TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Primary splenic diffuse large B-cell lymphoma presenting as a splenic abscess.

Diffuse large B-cell lymphoma (DLBCL) arising in the spleen, also known as primary splenic DLBCL (PS-DLBCL), is a rare form of malignant lymphoma. It is defined as a lymphoma confined to the spleen or involving splenic hilar lymph nodes. Here we report a case of PS-DLBCL with CD30. The patient was a 62-year-old who presented with 2 weeks of left flank pain, chills, and abdominal distension. Computed tomography identified an 8-cm splenic mass with central necrosis interpreted as an abscess. A drain was placed, yielding purulent necrotic material; cytologically, only neutrophils were identified. However, purulent drainage continued for 28 days without resolution, prompting splenectomy. Pathological dissection revealed a multinodular mass with central necrosis. Microscopic examination revealed extensive karyorrhexis, abundant ghosts of large cells, and scattered large cells with pleomorphic, multilobated, and vesicular nuclei with moderately abundant cytoplasm. Immunohistochemical staining revealed large, atypical cells positive for CD20, CD30, CD45, PAX5, MYC (>40%), MUM1 (>30%), and p53 (focally). The large cells were negative for CD3 (polyclonal), CD4, CD5, CD8, CD10, CD15, CD34, BCL2, BCL6, AE1/AE3, S100, HHV8, and ALK. The Ki-67 proliferation rate was approximately 80% in large cells. Notably, this PS-DLBCL was positive for CD30, an unusual finding among non-Hodgkin B-cell lymphomas, which, coupled with the Reed-Sternberg-like morphology, raised the possibility of classic Hodgkin lymphoma. Therefore, we reviewed the literature to confirm the unique features of this large B-cell lymphoma, its abscess-like appearance, and its expression of CD30.

Two cases of mycosis fungoides with large cell transformation with KMT2A rearrangements.

Cutaneous T-cell lymphomas (CTCL) are a clinically and molecularly heterogeneous class of lymphomas of the skin-homing T cell, and their genetic profiles are not fully characterized. Previously, rearrangements of the Lysine Methyltransferase 2A (KMT2A) gene have been identified as driver mutations only in acute leukemias. KMT2A plays a role in epigenetic regulation, and cancers with such rearrangements are responsive to epigenetic therapy including hypomethylating agents. Here, we report two cases of CTCL with novel genetic profiles. KMT2A rearrangements were identified in two aggressive cases of mycosis fungoides with large cell transformation. A KMT2A::DSCAML1 gene rearrangement was seen in Case 1, while a KMT2A::MAPRE1 fusion was identified in Case 2. These cases demonstrate that KMT2A rearrangements can be found in primary CTCLs rather than solely acute leukemias, illustrating the importance of correlating molecular findings with clinical and histologic features in diagnosis. Additionally, this finding suggests that the subset of CTCLs driven by aberrancy of the KMT2A pathway may be responsive to therapy with hypomethylating agents or menin inhibitors, as seen in acute leukemias.

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation.

Voltage-gated Na+ (Nav) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Nav channel α subunit that have been described (Nav1.1-Nav1.9), Nav1.1, Nav1.2, and Nav1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Nav channel isoforms display differential expression across neuronal cell types and diverge with respect to their subcellular distributions. Considering these differences in terms of their localization, the CNS Nav channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Nav channels lack selectivity, which results in deleterious side effects due to modulation of off-target Nav channel isoforms. Among the structural components of the Nav channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Nav channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein-protein interactions (PPIs) with Nav channel auxiliary proteins. In medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a critical brain region of the mesocorticolimbic circuit, the PPI between the CTD of the Nav1.6 channel and its auxiliary protein fibroblast growth factor 14 (FGF14) is central to the generation of electrical outputs, underscoring its potential value as a site for targeted neuromodulation. Focusing on this PPI, we previously developed a peptidomimetic derived from residues of FGF14 that have an interaction site on the CTD of the Nav1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Nav1.6 channels in heterologous cells, the compound does not affect Nav1.1 or Nav1.2 channels at comparable concentrations. In addition, we show that the compound correspondingly modulates the action potential discharge and the transient Na+ of MSNs of the NAc. Overall, these results demonstrate that pharmacologically targeting the FGF14 interaction site on the CTD of the Nav1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Nav channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.

Development of Allosteric Modulators of Voltage-Gated Na+ Channels: A Novel Approach for an Old Target.

Given their primacy in governing the action potential (AP) of excitable cells, voltage-gated Na+ (Nav) channels are important pharmacological targets of therapeutics for a diverse array of clinical indications. Despite historically being a traditional drug target, therapeutics targeting Nav channels lack isoform selectivity, giving rise to off-target side effects. To develop isoform-selective modulators of Nav channels with improved target-specificity, the identification and pharmacological targeting of allosteric sites that display structural divergence among Nav channel isoforms represents an attractive approach. Despite the high homology among Nav channel α subunit isoforms (Nav1.1-Nav1.9), there is considerable amino acid sequence divergence among their constituent C-terminal domains (CTD), which enables structurally and functionally specific protein: protein interactions (PPI) with auxiliary proteins. Although pharmacological targeting of such PPI interfaces between the CTDs of Nav channels and auxiliary proteins represents an innovate approach for developing isoform-selective modulators of Nav channels, appreciable modulation of PPIs using small molecules has conventionally been difficult to achieve. After briefly discussing the challenges of modulating PPIs using small molecules, this current frontier review that follows subsequently expounds on approaches for circumventing such difficulties in the context of developing small molecule modulators of PPIs between transmembrane ion channels and their auxiliary proteins. In addition to broadly discussing such approaches, the implementation of such approaches is specifically discussed in the context of developing small molecule modulators between the CTD of Nav channels and auxiliary proteins. Developing allosteric modulators of ion channels by targeting their PPI interfaces with auxiliary proteins represents an innovative and promising strategy in ion channel drug discovery that could expand the "druggable genome" and usher in first-in-class PPI-targeting therapeutics for a multitude of channelopathies.

Bioluminescence Methodology for Ion Channel Studies.

As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein-protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na+ (Nav) channel Nav1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.

Design, Synthesis, and Pharmacological Evaluation of Analogues Derived from the PLEV Tetrapeptide as Protein-Protein Interaction Modulators of Voltage-Gated Sodium Channel 1.6.

The voltage-gated Na+ (Nav) channel is the molecular determinant of excitability. Disruption of protein-protein interactions (PPIs) between Nav1.6 and fibroblast growth factor 14 (FGF14) leads to impaired excitability of neurons in clinically relevant brain areas associated with channelopathies. Here, we designed, synthesized, and pharmacologically characterized new peptidomimetics based on a PLEV tetrapeptide scaffold derived from the FGF14:Nav1.6 PPI interface. Addition of an N-terminal 1-adamantanecarbonyl pharmacophore significantly improved peptidomimetic inhibitory potency. Surface plasmon resonance studies revealed that while this moiety was sufficient to confer binding to FGF14, altering the C-terminal moiety from methoxy (21a) to π bond-containing (23a and 23b) or cycloalkane substituents (23e) abrogated the binding to Nav1.6. Whole-cell patch-clamp electrophysiology subsequently revealed that 21a had functionally relevant interactions with both the C-terminal tail of Nav1.6 and FGF14. Collectively, these findings support that 21a (PW0564) may serve as a promising lead to develop target-selective neurotherapeutics by modulating protein-channel interactions.

Mobility but Not Balance Limitations Are Associated With Cognitive Decline among Older Hispanics.

Aging is associated with changes in lower-body functioning. The extent to which lower-body function is associated with cognitive changes over time is unclear, especially among older Hispanics, a high-risk population for declines in physical and cognitive functioning. We sought to determine if the association between lower-body functioning and cognitive decline over 9-years differentially varied with respect to balance, gait speed, lower-body strength (chair stands), or a summary score of the three measures. This retrospective cohort study used clinical performance data from the Hispanic Established Populations for the Epidemiologic Study of the Elderly (H-EPESE). Cognitive function was measured using the Mini-Mental Status Exam. Linear mixed modeling was used to investigate the association between lower-body function and cognitive decline, controlling for patients' demographic and health characteristics. We found that gait speed and timed chair stands but not balance were associated with accelerated cognitive decline in Mexican-Americans age 75 years and older. These parameters of lower-body function can be feasibly measured in any clinic. As limitations in lower-body functioning may be an early marker of cognitive decline, this suggests an opportunity for the development of interventions to slow cognitive and physical disablement and promote successful aging among persons older than 75 years.

Mapping of the FGF14:Nav1.6 complex interface reveals FLPK as a functionally active peptide modulating excitability.

The voltage-gated sodium (Nav) channel complex is comprised of pore-forming α subunits (Nav1.1-1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C-terminal tail binds directly to FGF14 and this interaction modifies Nav1.6-mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14V160 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14V160 to probe for the FGF14 interaction with the Nav1.6 C-terminal tail and to evaluate the activity of the peptide on Nav1.6-mediated currents. In silico docking predicts FLPK to bind to FGF14V160 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split-luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole-cell patch-clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14-dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N-terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na+ currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.

Bidirectional Modulation of the Voltage-Gated Sodium (Nav1.6) Channel by Rationally Designed Peptidomimetics.

Disruption of protein:protein interactions (PPIs) that regulate the function of voltage-gated Na+ (Nav) channels leads to neural circuitry aberrations that have been implicated in numerous channelopathies. One example of this pathophysiology is mediated by dysfunction of the PPI between Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). Thus, peptides derived from FGF14 might exert modulatory actions on the FGF14:Nav1.6 complex that are functionally relevant. The tetrapeptide Glu-Tyr-Tyr-Val (EYYV) mimics surface residues of FGF14 at the ß8-ß9 loop, a structural region previously implicated in its binding to Nav1.6. Here, peptidomimetics derived from EYYV (6) were designed, synthesized, and pharmacologically evaluated to develop probes with improved potency. Addition of hydrophobic protective groups to 6 and truncation to a tripeptide (12) produced a potent inhibitor of FGF14:Nav1.6 complex assembly. Conversely, addition of hydrophobic protective groups to 6 followed by addition of an N-terminal benzoyl substituent (19) produced a potentiator of FGF14:Nav1.6 complex assembly. Subsequent functional evaluation using whole-cell patch-clamp electrophysiology confirmed their inverse activities, with 12 and 19 reducing and increasing Nav1.6-mediated transient current densities, respectively. Overall, we have identified a negative and positive allosteric modulator of Nav1.6, both of which could serve as scaffolds for the development of target-selective neurotherapeutics.

JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation.

BACKGROUND: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons. METHODS: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology. RESULTS: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14Y158, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14Y158A mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14. CONCLUSIONS: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.

High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na+ channel complex.

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

Sex-Specific Proteomic Changes Induced by Genetic Deletion of Fibroblast Growth Factor 14 (FGF14), a Regulator of Neuronal Ion Channels.

Fibroblast growth factor 14 (FGF14) is a member of the intracellular FGFs, which is a group of proteins involved in neuronal ion channel regulation and synaptic transmission. We previously demonstrated that male Fgf14 -/- mice recapitulate the salient endophenotypes of synaptic dysfunction and behaviors that are associated with schizophrenia (SZ). As the underlying etiology of SZ and its sex-specific onset remain elusive, the Fgf14 -/- model may provide a valuable tool to interrogate pathways related to disease mechanisms. Here, we performed label-free quantitative proteomics to identify enriched pathways in both male and female hippocampi from Fgf14 +/+ and Fgf14 -/- mice. We discovered that all of the differentially expressed proteins measured in Fgf14 -/- animals, relative to their same-sex wildtype counterparts, are associated with SZ based on genome-wide association data. In addition, measured changes in the proteome were predominantly sex-specific, with the male Fgf14 -/- mice distinctly enriched for pathways associated with neuropsychiatric disorders. In the male Fgf14-/- mouse, we found molecular characteristics that, in part, may explain a previously described neurotransmission and behavioral phenotype. This includes decreased levels of ALDH1A1 and protein kinase A (PRKAR2B). ALDH1A1 has been shown to mediate an alternative pathway for gamma-aminobutyric acid (GABA) synthesis, while PRKAR2B is essential for dopamine 2 receptor signaling, which is the basis of current antipsychotics. Collectively, our results provide new insights in the role of FGF14 and support the use of the Fgf14 -/- mouse as a useful preclinical model of SZ for generating hypotheses on disease mechanisms, sex-specific manifestation, and therapy.

Intracellular Fibroblast Growth Factor 14: Emerging Risk Factor for Brain Disorders.

The finely tuned regulation of neuronal firing relies on the integrity of ion channel macromolecular complexes. Minimal disturbances of these tightly regulated networks can lead to persistent maladaptive plasticity of brain circuitry. The intracellular fibroblast growth factor 14 (FGF14) belongs to the nexus of proteins interacting with voltage-gated Na+ (Nav) channels at the axonal initial segment. Through isoform-specific interactions with the intracellular C-terminal tail of neuronal Nav channels (Nav1.1, Nav1.2, Nav1.6), FGF14 controls channel gating, axonal targeting and phosphorylation in neurons effecting excitability. FGF14 has been also involved in synaptic transmission, plasticity and neurogenesis in the cortico-mesolimbic circuit with cognitive and affective behavioral outcomes. In translational studies, interest in FGF14 continues to rise with a growing list of associative links to diseases of the cognitive and affective domains such as neurodegeneration, depression, anxiety, addictive behaviors and recently schizophrenia, suggesting its role as a converging node in the etiology of complex brain disorders. Yet, a full understanding of FGF14 function in neurons is far from being complete and likely to involve other functions unrelated to the direct regulation of Nav channels. The goal of this Mini Review article is to provide a summary of studies on the emerging role of FGF14 in complex brain disorders.

Systematic identification of single amino acid variants in glioma stem-cell-derived chromosome 19 proteins.

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.