9 research outputs found
Liquid-Liquid Phase Separation in Physiology and Pathophysiology of the Nervous System
Molecules within cells are segregated into functional domains to form various organelles. While some of those organelles are delimited by lipid membranes demarcating their constituents, others lack a membrane enclosure. Recently, liquid-liquid phase separation (LLPS) revolutionized our view of how segregation of macromolecules can produce membraneless organelles. While the concept of LLPS has been well studied in the areas of soft matter physics and polymer chemistry, its significance has only recently been recognized in the field of biology. It occurs typically between macromolecules that have multivalent interactions. Interestingly, these features are present in many molecules that exert key functions within neurons. In this review, we cover recent topics of LLPS in different contexts of neuronal physiology and pathology
A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease
Background
Alzheimer’s disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer’s disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.
Results
We have investigated the formation of dityrosine cross-links in Aβ42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aβ oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aβ in cell cultures treated with oligomeric Aβ42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients.
Conclusions
Aβ dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer’s disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aβ and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD
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Aß’s effect on long term memory: a top-down approach in Lymnaea stagnalis
Amyloid ß(Aß)-induced synaptic and neuronal degeneration has been linked to the memory loss observed in Alzheimer’s disease (AD). Although Aß-induced impairment of synaptic and nonsynaptic plasticity is known to occur before any cell death, the links between these neurophysiological changes and the loss of specific types of behavioural memory are not fully understood. This thesis introduces a behaviourally and physiologically tractable animal model to the Aß field for the first time, allowing for an in-depth approach to investigating Aß-induced memory loss to be explored. In Aß 1-42- and Aß 25-35-treated Lymnaea stagnalis, retrieval of consolidated memory is disrupted after single-trial conditioning and single-injection of synthetic peptide. All succeeding work builds upon these findings using a top-down approach to investigate how Aß disrupts retrieval of consolidated memory. Neuronal and synaptic health were monitored over a 24 hour in vivo incubation period and other memory stages were considered to determine time points of memory vulnerability. In brains that displayed healthy neurons and degenerating synapses, only animals that were exposed to Aß during the 24-48 hour post-training time points exhibited any behavioural deficits. All other behavioural responses remained normal. Focus then shifted to investigate the peptide, as opposed to behaviour, involved in the above mentioned experiments. After systemic injection, Aß was found to penetrate the ganglia, enter cells, and localise to specific organelles by 24 hours exposure. Aß morphology and structure were also monitored over the 24 hour incubation period, using transmission electron microscopy (TEM), formic acid extraction, silver stain, and western blot. A large distinction between the two peptides, Aß 1-42 and Aß 25-35, became apparent at this point and even when peptides were prepared using the same procedure, their effects on behaviour became drastically different. However, it is interesting to note that although the two peptides used are very different, under different preparation procedures they will both produce predominantly tetramer species after 24 hour in vivo incubation. Finally, investigations into disruptions of molecular signalling cascades were considered in order to correlate these disruptions to the observed Aß-induced behavioural deficits. Specifically, molecular, pharmacological, and biochemical techniques were used to measure protein alterations and post-translational modifications, and to inhibit key protein components, involved in cAMP response element binding protein (CREB)-signalling pathways in Lymnaea brain after 24 hour in vivo incubation of Aß. Phosphorylated CREB was found to be decreased in both Aß-treated groups; this decrease pattern was also found in active protein kinase A (PKA) experiments. These experiments correlate memory deficits to Aß-induced disruptions in PKA and CREB activity; however, PKA inhibition experiments indicate that this molecular cascade disruption is not sufficient to cause the observed behavioural deficits. Taken together, this work correlates Aß-induced changes from a wide range of components involved in learning and memory, with Aß-disrupted memory recall. Importantly as well, this work develops Lymnaea stagnalis as a novel model for Aß research and continues to distinguish the two commonly used peptides, Aß 1-42 and Aß 25-35. By linking the effects of Aß on defined neuronal circuits to behavioural deficits in a novel model, the Aß field has been further developed in an important and unique way
Lyme Neuroborreliosis: Mechanisms of B. burgdorferi Infection of the Nervous System
Lyme borreliosis is the most prevalent tick-borne disease in the United States, infecting ~476,000 people annually. Borrelia spp. spirochetal bacteria are the causative agents of Lyme disease in humans and are transmitted by Ixodes spp ticks. Clinical manifestations vary depending on which Borrelia genospecies infects the patient and may be a consequence of distinct organotropism between species. In the US, B. burgdorferi sensu stricto is the most commonly reported genospecies and infection can manifest as mild to severe symptoms. Different genotypes of B. burgdorferi sensu stricto may be responsible for causing varying degrees of clinical manifestations. While the majority of Lyme borreliae-infected patients fully recover with antibiotic treatment, approximately 15% of infected individuals experience long-term neurological and psychological symptoms that are unresponsive to antibiotics. Currently, long-term antibiotic treatment remains the only FDA-approved option for those suffering from these chronic effects. Here, we discuss the current knowledge pertaining to B. burgdorferi sensu stricto infection in the central nervous system (CNS), termed Lyme neuroborreliosis (LNB), within North America and specifically the United States. We explore the molecular mechanisms of spirochete entry into the brain and the role B. burgdorferi sensu stricto genotypes play in CNS infectivity. Understanding infectivity can provide therapeutic targets for LNB treatment and offer public health understanding of the B. burgdorferi sensu stricto genotypes that cause long-lasting symptoms
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Structure dependent effects of Amyloid-ß on long-term memory in Lymnaea stagnalis
Amyloid-ß (Aß) peptides are implicated in the causation of memory loss, neuronal impairment, and neurodegeneration in Alzheimer's disease. Our recent work revealed that Aß 1–42 and Aß 25–35 inhibit long-term memory (LTM) recall in Lymnaea stagnalis (pond snail) in the absence of cell death. Here, we report the characterization of the active species prepared under different conditions, describe which Aß species is present in brain tissue during the behavioral recall time point and relate the sequence and structure of the oligomeric species to the resulting neuronal properties and effect on LTM. Our results suggest that oligomers are the key toxic Aß1–42 structures, which likely affect LTM through synaptic plasticity pathways, and that Aß 1–42 and Aß 25–35 cannot be used as interchangeable peptides
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Effects of Aß exposure on longterm associative memory and its neuronal mechanisms in a defined neuronal network
Amyloid beta (Aß ) induced neuronal death has been linked to memory loss, perhaps the most devastating symptom of Alzheimer’s disease (AD). Although Aß -induced impairment of synaptic or intrinsic plasticity is known to occur before any cell death, the links between these neurophysiological changes and the loss of specific types of behavioral memory are not fully understood. Here we used a behaviorally and physiologically tractable animal model to investigate Aß -induced memory loss and electrophysiological changes in the absence of neuronal death in a defined network underlying associative memory. We found similar behavioral but different neurophysiological effects for Aß 25-35 and Aß 1-42 in the feeding circuitry of the snail Lymnaea stagnalis. Importantly, we also established that both the behavioral and neuronal effects were dependent upon the animals having been classically conditioned prior to treatment, since Aß application before training caused neither memory impairment nor underlying neuronal changes over a comparable period of time following treatment
Impact of Detergents on Membrane Protein Complex Isolation
Detergents
play an essential role during the isolation of membrane
protein complexes. Inappropriate use of detergents may affect the
native fold of the membrane proteins, their binding to antibodies,
or their interaction with partner proteins. Here we used cadherin-11
(Cad11) as an example to examine the impact of detergents on membrane
protein complex isolation. We found that mAb 1A5 could immunoprecipitate
Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but
not by octylglucoside, suggesting that octylglucoside interferes with
Cad11–mAb 1A5 interaction. Furthermore, we compared the effects
of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy
BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation.
We found that all detergents except Brij-35 could solubilize Cad11
from the membrane. Upon immunoprecipitation, we found that β-catenin,
a known cadherin-interacting protein, was present in Cad11 immune
complex among the detergents tested except Brij-35. However, the association
of p120 catenin with Cad11 varied depending on the detergents used.
Using isobaric tag for relative and absolute quantitation (iTRAQ)
to determine the relative levels of proteins in Cad11 immune complexes,
we found that DDM and Triton X-100 were more efficient than cholate
in solubilization and immunoprecipitation of Cad11 and resulted in
the identification of both canonical and new candidate Cad11-interacting
proteins