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Background: Natalizumab (TYSABRI®) 300 mg administered intravenously every-4-weeks (Q4W) is approved for treatment of relapsing-remitting multiple sclerosis but is associated with increased risk of progressive multifocal leukoencephalopathy (PML). Extended natalizumab dosing intervals of approximately every-6-weeks (Q6W) are associated with a lower risk of PML. Primary and secondary clinical outcomes from the NOVA randomized clinical trial (NCT03689972) suggest that effective disease control is maintained in patients who were stable during treatment with natalizumab Q4W for ≥12 months and who then switched to Q6W dosing. We compared additional exploratory clinical and patient-reported outcomes (PROs) from NOVA to assess the efficacy of Q6W dosing. Methods: Prespecified exploratory clinical efficacy endpoints in NOVA included change from baseline in Expanded Disability Status Scale (EDSS) score, Timed 25-Foot Walk (T25FW), dominant- and nondominant-hand 9-Hole Peg Test (9HPT), and Symbol Digit Modalities Test (SDMT). Exploratory patient-reported outcome (PRO) efficacy endpoints included change from baseline in the Treatment Satisfaction Questionnaire for Medication (TSQM), Neuro-QoL fatigue questionnaire, Multiple Sclerosis Impact Scale (MSIS-29), EuroQol 5 Dimensions (EQ-5D-5 L) index score, Clinical Global Impression (CGI)–Improvement (patient- and clinician-assessed) and CGI-Severity (clinician-assessed) rating scales. Estimated proportions of patients with confirmed EDSS improvement were based on Kaplan-Meier methods. Estimates of mean treatment differences for Q6W versus Q4W in other outcomes were assessed by least squares mean (LSM) and analyzed using a linear mixed model of repeated measures or ordinal logistic regression (CGI-scale). Results: Exploratory clinical and patient-reported outcomes were assessed in patients who received ≥1 dose of randomly assigned study treatment and had ≥1 postbaseline efficacy assessment (Q6W group, n = 247, and Q4W group, n = 242). Estimated proportions of patients with EDSS improvement at week 72 were similar for Q6W and Q4W groups (11.7% [19/163] vs 10.8% [17/158]; HR 1.02 [95% confidence interval [CI], 0.53–1.98]; P = 0.9501). At week 72, there were no significant differences between Q6W and Q4W groups in LSM change from baseline for T25FW (0.00, P = 0.975), 9HPT (dominant [0.22, P = 0.533] or nondominant [0.09, P = 0.862] hand), or SDMT (−1.03, P = 0.194). Similarly, there were no significant differences between Q6W and Q4W groups in LSM change from baseline for any PRO (TSQM, −1.00, P = 0.410; Neuro-QoL fatigue, 0.52, P = 0.292; MSIS-29 Psychological, 0.67, P = 0.572; MSIS-29 Physical, 0.74, P = 0.429; EQ-5D-5 L, 0.00, P = 0.978). For the EQ-5D-5 L, a higher proportion of Q6W patients than Q4W patients demonstrated worsening (≥0.5 standard deviation increase in the EQ-5D-5 L index score; P = 0.0475). From baseline to week 72 for Q6W versus Q4W, odds ratio (ORs) of LSM change in CGI scores did not show meaningful differences between groups (CGI-Improvement [patient]: OR [95% CI] 1.2 [0.80–1.73]; CGI-Improvement [physician]: 0.8 [0.47–1.36]; CGI-Severity [physician]: 1.0 [0.71–1.54]). Conclusions: No significant differences were observed in change from baseline to week 72 between natalizumab Q6W and Q4W groups for all exploratory clinical or PRO-related endpoints assessed. For the EQ-5D-5 L, a higher proportion of Q6W than Q4W patients demonstrated worsening.

Aim: Patients with relapsing–remitting multiple sclerosis (RRMS) treated with natalizumab have anecdotally reported a ‘feel-good experience’ (FGE). The authors characterized the FGE using survey data from patients with RRMS treated with natalizumab or other disease-modifying therapies (other-DMT). Methods: Questionnaire data from RRMS patients who use MyMSTeam, an online patient social network, were analyzed. Results: The survey included 347 patients (95 natalizumab; 252 other-DMT). More natalizumab than other-DMT patients self-reported having an FGE (62.1 vs 44.8%; p = 0.001) as well as other physical, emotional and cognitive benefits. Conclusion: This study demonstrates that physical, emotional and cognitive benefits were more commonly reported by patients treated with natalizumab than those treated with other disease-modifying therapies and helps characterize patient-reported factors associated with the FGE.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca2+-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamatein vivo. In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENTLoss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in 'Big Pharma,' 'undruggable' for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in ‘Big Pharma,’ ‘undruggable’ for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

A decrease in the excitability of CA1 pyramidal neurons contributes to the age related decrease in hippocampal function and memory decline. Decreased neuronal excitability in aged neurons can be observed as an increase in the Ca2+- activated K+- mediated post burst afterhyperpolarization (AHP). In this study, we demonstrate that the slow component of AHP (sAHP) in aged CA1 neurons (aged-sAHP) is decreased ∼50% by application of the reducing agent dithiothreitol (DTT). The DTT-mediated decrease in the sAHP was age specific, such that it was observed in CA1 pyramidal neurons of aged (20–25 mo), but not young (6–9 mo) F344 rats. The effect of DTT on the aged-sAHP was blocked following depletion of intracellular Ca2+ stores (ICS) by thapsigargin or blockade of ryanodine receptors (RyRs) by ryanodine, suggesting that the age-related increase in the sAHP was due to release of Ca2+ from ICS through redox sensitive RyRs. The DTT-mediated decrease in the aged-sAHP was not blocked by inhibition of L-type voltage gated Ca2+ channels (L-type VGCC), inhibition of Ser/Thr kinases, or inhibition of the large conductance BK potassium channels. The results add support to the idea that a shift in the intracellular redox state contributes to Ca2+ dysregulation during aging.

Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction.

Alcohol (ethanol) produces a wide range of pharmacological effects on the nervous system through its actions on ion channels. The molecular mechanism underlying ethanol modulation of ion channels is poorly understood. Here we used a unique method of alcohol-tagging to demonstrate that alcohol activation of a G-protein–gated inwardly rectifying potassium (GIRK or Kir3) channel is mediated by a defined alcohol pocket through changes in affinity for the membrane phospholipid signaling molecule phosphatidylinositol 4,5-bisphosphate. Surprisingly, hydrophobicity and size, but not the canonical hydroxyl, were important determinants of alcohol-dependent activation. Altering levels of G protein Gβγ subunits, conversely, did not affect alcohol-dependent activation, suggesting a fundamental distinction between receptor and alcohol gating of GIRK channels. The chemical properties of the alcohol pocket revealed here might extend to other alcohol-sensitive proteins, revealing a unique protein microdomain for targeting alcohol-selective therapeutics in the treatment of alcoholism and addiction.

We previously identified a set of 50 genes that were differentially transcribed in the hippocampal CA1 region of aged, learning-impaired rats compared to aged, superior learning animals during a Morris water maze paradigm. In the current study, we expressed three of these genes (Pctk1, Tcf12 and Ccnd1), which had shown increased transcription in aged, learning impaired rats, in the hippocampus of young rats using viral gene transfer and tested for learning and memory deficits at age 7–14 months. Pctk1 injected animals displayed a modest deficit in acquiring latency in both the Morris water maze and the reverse Morris maze. In the radial arm water maze paradigm, Pctk1, Tcf12 and Ccnd1 expressing animals all showed significant deficits in spatial working memory compared to controls. Rats injected with Ccnd1 and Tcf12, but not Pctk1, also showed a significant deficit in spatial reference memory in the radial arm water maze. Electrophysiological experiments revealed no difference in LTP in Ccnd1 and Pctk1 animals. However, LTD induced by low frequency stimulation was observed in control and Ccnd1 animals, but not in Pctk1 treated animals. In addition, neither Ccnd1 nor Pctk1 expression produced any detectable neuropathology. In contrast Tcf12 expressing animals displayed significant neurodegeneration in both CA1 and dentate gyrus. Several Tcf12 animals also developed tumors that appeared to be glioblastomas, suggesting that aberrant Tcf12 expression in the hippocampus is tumorigenic. Thus, behavioral experiments suggested that overexpression of Pctk1 and Ccnd1 produce a deficit in learning and memory, but electrophysiological experiments do not point to a simple mechanism. In contrast, the learning and memory deficits in Tcf12 animals are likely due to neuropathology associated with Tcf12 gene expression.

The contribution of the NMDA receptors (NMDARs) to synaptic plasticity declines during aging, and the decline is thought to contribute to memory deficits. Here, we demonstrate that an age-related shift in intracellular redox state contributes to the decline in NMDAR responses through Ca2+/calmodulin-dependent protein kinase II (CaMKII). The oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR-mediated synaptic responses at hippocampal CA3–CA1 synapses in slices from young (3–8 months) but not aged (20–25 months) rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced NMDAR response to a greater extent in aged hippocampal slices. The enhancement of NMDAR responses facilitated induction of long-term potentiation in aged but not young animals. The DTT-mediated growth in the NMDAR response was not observed for the AMPA receptor-mediated synaptic responses. A similar increase was observed by intracellular application of the membrane-impermeable reducing agent,l-glutathione (l-GSH), through the intracellular recording pipette, indicating that the increased NMDAR response was dependent on intracellular redox state. DTT enhancement of the NMDAR response was dependent on CaMKII activity and was blocked by the CaMKII inhibitor—myristoylated autocamtide-2-related inhibitory peptide (myr-AIP)—but not by inhibition of the activity of protein phosphatases—PP1 and calcineurin (CaN/PP2B) or protein kinase C. CaMKII activity assays established that DTT increased CaMKII activity in CA1 cytosolic extracts in aged but not in young animals. These findings indicate a link between oxidation of CaMKII during aging, a decline in NMDAR responses, and altered synaptic plasticity.

Calcium (Ca(2+)) is a highly versatile intracellular signaling molecule that is essential for regulating a variety of cellular and physiological processes ranging from fertilization to programmed cell death. Research has provided ample evidence that brain aging is associated with altered Ca(2+) homeostasis. Much of the work has focused on the hippocampus, a brain region critically involved in learning and memory, which is particularly susceptible to dysfunction during senescence. The current review takes a broader perspective, assessing age-related changes in Ca(2+) sources, Ca(2+) sequestration, and Ca(2+) binding proteins throughout the nervous system. The nature of altered Ca(2+) homeostasis is cell specific and may represent a deficit or a compensatory mechanism, producing complex patterns of impaired cellular function. Incorporating the knowledge of the complexity of age-related alterations in Ca(2+) homeostasis will positively shape the development of highly effective therapeutics to treat brain disorders.

Fragile X syndrome (FXS) is caused by a mutation that silences the fragile X mental retardation gene (FMR1), which encodes the fragile X mental retardation protein (FMRP). To determine whether FMRP replacement can rescue phenotypic deficits in a fmr1-knockout (KO) mouse model of FXS, we constructed an adeno-associated virus-based viral vector that expresses the major central nervous system (CNS) isoform of FMRP. Using this vector, we tested whether FMRP replacement could rescue the fmr1-KO phenotype of enhanced long-term depression (LTD), a form of synaptic plasticity that may be linked to cognitive impairments associated with FXS. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synaptic contacts in hippocampal slices from wild-type (WT) and fmr1-KO mice in the presence of AP-5 and anisomycin. Paired-pulse low-frequency stimulation (PP-LFS)-induced LTD is enhanced in slices obtained from fmr1 KO compared with WT mice. Analyses of hippocampal synaptic function in fmr1-KO mice that received hippocampal injections of vector showed that the PP-LFS-induced LTD was restored to WT levels. These results indicate that expression of the major CNS isoform of FMRP alone is sufficient to rescue this phenotype and suggest that post-developmental protein replacement may have the potential to improve cognitive function in FXS.

Translation initiation of hepatitis C virus RNA occurs via ribosome binding to an ‘internal ribosome entry site (IRES)’ located in the 5′untranslated region of the viral RNA. The principle interaction between the 40S ribosomal subunit and the HCV IRES has been shown to be largely factor independent, which is followed by the joining of the 60S ribosomal subunit to form functional 80S complex. However several additional cellular proteins have been reported to bind to HCV IRES and enhance the initiation of translation. In order to identify novel factors involved in the ribosome assembly during internal initiation of HCV RNA, northwestern screening of a HeLa cDNA expression library was performed, using HCV IRES RNA as probe. We demonstrate here, that human ribosomal protein L18a, a constituent of 60S subunit, interacts with HCV IRES RNA. This interaction was further confirmed by using a recombinant protein similar to L18a (sL18a), cloned from human blood. Interestingly, addition of increasing concentration of the purified recombinant sL18a protein, showed moderate stimulation of HCV IRES activity in the in vitro translation assay. These observations suggest that the human L18a might influence the HCV IRES mediated translation.

G-protein gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse. Ethanol directly activates GIRKs through interaction with a discrete alcohol-binding pocket that we identified in the cytoplasmic domain. The structural mechanism underlying ethanol-dependent activation, however, is not well understood. Here, we hypothesized that chemically modifying the alcohol pocket with alcohol-like reagents would activate the channel. To examine this, we engineered GIRK2 containing a single cytoplasmic cysteine substitution in the βD-βE domain of the alcohol pocket; S246 is located near the edge of the pocket, and L257 is located within the pocket. All Cys substitutions were introduced into GIRK2c lacking four native Cys residues (GIRK2c(cys-)) and transiently expressed in HEK293T cells. We studied the effect of 2-Hydroxyethyl MTS (MTS-HE, alcohol-like) and two hydrophobic MTS reagents- Benzyl-MTS (MTS-Bn), and 4-hydroxy benzyl-MTS (MTS-Y), on basal (Ba2+-sensitive) and alcohol-activated GIRK currents. MTS-HE (100 μM) increased basal GIRK currents for S246C (34% ± 12%, n=7) and L257C (120% ± 24%, n=7). Similarly, MTS-Bn (10 μM) modification increased basal currents by 74% ± 18% (n=7) for S246C, and by 333% ± 31% (n=4) for L257C. MTS-Y (100 μM) increased the basal currents by 102% ± 25% (n= 5) for L257C but did not affect S246C. In addition, MTS modification decreased MPD-activated current. For L257C, we confirmed that intracellular application of MTS-HE produced a concentration-dependent increase in the time course of channel activation, having a rate constant of 90.6±17.2 M−1s−1 (n=11). Taken together, these data demonstrate that MTS modification of a single amino acid in the alcohol pocket can engage channel activation, and limit the access of bulkier diols (e.g., MPD). These results reveal novel mechanisms for chemical activation of GIRKs and underscore the importance of hydrophobic interactions in alcohol-mediated activation of GIRKs.

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.

G-protein-gated potassium inwardly rectifying (GIRK) channels play an important role in regulating neuronal excitability in the brain. We have previously shown that trafficking and surface expression of GIRK channels are regulated by sorting nexin 27 (SNX27). SXN27 contains three domains; a phosphoinositide-binding Phox (PX) domain that targets SNX27 preferentially to early endosomes, a PDZ domain that associates directly with carboxyl-terminal tailof GIRK3 and GIRK2c subunits, and a Ras-associated RA domain that could potentially bind to small monomeric G proteins. The functional consequence of this interaction with the RA domain was unknown. Here we show for the first time that expression of a SNX27 mutant devoid of the RA domain (SNX27ΔRA) in HEK293T cells abrogates the down-regulation of baclofen-induced GIRK2c/3 currents that is typically observed with wild-type SNX27 (12.4±3.6 pA/pF (SNX27 WT group) vs. 55.8±8.2 pA/pF (SNX27ΔRA) vs. 47.1±6.7pA/pF (control group without SNX27); mean current density±SEM). Using bimolecular fluorescence complementation (BiFC) technique to follow the intracellular localization of GIRK2c/3 heterotetramers, we observed that SNX27 but not SNX27ΔRA increases the number of GIRK2c/3 heterotetramers in early endosomes. Moreover, a single point mutation within the RA domain (K305A) was sufficient to disrupt SNX27-dependent clustering of GIRK2c/3 heterotetramers, mimicking the functional effects of the RA deletion. Recently, it was shown that the RA domain of SNX27 interacts with H-Ras in vitro. We postulate that K305A disrupts RA interaction with H-Ras. In conclusion, we provide the first functional evidence that intact SNX27 RA domain and interaction with small monomeric G proteins is necessary for the regulation of GIRK channels. These new findings suggest that H-Ras signaling directly affects the magnitude of GIRK currents through regulation of SNX27.

G protein-gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse and addiction. We discovered a discrete alcohol-binding pocket in the channel mediating ethanol-dependent activation. Here, we investigated the role of G proteins and PIP2 in ethanol-dependent gating. We engineered GIRK2 with single, modifiable cysteine at L257 in alcohol pocket and found that alcohol-like methanthiosulfonate (MTS) reagents activate GIRK2-L257C, similar to ethanol-dependent activation. We assessed role of G proteins in alcohol activation by either increasing levels of Gβγ (+Gβ1γ2) or decreasing Gβγ through chelation with membrane bound Phosducin (+mPhos). Neither Gβ1γ2 nor mPhos altered the rate of MTS-HE-mediated GIRK2-L257C activation. For comparison, we examined GIRK2-L344C, a key site for Gβγ activation that is inhibited by MTS modification. In contrast to L257C, rate of MTS modification showed a clear dependence on Gβγ levels. These results suggest that alcohol activation of GIRK channel is independent of G proteins.To investigate the role of PIP2, we used voltage-sensitive phosphatase DR-VSP to transiently deplete PIP2 in the membrane. Activation of DR-VSP completely reversed MTS-HE activated current of GIRK2-L257C. Furthermore, MTS-HE treatment significantly slowed the rate of GIRK2-L257C current inhibition following DR-VSP activation, suggesting an increase in apparent affinity for PIP2 and GIRK2-L257C channels modified by MTS-HE. Lastly, we examined the role of PIP2 on alcohol-dependent activation of wild-type GIRK2. Addition of propanol significantly slowed the rate of wild-type GIRK2 current inhibition following PIP2 depletion. Taken together, these data demonstrate that alcohol-dependent activation of GIRK involves an increase in apparent affinity for PIP2, with little influence of Gβγ subunits. The fundamental dichotomy between alcohol and Gβγ arises from distinct gating mechanisms converging on PIP2-dependent opening, revealing novel pathways for antagonizing alcohol's effects on ion channels.