Increased Expression of Rat Synuclein in the Substantia Nigra Pars Compacta Identified by mRNA Differential Display in a Model of Developmental Target Injury *Nikolai G. Kholodilov, *Michael Neystat, *Tinmarla F. Oo, *Steven E. Lo, *†Kristin E. Larsen, *†David Sulzer, and *‡Robert E. Burke Departments of *Neurology and ‡Pathology; and †Departments of Psychiatry and Neuroscience, New York State Psychiatric Institute, Columbia University, College of Physicians and Surgeons, New York, New York, U.S.A. Abstract: Human -synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer’s disease, and it is the locus of mutations in some familial forms of Parkinson’s disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular re- sponses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental in- jury to their target, the striatum. We find by mRNA differ- ential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than ap- optotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neu- rons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive. Key Words: Synuclein—Programmed cell death—Apo- ptosis—Substantia nigra—Dopaminergic neurons—Par- kinson’s disease. J. Neurochem. 73, 2586 –2599 (1999). Human -synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer’s disease. A 35-amino acid peptide fragment was named the non-A component of amyloid, or NAC, and its 140-amino acid precursor was termed NACP (Ueda et al., 1993). NACP was also subsequently iden- tified on the basis of its cross-reactivity with a monoclo- nal antibody that recognized phosphorylated tau protein (Jakes et al., 1994) and named -synuclein, given its homology to synucleins that had been isolated from Torpedo electroplaques and identified in rat brain (Maro- teaux et al., 1988; Maroteaux and Scheller, 1991). It is now recognized that in addition to human and rat -synucleins, there are other members of a rapidly ex- panding family of synuclein-related proteins (for re- views, see Clayton and George, 1998; Lavedan, 1998). Nakajo et al. (1993, 1994) identified a 14-kDa protein, termed phosphoneuroprotein 14, that is homologous to -synuclein and that, in rat brain, shares a similar distri- bution in axon terminals. Jakes et al. (1994) identified the 134-amino acid human homologue of this protein and termed it -synuclein. More recently, another homo- logue has been isolated from breast cancer tissue (BCSG1) (Ji et al., 1997), and given its homology to - and -synucleins, the terminology -synuclein has been proposed (Lavedan et al., 1998). A phylogenetic analysis suggests that the first synuclein identified, that in Tor- pedo, by Maroteaux et al. (1988), is a member of this subfamily (Lavedan, 1998). A recently reported new member of the synuclein family, synoretin, identified on the basis of protein interaction with guanylate cyclase- activating protein, is also a member of this subfamily (Surguchov et al., 1999). In relation to nomenclature, it is important to recog- nize that the proposed , , and  designations are Received June 25, 1999; revised manuscript received August 2, 1999; accepted August 2, 1999. Address correspondence and reprint requests to Dr. R. E. Burke at Department of Neurology, Columbia University College of Physicians and Surgeons, Black Building, Room 308, 650 West 168th Street, New York, NY 10032, U.S.A. The synuclein sequence in this study has been deposited with Gen- Bank under accession number AF007758. Abbreviations used: aRNA, antisense RNA; GAPDH, glyceralde- hyde 3-phosphate dehydrogenase; HO2, hemoxygenase-2; IST, in situ transcription; NAC, non-A component of amyloid; NACP, 140-amino acid precursor of non-A component of amyloid; PBS, phosphate- buffered saline; PND, postnatal day; QA, quinolinic acid; SDS, sodium dodecyl sulfate; SN, substantia nigra; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; SSC, saline–sodium citrate; TH, tyrosine hydroxylase; VMAT, vesicular monoamine trans- porter; VTA, ventral tegmental area. 2586 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry