Jennifer C. Lee

The C-terminal domain of TAR DNA-binding protein 43 (TDP-43_CTD) drives both liquid-liquid phase separation (LLPS) and amyloid formation. Understanding how TDP-43_CTD droplets convert into amyloid aggregates, a process implicated in amyotrophic lateral sclerosis and frontotemporal dementia, requires methodology capable of site-specific structural characterization with spatial resolution. Here, we used confocal Raman spectroscopy in conjunction with an alkyne-modified amino acid (4-ethynyl-l-phenylalanine, F_CC) to probe aging in individual TDP-43_CTD droplets at seven aromatic sites. While nascent droplets are composed of disordered proteins, β-sheet conformers develop in aged droplets and amyloid aggregates. All three states are spectrally distinct via the alkyne stretching band, with sensitivity that varies depending on the aromatic site probed. C-terminal sites (Y374F_CC, W385F_CC, and F397F_CC) are highly sensitive amyloid probes, revealing multiple polymorphs at the single-residue level that are not resolvable by global secondary structure or morphological characterization alone. Strikingly, while W334F_CC abolishes β-sheet formation in droplets, de novo aggregation still occurs, demonstrating that droplet aging is not required for amyloid formation. Given its broad applicability to other proteins and compatibility with cellular imaging, this work establishes a generalizable approach for investigating conformational changes underlying LLPS and amyloid formation in cellulo.

Cytosolic inclusions of aggregated TAR DNA-binding protein 43 (TDP-43) are hallmarks of neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. A prevailing hypothesis suggests that TDP-43 condensates undergo a liquid-to-solid transition during maturation, involving the formation of β-sheet–rich, amyloid-like aggregates. To test this hypothesis, we sought to study the temporal and spatial evolution of protein secondary structure within individual condensates by Raman spectroscopy. We measured in vitro β-sheet development of the C-terminal domain of TDP-43 (TDP-43_CTD) at the single-condensate level under physiological solution conditions. All condensates showed apparent single-exponential kinetics (k = 1.6 × 10^–5 s^–1) for the disordered-to-β-sheet transformation, as indicated by increased amide-I intensity and a shift of the amide-III band to lower energy. Interestingly, the water bend-libration band exhibited a slower rate (k = 4.0 × 10^–6 s^–1), suggesting that changes in the water environment lag behind protein conformational rearrangement. Further, Raman maps revealed that protein density is highest near the condensate center, whereas β-sheet content is mostly uniform in the interior of the condensate. The unexpected difference between the spatial distributions of β-sheet content and protein density challenges the typical concentration-dependent model of protein aggregation. Importantly, rare events were captured where condensates exhibited spatially asymmetric β-sheet development, revealing localized structural heterogeneity not detectable by ensemble measurements. Collectively, these results provide insight into the temporal and spatial dynamics of protein structure within TDP-43_CTD condensates and demonstrate the utility of Raman spectral imaging for tracking condensate maturation.

Gangliosides are sialic acid-containing glycosphingolipids integral to the cell membrane, and they are particularly abundant in the nervous system. Aberrant ganglioside metabolism contributes to pathological conditions, including neurodegenerative diseases, lysosomal storage disorders, and cancer. A critical precursor for sialic acid biosynthesis is N-acetyl-D-mannosamine (ManNAc), which can be epimerized from the corresponding UDP-GlcNAc or exogenously supplied through ManNAc derivatives. Currently, tools to visualize and detect gangliosides are very limited and non-specific. Here, we describe a dual fluorescent and Raman-active ManNAlk derivative, phenanthrene-9-Pr₄ManNAlk (MM-JH-2), capable of one-step selective labeling of gangliosides in cells. This modified ManNAlk derivative produces a biologically unique Raman spectral signature, which arises from the carbon-carbon triple bond augmented by conjugation to a fluorescent phenanthrene moiety. Raman maps generated using the alkyne stretching frequency indicate a distribution of MM-JH-2 overlapping with intracellular membrane lipids. Using confocal fluorescence imaging, the cellular transport of labeled gangliosides was tracked. Notably, MM-JH-2 can differentiate between cells that differ in ganglioside biosynthetic flux, such as malignant and nonmalignant cells, as well as distinguish between B cells and T cells. Thus, MM-JH-2 is a next-generation metabolic chemical reporter (MCR) that is Raman-active, fluorescent, and can be broadly applied to cellular studies investigating ganglioside biosynthetic flux.

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are disorders characterized by the presence of cytosolic α‐synuclein (SNCA) amyloids. The gene SNCA is alternatively spliced, generating three variants of SNCA, missing exon 3 (SNCAΔ3) or 5 (SNCAΔ5), or both exons (SNCAΔ3Δ5). Despite purported upregulation in disease states, their pathological relevance is ill‐defined. Here, we investigated the amyloid formation of alternatively spliced variants under physiological conditions. Aggregation kinetics, secondary structure, and fibril morphology of N‐terminally acetylated SNCAΔ3, SNCAΔ5, and SNCAΔ3Δ5 were assessed by thioflavin‐T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. Compared to SNCA, both SNCAΔ5 and SNCAΔ3Δ5 aggregate faster and adopt a more twisted fibril morphology, whereas SNCAΔ3 is more sensitive to solution conditions, exhibiting similar or modestly faster aggregation kinetics compared to SNCA. Cross‐seeding experiments using spliced‐variant fibrils and soluble SNCA showed that despite fibril morphological differences, SNCAΔ5 were competent seeds for SNCA, which is explained by their similar protease‐K resistant regions. Contrastingly, neither SNCAΔ3 nor SNCAΔ3Δ5 fibrils cross‐seed SNCA, indicating exon 3 (residues 41–54) is essential in modulating fibril structure. Notably, SNCA aggregation is stimulated by sub‐stoichiometric amounts of soluble SNCAΔ5 and SNCAΔ3Δ5, but not SNCAΔ3, suggesting that exon 5 (residues 103–130) is more important in modulating aggregation kinetics. Taken together, we propose that alternatively spliced variants are pathogenic by exacerbating aggregation of the main SNCA isoform.

Gangliosides are sialic acid-containing glycosphingolipids integral to the cell membrane and are particularly abundant in the nervous system. Aberrant ganglioside metabolism contributes to pathological conditions including neurodegenerative diseases, lysosomal storage disorders, and cancer. Currently, tools to visualize and detect gangliosides are very limited and non-specific. Here, we describe a dual fluorescent and Raman-active ManNAlk derivative, phenanthrene-9-Pr4ManNAlk (MM-JH-2), capable of one-step selective labeling of gangliosides in cells. This modified ManNAlk derivative produces a biologically unique Raman spectral signature, which arise from the carbon-carbon triple bond augmented by conjugation to a fluorescent phenanthrene moiety. By using this dual probe, unambiguous identification is achieved within cells. Raman maps generated using the alkyne stretching frequency indicate a distribution of MM-JH-2 overlapping with intracellular membrane lipids. Using confocal fluorescence imaging, the cellular transport of labeled gangliosides was tracked from synthesis to recycling in the acidic compartments. Notably, MM-JH-2 can differentiate between cells that differ in ganglioside biosynthetic flux, such as malignant and nonmalignant cells, as well as distinguish between B cells and T cells. Thus, MM-JH-2 is a novel next-generation metabolic chemical reporter (MCR) that is Raman-active, fluorescent, and can be broadly applied to cellular studies investigating ganglioside biosynthetic flux.

N- and C-terminal α-synuclein (α-syn) truncations are prevalent in Parkinson’s disease. Effects of the N- and C-terminal residues on α-syn aggregation and fibril propagation are distinct, where the N-terminus dictates fibril structure. Here, the majority of α-syn truncations are assigned by intact mass spectrometry to lysosomal activity. To delineate essential charged residues in fibril formation, we selected an N-terminal truncation (66–140) that is generated solely from soluble α-syn by asparagine endopeptidase. Ala-substitutions at K80 and E83 impact aggregation kinetics, revealing their vital roles in defining fibril polymorphism. K80, E83, and K97 are identified to be critical for fibril elongation. Based on solid-state NMR, mutational and Raman studies, and molecular dynamics simulations, a E83–K97 salt bridge is proposed. Finally, participation of C-terminal Lys residues in the full-length α-syn fibril assembly process is also shown, highlighting that individual residues can be targeted for therapeutic intervention.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, β-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.