Neurofibrillary tangle-bearing neurons have reduced risk of cell death in mice with Alzheimer's pathology.

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer's disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer's donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer's.

Instability of excitatory synapses in experimental autoimmune encephalomyelitis and the outcome for excitatory circuit inputs to individual cortical neurons.

Synapses are lost on a massive scale in the brain and spinal cord of people living with multiple sclerosis (PwMS), and this synaptic loss extends far beyond demyelinating lesions. Post-mortem studies show the long-term consequences of multiple sclerosis (MS) on synapses but do not inform on the early impacts of neuroinflammation on synapses that subsequently lead to synapse loss. How excitatory circuit inputs are altered across the dendritic tree of individual neurons under neuroinflammatory stress is not well understood. Here, we directly assessed the structural dynamics of labeled excitatory synapses in experimental autoimmune encephalomyelitis (EAE) as a model of immune-mediated cortical neuronal damage. We used in vivo two-photon imaging and a synthetic tissue-hydrogel super-resolution imaging technique to reveal the dynamics of excitatory synapses, map their location across the dendritic tree of individual neurons, and examine neurons at super-resolution for synaptic loss. We found that excitatory synapses are destabilized but not lost from dendritic spines in EAE, starting with the earliest imaging session before symptom onset. This led to changes in excitatory circuit inputs to individual cells. In EAE, stable synapses are replaced by synapses that appear or disappear across the imaging sessions or repeatedly change at the same location. These unstable excitatory inputs occur closer to one another in EAE than in healthy controls and are distributed across the dendritic tree. When imaged at super-resolution, we found that a small proportion of dendritic protrusions lost their presynapse and/or postsynapse. Our finding of diffuse destabilizing effects of neuroinflammation on excitatory synapses across cortical neurons may have significant functional consequences since normal dendritic spine dynamics and clustering are essential for learning and memory.

Instability of excitatory synapses in experimental autoimmune encephalomyelitis and the outcome for excitatory circuit inputs to individual cortical neurons.

Synapses are lost on a massive scale in the brain and spinal cord of people living with multiple sclerosis (PwMS), and this synaptic loss extends far beyond demyelinating lesions. Post-mortem studies show the long-term consequences of multiple sclerosis (MS) on synapses but do not inform on the early impacts of neuroinflammation on synapses that subsequently lead to synapse loss. How excitatory circuit inputs are altered across the dendritic tree of individual neurons under neuroinflammatory stress is not well understood. Here, we directly assessed the structural dynamics of labeled excitatory synapses in experimental autoimmune encephalomyelitis (EAE) as a model of immune-mediated cortical neuronal damage. We used in vivo two-photon imaging and a synthetic tissue-hydrogel super-resolution imaging technique to reveal the dynamics of excitatory synapses, map their location across the dendritic tree of individual neurons, and examine neurons at super-resolution for synaptic loss. We found that excitatory synapses are destabilized but not lost from dendritic spines in EAE, starting with the earliest imaging session before symptom onset. This led to dramatic changes in excitatory circuit inputs to individual cells. In EAE, stable synapses are replaced by synapses that appear or disappear across the imaging sessions or repeatedly change at the same location. These unstable excitatory inputs occur closer to one another in EAE than in healthy controls and are distributed across the dendritic tree. When imaged at super-resolution, we found that a small proportion of dendritic protrusions lost their presynapse and/or postsynapse. Our finding of diffuse destabilizing effects of neuroinflammation on excitatory synapses across cortical neurons may have significant functional consequences since normal dendritic spine dynamics and clustering are essential for learning and memory.

Biochemically functionalized probes for cell-type-specific targeting and recording in the brain.

Selective targeting and modulation of distinct cell types and neuron subtypes is central to understanding complex neural circuitry and could enable electronic treatments that target specific circuits while minimizing off-target effects. However, current brain-implantable electronics have not yet achieved cell-type specificity. We address this challenge by functionalizing flexible mesh electronic probes, which elicit minimal immune response, with antibodies or peptides to target specific cell markers. Histology studies reveal selective association of targeted neurons, astrocytes, and microglia with functionalized probe surfaces without accumulating off-target cells. In vivo chronic electrophysiology further yields recordings consistent with selective targeting of these cell types. Last, probes functionalized to target dopamine receptor 2 expressing neurons show the potential for neuron-subtype-specific targeting and electrophysiology.

Biochemically-functionalized probes for cell type-specific targeting and recording in the brain.

Selective targeting and modulation of distinct cell types and neuron subtypes is central to understanding complex neural circuitry, and could enable electronic treatments that target specific circuits while minimizing off-target effects. However, current brain-implantable electronics have not yet achieved cell-type specificity. We address this challenge by functionalizing flexible mesh electronic probes, which elicit minimal immune response, with antibodies or peptides to target specific cell markers. Histology studies reveal selective association of targeted neurons, astrocytes and microglia with functionalized probe surfaces without accumulating off-target cells. In vivo chronic electrophysiology further yields recordings consistent with selective targeting of these cell types. Last, probes functionalized to target dopamine 2 receptor expressing neurons show the potential for neuron subtype specific targeting and electrophysiology.

Laminin-coated electronic scaffolds with vascular topography for tracking and promoting the migration of brain cells after injury.

In the adult brain, neural stem cells are largely restricted into spatially discrete neurogenic niches, and hence areas of neuron loss during neurodegenerative disease or following a stroke or traumatic brain injury do not typically repopulate spontaneously. Moreover, understanding neural activity accompanying the neural repair process is hindered by a lack of minimally invasive devices for the chronic measurement of the electrophysiological dynamics in damaged brain tissue. Here we show that 32 individually addressable platinum microelectrodes integrated into laminin-coated branched polymer scaffolds stereotaxically injected to span a hydrogel-filled cortical lesion and deeper regions in the brains of mice promote neural regeneration while allowing for the tracking of migrating host brain cells into the lesion. Chronic measurements of single-unit activity and neural-circuit analyses revealed the establishment of spiking activity in new neurons in the lesion and their functional connections with neurons deeper in the brain. Electronic implants mimicking the topographical and surface properties of brain vasculature may aid the stimulation and tracking of neural-circuit restoration following injury.

Spatial characterization of tangle-bearing neurons and ghost tangles in the human inferior temporal gyrus with three-dimensional imaging.

Studies of post-mortem human tissue provide insight into pathological processes, but are inherently limited by practical considerations that limit the scale at which tissue can be examined, and the obvious issue that the tissue reflects only one time point in a continuous disease process. We approached this problem by adapting new tissue clearance techniques to an entire cortical area of human brain, which allows surveillance of hundreds of thousands of neurons throughout the depth of the entire cortical thickness. This approach allows detection of 'rare' events that may be difficult to detect in standard 5 micrometre-thick paraffin sections. For example, it is well established that neurofibrillary tangles begin within a neuron, and ultimately, in at least some instances, persist in the brain even after the neuron has died. These are referred to as 'ghost tangles', a term that appropriately implies their 'difficult to see' ephemeral qualities. We set out to find ghost tangles as one example of the power of the tissue clearance/image analysis techniques to detect rare events, and to learn what happens at the end-point of a tangle's life history. We were able to identify 8103 tau tangles, 132 465 neurons and 299 640 nuclei in tissue samples from three subjects with severe Alzheimer's disease (Braak V-VI) and 4 tau tangles, 200 447 neurons and 462 715 nuclei in tissue samples from three subjects with no significant tau pathology (Braak 0-I). Among these data, we located 57 ghost tangles, which makes them only 0.7% of the total tau tangles observed. We found that ghost tangles are more likely to be found in cortical layers 3 and 5 (49/57), with a select few scattered across other layers 1, 2, 4 and 6. This ability to find rare events, such as ghost tangles, in large enough quantities to statistically test their distribution exemplifies how tissue clearing can be used as a powerful tool for studying selective vulnerability or resilience to pathology across brain regions.

Tissue libraries enable rapid determination of conditions that preserve antibody labeling in cleared mouse and human tissue.

Difficulty achieving complete, specific, and homogenous staining is a major bottleneck preventing the widespread use of tissue clearing techniques to image large volumes of human tissue. In this manuscript, we describe a procedure to rapidly design immunostaining protocols for antibody labeling of cleared brain tissue. We prepared libraries of 0.5-1.0 mm thick tissue sections that are fixed, pre-treated, and cleared via similar, but different procedures to optimize staining conditions for a panel of antibodies. Results from a library of mouse tissue correlate well with results from a similarly prepared library of human brain tissue, suggesting mouse tissue is an adequate substitute for protocol optimization. These data show that procedural differences do not influence every antibody-antigen pair in the same way, and minor changes can have deleterious effects, therefore, optimization should be conducted for each target. The approach outlined here will help guide researchers to successfully label a variety of targets, thus removing a major hurdle to accessing the rich 3D information available in large, cleared human tissue volumes.

Bioinspired neuron-like electronics.

As an important application of functional biomaterials, neural probes have contributed substantially to studying the brain. Bioinspired and biomimetic strategies have begun to be applied to the development of neural probes, although these and previous generations of probes have had structural and mechanical dissimilarities from their neuron targets that lead to neuronal loss, neuroinflammatory responses and measurement instabilities. Here, we present a bioinspired design for neural probes-neuron-like electronics (NeuE)-where the key building blocks mimic the subcellular structural features and mechanical properties of neurons. Full three-dimensional mapping of implanted NeuE-brain interfaces highlights the structural indistinguishability and intimate interpenetration of NeuE and neurons. Time-dependent histology and electrophysiology studies further reveal a structurally and functionally stable interface with the neuronal and glial networks shortly following implantation, thus opening opportunities for next-generation brain-machine interfaces. Finally, the NeuE subcellular structural features are shown to facilitate migration of endogenous neural progenitor cells, thus holding promise as an electrically active platform for transplantation-free regenerative medicine.

Effective Distance for DNA-Mediated Charge Transport between Repair Proteins.

The stacked aromatic base pairs within the DNA double helix facilitate charge transport down its length in the absence of lesions, mismatches, and other stacking perturbations. DNA repair proteins containing [4Fe4S] clusters can take advantage of DNA charge transport (CT) chemistry to scan the genome for mistakes more efficiently. Here we examine the effective length over which charge can be transported along DNA between these repair proteins. We define the effective CT distance as the length of DNA within which two proteins are able to influence their ensemble affinity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with DNA duplexes of different lengths (1.5-9 kb), and atomic force microscopy was used to quantify the binding of proteins to these duplexes to determine how the relative protein affinity changes with increasing DNA length. A sharp change in binding slope is observed at 3509 base pairs, or about 1.2 µm, that supports the existence of two regimes for protein binding, one within the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA effectively decreases the ensemble binding affinity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A, which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4 °C or including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA floppiness, leads to extending the effective length for DNA charge transport to ∼5300 bp or 1.8 µm. These results thus support DNA charge transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins to search the genome quickly and efficiently using DNA charge transport chemistry.

Sensing DNA through DNA Charge Transport.

DNA charge transport chemistry involves the migration of charge over long molecular distances through the aromatic base pair stack within the DNA helix. This migration depends upon the intimate coupling of bases stacked one with another, and hence any perturbation in that stacking, through base modifications or protein binding, can be sensed electrically. In this review, we describe the many ways DNA charge transport chemistry has been utilized to sense changes in DNA, including the presence of lesions, mismatches, DNA-binding proteins, protein activity, and even reactions under weak magnetic fields. Charge transport chemistry is remarkable in its ability to sense the integrity of DNA.

A Compass at Weak Magnetic Fields Using Thymine Dimer Repair.

How birds sense the variations in Earth's magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by photolyase, mutants of photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth's magnetic field.

Tissue-like Neural Probes for Understanding and Modulating the Brain.

Electrophysiology tools have contributed substantially to understanding brain function, yet the capabilities of conventional electrophysiology probes have remained limited in key ways because of large structural and mechanical mismatches with respect to neural tissue. In this Perspective, we discuss how the general goal of probe design in biochemistry, that the probe or label have a minimal impact on the properties and function of the system being studied, can be realized by minimizing structural, mechanical, and topological differences between neural probes and brain tissue, thus leading to a new paradigm of tissue-like mesh electronics. The unique properties and capabilities of the tissue-like mesh electronics as well as future opportunities are summarized. First, we discuss the design of an ultraflexible and open mesh structure of electronics that is tissue-like and can be delivered in the brain via minimally invasive syringe injection like molecular and macromolecular pharmaceuticals. Second, we describe the unprecedented tissue healing without chronic immune response that leads to seamless three-dimensional integration with a natural distribution of neurons and other key cells through these tissue-like probes. These unique characteristics lead to unmatched stable long-term, multiplexed mapping and modulation of neural circuits at the single-neuron level on a year time scale. Last, we offer insights on several exciting future directions for the tissue-like electronics paradigm that capitalize on their unique properties to explore biochemical interactions and signaling in a "natural" brain environment.

The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins.

A central question important to understanding DNA repair is how certain proteins are able to search for, detect, and fix DNA damage on a biologically relevant time scale. A feature of many base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for lesions. In this paper, we establish the importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an O2-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying [4Fe4S]3+ clusters bind to DNA 550 times more tightly than those with [4Fe4S]2+ clusters. The measured increase in DNA-binding affinity matches the calculated affinity change associated with the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S] cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.

Helix-Dependent Spin Filtering through the DNA Duplex.

Recent work suggests that electrons can travel through DNA and other chiral molecules in a spin-selective manner, but little is known about the origin of this spin selectivity. Here we describe experiments on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. Our results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.

Accounting for unintended binding events in the analysis of quartz crystal microbalance kinetic data.

Previous methods for analyzing protein-ligand binding events using the quartz crystal microbalance with dissipation monitoring (QCM-D) fail to account for unintended binding that inevitably occurs during surface measurements and obscure kinetic information. In this article, we present a system of differential equations that accounts for both reversible and irreversible unintended interactions. This model is tested on three protein-ligand systems, each of which has different features, to establish the feasibility of using the QCM-D for protein binding analysis. Based on this analysis, we were able to obtain kinetic information for the intended interaction that is consistent with those obtained in literature via bulk-phase methods. In the appendix, we include a method for decoupling these from the intended binding events and extracting relevant affinity information.

DNA-mediated signaling by proteins with 4Fe-4S clusters is necessary for genomic integrity.

Iron-sulfur clusters have increasingly been found to be associated with enzymes involved in DNA processing. Here we describe a role for these redox clusters in DNA-mediated charge-transport signaling in E. coli between DNA repair proteins from distinct pathways. DNA-modified electrochemistry shows that the 4Fe-4S cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III (EndoIII), a base excision repair enzyme, cooperate at long-range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Silencing the gene encoding EndoIII in a strain of E. coli where repair by DinG is essential results in a significant growth defect that is rescued by complementation with EndoIII but not with an EndoIII mutant that is enzymatically active but unable to carry out DNA charge transport. This work thus elucidates a fundamental mechanism to coordinate the activities of DNA repair enzymes across the genome.

Elucidation of the molecular interaction between cisplatin and flavonol(s) and their effect on DNA binding.

Combination therapy of cisplatin with flavonols is a promising treatment for increasing the efficacy of cisplatin when combating cancer. However, little is known about the molecular interactions between cisplatin and flavonols. The data herein helps to elucidate this interaction. Spectrophotometric data in the UV-visible range indicates that hydroxyl groups on the B-ring of flavonols are essential for reactivity with cisplatin. The use of a quartz crystal microbalance with dissipation monitoring approach clearly supports the critical role played by B-ring hydroxyls in their interactions with a cisplatin-bound double-stranded DNA surface; an increase in the number of hydroxyl groups on the B-ring of flavonols parallels the increase in their reaction rates with cisplatin and correlates well with their reported effects on leukemia cell apoptosis efficacy. This study underscores the importance of B-ring hydroxyls in cisplatin's toxicity and may be used to better understand and improve combination therapies of flavonols with cisplatin.

Desolvation of BSA-ligand complexes measured using the quartz crystal microbalance and dual polarization interferometer.

By taking advantage of their unique difference in hydration sensitivity, we have shown that dual polarization interferometer (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) measurements can be used together to explore the degree of desolvation involved in the binding of small drug molecules to an immobilized bovine serum albumin film in real time. Results with DPI and QCM-D show significantly different mass values for three ligands of varying hydrophobicities that may be attributed to changes in the degree of hydration of the ligand-protein complexes in accordance with the physicochemical properties of the ligands. Furthermore, our data suggest that masses measured by QCM-D can be overwhelmed by changes in water content of ligand-protein, binary complexes, which has important consequences for future studies using mechanical resonators to study protein-binding events.

Multiphasic DNA adsorption to silica surfaces under varying buffer, pH, and ionic strength conditions.

Reversible interactions between DNA and silica are utilized in the solid phase extraction and purification of DNA from complex samples. Chaotropic salts commonly drive DNA binding to silica but inhibit DNA polymerase amplification. We studied DNA adsorption to silica using conditions with or without chaotropic salts through bulk depletion and quartz crystal microbalance (QCM) experiments. While more DNA adsorbed to silica using chaotropic salts, certain buffer conditions without chaotropic salts yielded a similar amount of eluted DNA. QCM results indicate that under stronger adsorbing conditions the adsorbed DNA layer is initially rigid but becomes viscoelastic within minutes. These results qualitatively agreed with a mathematical model for a multiphasic adsorption process. Buffer conditions that do not require chaotropic salts can simplify protocols for nucleic acid sample preparation. Understanding how DNA adsorbs to silica can help optimize nucleic acid sample preparation for clinical diagnostic and research applications.