P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma.

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependent kinase inhibitor 2A, isoform INK4a). We further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and in human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis.

Idealized multiple-timescale model of cortical spreading depolarization initiation and pre-epileptic hyperexcitability caused by NaV1.1/SCN1A mutations.

NaV1.1 (SCN1A) is a voltage-gated sodium channel mainly expressed in GABAergic neurons. Loss of function mutations of NaV1.1 lead to epileptic disorders, while gain of function mutations cause a migraine in which cortical spreading depolarizations (CSDs) are involved. It is still debated how these opposite effects initiate two different manifestations of neuronal hyperactivity: epileptic seizures and CSD. To investigate this question, we previously built a conductance-based model of two neurons (GABAergic and pyramidal), with dynamic ion concentrations (Lemaire et al. in PLoS Comput Biol 17(7):e1009239, 2021. https://doi.org/10.1371/journal.pcbi.1009239 ). When implementing either NaV1.1 migraine or epileptogenic mutations, ion concentration modifications acted as slow processes driving the system to the corresponding pathological firing regime. However, the large dimensionality of the model complicated the exploitation of its implicit multi-timescale structure. Here, we substantially simplify our biophysical model to a minimal version more suitable for bifurcation analysis. The explicit timescale separation allows us to apply slow-fast theory, where slow variables are treated as parameters in the fast singular limit. In this setting, we reproduce both pathological transitions as dynamic bifurcations in the full system. In the epilepsy condition, we shift the spike-terminating bifurcation to lower inputs for the GABAergic neuron, to model an increased susceptibility to depolarization block. The resulting failure of synaptic inhibition triggers hyperactivity of the pyramidal neuron. In the migraine scenario, spiking-induced release of potassium leads to the abrupt increase of the extracellular potassium concentration. This causes a dynamic spike-terminating bifurcation of both neurons, which we interpret as CSD initiation.

Dynamic branching in a neural network model for probabilistic prediction of sequences.

An important function of the brain is to predict which stimulus is likely to occur based on the perceived cues. The present research studied the branching behavior of a computational network model of populations of excitatory and inhibitory neurons, both analytically and through simulations. Results show how synaptic efficacy, retroactive inhibition and short-term synaptic depression determine the dynamics of selection between different branches predicting sequences of stimuli of different probabilities. Further results show that changes in the probability of the different predictions depend on variations of neuronal gain. Such variations allow the network to optimize the probability of its predictions to changing probabilities of the sequences without changing synaptic efficacy.

Modeling NaV1.1/SCN1A sodium channel mutations in a microcircuit with realistic ion concentration dynamics suggests differential GABAergic mechanisms leading to hyperexcitability in epilepsy and hemiplegic migraine.

Loss of function mutations of SCN1A, the gene coding for the voltage-gated sodium channel NaV1.1, cause different types of epilepsy, whereas gain of function mutations cause sporadic and familial hemiplegic migraine type 3 (FHM-3). However, it is not clear yet how these opposite effects can induce paroxysmal pathological activities involving neuronal networks' hyperexcitability that are specific of epilepsy (seizures) or migraine (cortical spreading depolarization, CSD). To better understand differential mechanisms leading to the initiation of these pathological activities, we used a two-neuron conductance-based model of interconnected GABAergic and pyramidal glutamatergic neurons, in which we incorporated ionic concentration dynamics in both neurons. We modeled FHM-3 mutations by increasing the persistent sodium current in the interneuron and epileptogenic mutations by decreasing the sodium conductance in the interneuron. Therefore, we studied both FHM-3 and epileptogenic mutations within the same framework, modifying only two parameters. In our model, the key effect of gain of function FHM-3 mutations is ion fluxes modification at each action potential (in particular the larger activation of voltage-gated potassium channels induced by the NaV1.1 gain of function), and the resulting CSD-triggering extracellular potassium accumulation, which is not caused only by modifications of firing frequency. Loss of function epileptogenic mutations, on the other hand, increase GABAergic neurons' susceptibility to depolarization block, without major modifications of firing frequency before it. Our modeling results connect qualitatively to experimental data: potassium accumulation in the case of FHM-3 mutations and facilitated depolarization block of the GABAergic neuron in the case of epileptogenic mutations. Both these effects can lead to pyramidal neuron hyperexcitability, inducing in the migraine condition depolarization block of both the GABAergic and the pyramidal neuron. Overall, our findings suggest different mechanisms of network hyperexcitability for migraine and epileptogenic NaV1.1 mutations, implying that the modifications of firing frequency may not be the only relevant pathological mechanism.

Modeling cortical spreading depression induced by the hyperactivity of interneurons.

Cortical spreading depression (CSD) is a wave of transient intense neuronal firing leading to a long lasting depolarizing block of neuronal activity. It is a proposed pathological mechanism of migraine with aura. Some forms of migraine are associated with a genetic mutation of the Nav1.1 channel, resulting in its gain of function and implying hyperexcitability of interneurons. This leads to the counterintuitive hypothesis that intense firing of interneurons can cause CSD ignition. To test this hypothesis in silico, we developed a computational model of an E-I pair (a pyramidal cell and an interneuron), in which the coupling between the cells in not just synaptic, but takes into account also the effects of the accumulation of extracellular potassium caused by the activity of the neurons and of the synapses. In the context of this model, we show that the intense firing of the interneuron can lead to CSD. We have investigated the effect of various biophysical parameters on the transition to CSD, including the levels of glutamate or GABA, frequency of the interneuron firing and the efficacy of the KCC2 co-transporter. The key element for CSD ignition in our model was the frequency of interneuron firing and the related accumulation of extracellular potassium, which induced a depolarizing block of the pyramidal cell. This constitutes a new mechanism of CSD ignition.

Time-coded neurotransmitter release at excitatory and inhibitory synapses.

Communication between neurons at chemical synapses is regulated by hundreds of different proteins that control the release of neurotransmitter that is packaged in vesicles, transported to an active zone, and released when an input spike occurs. Neurotransmitter can also be released asynchronously, that is, after a delay following the spike, or spontaneously in the absence of a stimulus. The mechanisms underlying asynchronous and spontaneous neurotransmitter release remain elusive. Here, we describe a model of the exocytotic cycle of vesicles at excitatory and inhibitory synapses that accounts for all modes of vesicle release as well as short-term synaptic plasticity (STSP). For asynchronous release, the model predicts a delayed inertial protein unbinding associated with the SNARE complex assembly immediately after vesicle priming. Experiments are proposed to test the model's molecular predictions for differential exocytosis. The simplicity of the model will also facilitate large-scale simulations of neural circuits.

Complement factor H genotypes impact risk of age-related macular degeneration by interaction with oxidized phospholipids.

The rs1061170T/C variant encoding the Y402H change in complement factor H (CFH) has been identified by genome-wide association studies as being significantly associated with age-related macular degeneration (AMD). However, the precise mechanism by which this CFH variant impacts the risk of AMD remains largely unknown. Oxidative stress plays an important role in many aging diseases, including cardiovascular disease and AMD. A large amount of oxidized phospholipids (oxPLs) are generated in the eye because of sunlight exposure and high oxygen content. OxPLs bind to the retinal pigment epithelium and macrophages and strongly activate downstream inflammatory cascades. We hypothesize that CFH may impact the risk of AMD by modulating oxidative stress. Here we demonstrate that CFH binds to oxPLs. The CFH 402Y variant of the protective rs1061170 genotype binds oxPLs with a higher affinity and exhibits a stronger inhibitory effect on the binding of oxPLs to retinal pigment epithelium and macrophages. In addition, plasma from non-AMD subjects with the protective genotype has a lower level of systemic oxidative stress measured by oxPLs per apolipoprotein B (oxPLs/apoB). We also show that oxPL stimulation increases expression of genes involved in macrophage infiltration, inflammation, and neovascularization in the eye. OxPLs colocalize with CFH in drusen in the human AMD eye. Subretinal injection of oxPLs induces choroidal neovascularization in mice. In addition, we show that the CFH risk allele confers higher complement activation and cell lysis activity. Together, these findings suggest that CFH influences AMD risk by modulating oxidative stress, inflammation, and abnormal angiogenesis.

Adaptation and shunting inhibition leads to pyramidal/interneuron gamma with sparse firing of pyramidal cells.

Gamma oscillations are a prominent phenomenon related to a number of brain functions. Data show that individual pyramidal neurons can fire at rate below gamma with the population showing clear gamma oscillations and synchrony. In one kind of idealized model of such weak gamma, pyramidal neurons fire in clusters. Here we provide a theory for clustered gamma PING rhythms with strong inhibition and weaker excitation. Our simulations of biophysical models show that the adaptation of pyramidal neurons coupled with their low firing rate leads to cluster formation. A partially analytic study of a canonical model shows that the phase response curves with a near zero flat region, caused by the presence of the slow adaptive current, are the key to the formation of clusters. Furthermore we examine shunting inhibition and show that clusters become robust and generic.

Mixed-mode bursting oscillations: dynamics created by a slow passage through spike-adding canard explosion in a square-wave burster.

This article concerns the phenomenon of Mixed-Mode Bursting Oscillations (MMBOs). These are solutions of fast-slow systems of ordinary differential equations that exhibit both small-amplitude oscillations (SAOs) and bursts consisting of one or multiple large-amplitude oscillations (LAOs). The name MMBO is given in analogy to Mixed-Mode Oscillations, which consist of alternating SAOs and LAOs, without the LAOs being organized into burst events. In this article, we show how MMBOs are created naturally in systems that have a spike-adding bifurcation or spike-adding mechanism, and in which the dynamics of one (or more) of the slow variables causes the system to pass slowly through that bifurcation. Canards are central to the dynamics of MMBOs, and their role in shaping the MMBOs is two-fold: saddle-type canards are involved in the spike-adding mechanism of the underlying burster and permit one to understand the number of LAOs in each burst event, and folded-node canards arise due to the slow passage effect and control the number of SAOs. The analysis is carried out for a prototypical fourth-order system of this type, which consists of the third-order Hindmarsh-Rose system, known to have the spike-adding mechanism, and in which one of the key bifurcation parameters also varies slowly. We also include a discussion of the MMBO phenomenon for the Morris-Lecar-Terman system. Finally, we discuss the role of the MMBOs to a biological modeling of secreting neurons.

Antinociceptive and Anti-Inflammatory Effects of Recombinant Crotamine in Mouse Models of Pain.

Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus, has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04-1.2 mg kg-1) or intraplantarly (0.9-7.5 µg 10 µL-1) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg-1) or intraplantar naloxone (5 µg 10 µL-1) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg-1) or intraplantar (2.5 and 7.5 µg 10 µL-1) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.

Initiation of migraine-related cortical spreading depolarization by hyperactivity of GABAergic neurons and NaV1.1 channels.

Spreading depolarizations (SDs) are involved in migraine, epilepsy, stroke, traumatic brain injury, and subarachnoid hemorrhage. However, the cellular origin and specific differential mechanisms are not clear. Increased glutamatergic activity is thought to be the key factor for generating cortical spreading depression (CSD), a pathological mechanism of migraine. Here, we show that acute pharmacological activation of NaV1.1 (the main Na+ channel of interneurons) or optogenetic-induced hyperactivity of GABAergic interneurons is sufficient to ignite CSD in the neocortex by spiking-generated extracellular K+ build-up. Neither GABAergic nor glutamatergic synaptic transmission were required for CSD initiation. CSD was not generated in other brain areas, suggesting that this is a neocortex-specific mechanism of CSD initiation. Gain-of-function mutations of NaV1.1 (SCN1A) cause familial hemiplegic migraine type-3 (FHM3), a subtype of migraine with aura, of which CSD is the neurophysiological correlate. Our results provide the mechanism linking NaV1.1 gain of function to CSD generation in FHM3. Thus, we reveal the key role of hyperactivity of GABAergic interneurons in a mechanism of CSD initiation, which is relevant as a pathological mechanism of Nav1.1 FHM3 mutations, and possibly also for other types of migraine and diseases in which SDs are involved.

The response of a classical Hodgkin-Huxley neuron to an inhibitory input pulse.

A population of uncoupled neurons can often be brought close to synchrony by a single strong inhibitory input pulse affecting all neurons equally. This mechanism is thought to underlie some brain rhythms, in particular gamma frequency (30-80 Hz) oscillations in the hippocampus and neocortex. Here we show that synchronization by an inhibitory input pulse often fails for populations of classical Hodgkin-Huxley neurons. Our reasoning suggests that in general, synchronization by inhibitory input pulses can fail when the transition of the target neurons from rest to spiking involves a Hopf bifurcation, especially when inhibition is shunting, not hyperpolarizing. Surprisingly, synchronization is more likely to fail when the inhibitory pulse is stronger or longer-lasting. These findings have potential implications for the question which neurons participate in brain rhythms, in particular in gamma oscillations.

Gamma oscillations as a mechanism for selective information transmission.

In the past decades, many studies have focussed on the relation between the input and output of neurons with the aim to understand information processing by neurons. A particular aspect of neuronal information, which has not received much attention so far, concerns the problem of information transfer when a neuron or a population of neurons receives input from two or more (populations of) neurons, in particular when these (populations of) neurons carry different types of information. The aim of the present study is to investigate the responses of neurons to multiple inputs modulated in the gamma frequency range. By a combination of theoretical approaches and computer simulations, we test the hypothesis that enhanced modulation of synchronized excitatory neuronal activity in the gamma frequency range provides an advantage over a less synchronized input for various types of neurons. The results of this study show that the spike output of various types of neurons [i.e. the leaky integrate and fire neuron, the quadratic integrate and fire neuron and the Hodgkin-Huxley (HH) neuron] and that of excitatory-inhibitory coupled pairs of neurons, like the Pyramidal Interneuronal Network Gamma (PING) model, is highly phase-locked to the larger of two gamma-modulated input signals. This implies that the neuron selectively responds to the input with the larger gamma modulation if the amplitude of the gamma modulation exceeds that of the other signals by a certain amount. In that case, the output of the neuron is entrained by one of multiple inputs and that other inputs are not represented in the output. This mechanism for selective information transmission is enhanced for short membrane time constants of the neuron.

Neuronal mechanisms for sequential activation of memory items: Dynamics and reliability.

In this article we present a biologically inspired model of activation of memory items in a sequence. Our model produces two types of sequences, corresponding to two different types of cerebral functions: activation of regular or irregular sequences. The switch between the two types of activation occurs through the modulation of biological parameters, without altering the connectivity matrix. Some of the parameters included in our model are neuronal gain, strength of inhibition, synaptic depression and noise. We investigate how these parameters enable the existence of sequences and influence the type of sequences observed. In particular we show that synaptic depression and noise drive the transitions from one memory item to the next and neuronal gain controls the switching between regular and irregular (random) activation.

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation.

Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.

Frequency-selectivity of a thalamocortical relay neuron during Parkinson's disease and deep brain stimulation: a computational study.

In this computational study, we investigated (i) the functional importance of correlated basal ganglia (BG) activity associated with Parkinson's disease (PD) motor symptoms by analysing the effects of globus pallidus internum (GPi) bursting frequency and synchrony on a thalamocortical (TC) relay neuron, which received GABAergic projections from this nucleus; (ii) the effects of subthalamic nucleus (STN) deep brain stimulation (DBS) on the response of the TC relay neuron to synchronized GPi oscillations; and (iii) the functional basis of the inverse relationship that has been reported between DBS frequency and stimulus amplitude, required to alleviate PD motor symptoms [A. L. Benabid et al. (1991)Lancet, 337, 403-406]. The TC relay neuron selectively responded to and relayed synchronized GPi inputs bursting at a frequency located in the range 2-25 Hz. Input selectivity of the TC relay neuron is dictated by low-threshold calcium current dynamics and passive membrane properties of the neuron. STN-DBS prevented the TC relay neuron from relaying synchronized GPi oscillations to cortex. Our model indicates that DBS alters BG output and input selectivity of the TC relay neuron, providing an explanation for the clinically observed inverse relationship between DBS frequency and stimulus amplitude.

Technetium-99m pyrophosphate cardiac SPECT in endomyocardial biopsy negative cardiac amyloidosis.

Cardiac amyloidosis is an under-appreciated cause of heart failure. Establishing a diagnosis is important because traditional heart failure treatment regimens can worsen left ventricular failure in this disease. Endomyocardial biopsy is the gold standard for diagnosis; however, scintigraphy with radiolabeled phosphate derivatives and cardiac magnetic resonance imaging have been shown to have high sensitivity and specificity in diagnosing cardiac amyloidosis. Furthermore, cardiac scintigraphy can reliably differentiate amyloid subtypes. We present a case of transthyretin-related cardiac amyloidosis with a negative endomyocardial biopsy but positive 99m-technetium pyrophosphate single photon emission computed tomography scan and cardiac magnetic resonance imaging. We discuss the utility of 99m-technetium pyrophosphate imaging in cardiac amyloidosis and the role of single photon emission computed tomography. Finally, we review the several forms of cardiac amyloidosis and how they pertain to cardiac scintigraphy.

Ipilimumab cystic hypophysitis mimicking metastatic melanoma.

Ipilimumab is an immunotherapeutic agent used in the treatment of metastatic melanoma, and is known to cause hypophysitis in some patients. Magnetic resonance imaging of ipilimumab-induced hypophysitis typically shows diffuse enlargement of the pituitary gland with variable enhancement or enlargement of the infundibulum. This often produces a diagnostic dilemma as melanoma not uncommonly metastasizes to the pituitary gland due to the rich vascular plexus of the hypophyseal portal system, and has a similar imaging appearance to autoimmune hypophysitis. We present a case of a 49-year-old man with a Clark level 4 melanoma of the left calf with inguinal nodal metastases that was treated with resection and 2 cycles of ipilimumab, and subsequently developed a "cystic" pituitary mass. To our knowledge, all of the described cases of ipilimumab-induced hypophysitis to date have shown solid enhancement on imaging. Because metastatic melanoma to the pituitary gland often has internal hemorrhage that produces a "cystic" appearance, and ipilimumab-induced hypophysitis is typically a solidly enhancing abnormality, this presented a significant diagnostic and therapeutic dilemma. Our patient's symptoms, although significant, did not necessitate immediate surgical intervention, and a conservative approach of withholding the ipilimumab and administering therapeutic corticosteroids was pursued. The patient's symptoms abated and follow-up magnetic resonance imaging 1 month later showed near complete resolution of the pituitary abnormalities. As such, this is a unique case of ipilimumab-induced hypophysitis presenting as a "cystic" pituitary mass.

Leptomeningeal carcinomatosis and bilateral internal auditory canal metastases from ovarian carcinoma.

Ovarian cancer, a leading cause of death in women, typically spreads locally and rarely metastasizes to the brain or seeds the leptomeninges. We present a case of a 62-year-old woman with a history of treated ovarian cell carcinoma who developed bilateral sensorineural deafness and right-sided facial weakness and on imaging was found to have bilateral internal auditory canal (IAC) masses and leptomeningeal carcinomatosis, pathologically proven by cerebrospinal fluid cytology. We discuss her magnetic resonance imaging and positron emission tomography-computed tomography findings and review the imaging characteristics of IAC metastases. Finally, we review the literature on leptomeningeal carcinomatosis from ovarian cancer and discuss the high incidence of bilateral IAC metastases in patients with leptomeningeal carcinomatosis.