Raman spectroscopy in the study of amyloid formation and phase separation.

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, ß-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.

Regenerative Peripheral Nerve Interface Surgery for the Management of Chronic Posttraumatic Neuropathic Pain.

Chronic pain resulting from peripheral nerve injury remains a common issue in the United States and affects 7 to 10% of the population. Regenerative Peripheral Nerve Interface (RPNI) surgery is an innovative surgical procedure designed to treat posttraumatic neuropathic pain, particularly when a symptomatic neuroma is present on clinical exam. RPNI surgery involves implantation of a transected peripheral nerve into an autologous free muscle graft to provide denervated targets to regenerating axons. RPNI surgery has been found in animal and human studies to be highly effective in addressing postamputation pain. While most studies have reported its uses in the amputation patient population for the treatment of neuroma and phantom limb pain, RPNI surgery has recently been used to address refractory headache, postmastectomy pain, and painful donor sites from the harvest of neurotized flaps. This review summarizes the current understanding of RPNI surgery for the treatment of chronic neuropathic pain.

Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import.

Intracellular aggregation of transactive response DNA binding protein of 43 kDa (TDP-43) is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis. While primarily a nuclear protein, TDP-43 translocates to the cytosol during cellular stress. Consequences of cytosolic accumulation of TDP-43 is difficult to evaluate in the absence of exogenous toxins. Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (ortho-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells. Translocation of this cytosolic construct is photo-triggerable in a dose-dependent manner with 355 nm light. Interestingly, both fluid- and solid-like puncta were found based on fluorescence recovery after photobleaching experiments, similar to what is expected of stress granules and intracellular aggregates, respectively. This optogenetic method is advantageous as it is minimally perturbative and broadly applicable to other studies of protein translocation between cellular compartments.

Agreement between Patient-reported Pain Medication Use and Electronic Medical Record Data in Surgical Amputation Patients.

Background: Opioid misuse after surgery remains a public health crisis in the United States. Recent efforts have focused on tracking pain medication use in surgical populations. However, accurate interpretations of medication use remain quite challenging given inconsistent usage of different datasets. The purpose of this study was to investigate the agreement between electronic medical records (EMR) versus patient self-reported use of pain medications in a surgical amputation population. Methods: Patients undergoing major lower extremity amputation or amputation-related procedures were included in this study. Both self-reported and EMR data for pain medication intake were obtained for each patient at three time points (preoperatively, 4 months postoperatively, and 12 months postoperatively). Percentage agreement and the kappa statistic were calculated for both usage (yes/no) and dose categories. Results: Forty-five patients were included in this study, resulting in 108 pairs of self-reported and EMR datasets. Substantial levels of agreement (>70% agreement, kappa >0.61) for opioid use was seen at preoperative and 12 months postoperative. However, agreement dropped at 4 months postoperatively. Anticonvulsant medication showed high levels, whereas acetaminophen showed lower levels of agreements at all time points. Conclusions: Either self-reported or EMR data may be used in research and clinical settings for preoperative or 12-month postoperative patients with little concern for discrepancies. However, at time points immediately following the expected end of acute surgical pain, self-reported data may be needed for more accurate medication reporting. With these findings in mind, usage of datasets should be driven by study objectives and the dataset's strength (eg, accuracy, ease, lack of bias).

Water bend-libration as a cellular Raman imaging probe of hydration.

Water is a ubiquitous and vital component of living systems. Hydration, which is the interaction between water and intracellular biomolecules, plays an important role in cellular processes. However, it is technically challenging to study water structure within cells directly. Here, we demonstrate the utility and power of the water bend-libration combination band as a unique Raman spectral imaging probe of cellular hydration. Hydration maps reveal distinct water environments within subcellular compartments (e.g., nucleolus and lipid droplet) due to the spectral sensitivity of this coupled vibrational band. Spectroscopic studies using the water bend-libration are broadly applicable, offering the potential to capture the chemical complexity of hydration in numerous systems.

Genetically Encoded Aryl Alkyne for Raman Spectral Imaging of Intracellular α-Synuclein Fibrils.

α-Synuclein (α-syn) is an intrinsically disordered protein involved in a group of diseases collectively termed synucleinopathies, characterized by the aggregation of α-syn to form insoluble, ß-sheet-rich amyloid fibrils. Amyloid fibrils are thought to contribute to disease progression through cell-to-cell transmission, templating and propagating intracellular amyloid formation. Raman spectral imaging offers a direct characterization of protein secondary structure via the amide-I backbone vibration; however, specific detection of α-syn conformational changes against the background of other cellular components presents a challenge. Here, we demonstrate the ability to unambiguously identify cellularly internalized α-syn fibrils by coupling Raman spectral imaging with the use of a genetically encoded aryl alkyne, 4-ethynyl-l-phenylalanine (FCC), through amber codon suppression. The alkyne stretch (CC) of FCC provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of FCC-α-syn fibrils formed in vitro was visualized in cultured human SH-SY5Y neuroblastoma cells by Raman spectral imaging. Fibrils appear as discrete cytosolic clusters of varying sizes, found often at the cellular periphery. Raman spectra of internalized fibrils exhibit frequency shifts and spectral narrowing relative to in vitro fibrils, highlighting the environmental sensitivity of the alkyne vibration. Interestingly, spectral analysis reveals variations in lipid and protein recruitment to these aggregates, and in some cases, secondary structural changes in the fibrils are observed. This work sets the groundwork for future Raman spectroscopic investigations using a similar approach of an evolved aminoacyl-tRNA synthetase/tRNA pair to incorporate FCC into endogenous amyloidogenic proteins to monitor their aggregation in cells.

Shadow education, pandemic style: Social class, race, and supplemental education during Covid-19.

Research on shadow education-i.e., one-on-one or group learning intended to supplement children's experiences in school-has documented persistent social class and racial/ethnic inequalities. Yet, as with many things during the Covid-19 pandemic, the nature of shadow education changed dramatically. Much supplemental education shifted online, potentially increasing accessibility; many universities became testoptional, potentially reducing the demand for the shadow education industry; and a new form of shadow education-learning pods-emerged to take pandemic schooling from a more individual to a more collective experience. In this article, we use data from a sample of U.S. parents of K-12 students stratified by race/ethnicity (N = 1911) to assess social class and racial/ethnic inequalities in shadow education in 2020-21, the first full academic year of the Covid-19 pandemic. We are also the first scholars, to our knowledge, to assess high-quality data on the use of learning pods. We find that during the pandemic, African American and South Asian students were more likely than White student to use test preparation services and online supplemental education, and that African Americans, East Asians and Latinx were more likely to utilize private tutoring. We find few disparities by family income, however, thus supporting the idea that some forms of shadow education have become more accessible than they once were. Regarding learning pods, we find that pods were most common among African American families and families with parents who were less educated and worked fulltime. Thus, most learning pods were not a means of "opportunity hoarding," as some scholars originally feared, but instead provided sorely needed childcare and support during a time of social turbulence.

Monitoring Kinetics of pH-Dependent Aggregation and Disaggregation of the Pmel17 Repeat Domain.

The premelanosomal protein (Pmel17) is a human functional amyloid that promotes pigmentation by serving as a scaffold for melanin polymerization. This occurs within the melanosome, where Pmel17 is first proteolyzed into smaller domain(s) that are responsible for fibril formation. Our work has shown that the Pmel17 repeat domain (RPT, residues 315-444) forms amyloid fibrils in vitro under acidic conditions similar to those found in melanosomes. Mechanistically, this is driven by the protonation of acidic residues, resulting in charge neutralization and subsequent aggregation. Interestingly, the deprotonation of acidic residues leads to rapid disaggregation, highlighting a reversible mechanism of fibril formation and dissolution thus far only observed for functional amyloid proteins. In this chapter, we describe how to monitor pH-dependent RPT aggregation and disaggregation using extrinsic thioflavin-T and intrinsic tryptophan fluorescence, respectively. These methods can also be adapted more broadly to investigate the reversibility of other amyloid systems, both functional and pathogenic.

Sensory nerve regeneration and reinnervation in muscle following peripheral nerve injury.

Sensory afferent fibers are an important component of motor nerves and compose the majority of axons in many nerves traditionally thought of as "pure" motor nerves. These sensory afferent fibers innervate special sensory end organs in muscle, including muscle spindles that respond to changes in muscle length and Golgi tendons that detect muscle tension. Both play a major role in proprioception, sensorimotor extremity control feedback, and force regulation. After peripheral nerve injury, there is histological and electrophysiological evidence that sensory afferents can reinnervate muscle, including muscle that was not the nerve's original target. Reinnervation can occur after different nerve injury and muscle models, including muscle graft, crush, and transection injuries, and occurs in a nonspecific manner, allowing for cross-innervation to occur. Evidence of cross-innervation includes the following: muscle spindle and Golgi tendon afferent-receptor mismatch, vagal sensory fiber reinnervation of muscle, and cutaneous afferent reinnervation of muscle spindle or Golgi tendons. There are several notable clinical applications of sensory reinnervation and cross-reinnervation of muscle, including restoration of optimal motor control after peripheral nerve repair, flap sensation, sensory protection of denervated muscle, neuroma treatment and prevention, and facilitation of prosthetic sensorimotor control. This review focuses on sensory nerve regeneration and reinnervation in muscle, and the clinical applications of this phenomena. Understanding the physiology and limitations of sensory nerve regeneration and reinnervation in muscle may ultimately facilitate improvement of its clinical applications.

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy.

Liquid-liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis-related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of ß-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.

The N terminus of α-synuclein dictates fibril formation.

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson's disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41-140). This behavior can be explained by the unique fibril structure that is adopted by 41-140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent ß-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended ß-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Purification and characterization of an amyloidogenic repeat domain from the functional amyloid Pmel17.

The pre-melanosomal protein (Pmel17) is a human functional amyloid that supports melanin biosynthesis within melanocytes. This occurs in the melanosome, a membrane-bound organelle with an acidic intraluminal pH. The repeat region of Pmel17 (RPT, residues 315-444) has been previously shown to form amyloid aggregates under acidic melanosomal conditions, but not under neutral cytosolic conditions, when expressed and purified using a C-terminal hexa-histidine tag (RPT-His). Given the importance of protonation states in RPT-His aggregation, we questioned whether the histidine tag influenced the pH-dependent behavior. In this report, we generated a tagless RPT by inserting a tobacco etch virus (TEV) protease recognition sequence (ENLYGQ(G/S)) immediately upstream of a native glycine residue at position 312 in Pmel17. After purification of the fusion construct using a histidine tag, cleavage with TEV protease generated a fully native RPT (nRPT) spanning resides 312-444. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH ≤ 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/ß-sheet at pH 4 to canonical ß-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.

Coupling chemical biology and vibrational spectroscopy for studies of amyloids in vitro and in cells.

Amyloid diseases are characterized by the aggregation of various proteins to form insoluble ß-sheet-rich fibrils leading to cell death. Vibrational spectroscopies have emerged as attractive methods to study this process because of the rich structural information that can be extracted without large, perturbative probes. Importantly, specific vibrations such as the amide-I band directly report on secondary structure changes, which are key features of amyloid formation. Beyond intrinsic vibrations, the incorporation of unnatural vibrational probes can improve sensitivity for secondary structure determination (e.g. isotopic labeling), can provide residue-specific information of the surrounding polarity (e.g. unnatural amino acid), and are translatable into cellular studies. Here, we review the latest studies that have leveraged tools from chemical biology for the incorporation of novel vibrational probes into amyloidogenic proteins for both mechanistic and cellular studies.

Linking Parkinson's Disease and Melanoma: Interplay Between α-Synuclein and Pmel17 Amyloid Formation.

Parkinson's disease (PD) is a neurodegenerative disorder associated with the death of dopaminergic neurons within the substantia nigra of the brain. Melanoma is a cancer of melanocytes, pigmented cells that give rise to skin tone, hair, and eye color. Although these two diseases fundamentally differ, with PD leading to cell degeneration and melanoma leading to cell proliferation, epidemiological evidence has revealed a reciprocal relationship where patients with PD are more susceptible to melanoma and patients with melanoma are more susceptible to PD. The hallmark pathology observed in PD brains is intracellular inclusions, of which the primary component is proteinaceous α-synuclein (α-syn) amyloid fibrils. α-Syn also has been detected in cultured melanoma cells and tissues derived from patients with melanoma, where an inverse correlation exists between α-syn expression and pigmentation. Although this has led to the prevailing hypothesis that α-syn inhibits enzymes involved in melanin biosynthesis, we recently reported an alternative hypothesis in which α-syn interacts with and modulates the aggregation of Pmel17, a functional amyloid that serves as a scaffold for melanin biosynthesis. In this perspective, we review the literature describing the epidemiological and molecular connections between PD and melanoma, presenting both the prevailing hypothesis and our amyloid-centric hypothesis. We offer our views of the essential questions that remain unanswered to motivate future investigations. Understanding the behavior of α-syn in melanoma could not only provide novel approaches for treating melanoma but also could reveal insights into the role of α-syn in PD. © 2021 International Parkinson and Movement Disorder Society.

Tryptophan Probes of TDP-43 C-Terminal Domain Amyloid Formation.

Aggregated TAR DNA-binding protein 43 (TDP-43) forms the cytoplasmic hallmarks associated with patients suffering from amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin. Under normal conditions, TDP-43 is a 414-amino acid protein; however, aggregates are enriched with N-terminal truncations which contain residues 267-414, known as the C-terminal domain of TDP-43 (TDP-43CTD). To gain residue-specific information on the aggregation process of TDP-43CTD, we created three single-Trp containing mutants (W385F/W412F, W334F/W412F, and W334F/W385F) by substituting two of the three native Trp residues with Phe, yielding fluorescent probes at W334, W385, and W412, respectively. Aggregation kinetics, secondary structure, and fibril morphology were compared to the wild-type protein using thioflavin-T fluorescence, Raman spectroscopy, and transmission electron microscopy, respectively. While only W334 is determined to be in the proteinase-K resistant core, all three sites are sensitive reporters of aggregation, revealing site-specific differences. Interestingly, W334 exhibited unusual multistep Trp kinetics, pinpointing a distinctive role for W334 and its nearby region during aggregation. This behavior is retained even upon seeding, suggesting the observed spectral change is related to fibril growth. This work provides new insights into the aggregation mechanism of TDP-43CTD and exemplifies the advantages of Trp as a site-specific environmentally sensitive fluorescent probe.

Membrane Interactions of α-Synuclein Probed by Neutrons and Photons.

α-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in ß-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Raman spectral imaging of 13C2H15N-labeled α-synuclein amyloid fibrils in cells.

Parkinson's disease is characterized by the intracellular accumulation of α-synuclein (α-syn) amyloid fibrils, which are insoluble, ß-sheet-rich protein aggregates. Raman spectroscopy is a powerful technique that reports on intrinsic molecular vibrations such as the coupled vibrational modes of the polypeptide backbone, yielding secondary structural information. However, in order to apply this method in cells, spectroscopically unique frequencies are necessary to resolve proteins of interest from the cellular proteome. Here, we report the use of 13C2H15N-labeled α-syn to study the localization of preformed fibrils fed to cells. Isotopic labeling shifts the amide-I (13CO) band away from endogenous 12CO vibrations, permitting secondary structural analysis of internalized α-syn fibrils. Similarly, 13C2H stretches move to lower energies in the "cellular quiet" region, where there is negligible biological spectral interference. This combination of well-resolved, distinct vibrations allows Raman spectral imaging of α-syn fibrils across a cell, which provides conformational information with spatial context.

Lipid-Chaperone Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins.

An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-ß, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-ß, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-ß, and α-synuclein) and nonamyloidogenic (rat IAPP, ß-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning.

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish's color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.