Sex differences in single neuron function and proteomics profiles examined by patch-clamp and mass spectrometry in the locus coeruleus of the adult mouse.

AIMS: This study aimed to characterize the properties of locus coeruleus (LC) noradrenergic neurons in male and female mice. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. METHODS: Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal functional properties were assessed using patch-clamp recordings. Proteomic analyses of individual LC neuron soma was conducted using mass spectrometry to discern protein expression profiles. Data are available via ProteomeXchange with identifier PXD045844. RESULTS: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. CONCLUSIONS: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Levels of Soluble Interleukin 6 Receptor and Asp358Ala Are Associated With Cognitive Performance and Alzheimer Disease Biomarkers.

BACKGROUND AND OBJECTIVES: Alzheimer disease (AD) is a neurodegenerative disease process manifesting clinically with cognitive impairment and dementia. AD pathology is complex, and in addition to plaques and tangles, neuroinflammation is a consistent feature. Interleukin (IL) 6 is a multifaceted cytokine involved in a plethora of cellular mechanisms including both anti-inflammatory and inflammatory processes. IL6 can signal classically through the membrane-bound receptor or by IL6 trans-signaling forming a complex with the soluble IL6 receptor (sIL6R) and activating membrane-bound glycoprotein 130 on cells not expressing IL6R. IL6 trans-signaling has been demonstrated as the primary mechanism of IL6-mediated events in neurodegenerative processes. In this study, we performed a cross-sectional analysis to investigate whether inheritance of a genetic variation in the IL6R gene and associated elevated sIL6R levels in plasma and CSF were associated with cognitive performance. METHODS: We genotyped the IL6R rs2228145 nonsynonymous variant (Asp358Ala) and assayed IL6 and sIL6R concentrations in paired samples of plasma and CSF obtained from 120 participants with normal cognition, mild cognitive impairment, or probable AD enrolled in the Wake Forest Alzheimer's Disease Research Center's Clinical Core. IL6 rs2228145 genotype and measures of plasma IL6 and sIL6R were assessed for relationships with cognitive status and clinical data, including the Montreal Cognitive Assessment (MoCA), modified Preclinical Alzheimer's Cognitive Composite (mPACC), cognitive domain scores obtained from the Uniform Data Set, and CSF concentrations of phosphoTauT181 (pTau181), ß-amyloid (Aß) Aß40 and Aß42 concentrations. RESULTS: We found that inheritance of the IL6R Ala358 variant and elevated sIL6R levels in plasma and CSF were correlated with lower mPACC, MoCA and memory domain scores, increases in CSF pTau181, and decreases in the CSF Aß42/40 ratio in both unadjusted and covariate-adjusted statistical models. DISCUSSION: These data suggest that IL6 trans-signaling and the inheritance of the IL6R Ala358 variant are related to reduced cognition and greater levels of biomarkers for AD disease pathology. Follow-up prospective studies are necessary, as patients who inherit IL6R Ala358 may be identified as ideally responsive to IL6 receptor-blocking therapies.

A nutraceutical product, extracted from Cannabis sativa, modulates voltage-gated sodium channel function.

BACKGROUND: Purified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (NaV) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that NaV channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of NaV channels is unknown. METHODS: Using automated-planar patch-clamp technology, we profile a hemp-derived nutraceutical product (NP) against human NaV1.1-NaV1.8 expressed in mammalian cells to examine effects on the biophysical properties of channel conductance, steady-state fast inactivation and recovery from fast inactivation. RESULTS: NP modifies peak current amplitude of the NaV1.1-NaV1.7 subtypes and has variable effects on the biophysical properties for all channel subtypes tested. NP potently inhibits NaV channels revealing half-maximal inhibitory concentration (IC50) values of between 1.6 and 4.2 µg NP/mL. Purified CBD inhibits NaV1.1, NaV1.2, NaV1.6 and NaV1.7 to reveal IC50 values in the micromolar range. The CBD content of the product equates to IC50 values (93-245 nM), which are at least an order of magnitude lower than purified CBD. Unlike NP, hemp seed oil vehicle alone did not inhibit NaV channels, suggesting that the inhibitory effects of NP are independent of hemp seed oil. CONCLUSIONS: This CBD-dominant NP potently inhibits NaV channels. Future study of the individual elements of NP, including phytocannabinoids and terpenes, may reveal a potent individual component or that its components interact to modulate NaV channels.

Neurovascular unit pathology is observed very early in disease progression in the mutant SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by motor neuron degeneration that causes neuromuscular denervation, resulting in muscle weakness and atrophy. Work over the decades using ALS mouse models has revealed that while initial pathology may occur within motor neurons, disease pathology is cell non-autologous. Impairment of the blood-spinal cord barrier (BSCB) occurs before motor neuron frank degeneration; however, precisely when the early pathogenesis of the neurovascular units occurs is not fully understood. Here we examine changes in morphology of neurovascular units, associated gene and protein expression in the lumbar spinal cord of SOD1G93A, and wild-type mice and correlate results with previous reports of early pathological events. Using RNA-sequencing and immunolabeling, we also show that both the neurovascular units and the vasculature of the SOD1G93A lumbar spinal cord present important modifications throughout the disease. Genes relevant for the neurovascular unit and immune cells were differentially expressed in the SOD1G93A ventral lumbar spinal cord compared to wild-type. A reduction in capillary density and tight junction (TJ) with overt BSCB breakdown was observed in the SOD1G93A lumbar spinal cord and ultrastructural observation revealed intact TJ. Additionally, thickened basement membrane, increased pericytes, and string vessels were observed. These alterations in neurovascular units and the vasculature are observed prior to reports of initial neuromuscular junction denervation. The identification of early pathogenesis may be critical to develop diagnostic tests and development of novel treatment strategies that target these early events.

Tocilizumab is safe and tolerable and reduces C-reactive protein concentrations in the plasma and cerebrospinal fluid of ALS patients.

INTRODUCTION/AIMS: We tested safety, tolerability, and target engagement of tocilizumab in amyotrophic lateral sclerosis (ALS) patients. METHODS: Twenty-two participants, whose peripheral blood mononuclear cell (PBMC) gene expression profile reflected high messenger ribonucleic acid (mRNA) expression of inflammatory markers, were randomized 2:1 to three tocilizumab or placebo treatments (weeks 0, 4, and 8; 8 mg/kg intravenous). Participants were followed every 4 wk in a double-blind fashion for 16 wk and assessed for safety, tolerability, plasma inflammatory markers, and clinical measures. Cerebrospinal fluid (CSF) was collected at baseline and after the third treatment. Participants were genotyped for Asp358 Ala polymorphism of the interleukin 6 receptor (IL-6R) gene. RESULTS: Baseline characteristics, safety, and tolerability were similar between treatment groups. One serious adverse event was reported in the placebo group; no deaths occurred. Mean plasma C-reactive protein (CRP) level decreased by 88% in the tocilizumab group and increased by 4% in the placebo group (-3.0-fold relative change, P < .001). CSF CRP reduction (-1.8-fold relative change, P = .01) was associated with IL-6R C allele count. No differences in PBMC gene expression or clinical measures were observed between groups. DISCUSSION: Tocilizumab treatment was safe and well tolerated. PBMC gene expression profile was inadequate as a predictive or pharmacodynamic biomarker. Treatment reduced CRP levels in plasma and CSF, with CSF effects potentially dependent on IL-6R Asp358 Ala genotype. IL-6 trans-signaling may mediate a distinct central nervous system response in individuals inheriting the IL-6R C allele. These results warrant further study in ALS patients where IL-6R genotype and CRP levels may be useful enrichment biomarkers.

The Cellular Senescence Stress Response in Post-Mitotic Brain Cells: Cell Survival at the Expense of Tissue Degeneration.

In 1960, Rita Levi-Montalcini and Barbara Booker made an observation that transformed neuroscience: as neurons mature, they become apoptosis resistant. The following year Leonard Hayflick and Paul Moorhead described a stable replicative arrest of cells in vitro, termed "senescence". For nearly 60 years, the cell biology fields of neuroscience and senescence ran in parallel, each separately defining phenotypes and uncovering molecular mediators to explain the 1960s observations of their founding mothers and fathers, respectively. During this time neuroscientists have consistently observed the remarkable ability of neurons to survive. Despite residing in environments of chronic inflammation and degeneration, as occurs in numerous neurodegenerative diseases, often times the neurons with highest levels of pathology resist death. Similarly, cellular senescence (hereon referred to simply as "senescence") now is recognized as a complex stress response that culminates with a change in cell fate. Instead of reacting to cellular/DNA damage by proliferation or apoptosis, senescent cells survive in a stable cell cycle arrest. Senescent cells simultaneously contribute to chronic tissue degeneration by secreting deleterious molecules that negatively impact surrounding cells. These fields have finally collided. Neuroscientists have begun applying concepts of senescence to the brain, including post-mitotic cells. This initially presented conceptual challenges to senescence cell biologists. Nonetheless, efforts to understand senescence in the context of brain aging and neurodegenerative disease and injury emerged and are advancing the field. The present review uses pre-defined criteria to evaluate evidence for post-mitotic brain cell senescence. A closer interaction between neuro and senescent cell biologists has potential to advance both disciplines and explain fundamental questions that have plagued their fields for decades.

Interleukin 6 (IL6) level is a biomarker for functional disease progression within IL6R358Ala variant groups in amyotrophic lateral sclerosis patients.

Interleukin-6 (IL6) expression increases in atrophying muscles and lung tissue during compromised function. Considering ALS patients undergo these same pathological changes, IL6 levels may be relevant for prognostication and treatment. The amount of soluble IL6 receptor, dictated by the IL6R358Ala variant, and local tissue environment in which IL6 signaling occurs is known to influence the ultimate effects of IL6 in multiple diseases. In this longitudinal study, we show that serum IL6 levels negatively correlate both with the patient's functional status as measured by the overall ALSFRS-R and subscores, and with respiratory function as measured by the percent predicted FVC (ppFVC). The correlations are only present in the two-thirds of patients who carry the IL6R358Ala variant that mediates pro-inflammatory transsignaling in the cases of ALSFRS-R limb and respiratory subscores and ppFVC. These results suggest that some observed associations between IL6 and ALS are driven by the subset of patients carrying the IL6R358Ala variant and thus that any IL6-targeted therapeutic approaches may be more advantageous when aimed at this group. Specifically, with relation to respiratory decline, these patients may benefit from closer respiratory follow-up and early initiation of noninvasive ventilation.

Utilising Automated Electrophysiological Platforms in Epilepsy Research.

Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability. Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic. This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

Automated Planar Patch-Clamp Recording of P2X Receptors.

P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5'-triphosphate (ATP) signaling in diverse physiological and pathophysiological processes. For several decades, the "manual" patch-clamp technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-clamp technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.

IL6 receptor358Ala variant and trans-signaling are disease modifiers in amyotrophic lateral sclerosis.

OBJECTIVE: To test the hypothesis that patients with amyotrophic lateral sclerosis (ALS) inheriting the common interleukin 6 receptor (IL6R) coding variant (Asp358Ala, rs2228145, C allele) have associated increases in interleukin 6 (IL6) and IL6R levels in serum and CSF and faster disease progression than noncarriers. METHODS: An observational, case-control study of paired serum and CSF of 47 patients with ALS, 46 healthy, and 23 neurologic disease controls from the Northeastern ALS Consortium Biofluid Repository (cohort 1) was performed to determine serum levels of IL6, sIL6R, and soluble glycoprotein 130 and compared across groups and IL6R genotype. Clinical data regarding disease progression from a separate cohort of 35 patients with ALS from the Wake Forest ALS Center (cohort 2) were used to determine change in ALSFRS-R scores by genotype. RESULTS: Patients with ALS had increased CSF IL6 levels compared with healthy (p < 0.001) and neurologic (p = 0.021) controls. Patients with ALS also had increased serum IL6 compared with healthy (p = 0.040) but not neurologic controls. Additive allelic increases in serum IL6R were observed in all groups (average increase of 52% with the presence of the IL6R C allele; p < 0.001). However, only subjects with ALS had significantly increased CSF sIL6R levels compared with controls (p < 0.001). When compared across genotypes, only patients with ALS inheriting the IL6R C allele exhibit increased CSF IL6. ALSFRS-R scores decreased more in patients with ALS with the IL6R C allele than in those without (p = 0.019). CONCLUSIONS: Theses results suggest that for individuals inheriting the IL6R C allele, the cytokine exerts a disease- and location-specific role in ALS. Follow-up, prospective studies are necessary, as this subgroup of patients may be identified as ideally responsive to IL6 receptor-blocking therapies.

Extracellular heat shock proteins in neurodegenerative diseases: New perspectives.

One pathological hallmark of neurodegenerative diseases and CNS trauma is accumulation of insoluble, hydrophobic molecules and protein aggregations found both within and outside cells. These may be the consequences of an inadequate or overburdened cellular response to stresses resulting from potentially toxic changes in extra- and intracellular environments. The upregulated expression of heat shock proteins (HSPs) is one example of a highly conserved cellular response to both internal and external stress. Intracellularly these proteins act as chaperones, playing vital roles in the folding of nascent polypeptides, the translocation of proteins between subcellular locations, and the disaggregation of misfolded or aggregated proteins in an attempt to maintain cellular proteostasis during both homeostatic and stressful conditions. While the predominant study of the HSPs has focused on their intracellular chaperone functions, it remains unclear if all neuronal populations can mount a complete stress response. Alternately, it is now well established that some members of this family of proteins can be secreted by nearby, non-neuronal cells to act in the extracellular environment. This review addresses the current literature detailing the use of exogenous and extracellular HSPs in the treatment of cellular and animal models of neurodegenerative disease. These findings offer a new measure of therapeutic potential to the HSPs, but obstacles must be overcome before they can be efficiently used in a clinical setting.

Racial differences in intervention rates in individuals with ALS: A case-control study.

OBJECTIVE: This study was conducted to determine whether longer lifespans in African Americans with amyotrophic lateral sclerosis (ALS), compared to white non-Hispanics, are secondary to higher rates of tracheostomy and invasive ventilation (TIV) in African Americans. METHODS: A retrospective case-control study was conducted with 49 African Americans with ALS matched by age, gender, and site of onset to 137 white persons with ALS. RESULTS: African Americans had longer survival than whites when the outcome was death (p = 0.016), but this was no longer significant when the outcome was death or TIV (p = 0.100). African Americans also had a lower rate of noninvasive ventilation use compared to whites (27 [55%] vs 96 [70%], p = 0.015) and a higher rate of TIV (8 [16%] vs 7 [5%], p = 0.016), but after controlling for baseline severity, only the noninvasive ventilation difference (p = 0.036), and not the TIV difference (p = 0.115), remained significant. CONCLUSION: African Americans with ALS live longer than white persons with ALS, and this may be secondary to higher rates of TIV use among African Americans.

A pilot study of neuromuscular ultrasound as a biomarker for amyotrophic lateral sclerosis.

INTRODUCTION: This study explores the reliability and responsiveness of neuromuscular ultrasound in amyotrophic lateral sclerosis (ALS). METHODS: Investigations were conducted with 10 healthy controls, 10 patients with ALS (single point in time), and 10 different patients with ALS (followed over 6 months; 4 completed follow-up). Ultrasound was used to measure the thickness of the geniohyoid, bilateral biceps/brachialis, bilateral tibialis anterior, and bilateral hemidiaphragms (at inspiration and expiration). Interrater and intrarater reliability and change in muscle thickness over 6 months were measured. RESULTS: Interrater correlation coefficients ranged between 0.80 and 0.99 in healthy controls and between 0.78 and 0.97 in patients with ALS. Intrarater correlation coefficients ranged between 0.83 and 0.98 in healthy controls. The mean percentage decline in muscle thickness over 6 months was 20.25%. DISCUSSION: Muscle ultrasound appears to be a reliable technique for measuring important muscles in patients with ALS. Larger studies with age-matched controls should be conducted to assess further the responsiveness of this biomarker in ALS. Muscle Nerve 59:181-186, 2019.

Inflammation, Immunity, and amyotrophic lateral sclerosis: I. Etiology and pathology.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating disease characterized by progressive degeneration of motor neurons. Charcot first described ALS in 18691 ; however, its pathogenesis remains unknown, and effective treatments remain elusive. It is apparent that new paradigms must be investigated to understand the effectors of ALS, including inflammation, immune responses, and the body's response to stress and injury. Herein we discuss the potential role of the immune system in ALS pathogenesis and critically review evidence from patient and animal studies. Although immune system components may indeed play a role in ALS pathogenesis, studies implicating immune cells, antibodies, and cytokines in early disease pathology are limited. We propose more focused studies that examine the role of the immune system together with characterized pathogenesis to determine when, where, and if immune and inflammatory processes are critical to disease progression, and thus worthy targets of intervention. Muscle Nerve 59:10-22, 2019.

Inflammation, immunity, and amyotrophic lateral sclerosis: II. immune-modulating therapies.

With the emerging popularity of immune-modulatory therapies to treat human diseases there is a need to step back from hypotheses aimed at assessing a condition in a single-system context and instead take into account the disease pathology as a whole. In complex diseases, such as amyotrophic lateral sclerosis (ALS), the use of these therapies to treat patients has been largely unsuccessful and likely premature given our lack of understanding of how the immune system influences disease progression and initiation. In addition, we still have an incomplete understanding of the role of these responses in our model systems and how this may translate clinically to human patients. In this review we discuss preclinical evidence and clinical trial results for a selection of recently conducted studies in ALS. We provide evidence-based reasoning for the failure of these trials and offer suggestions to improve the design of future investigations. Muscle Nerve 59:23-33, 2019.

Selective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death.

Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.

KCTD12 modulation of GABA(B) receptor function.

The molecular composition and functional diversity of native GABAB receptors (GABABR) are still poorly understood, thus hindering development of selective GABABR ligands. Potassium channel tetramerization domain-containing protein (KCTD) 12 is a GABABR auxiliary subunit and mouse KCTD12 can alter GABABR function. In this study, we sought to characterize the effects of human KCTD12 on GABABR kinetics and pharmacology, using an automated electrophysiological assay. Seizure susceptibility and ethanol consumption were also investigated in a KCTD12 knockout mouse model. Human KCTD12 co-expression altered the kinetics of GABABR-mediated GIRK channels, speeding rates of both activation and desensitization. Analysis of concentration-response curves showed that KCTD12 coexpression did not alter effects of the agonists GABA or baclofen on GABABR. KCTD12 coexpression enhanced the potentiating effects of the positive allosteric modulator CGP7930, and its effects on GABABR activation and desensitization. The function of KCTD12 in vivo was examined, using the KCTD12 knockout mouse model. The knockout mice were more resistant to a pentylenetetrazole proconvulsant challenge suggesting reduced seizure susceptibility. In the two bottle preference test, KCTD12 knockout mice demonstrated a reduced consumption at high ethanol concentrations. In summary, human KCTD12 accelerated the kinetics of GABABR in vitro, in a manner possibly sensitive to allosteric pharmacological modulation. This study also provides novel in vivo evidence that the interaction between KCTD12 and GABABR is of physiological significance, and may be a mechanism to more selectively modulate GABABR.

Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+ channel properties.

OBJECTIVE: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. METHODS: We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV 3.1, increasing channel availability. INTERPRETATION: MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.