Transcriptomic Profiling of Early Synucleinopathy in Rats Induced with Preformed Fibrils

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson’s disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.在包涵体已形成但神经退行性变尚远未发生的突触核蛋白病（synucleinopathy）早期阶段开展研究，对于阐明疾病进展机制以及开发疾病修饰治疗手段均至关重要。大鼠α-突触核蛋白（alpha-synuclein, α-syn）预成型纤维（preformed fibril, PFF）模型可诱导同步化的突触核蛋白病，重现帕金森病（Parkinson’s disease, PD）的病理特征，可用于研究突触核蛋白病的进展过程。在该模型中，黑质致密部（substantia nigra pars compacta, SNpc）内携带磷酸化α-突触核蛋白（pSyn）包涵体的神经元与反应性小胶质细胞（主要组织相容性复合体-II免疫反应阳性）的丰度峰值，较可观测到的神经退行性变提前数月出现。然而，目前仍不明确究竟是哪些特定基因驱动了这些表型变化。为识别与早期突触核蛋白病相关的转录组变化，我们采用激光捕获显微切割（laser capture microdissection）结合RNA测序（RNA sequencing, RNASeq）的实验策略。对黑质致密部的精准取材，使得我们能够评估黑质多巴胺能神经元及邻近胶质细胞的差异转录表达情况。早期突触核蛋白病中上调的转录本主要与免疫应答相关，而下调的转录本则主要与神经传递及多巴胺能通路相关。我们在另一组雌雄大鼠队列中验证了29个与神经传递/囊泡释放及多巴胺能通路相关的转录本子集，以确认实验结果的可重复性。在该转录本子集中，我们利用荧光原位杂交（fluorescent in situ hybridization, FISH）技术将Syt1与Slc6a3转录本的表达下调定位至携带pSyn包涵体的神经元中。本研究鉴定出的早期突触核蛋白病相关转录变化，为阐明驱动神经退行性变的分子机制提供了新的见解。