| Parkinson disease (PD) is the second common neurodegenerative movement disorder, affecting more than 1 in 100 individuals over the age of 65, with clinical symptoms of dyskinesia, bradykinesia, tremor and rigor. alpha-synuclein(α-SYN) was initially found in 1988, which was isolated from Torpedo California as a protein localized in the nuclear envelope of neurons and pre-synaptical nerve terminals. Its relevance to Parkinson's disease (PD) is based on mutations found in familial cases of the disease and its presence in filaments of Lewy body (LB). The deposition ofα-SYN in neurons or glial cells is a hallmark lesion in a subset of neurodegenerative disorders, including PD, dementia with Lewy bodies (DLB) and multiple system atrophy, collectively referred to as synucleinopathies.As to the developmental expression ofα-SYN in the mouse, it is reported thatα-SYN was firstly found in the marginal zone of the neocortex in E12, and later the expression became strong in both marginal zone and growth plate areas of the cortex.α-SYN expression has been detected in olfactory mucosa with no difference between controls and patients with neurodegenerative diseases, thus it is uncertain if the accumulation ofα-SYN contributed to the common occurrence of olfactory dysfunction in AD, PD, MSA and other neurodegenerative disorders. Up to now, no published data revealed ifα-SYN involved in the development of the olfactory mucosa. Although the peripheral nervous system has not been carefully analyzed,α-SYN was found in the neurons of the dorsal root ganglia in adult mice. To explore its developmental function more clearly, it is necessary to reveal the detailed distribution ofα-SYN during the development of mouse.Engrailed(EN) is a group of the homeodomain-containing transcription factors, including Engrailed-1(EN1) and Engrailed-2(EN2). EN has numerous roles during the development of the vertebrates, it was first characterized as a Drosophila mutation that results in a failure of the border that normally divides the posterior and anterior wing compartments, later it was thought to be important in the neural development especially in the development of the mid/hindbrain junction. During these years, more and more function of EN has been revealed, it is reported that EN may control the fate of midbrain dopaminergic neurons, and there may be a link between EN and Parkinson's disease. As to the expression of EN during the development of mouse, most of the previous studies were focus on the early development of CNS, and few studies were found about the late stages and out of the CNS during the development. However, antisense oligonucleotide targeting of EN1 in early somite mouse embryos resulted in abnormalities of the brain, face, and heart, as well as the shortening of the embryonic axis. The EN1 homozygous mutant mice died shortly after birth and exhibited multiple developmental defects in the mid/hindbrain. The replacement of the mouse EN1 coding sequence with EN2 by gene targeting leads to viable fertile animals and to a complete rescue of the brain defects, but can not able to rescue limb abnormalities. These suggest that EN1 may have acquired some more important biochemical specificities during evolution which can not be replaced by EN2. To explore the function of EN1 during the development more clearly, it is essential to detect the developmental expression of EN1 in detail.As it is reported that EN1 was involved in the development and the survival of the midbrain dopaminergic neurons; and the aggregation ofα-SYN in the dopaminergic neurons of PD patients may be the cause of the cell death. To explore the relationship between EN1 andα-SYN with dopaminergic neurons, we carried out intra-striatal 6-OHDA injections in the mouse brain, which lead to gradually dopaminergic cell loss and imitate the progressive degenerative disease of PD. At last we evaluate the function of EN1 andα-SYN acting on the PD according to their changes during the proceeding of the cell death of dopaminergic neurons.In this study, the developmental expression of EN1 andα-SYN in the mouse has been revealed in detail by IHC and Western Blot analysis, and we have preliminarily explored their association with PD.1. Expression ofα-SYN during the development of mouseWe have described the expression pattern ofα-SYN during the development of mice from E9.5 to adulthood by IHC. As a result,α-SYN was detected as early as E9.5. During the embryonic stages,α-SYN was dynamically expressed in several regions of the brain. In the neocortex, expression was detected in the marginal zone (MZ) in the early stages and was later condensed in the MZ and in the subplate (SP); in the cerebellum, expression was initially detected in the deep cerebellar nuclei (DCN) and was later condensed in the Purkinje cells. These spatio-temporal expression patterns matched the neuronal migratory pathways and the formation of the synapse connections. Additionally,α-SYN was detected in the sensory systems, including the nasal mucosa, the optic cup, the sensory ganglia, and their dominating nerve fibers. Furthermore, the nuclear location ofα-SYN protein was found in developing neurons in the early stages, and later it was mostly found in the non-nuclear compartments. This finding was further confirmed by Western blot analysis. These results suggest thatα-SYN may be involved not only in the migration of neurons and in the synaptogenesis of the central nervous system (CNS) but also in the establishment of the sensory systems.α-SYN was expressed strongly during the development of the dopaminergic neurons, located in the whole cell bodies in the early stages and later in the non-nuclear compartments. This suggestsα-SYN may play different roles during the development of dopaminergic neurons.2. Expression of EN1 during the development of mouseBy IHC detection, EN1 was found in the central nervous system (CNS) and was limited to mid/hindbrain junction at E9.5 and to parts of those structures that later developed from it; the midbrain, cerebellum, pons, periaqueductal gray, and colliculi. During late stages of development, expression of EN1 was strongest in the midbrain dopaminergic neurons, and this expression pattern persisted to adulthood. Outside the CNS, EN1 is dynamically expressed in several neural crest-associated structures during the mouse embryonic development, including the cranial mesenchyme, the mandibular arches, the vagus nerve, the dorsal root ganglia, the sympathetic ganglia, the somites, the heart and the cloaca. Additionally, by IHC and Western Blot analysis, we found that in the CNS, most of the EN1 was located in the nuclei, while outside the CNS, EN1 was mainly expressed in the cytoplasm. These findings suggest that EN1 may be important for the development and survival of the midbrain dopaminergic neurons, and it may be involved in the development of neural crest cells (NCCs).3. Experimental study ofα-SYN and EN1 on the PD modelMice received a 6-OHDA injection on the right side and a sham injection on the contralateral side, which lead to gradually dopaminergic cell loss in the right side and imitate the progressive degenerative disease of PD. At last we evaluate the function of EN1 andα-SYN acting on the PD according to their changes during the proceeding of the cell death of dopaminergic neurons. As a result, in the SN and VTA, following unilateral injection of 6-OHDA in the striatum, a long-lasting loss of TH+ cells was observed, with the most important decline occurring within the first 7 days, at the same time the astrocytes proliferated significantly. After 1 day and 3 days of 6-OHDA injection,α-SYN markedly aggregated in the cell bodies of the dopaminergic neurons. After 7 days, when the cell death became slight, the cells which expressedα-SYN strongly decrease. Western Blot analysis also found that the expression ofα-SYN increased 1 day and 3 days after 6-OHDA injection, and decreased to normal level after 7 days. These suggest that the aggregation ofα-SYN may be the cause of cell death of dopaminergic neurons.After 1 day of 6-OHDA injection, when the decrease of dopaminergic neurons has not yet been significant, EN1 positive cells decreased significantly, andα-SYN became aggregated in the cell bodies of the dopaminergic neurons. Previous studies showed that the expression ofα-SYN may be controlled by the transcription factor EN1, and EN1 can act as transcriptional activation or inhibition factors according to the different binding targets. Thus we propose the hypothesis that EN1 may be a transcriptional activation factor toα-SYN during the early developmental stages, and may be a transcriptional inhibition factor toα-SYN during late developmental stages and adulthood.In conclusion, we have elucidated in detail the expression pattern of EN1 andα-SYN during the development of mouse. By establishing the PD model which led to the progressive cell death of dopaminergic neurons, we have explored the roles of EN1 andα-SYN during the process of the death of dopaminergic neurons. This study might provide some primary data for further understanding the precaution and therapy target of PD.
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