation, or programmed cell death is not affected by the absence of γ-synuclein. Consistently, survival of γ-synuclein-deficient neurons in primary culture is not different from survival of wild-type neurons. Moreover, the absence of γ-synuclein does not render these neurons either more or less sensitive to any of the survival-affecting factors studied so far. Because mid-brain dopaminergic neurons seem to be most vulnerable to changes of synuclein metabolism (references 10
, and 33
and references therein), it is feasible that this neuronal population might be more sensitive to changes in the synuclein ratio than motoneurons and sensory neurons. We are currently testing whether the absence of γ-synuclein affects survival of dopaminergic neurons in the substantia nigra and ventral tegmental area of null mutant mice.
It has been suggested previously that γ-synuclein could be involved in axonal growth and stabilization of axon architecture (6
). However, we did not find differences in the morphology and number of myelinated or unmyelinated fibers in the saphenous nerves of mutant and wild-type mice. Sensory reflex thresholds were also intact in γ-synuclein
null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Normalization of sensory function after nerve injury is believed to be associated with neuronal plasticity in the spinal cord and axonal regeneration processes in the injured peripheral nerve (9
). These processes require remodeling of the axonal cytoskeleton, including the neurofilament network, and involvement of γ-synuclein in regulation of neurofilament network integrity has been suggested previously (6
). However, the time course of sensory recovery after CCI in γ-synuclein
null mutant mice is the same as that in wild-type mice, which suggested that nerve regeneration and plasticity of early somatosensory pathways in γ-synuclein
mutant mice were unaffected.
The most straightforward explanation of our results is that despite high levels of expression, γ-synuclein is not essential for the development and function of motor and peripheral sensory neurons. Nevertheless an alternative explanation is also possible. This function(s) could be vital for vertebrate organisms, and therefore effective mechanisms of protection against its loss have been developed in evolution. The presence of three closely related synucleins in all vertebrates and substantial overlapping of their expression patterns readily suggest that they are able to compensate for each other's function(s). It is unlikely that compensation for the loss of one synuclein function in mice is achieved by a simple increase of expression of other synucleins. We found no difference in the levels of mRNAs encoding the two remaining synucleins in several neuronal populations of γ-synuclein
null mutant mice, and the same has been demonstrated before for α-synuclein
null mutant mice (1
). However, it is possible that it is not necessary to boost an already high level of synuclein expression, because changes in compartmentalization, posttranslational modifications, or interaction with other macromolecules could be required and sufficient for functional compensation. Detailed studies of these processes in synuclein null mutant mice as well as studies of double and triple synuclein mutants should shed more light on this problem and finally reveal the normal functions of these proteins.