Synphilin in normal human brains and in synucleinopathies: studies with new antibodies

Mutations in the gene encoding alpha-synuclein (alpha-syn) have recently been linked to rare hereditary forms of Parkinson's disease. A yeast two-hybrid screen with alpha-synuclein (alpha-syn) identified synphilin as an alpha-syn-interacting protein, potentially implicating synphilin in the pathogenesis of synucleinopathies. Co-transfection of synphilin and the central (NAC) region of alpha-syn in HEH293 cells resulted in synuclein inclusions. Furthermore, synphilin immunoreactivity has been observed in Lewy bodies (LBs) and glial cytoplasmic inclusions of synucleinopathies. To further characterize synphilin, we utilized two new anti-synphilin antibodies for biochemical and immunohistochemical studies in normal and disease brain tissues. In normal brain tissue, synphilin localized predominantly to large neurons, such as substantia nigra neurons, hippocampal pyramidal and cerebellar Purkinje cells. However, in a few pathological cases synphilin immunoreactivity was present in glial cells and a small percentage of cortical and nigral LBs. In brain extracts, synphilin was observed primarily as a 90-kDa band but protein bands of 50 and 65 kDa were also present in both soluble (high salt) and lipid (Triton X-100) fractions. Additionally, less abundant higher molecular mass species, including a 120-kDa band of similar size to that of synphilin expressed in transiently transfected cells were recovered in 8 M urea-solubilized pellets after sequential extraction of brain tissue with buffers of increasing strengths. The presence of the synphilin of higher molecular mass was detected regardless of alpha-syn pathology and may represent an immature form of synphilin. Thus, although synphilin may be an alpha-syn-interacting protein present in some alpha-syn lesions, it still remains to be determined whether synphilin plays a critical role in mechanisms of brain degeneration in human synucleinopathies.