Axonal Membrane Research Articles

Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

Apoptotic cells expose Phosphatidylserine (PS), that serves as an “eat me” signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.

White matter microstructural correlates of relapse in alcohol dependence

Identification of neural correlates of relapse to alcohol after treatment is clinically important as it may inform better substance abuse treatment. Few studies have specifically analyzed the white matter microstructure in treatment seekers as it might relate to relapse risk versus long-term abstinence. Using 4 Tesla diffusion tensor imaging, we compared two groups of one-month-abstinent treatment-seekers, who were classified based on their drinking status between six and nine months after treatment initiation. We hypothesized that subsequent relapsers had greater white matter microstructural deficits in specific brain regions than long-term abstainers. At one month of abstinence, 37 future relapsers versus 25 future abstainers had lower fractional anisotropy (a measure of axonal organization and membrane integrity) in the corpus callosum and right stria terminalis/fornix, higher diffusivity in the genu of the corpus callosum, left and right stria terminalis/fornix, and lower diffusivity in left anterior corona radiata. These differences existed despite similar lifetime and recent drinking and smoking histories in the groups. Longer smoking duration in relapsers was associated with lower fractional anisotropy in right stria terminalis/fornix. The study identified specific microstructural biomarkers of alcohol relapse risk in adults, contributing to the definition of a neurobiological relapse risk profile in alcohol use disorder.

Dysregulation of schizophrenia-related aquaporin 3 through disruption of paranode influences neuronal viability.

Myelinated axons segregate the axonal membrane into four defined regions: the node of Ranvier, paranode, juxtaparanode, and internode. The paranodal junction consists of specific component proteins, such as neurofascin155 (NF155) on the glial side, and Caspr and Contactin on the axonal side. Although paranodal junctions are thought to play crucial roles in rapid saltatory conduction and nodal assembly, the role of their interaction with neurons is not fully understood. In a previous study, conditional NF155 knockout in oligodendrocytes led to disorganization of the paranodal junctions. To examine if disruption of paranodal junctions affects neuronal gene expression, we prepared total RNA from the retina of NF155 conditional knockout, and performed expression analysis. We found that the expression level of 433 genes changed in response to paranodal junction ablation. Interestingly, expression of aquaporin 3 (AQP3) was significantly reduced in NF155 conditional knockout mice, but not in cerebroside sulfotransferase knockout (CST-KO) mice, whose paranodes are not originally formed during development. Copy number variations have an important role in the etiology of schizophrenia (SCZ). We observed rare duplications of AQP3 in SCZpatients, suggesting a correlation between abnormal AQP3 expression and SCZ. To determine if AQP3 over-expression in NF155 conditional knockout mice influences neuronal function, we performed adeno-associated virus (AAV)-mediated over-expression of AQP3 in the motor cortex of mice and found a significant increase incaspase 3-dependent neuronal apoptosis in AQP3-transduced cells. This study may provide new insights into therapeutic approaches for SCZ by regulating AQP3 expression, which is associated with paranodal disruption.

Fusogens: Chemical Agents That Can Rapidly Restore Function After Nerve Injury

BackgroundRestoring function after nerve injury remains one of medicine's greatest challenges. The current approach of epineurial coaptation does not address the fundamental insult at the molecular level: a discontinuity in the axonal membranes. Membrane fusion is possible through agents collectively called chemical fusogens, which are heterogeneous in structure and mechanism of action. We sought a unifying system for classifying fusogens to better understand their role in cell fusion. Materials and methodsWe conducted a comprehensive literature review to identify the most commonly cited chemical fusogens, their structures, mechanisms of actions, and clinical applications to date. We identified seven chemical fusogens (polyethylene glycol, chitosan, dextran sulfate, n-nonyl bromide, calcium, sodium nitrate, and H-α-7), which have each been studied to different extents in protoplasts, animals, and humans. ResultsChemical fusogens achieve cell fusion by one of two ways: bringing cells in close enough proximity to each other so the inherent fluidity of the phospholipid membrane allows for their rearrangement or modifying the surface charges of the membranes to diminish repellent charges. Sowers initially put forth a classification system that identified these agents as cell aggregators and membrane modifiers, respectively. We adapted this classification system in the setting of axonal membrane fusion and hypothesized that the most effective approach to axonal membrane repair is likely combination of both. ConclusionsChemical fusogens could be grouped into two mechanistic categories—cell aggregators and membrane modifiers. For axonal membrane fusion, a combination of both mechanisms can significantly contribute to advancing outcomes in peripheral nerve repair via a chemical-surgical intervention.

Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy

Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of Kv7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to Kv7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), revealing novel PIP2 binding residues. While these mutations disrupted Kv7 function to suppress excitability, hyperexcitability was observed in neurons expressing Kv7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in Kv7 channels by severely reducing their CaM binding, K+ currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of Kv7.2, whereas the presence of Kv7.3 blocked this degradation. Furthermore, expression of Kv7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of Kv7.2 cause abnormal Kv7 expression and function by disrupting Kv7.2 binding to CaM and/or modulation by PIP2. We propose that such multiple Kv7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

Correlation between markers of peripheral nerve function and structure in type 1 diabetes.

Clinical and experimental studies in patients with type 1 and type 2 diabetes have demonstrated changes in ion channel function and nerve structure. In this study, we investigated the relationship between axonal dysfunction and morphological change in diabetic polyneuropathy by using neuromuscular ultrasound and nerve excitability techniques. We also explored possible differences in this relationship between type 1 and type 2 diabetes. Nerve ultrasound and corresponding motor excitability studies were undertaken in 110 diabetes patients (50 type 1; 60 type 2) and 60 age-matched controls (30 for each group). Neuropathy severity was assessed by using total neuropathy score. Median and tibial nerve cross-sectional areas were measured at nonentrapment sites by using high-resolution linear probe. Median and tibial nerve cross-sectional areas were significantly higher in diabetes patients compared with controls: type 1 (median=7.6±0.2mm2 vs 6.3±0.1mm2 ; tibial=14.5±0.7mm2 vs 10.8±0.3mm2 , P<.05) and type 2 (median=9.1±0.3mm2 vs 7.2±0.1mm2 ; tibial=18.5±1.0mm2 vs 12.8±0.5mm2 , P<.05). In the type 1 cohort, significant correlations were found between nerve cross-sectional area and excitability parameters including resting current-threshold slope (median: r=0.523, P<.0001; tibial: r=-0.571, P=.004) and depolarizing threshold electrotonus at 90 to 100ms (median: 0.424, P<.01; tibial: r=0.435, P=.030). In contrast, there was no relationship between excitability values and nerve cross-sectional area in the type 2 cohort. This study has identified correlation between markers of axonal membrane function and structural abnormalities in peripheral nerves of type 1 diabetes patients. The differential relationship in nerve function and structure between type 1 and type 2 diabetes provides clinical evidence that different pathophysiological mechanisms underlie the development of neuropathy in these patient groups.

Abstract 1083: GAP43, a novel potential metastases promoter in non-small cell lung cancer

Abstract Metastasis is a serious sequela with dismal prognosis in non-small cell lung cancer (NSCLC). To identify novel biomarkers for predicting NSCLC metastasis, we performed high-throughput Luminex assays to profile the transcriptional level of 36 genes in 37 operable NSCLC patients who had distant metastases within 3 years after surgery and 33 paired patients without relapse after operation. Among the 36 genes, growth associated protein 43 (GAP43), an axonal membrane protein, was identified related to distant metastases. Multivariate cox regression analysis showed that NSCLC patients with elevated GAP43 had a 3.71-fold increase in risk for metastasis than those with low levels (95% confidence interval 1.69-8.14, P = 0.001). Analysis of a patient cohort of 1,926 NSCLC revealed that patients with high levels of GAP43 had worse progression-free and overall survival. In vitro experiments showed that GAP43 knockdown suppressed NSCLC cell migration and invasion, while GAP43 overexpression facilitated their migration and invasion. Immunofluorescence assay indicated that the linear polymer microfilament F-actin became cracked after GAP43 knockdown. Furthermore, animal studies using left ventrical injection demonstrated that GAP43 silenced NSCLC cells were less likely to metastasize to brain and bone than the controls. Together, our findings suggest that GAP43 may be an independent predictor for NSLCL metastasis, and it may facilitate distant metastasis through targeting F-actin. Citation Format: Fanrong Zhang, Jiaoyue Jin, Junzhou Wu, Kaiyan Chen, Minran Huang, Herbert Yu, Lisha Ying, Dan Su. GAP43, a novel potential metastases promoter in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1083.

Sodium Channel-Dependent and -Independent Mechanisms Underlying Axonal Afterdepolarization at Mouse Hippocampal Mossy Fibers.

Action potentials propagating along axons are often followed by prolonged afterdepolarization (ADP) lasting for several tens of milliseconds. Axonal ADP is thought to be an important factor in modulating the fidelity of spike propagation during repetitive firings. However, the mechanism as well as the functional significance of axonal ADP remain unclear, partly due to inaccessibility to small structures of axon for direct electrophysiological recordings. Here, we examined the ionic and electrical mechanisms underlying axonal ADP using whole-bouton recording from mossy fiber terminals in mice hippocampal slices. ADP following axonal action potentials was strongly enhanced by focal application of veratridine, an inhibitor of Na+ channel inactivation. In contrast, tetrodotoxin (TTX) partly suppressed ADP, suggesting that a Na+ channel-dependent component is involved in axonal ADP. The remaining TTX-resistant Na+ channel-independent component represents slow capacitive discharge reflecting the shape and electrical properties of the axonal membrane. We also addressed the functional impact of axonal ADP on presynaptic function. In paired-pulse stimuli, we found that axonal ADP minimally affected the peak height of subsequent action potentials, although the rising phase of action potentials was slightly slowed, possibly due to steady-state inactivation of Na+ channels by prolonged depolarization. Voltage clamp analysis of Ca2+ current elicited by action potential waveform commands revealed that axonal ADP assists short-term facilitation of Ca2+ entry into the presynaptic terminals. Taken together, these data show that axonal ADP maintains reliable firing during repetitive stimuli and plays important roles in the fine-tuning of short-term plasticity of transmitter release by modulating Ca2+ entry into presynaptic terminals.

In vivo diffusion MRI detects early spinal cord axonal pathology in a mouse model of amyotrophic lateral sclerosis.

Diffusion magnetic resonance imaging (MRI) exhibits contrast that identifies macro- and microstructural changes in neurodegenerative diseases. Previous studies have shown that MR diffusion tensor imaging (DTI) can observe changes in spinal cord white matter in animals and humans affected with symptomatic amyotrophic lateral sclerosis (ALS). The goal of this preclinical work was to investigate the sensitivity of DTI for the detection of signs of tissue damage before symptoms appear. High-field MRI data were acquired using a 9.4-T animal scanner to examine the spinal cord of an ALS mouse model at pre- and post-symptomatic stages (days 80 and 120, respectively). The MRI results were validated using yellow fluorescent protein (YFP) via optical microscopy of spinal cord tissue slices collected from the YFP,G93A-SOD1 mouse strain. DTI maps of diffusion-weighted imaging (DWI) signal intensity, mean diffusivity (MD), fractional anisotropy (FA), axial diffusivity (AD) and radial diffusivity (RD) were computed for axial slices of the lumbar region of the spinal cord. Significant changes were observed in FA (6.7% decrease, p<0.01), AD (19.5% decrease, p<0.01) and RD (16.1% increase, p<0.001) at postnatal day 80 (P80). These differences were correlated with changes in axonal fluorescence intensity and membrane cellular markers. This study demonstrates the value of DTI as a potential tool to detect the underlying pathological progression associated with ALS, and may accelerate the discovery of therapeutic strategies for patients with this disease.

High-Fat Diet Induces Mitochondrial Dysfunction in Saphenous Nerve Axons—A Study by Confocal Microscopy In Vivo

Diabetic peripheral neuropathy (DPN) is a common, progressive and irreversible complication of diabetes (DM). Glucose regulation has no effect on the development and/or progression of the disease in type 2 DM and so, as the prevalence of DM increases, there is a growing unmet need for a mechanism-based therapy. We have examined the effect of high fat diet (HFD, 60% kcal fat) on axonal mitochondrial membrane potential and intra-axonal Ca2+ concentration [Ca2+]i in C56Bl/6 mice, in vivo, using confocal microscopy. The HFD resulted in obesity, hyperglycaemia and glucose intolerance. Mitochondria within saphenous nerve axons of mice fed a HFD for 16 or 36 weeks were significantly depolarised compared with those in mice fed a control diet. Furthermore, HFD mitochondria did not depolarise in response to high frequency electrical stimulation, in contrast to controls where depolarisation is associated with the increased energy demand of sustained impulse conduction. Confocal microscopy revealed that the number of mobile mitochondria was significantly greater in electrically active axons after 16 weeks of HFD, compared with controls, whereas after 36 weeks the number was significantly lower. Histologically, these changes were accompanied by significantly increased labelling for the mitochondrial biogenesis marker PGC1α in neuronal cell bodies at 16 weeks, but a reduction at 36 weeks, consistent with exhaustion of compensatory mechanisms activated early in the disease course. Interestingly, we found that [Ca2+]i was decreased in mice fed a HFD. We conclude that a HFD impairs axonal mitochondrial function early in the course of type 2 MD. Impaired ATP production, especially during sustained impulse activity, may be a mechanism in the development of DPN. Disclosure M. Sajic: None. A. Kanhai: None. G. Dentoni: None. S. Varatharajah: None. L.M. Hinder: None. A. Rumora: None. E.L. Feldman: None. K. Smith: None.

Axon Diameters and Myelin Content Modulate Microscopic Fractional Anisotropy at Short Diffusion Times in Fixed Rat Spinal Cord

Mapping tissue microstructure accurately and noninvasively is one of the frontiers of biomedical imaging. Diffusion Magnetic Resonance Imaging (MRI) is at the forefront of such efforts, as it is capable of reporting on microscopic structures orders of magnitude smaller than the voxel size by probing restricted diffusion. Double Diffusion Encoding (DDE) and Double Oscillating Diffusion Encoding (DODE) in particular, are highly promising for their ability to report on microscopic fractional anisotropy (μFA), a measure of the pore anisotropy in its own eigenframe, irrespective of orientation distribution. However, the underlying correlates of μFA have insofar not been studied. Here, we extract μFA from DDE and DODE measurements at ultrahigh magnetic field of 16.4T with the goal of probing fixed rat spinal cord microstructure. We further endeavor to correlate μFA with Myelin Water Fraction (MWF) derived from multiexponential T2 relaxometry, as well as with literature-based spatially varying axon diameter. In addition, a simple new method is presented for extracting unbiased μFA from three measurements at different b-values. Our findings reveal strong anticorrelations between μFA (derived from DODE) and axon diameter in the distinct spinal cord tracts; a moderate correlation was also observed between μFA derived from DODE and MWF. These findings suggest that axonal membranes strongly modulate μFA, which—owing to its robustness toward orientation dispersion effects—reflects axon diameter much better than its typical FA counterpart. μFA varied when measured via oscillating or blocked gradients, suggesting selective probing of different parallel path lengths and providing insight into how those modulate μFA metrics. Our findings thus shed light into the underlying microstructural correlates of μFA and are promising for future interpretations of this metric in health and disease.

Contactin-2, a synaptic and axonal protein, is reduced in cerebrospinal fluid and brain tissue in Alzheimer\u2019s disease

BackgroundSynaptic and axonal loss are two major mechanisms underlying Alzheimer’s disease (AD) pathogenesis, and biomarkers reflecting changes in these cellular processes are needed for early diagnosis and monitoring the progression of AD. Contactin-2 is a synaptic and axonal membrane protein that interacts with proteins involved in the pathology of AD such as amyloid precursor protein (APP) and beta-secretase 1 (BACE1). We hypothesized that AD might be characterized by changes in contactin-2 levels in the cerebrospinal fluid (CSF) and brain tissue. Therefore, we aimed to investigate the levels of contactin-2 in the CSF and evaluate its relationship with disease pathology.MethodsContactin-2 was measured in CSF from two cohorts (selected from the Amsterdam Dementia Cohort), comprising samples from controls (cohort 1, n = 28; cohort 2, n = 20) and AD (cohort 1, n = 36; cohort 2, n = 70) using an analytically validated commercial enzyme-linked immunosorbent assay (ELISA). The relationship of contactin-2 with cognitive decline (Mini-Mental State Examination (MMSE)) and other CSF biomarkers reflecting AD pathology were analyzed. We further characterized the expression of contactin-2 in postmortem AD human brain (n = 14) versus nondemented controls (n = 9).ResultsCSF contactin-2 was approximately 1.3-fold reduced in AD patients compared with controls (p < 0.0001). Overall, contactin-2 levels correlated with MMSE scores (r = 0.35, p = 0.004). We observed that CSF contactin-2 correlated with the levels of phosphorylated tau within the control (r = 0.46, p < 0.05) and AD groups (r = 0.31, p < 0.05). Contactin-2 also correlated strongly with another synaptic biomarker, neurogranin (control: r = 0.62, p < 0.05; AD: r = 0.60, p < 0.01), and BACE1, a contactin-2 processing enzyme (control: r = 0.64, p < 0.01; AD: r = 0.46, p < 0.05). Results were further validated in a second cohort (p < 0.01). Immunohistochemical analysis revealed that contactin-2 is expressed in the extracellular matrix. Lower levels of contactin-2 were specifically found in and around amyloid plaques in AD hippocampus and temporal cortex.ConclusionsTaken together, these data reveal that the contactin-2 changes observed in tissues are reflected in CSF, suggesting that decreased contactin-2 CSF levels might be a biomarker reflecting synaptic or axonal loss.

Abnormalities in thalamo-cortical connections in patients with first-episode schizophrenia: a two-tensor tractography study.

The "cognitive dysmetria" hypothesis suggests that impairments in cognition and behavior in patients with schizophrenia can be explained by disruptions in the cortico-cerebellar-thalamic-cortical circuit. In this study we examine thalamo-cortical connections in patients with first-episode schizophrenia (FESZ). White matter pathways are investigated that connect the thalamus with three frontal cortex regions including the anterior cingulate cortex (ACC), ventrolateral prefrontal cortex (VLPFC), and lateral oribitofrontal cortex (LOFC). We use a novel method of two-tensor tractography in 26 patients with FESZ compared to 31 healthy controls (HC), who did not differ on age, sex, or education. Dependent measures were fractional anisotropy (FA), Axial Diffusivity (AD), and Radial Diffusivity (RD). Subjects were also assessed using clinical functioning measures including the Global Assessment of Functioning (GAF) Scale, the Global Social Functioning Scale (GF: Social), and the Global Role Functioning Scale (GF: Role). FESZ patients showed decreased FA in the right thalamus-right ACC and right-thalamus-right LOFC pathways compared to healthy controls (HCs). In the right thalamus-right VLPFC tract, we found decreased FA and increased RD in the FESZ group compared to HCs. After correcting for multiple comparisons, reductions in FA in the right thalamus- right ACC and the right thalamus- right VLPC tracts remained significant. Moreover, reductions in FA were significantly associated with lower global functioning scores as well as lower social and role functioning scores. We report the first diffusion tensor imaging study of white matter pathways connecting the thalamus to three frontal regions. Findings of white matter alterations and clinical associations in the thalamic-cortical component of the cortico-cerebellar-thalamic-cortical circuit in patients with FESZ support the cognitive dysmetria hypothesis and further suggest the possible involvement of myelin sheath pathology and axonal membrane disruption in the pathogenesis of the disorder.

Excitability of sensory axons in amyotrophic lateral sclerosis

ObjectiveTo evaluate the excitability of sensory axons in patients with amyotrophic lateral sclerosis (ALS). MethodsComprehensive sensory nerve excitability studies were prospectively performed on 28 sporadic ALS patients, compared to age-matched controls. Sensory nerve action potentials were recorded from digit 2 following median nerve stimulation at the wrist. Disease severity was measured using motor unit number estimation (MUNE), the revised ALS Functional Rating Scale (ALSFRS-R) and the MRC scale. ResultsThere were no significant differences in standard and extended measures of nerve excitability between ALS patients and controls. These unchanged excitability measures included accommodation to long-lasting hyperpolarization and the threshold changes after two supramaximal stimuli during the recovery cycle. Excitability parameters did not correlate with MUNE, ALSFRS-R, APB MRC scale or disease duration. ConclusionsThis cross-sectional study has identified normal axonal membrane properties in myelinated sensory axons of ALS patients. Previously described sensory abnormalities could be the result of axonal fallout, possibly due to a ganglionopathy, or to involvement of central sensory pathways rostral to gracile and cuneate nuclei. SignificanceThese results demonstrate the absence of generalized dysfunction of the membrane properties of sensory axons in ALS in the face of substantial deficits in motor function.

Changes in the axo-glial junctions of the optic nerves of cuprizone-treated mice

Demyelination induced by cuprizone in mice has served a useful model system for the study of demyelinating diseases, such as multiple sclerosis. Severity of demyelination by cuprizone, however, varies across different regions of the central nervous system; the corpus callosum is sensitive, while the optic nerves are resistant. Here, we investigated the effects of cuprizone on optic nerves, focusing on the axo-glial junctions. Immunostaining for sodium channels, contactin-associated protein, neurofascins, and potassium channels revealed that there were no massive changes in the density and morphology of the axo-glial junctions in cuprizone-treated optic nerves. However, when we counted the number of incomplete junctional complexes, we observed increased numbers of isolated paranodes. These isolated paranodes were immunopositive for both axonal and glial membrane proteins, indicating that they were the contact sites between axons and glia. These were not associated with sodium channels or potassium channels, suggesting the absence of physiological functions. When teased axons from cuprizone-treated optic nerves were immunostained, the isolated paranodes were found at the internode region of the myelin. From these observations, we conclude that cuprizone induces new contacts between axons and myelins at the internode region.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Diffusion Tensor Imaging of Axonal and Myelin Changes in Classical Trigeminal Neuralgia.

Trigeminal neuralgia (TN) is commonly associated with pathologic factors of axonopathy and demyelination resulting from neurovascular compression at the trigeminal root entry zone (REZ). Decompression surgery can relieve TN pain, likely by resolving such structural abnormalities. To test this hypothesis, we used diffusion tensor imaging (DTI) to capture the full extent of trigeminal microarchitecture changes invivo in patients with TN. Twenty-four patients with TN were compared with 28 controls. DTI metrics of fractional anisotropy (FA) and mean, parallel, and perpendicular diffusivities (MD, λ||, and λ⊥, respectively) were calculated in isolation at each trigeminal REZ. In 6 patients with pain relief following decompression surgery, repeated studies were performed 2times (1 week and 4-6 months) after surgery to detect dynamic changes in FA, MD, λ||, and λ⊥. We observed significant FA reductions and increased diffusivity at the affected trigeminal REZ, corresponding to known underlying pathologic changes, including axonal edema and demyelination. Specifically, our results showed that these DTI-derived metrics are discriminating features for patients with TN according to the support vector machine approach. After effective treatment, diffusion recovery at 1 week was mainly due to the decrease in λ|| (consistent with axonal membrane stabilization), whereas at 4-6 months it was due to the predominant reduction in λ⊥ (consistent with remyelination). Together, these results support that DTI permits the noninvasive detection of the trigeminal microstructural abnormalities underlying TN invivo, and DTI-derived metrics could be considered surrogate markers of the axonal and myelin states for monitoring patients.

A Minimal Coarse-Grained Molecular Dynamics Model of Axon Plasma Membrane with its Implication on the Diffusion Behavior of Axon Membrane Proteins

The axon initial segment (AIS) in the neuron has two crucial functions: maintaining the polarity of neurons and initiation of action potentials. One extraordinary feature of the AIS is the separation of somatodendritic and axonal domains which was confirmed by measuring the trajectory of beads attached to various axonal membrane proteins that there exists a diffusion barrier in the AIS restricting the motion of membrane proteins and lipids. According to the “fences and pickets” model, the periodic cytoskeleton and the highly concentrated transmembrane proteins in the AIS are capable of hindering the diffusion of other proteins and lipids through steric interaction and hydrodynamic friction effect. In this work, we established the first coarse-grained molecular dynamics model of axon plasma membrane which includes both axon membrane skeleton and axonal membranes to investigate the diffusion behavior of membrane proteins and lipids in the AIS from a mechanics aspect. First, we found that transmembrane proteins and proteins in the inner lipid leaflet are confined within the areas between the actin rings. We suspected and proved from the computational model that the size of actin associated protein junctions, which connected to the lipid bilayers may restrict the motion of proteins in the outer leaflet. Then we showed that the spectrin-lipid attraction level will affect the circumferential mobility of membrane proteins. Finally, the highly dense membrane proteins slowed down the motion of all membrane proteins even the lipids. We conclude that actin rings and spectrin filaments restrict the motion of membrane proteins along and around the axon, respectively. However, the main mechanism forming the diffusion barrier in the AIS is the accumulation of concentrated transmembrane proteins.

Excitable Membrane, Ion Channel, Electrostatics: A Historical Sketch

The roughly one-tenth of a volt across an excitable membrane may not seem large, but across a membrane two lipid molecules thick it yields an electric field comparable to one causing atmospheric lightning. Early attempts to explain the physical basis of the nerve impulse modeled its ion currents as stemming from two processes: the movement of ionic charges by the electric field and the ion diffusion due to ion-concentration differences across the membrane. Hampered by its assumptions of constant properties, this classical electrodiffusion model couldn’t explain axonal membrane data. An eclectic approach combining an equation from electrodiffusion theory with elements of cable theory, wave theory and circuit theory—capacitor, batteries, variable conductors—transformed biophysical science with its successful fitting of the action potential's time course. The development of glass pipette electrodes that restrict ion flow to microscopic membrane patches led to the isolation of ion channels, glycoprotein macromolecules that selectively conduct ion currents. Because it's easy to visualize water-filled pores across these macromolecules, fitted with gates that block or allow ion currents across the membrane, they were named voltage-gated ion channels. This approach is challenged because of its: simplistic models of everyday devices, including screws and paddles; assumption of constant channel capacitance; and disregard of electrostatic repulsions between same-charge residues. Genetic engineering techniques brought the discovery of structural features of many such ion channels. One universal feature is a membrane-spanning segment with a regular array of positively charged amino-acid residues. All also contain multiple amino acids whose sidechains fork into two branches. An electrostatic model based on the ion channel's strong similarities to ferroelectric liquid crystals containing branched chains helps explain the depolarization-driven activation of voltage-sensitive ion channels. [See Leuchtag H R (2008) Voltage-Sensitive Ion Channels, Springer; (2017) Biophys. J. 112(3):544a; 112(3):543a-544a; 112(3):464a.]

Guillain-Barré syndrome, transverse myelitis and infectious diseases.

Guillain-Barré syndrome (GBS) and transverse myelitis (TM) both represent immunologically mediated polyneuropathies of major clinical importance. Both are thought to have a genetic predisposition, but as of yet no specific genetic risk loci have been clearly defined. Both are considered autoimmune, but again the etiologies remain enigmatic. Both may be induced via molecular mimicry, particularly from infectious agents and vaccines, but clearly host factor and co-founding host responses will modulate disease susceptibility and natural history. GBS is an acute inflammatory immune-mediated polyradiculoneuropathy characterized by tingling, progressive weakness, autonomic dysfunction, and pain. Immune injury specifically takes place at the myelin sheath and related Schwann-cell components in acute inflammatory demyelinating polyneuropathy, whereas in acute motor axonal neuropathy membranes on the nerve axon (the axolemma) are the primary target for immune-related injury. Outbreaks of GBS have been reported, most frequently related to Campylobacter jejuni infection, however, other agents such as Zika Virus have been strongly associated. Patients with GBS related to infections frequently produce antibodies against human peripheral nerve gangliosides. In contrast, TM is an inflammatory disorder characterized by acute or subacute motor, sensory, and autonomic spinal cord dysfunction. There is interruption of ascending and descending neuroanatomical pathways on the transverse plane of the spinal cord similar to GBS. It has been suggested to be triggered by infectious agents and molecular mimicry. In this review, we will focus on the putative role of infectious agents as triggering factors of GBS and TM.