Data Sheet 1_Fourier transform infrared spectroscopy detects distinct TAR DNA-binding protein 43 signatures in frontotemporal lobar degeneration.docx

BackgroundFrontotemporal lobar degeneration (FTLD) is a leading cause of cognitive impairment in young adults. A major pathological subtype of FTLD is characterized by positive expression of TAR DNA Binding Protein 43 (TDP-43), referred to as FTLD[TDP]. However, techniques that can be utilized to interrogate and distinguish between various subtypes of FTLD are limited. Herein, we evaluated the potential of Fourier transform infrared (FTIR) spectroscopy to inform on the biomolecular changes in FTLD and to discriminate this disease from other non-FTLD cases.
MethodsHistopathologically confirmed cases from FTLD[TDP] and Alzheimer’s disease (AD) autopsy cases were evaluated using FTIR spectroscopy. Formalin-fixed paraffin-embedded (FFPE) brain tissue sections from the superior and medial temporal lobes were obtained from a single control case, an AD case, an FTLD[TDP] case, and a comorbid FTLD[TDP] case that presented with AD pathology (tau and β-amyloid; FTLD[TDP] + AD). All samples were immunostained for pathological forms of tau, β-amyloid, and TDP-43. Myelin was assessed by proteolipid protein staining. Consecutive tissue sections were scanned by FTIR spectroscopy. Spectral maps were manually segmented, matching ten grey matter (GM) and ten white matter (WM) subregions per case for analysis. Peak-area ratios from lipid and amide functional groups as detected by FTIR spectroscopy were quantified and compared.
ResultsRelative to the control tissue, both FTLD cases and AD showed increased ratios of amide I/II, α-helix/unordered proteins, α-helix/phosphorylated proteins, and olefinic/lipid content in GM and WM. The α-helix/unordered ratio was significantly different between FTLD cases and AD, while α-helix/unordered and α-helix/phosphorylated ratios differed significantly between FTLD[TDP] and FTLD[TDP] + AD. Across all cases and brain subregions, FTIR spectroscopy-derived amide I/II, olefinic/lipid, and carboxyl/lipid ratios significantly correlated positively with TDP-43 and tau immunoreactivity (p-value < 0.05).
ConclusionFourier transform infrared spectroscopy of FFPE brain tissue sections from FTLD[TDP] and AD captures disease-specific changes in the composition of proteins, lipids, and secondary structures. These findings suggest that FTIR spectroscopy can serve as a rapid and cost-effective tool for mapping and quantitating biomolecular alterations in FTLD.

背景：额颞叶变性（Frontotemporal lobar degeneration, FTLD）是青年人群认知障碍的首要病因。FTLD的主要病理亚型以TAR DNA结合蛋白43（TAR DNA Binding Protein 43, TDP-43）阳性表达为特征，被称为FTLD[TDP]。然而，可用于检测和区分FTLD不同亚型的技术仍十分有限。本研究评估了傅里叶变换红外（Fourier transform infrared, FTIR）光谱技术在反映FTLD生物分子变化、以及区分该病与其他非FTLD病例方面的潜力。
方法：本研究采用FTIR光谱技术，对经组织病理学确认的FTLD[TDP]及阿尔茨海默病（Alzheimer’s disease, AD）尸检病例进行分析。研究获取了1例对照、1例AD病例、1例FTLD[TDP]病例以及1例合并AD病理（tau蛋白与β-淀粉样蛋白）的FTLD[TDP]共病病例（FTLD[TDP] + AD）的福尔马林固定石蜡包埋（Formalin-fixed paraffin-embedded, FFPE）大脑颞上叶和颞中叶组织切片。对所有样本的病理性tau蛋白、β-淀粉样蛋白及TDP-43进行免疫染色；采用蛋白脂质蛋白染色评估髓鞘状态。使用FTIR光谱对连续组织切片进行扫描，手动分割光谱图谱，为每个病例选取10个灰质（grey matter, GM）亚区与10个白质（white matter, WM）亚区进行分析。对FTIR光谱检测到的脂质与酰胺官能团的峰面积比进行定量并比较。
结果：与对照组织相比，FTLD病例与AD病例的灰质及白质中，酰胺I/II比值、α螺旋/无规卷曲蛋白比值、α螺旋/磷酸化蛋白比值以及烯烃/脂质含量比值均升高。FTLD病例与AD病例的α螺旋/无规卷曲蛋白比值存在显著差异；而FTLD[TDP]与FTLD[TDP] + AD病例的α螺旋/无规卷曲蛋白比值及α螺旋/磷酸化蛋白比值均存在显著差异。在所有病例及脑亚区中，FTIR光谱得到的酰胺I/II、烯烃/脂质及羧基/脂质比值均与TDP-43和tau蛋白免疫反应性呈显著正相关（p < 0.05）。
结论：对FTLD[TDP]及AD患者的FFPE脑组织切片进行傅里叶变换红外光谱检测，可捕捉到蛋白质、脂质及二级结构组成的疾病特异性变化。上述结果表明，FTIR光谱技术可作为一种快速且经济高效的工具，用于定位并定量FTLD中的生物分子改变。