Neuronal Transporter Research Articles

Interaction of Tau with Kinesin-1: Effect of Kinesin-1 Heavy Chain Elimination on Autophagy-Mediated Mutant Tau Degradation.

Natively unfolded tau has a low propensity to form aggregates, but in tauopathies, such as Alzheimer's disease (AD), tau aggregates into paired helical filaments (PHFs) and neurofibrillary tangles (NFTs). Multiple intracellular transport pathways utilize kinesin-1, a plus-end-directed microtubule-based motor. Kinesin-1 is crucial in various neurodegenerative diseases as it transports multiple cargoes along the microtubules (MT). Kinesin-1 proteins cannot progress along MTs due to an accumulation of tau on their surfaces. Although kinesin-1-mediated neuronal transport dysfunction is well-documented in other neurodegenerative diseases, its role in AD has received less attention. Very recently, we have shown that knocking down and knocking out of kinesin-1 heavy chain (KIF5B KO) expression significantly reduced the level and stability of tau in cells and tau transgenic mice, respectively. Here, we report that tau interacts with the motor domain of KIF5B in vivo and in vitro, possibly through its microtubule-binding repeat domain. This interaction leads to the inhibition of the ATPase activity of the motor domain. In addition, the KIF5B KO results in autophagy initiation, which subsequently assists in tau degradation. The mechanisms behind KIF5B KO-mediated tau degradation seem to involve its interaction with tau, promoting the trafficking of tau through retrograde transport into autophagosomes for subsequent lysosomal degradation of tau. Our results suggest how KIF5B removal facilitates the movement of autophagosomes toward lysosomes for efficient tau degradation. This mechanism can be enabled through the downregulation of kinesin-1 or the disruption of the association between kinesin-1 and tau, particularly in cases when neurons perceive disturbances in intercellular axonal transport.

A vesicular Warburg effect: Aerobic glycolysis occurs on axonal vesicles for local NAD+ recycling and transport.

In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.

Alzheimer’s disease biomarker‐related phospho‐tau 181 signals localize to demyelinated axons of parvalbumin‐positive GABAergic interneurons in App knock‐in mouse

AbstractBackgroundTau proteins phosphorylated at Thr181 (p‐tau 181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer’s disease (AD). An increase in p‐tau 181 level well correlates with Aβ pathology and precedes neurofibrillary tangle formation in the early stage of AD, though the relationship between p‐tau 181 and Aβ‐mediated pathology is less well understood. We have recently reported that p‐tau 181 signals are detected in the axonal structures and represent axonal abnormality around Aβ plaques in the cortex of App knock‐in mouse model of Aβ amyloidosis (AppNLGF mice). However, from which neuronal subtype(s) these p‐tau 181‐positive axons are originated remains elusive. In this study, we aimed to determine which neuronal subtype(s) express p‐tau 181 in the axons.MethodTo investigate the localization of p‐tau 181 signals in the brains of aged (24‐month‐old) AppNLGF and wild‐type mice, we prepared frozen brain sections and co‐immunostained with antibodies against p‐tau 181 and myeline basic protein (MBP), the glutamatergic neuronal vesicle marker vesicular glutamate transporter 1 (VGLUT1), the GABAergic neuronal vesicle marker vesicular GABA transporter (VGAT), the inhibitory neuron marker parvalbumin, the cholinergic neuronal vesicle marker vesicular acetylcholine transporter (VAChT), or the noradrenergic neuronal vesicle marker norepinephrine transporter (NET).ResultHistochemical analyses revealed that p‐tau 181 signals were not overlapped with axons of unmyelinated cholinergic nor noradrenergic neurons, but well colocalized with myelinated axons of parvalbumin‐positive GABAergic interneurons, but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, while that of glutamatergic, GABAergic or p‐tau 181‐positive axons was less affected. Instead, myelin sheaths surrounding p‐tau 181‐positive axons were significantly reduced in AppNLGF mice.ConclusionThis study suggests that p‐tau 181 signals may represent axonal abnormality of parvalbumin‐positive GABAergic interneurons with disrupted myelin sheaths in brains of mouse model of Aβ pathology.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology. ”

The relationship between [18F]FDG‐PET signal and key‐players of brain glucose metabolism

AbstractBackgroundNeuronal activity is supported by activity‐dependent lactate production from astrocytes, as postulated by the astrocyte–neuron lactate shuttle (ANLS) hypothesis. Indeed, astrocytic glutamate uptake is a main trigger for brain glucose consumption. [18F]fluorodeoxyglucose (FDG)‐PET showing a hypometabolic signature has been used as an index of neurodegeneration in Alzheimer’s disease (AD). In the early stages of the disease, soluble amyloid‐beta (Aβ) has been associated with neuronal hyperactivity due to decreased astrocytic glutamate uptake. It is hypothesized that this phenomenon occurs especially in regions with high ongoing baseline activity. Over time, neurodegeneration and hypometabolism in such regions may contribute to the progress of the AD continuum. Thus, we investigated whether the [18F]FDG‐PET signal in typical hypometabolic regions in AD is associated with the expression of ANLS transporters and enzymes in the healthy brain.MethodGene expression of GLUT1, GLUT3, MCT1, MCT2, MCT4, LDHA, and LDHB (ANLS genes) from postmortem brain tissue of healthy individuals were obtained from the Allen Human Brain Atlas. [18F]FDG‐PET data from cognitively unimpaired individuals were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Pearson correlations were performed between the mean gene expression and the mean [18F]FDG‐PET SUVr across typically hypometabolic brain regions in AD (precuneus, angular and temporal gyri, and parietal and cingulate regions) and non‐vulnerable regions (uncorrected p‐value < 0.05).ResultsThe expression of ANLS genes had a stronger positive correlation with [18F]FDG‐PET SUVr across the AD hypometabolic regions than the non‐vulnerable regions (p < 0.05). Of note, the astrocytic glucose transporter GLUT1 presented a stronger correlation with [18F]FDG‐PET than the neuronal transporter GLUT3 in AD‐vulnerable regions. Figure 1 presents all correlations.ConclusionOur results show that the physiological expression of transporters and enzymes responsible for the brain glucose metabolism correlate more with the [18F]FDG‐PET signal in typical brain regions of the AD hypometabolic signature. This suggests these regions present a higher baseline activity and may be more susceptible to Aβ‐induced hyperactivation in early AD, which could lead to a bioenergetic collapse in later disease stages, as shown in the [18F]FDG‐PET of AD individuals.

Altered anterograde axonal transport of mitochondria in cultured striatal neurons of a knock-in mouse model of Huntington's disease

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons.

Primary cilia-mediated regulation of microglial secretion in Alzheimer's disease.

Alzheimer's disease (AD) is a brain disorder manifested by a gradual decline in cognitive function due to the accumulation of extracellular amyloid plaques, disruptions in neuronal substance transport, and the degeneration of neurons. In affected neurons, incomplete clearance of toxic proteins by neighboring microglia leads to irreversible brain inflammation, for which cellular signaling is poorly understood. Through single-cell transcriptomic analysis, we discovered distinct regional differences in the ability of microglia to clear damaged neurites. Specifically, microglia in the septal region of wild type mice exhibited a transcriptomic signature resembling disease-associated microglia (DAM). These lateral septum (LS)-enriched microglia were associated with dense axonal bundles originating from the hippocampus. Further transcriptomic and proteomic approaches revealed that primary cilia, small hair-like structures found on cells, played a role in the regulation of microglial secretory function. Notably, primary cilia were transiently observed in microglia, and their presence was significantly reduced in microglia from AD mice. We observed significant changes in the secretion and proteomic profiles of the secretome after inhibiting the primary cilia gene intraflagellar transport particle 88 (Ift88) in microglia. Intriguingly, inhibiting primary cilia in the septal microglia of AD mice resulted in the expansion of extracellular amyloid plaques and damage to adjacent neurites. These results indicate that DAM-like microglia are present in the LS, a critical target region for hippocampal nerve bundles, and that the primary ciliary signaling system regulates microglial secretion, affecting extracellular proteostasis. Age-related primary ciliopathy probably contributes to the selective sensitivity of microglia, thereby exacerbating AD. Targeting the primary ciliary signaling system could therefore be a viable strategy for modulating neuroimmune responses in AD treatments.

Mitochondrial transport in neurons and evidence for its involvement in acute neurological disorders.

Ensuring mitochondrial quality is essential for maintaining neuronal homeostasis, and mitochondrial transport plays a vital role in mitochondrial quality control. In this review, we first provide an overview of neuronal mitochondrial transport, followed by a detailed description of the various motors and adaptors associated with the anterograde and retrograde transport of mitochondria. Subsequently, we review the modest evidence involving mitochondrial transport mechanisms that has surfaced in acute neurological disorders, including traumatic brain injury, spinal cord injury, spontaneous intracerebral hemorrhage, and ischemic stroke. An in-depth study of this area will help deepen our understanding of the mechanisms underlying the development of various acute neurological disorders and ultimately improve therapeutic options.

Cerebrospinal fluid solute transport associated with sensorimotor brain activity in rodents

Cerebrospinal fluid (CSF) is crucial for maintaining neuronal homeostasis, providing nutrition, and removing metabolic waste from the brain. However, the relationship between neuronal activity and CSF solute transport remains poorly understood. To investigate the effect of regional neuronal activity on CSF solute transport, Sprague–Dawley rats (all male, n = 30) under anesthesia received an intracisternal injection of a fluorescent tracer (Texas Red ovalbumin) and were subjected to unilateral electrical stimulation of a forelimb. Two groups (n = 10 each) underwent two different types of stimulation protocols for 90 min, one including intermittent 7.5-s resting periods and the other without rest. The control group was not stimulated. Compared to the control, the stimulation without resting periods led to increased transport across most of the cortical areas, including the ventricles. The group that received intermittent stimulation showed an elevated level of solute uptake in limited areas, i.e., near/within the ventricles and on the ventral brain surface. Interhemispheric differences in CSF solute transport were also found in the cortical regions that overlap with the forelimb sensorimotor area. These findings suggest that neuronal activity may trigger local and brain-wide increases in CSF solute transport, contributing to waste clearance.

FRI014 Exploring The Epigenetic Fingerprint Of Sedentary Behavior

Abstract Disclosure: A. Brener: None. D. Lorber: None. A. Reuveny: None. Y. Lebenthal: None. T. Volk: None. Eating habits and physical activity play a central role among the environmental factors that modulate biological aging. Sedentary lifestyle, characterized by increased waking time spent in minimal energy expenditure activity, is closely associated with increased cardiometabolic risk, a principal hallmark of aging. The biological mechanisms linking a sedentary lifestyle with metabolic derangements are incompletely understood. Exercise was reported as a stimulator of epigenetic modifications of the DNA in human muscle cell nuclei (myonuclei). Exploring the specific impact of sedentary behavior on epigenetics, especially among younger individuals, requires the exclusion of the effects of inflammatory and nutritional contributors to medical conditions. We developed an experimental animal model that imitates sedentary behavior in which a chromatin epigenetic landscape could be explored. We used third-instar Drosophila larvae carrying a temperature-sensitive mutation in the shibire gene coding for GTPase involved in neuronal synaptic vesicle transport. Shibire homozygous mutant larvae are paralyzed at restrictive temperature (>∼29°C) due to inhibition of neuromuscular junction vesicular transport, while muscle contraction is abolished without affecting other systems. Shibire and control (y,w) larvae were kept at 18°C or 30°C without food for six hours. They were then dissected, fixed and double-labeled with antibodies specific for epigenetic marks of active (H3K9ac) and repressive (H3K27me3) histone modifications. An anti-lamin fluorescent antibody served for nuclear membrane demarcation. Immunofluorescence images of epigenetic marks were acquired on a confocal microscope Zeiss LSM 800, after which a quantitative analysis of epigenetics marks was performed. At the restrictive temperature (30°C), the crawling motion of shibire mutant larvae ceased in contrast to control (shibire at 18°C). The crawling motion of the y,w larvae was similar at 18°C and 30°C. The mean repressive mark H3K27me3 decreased (p<0.001), while the mean active H3KPac increased (p<0.001) in the myonuclei of sedentary shibire larvae (30°C). The mean myonuclear active/repressed ratio of epigenetic marks (H3K9ac/H3K27me) was similar in both active shibire (18°C) and y,w at 18°C, while it increased significantly in sedentary shibire (30°C) (p<0.001). In contrast, this ratio decreased in the control larvae (y,w at 30°C) (p<0.001). The findings of this model, which was aimed at reflecting sedentary behavior in youth, revealed that muscle inactivity changed the epigenetic fingerprint in myonuclei, with a balance shift towards decreased chromatin methylation relative to acetylation. Our finding suggests a decline in the epigenetic control of gene transcription, reminiscent of decreased epigenetic repression described with aging. Presentation: Friday, June 16, 2023

Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.

Phase separation and pathologic transitions of RNP condensates in neurons: implications for amyotrophic lateral sclerosis, frontotemporal dementia and other neurodegenerative disorders.

Liquid-liquid phase separation results in the formation of dynamic biomolecular condensates, also known as membrane-less organelles, that allow for the assembly of functional compartments and higher order structures within cells. Multivalent, reversible interactions between RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1, and/or RNA (e.g., RBP-RBP, RBP-RNA, RNA-RNA), result in the formation of ribonucleoprotein (RNP) condensates, which are critical for RNA processing, mRNA transport, stability, stress granule assembly, and translation. Stress granules, neuronal transport granules, and processing bodies are examples of cytoplasmic RNP condensates, while the nucleolus and Cajal bodies are representative nuclear RNP condensates. In neurons, RNP condensates promote long-range mRNA transport and local translation in the dendrites and axon, and are essential for spatiotemporal regulation of gene expression, axonal integrity and synaptic function. Mutations of RBPs and/or pathologic mislocalization and aggregation of RBPs are hallmarks of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. ALS/FTD-linked mutations of RBPs alter the strength and reversibility of multivalent interactions with other RBPs and RNAs, resulting in aberrant phase transitions. These aberrant RNP condensates have detrimental functional consequences on mRNA stability, localization, and translation, and ultimately lead to compromised axonal integrity and synaptic function in disease. Pathogenic protein aggregation is dependent on various factors, and aberrant dynamically arrested RNP condensates may serve as an initial nucleation step for pathologic aggregate formation. Recent studies have focused on identifying mechanisms by which neurons resolve phase transitioned condensates to prevent the formation of pathogenic inclusions/aggregates. The present review focuses on the phase separation of neurodegenerative disease-linked RBPs, physiological functions of RNP condensates, and the pathologic role of aberrant phase transitions in neurodegenerative disease, particularly ALS/FTD. We also examine cellular mechanisms that contribute to the resolution of aberrant condensates in neurons, and potential therapeutic approaches to resolve aberrantly phase transitioned condensates at a molecular level.

Thinking about the action potential: the nerve signal as a window to the physical principles guiding neuronal excitability.

Ever since the work of Edgar Adrian, the neuronal action potential has been considered as an electric signal, modeled and interpreted using concepts and theories lent from electronic engineering. Accordingly, the electric action potential, as the prime manifestation of neuronal excitability, serving processing and reliable "long distance" communication of the information contained in the signal, was defined as a non-linear, self-propagating, regenerative, wave of electrical activity that travels along the surface of nerve cells. Thus, in the ground-breaking theory and mathematical model of Hodgkin and Huxley (HH), linking Nernst's treatment of the electrochemistry of semi-permeable membranes to the physical laws of electricity and Kelvin's cable theory, the electrical characteristics of the action potential are presented as the result of the depolarization-induced, voltage- and time-dependent opening and closure of ion channels in the membrane allowing the passive flow of charge, particularly in the form of Na+ and K+ -ions, into and out of the neuronal cytoplasm along the respective electrochemical ion gradient. In the model, which treats the membrane as a capacitor and ion channels as resistors, these changes in ionic conductance across the membrane cause a sudden and transient alteration of the transmembrane potential, i.e., the action potential, which is then carried forward and spreads over long(er) distances by means of both active and passive conduction dependent on local current flow by diffusion of Na+ ion in the neuronal cytoplasm. However, although highly successful in predicting and explaining many of the electric characteristics of the action potential, the HH model, nevertheless cannot accommodate the various non-electrical physical manifestations (mechanical, thermal and optical changes) that accompany action potential propagation, and for which there is ample experimental evidence. As such, the electrical conception of neuronal excitability appears to be incomplete and alternatives, aiming to improve, extend or even replace it, have been sought for. Commonly misunderstood as to their basic premises and the physical principles they are built on, and mistakenly perceived as a threat to the generally acknowledged explanatory power of the "classical" HH framework, these attempts to present a more complete picture of neuronal physiology, have met with fierce opposition from mainstream neuroscience and, as a consequence, currently remain underdeveloped and insufficiently tested. Here we present our perspective that this may be an unfortunate state of affairs as these different biophysics-informed approaches to incorporate also non-electrical signs of the action potential into the modeling and explanation of the nerve signal, in our view, are well suited to foster a new, more complete and better integrated understanding of the (multi)physical nature of neuronal excitability and signal transport and, hence, of neuronal function. In doing so, we will emphasize attempts to derive the different physical manifestations of the action potential from one common, macroscopic thermodynamics-based, framework treating the multiphysics of the nerve signal as the inevitable result of the collective material, i.e., physico-chemical, properties of the lipid bilayer neuronal membrane (in particular, the axolemma) and/or the so-called ectoplasm or membrane skeleton consisting of cytoskeletal protein polymers, in particular, actin fibrils. Potential consequences for our view of action potential physiology and role in neuronal function are identified and discussed.

Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control

Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). ED neural plasticity is driven by histone acetylation (HA) mediated epigenetic mechanisms that regulate dynamic activity-dependent gene transcription profiles in response to neuronal stimulation. Yet, how histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. We previously demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. Here, we focus on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. We used immunohistochemistry (IHC) to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, we found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in our well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that we identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. Our results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

Preliminary Evaluation of 18F-Labeled Benzylguanidine Analogs as NET Tracers for Myocardial Infarction Diagnosis.

Heart failure (HF) remains a major cause of late morbidity and mortality after myocardial infarction (MI). To date, no clinically established 18F-labeled sympathetic nerve PET tracers for monitoring myocardial infarction are available. Therefore, in this study, we synthesized a series of 18F-labeled benzyl guanidine analogs and evaluated their efficacy as cardiac neuronal norepinephrine transporter (NET) tracers for myocardial imaging. We also investigated the preliminary diagnostic capabilities of these tracers in myocardial infarction animal models, as well as the structure-activity relationship of these tracers. Three benzyl guanidine-NET tracers, including [18F]1, [18F]2, and [18F]3, were synthesized and evaluated in vivo as PET tracers in a myocardial infarction mouse model. [18F]LMI1195 was used as a positive control for the tracers. H&E staining of the isolated myocardial infarction heart tissue sections was performed to verify the efficacy of the selected PET tracer. Our data show that [18F]3 had a moderate decay corrected labeling yield (~10%) and high radiochemical purity (>95%) compared to other tracers. The uptake of [18F]3 in normal mouse hearts was 1.7±0.1%ID/cc at 1 h post-injection (p. i.), while it was 2.4±0.1, 2.6±0.9, and 2.1±0.4%ID/cc in the MI mouse hearts at 1, 2, and 3 days after surgery, respectively. Compared with [18F]LMI1195, [18F]3 had a better myocardial imaging effect in terms of the contrast between normal and MI hearts. The area of myocardial infarction shown by PET imaging corresponded well with the infarcted tissue demonstrated by H&E staining. With an obvious cardiac uptake contrast between normal mice and the myocardial infarction mouse model, [18F]3 appears to be a potential tool in the diagnosis of myocardial infarction. Therefore, it is necessary to conduct further structural modification studies on the chemical structure of [18F]3 to improve its in vivo stability and diagnostic detection ability to achieve reliable and practical imaging effects.

Targeting a xenobiotic transporter to ameliorate vincristine-induced sensory neuropathy.

Vincristine is a widely used chemotherapeutic drug for the treatment of multiple malignant diseases that causes a dose-limiting peripheral neurotoxicity. There is no clinically effective preventative treatment for vincristine-induced sensory peripheral neurotoxicity (VIPN), and mechanistic details of this side effect remain poorly understood. We hypothesized that VIPN is dependent on transporter-mediated vincristine accumulation in dorsal root ganglion neurons. Using a xenobiotic transporter screen, we identified OATP1B3 as a neuronal transporter regulating the uptake of vincristine. In addition, genetic or pharmacological inhibition of the murine orthologue transporter OATP1B2 protected mice from various hallmarks of VIPN - including mechanical allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes and neuronal morphology - without negatively affecting plasma levels or antitumor effects of vincristine. Finally, we identified α-tocopherol from an untargeted metabolomics analysis as a circulating endogenous biomarker of neuronal OATP1B2 function, and it could serve as a companion diagnostic to guide dose selection of OATP1B-type transport modulators given in combination with vincristine to prevent VIPN. Collectively, our findings shed light on the fundamental basis of VIPN and provide a rationale for the clinical development of transporter inhibitors to prevent this debilitating side effect.

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.