Peripherally targeted analgesia via AAV-mediated sensory neuron-specific inhibition of multiple pronociceptive sodium channels.

This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.

Impact of knockout of dual-specificity protein phosphatase 5 on structural and mechanical properties of rat middle cerebral arteries: implications for vascular aging.

Vascular aging influences hemodynamics, elevating risks for vascular diseases and dementia. We recently demonstrated that knockout (KO) of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This improvement correlates with elevated pPKC and pERK1/2 levels in the brain and kidneys. Additionally, we observed that Dusp5 KO modulates the passive mechanical properties of cerebral and renal arterioles, associated with increased myogenic tone at low pressure, enhanced distensibility, greater compliance, and reduced stiffness. The present study evaluates the structural and mechanical properties of the middle cerebral artery (MCA) in Dusp5 KO rats. We found that vascular smooth muscle cell layers and the collagen content in the MCA wall are comparable between Dusp5 KO and control rats. The internal elastic lamina in the MCA of Dusp5 KO rats exhibits increased thickness, higher autofluorescence intensity, smaller fenestrae areas, and fewer fenestrations. Despite an enhanced myogenic response and tone of the MCA in Dusp5 KO rats, other passive mechanical properties, such as wall thickness, cross-sectional area, wall-to-lumen ratio, distensibility, incremental elasticity, circumferential wall stress, and elastic modulus, do not significantly differ between strains. These findings suggest that while Dusp5 KO has a limited impact on altering the structural and mechanical properties of MCA, its primary role in ameliorating hemodynamics and cognitive functions is likely attributable to its enzymatic activity on cerebral arterioles. Further research is needed to elucidate the specific enzymatic mechanisms and explore potential clinical applications in the context of vascular aging.

Role of Dusp5 KO on Vascular Properties of Middle Cerebral Artery in Rats.

Vascular aging influences hemodynamics, elevating risks for vascular diseases and dementia. We recently demonstrated that knockout (KO) of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This improvement correlates with elevated pPKC and pERK1/2 levels in the brain and kidneys. Additionally, we observed that Dusp5 KO modulates the passive mechanical properties of cerebral and renal arterioles, associated with increased myogenic tone at low pressure, enhanced distensibility, greater compliance, and reduced stiffness. The present study evaluates the structural and mechanical properties of the middle cerebral artery (MCA) in Dusp5 KO rats. We found that vascular smooth muscle cell layers and the collagen content in the MCA wall are comparable between Dusp5 KO and control rats. The internal elastic lamina in the MCA of Dusp5 KO rats exhibits increased thickness, higher autofluorescence intensity, smaller fenestrae areas, and fewer fenestrations. Despite an enhanced myogenic response and tone of the MCA in Dusp5 KO rats, other passive mechanical properties, such as wall thickness, cross-sectional area, wall-to-lumen ratio, distensibility, incremental elasticity, circumferential wall stress, and elastic modulus, do not significantly differ between strains. These findings suggest that while Dusp5 KO has a limited impact on altering the structural and mechanical properties of MCA, its primary role in ameliorating hemodynamics and cognitive functions is likely attributable to its enzymatic activity on cerebral arterioles. Further research is needed to elucidate the specific enzymatic mechanisms and explore potential clinical applications in the context of vascular aging.

Selective RNAi-silencing of Schwann cell Piezo1 alleviates mechanical hypersensitization following peripheral nerve injury.

We previously reported functional Piezo1 expression in Schwann cells of the peripheral nervous system. This study is designed to further investigate the role of Schwann cell Piezo1 in peripheral nociception. We first developed an adeno-associated viral (AAV) vector that has primary Schwann cell tropism after delivery into the sciatic nerve. This was achieved by packing AAV-GFP transcribed by a hybrid CMV enhancer/chicken ß-actin (CBA) promoter using a capsid AAVolig001 to generate AAVolig001-CBA-GFP. Five weeks after intrasciatic injection of AAVolig001-CBA-GFP in naïve rats, GFP expression was detected selectively in the Schwann cells of the sciatic nerve. A short hairpin RNA against rat Piezo1 (PZ1shRNA) was designed that showed efficient physical and functional knockdown of Piezo1 in NG108 neuronal cells. A dual promoter and bidirectional AAV encoding a U6-driven PZ1shRNA and CBA-transcribed GFP was packed with capsid olig001 (AAVolig001-PZ1shRNA), and AAV was injected into unilateral sciatic nerve immediately after induction of common peroneal nerve injury (CPNI). Results showed that the development of mechanical hypersensitivity in the CPNI rats injected with AAVolig001-PZ1shRNA was mitigated, compared to rats subjected with AAVolig001-scramble. Selective in vivo Schwann cell transduction and functional block of Piezo1 channel activity of primary cultured Schwann cells was confirmed. Together, our data demonstrate that 1) AAVolig001 has unique and selective primary tropism to Schwann cells via intrasciatic delivery and 2) Schwann cell Piezo1 contributes to mechanical hypersensitivity following nerve injury.

Enhanced Cerebral Hemodynamics and Cognitive Function Via Knockout of Dual-Specificity Protein Phosphatase 5.

Alzheimer's Disease (AD) and Alzheimer's Disease-Related Dementias (ADRD) are neurodegenerative disorders. Recent studies suggest that cerebral hypoperfusion is an early symptom of AD/ADRD. Dual-specificity protein phosphatase 5 (DUSP5) has been implicated in several pathological conditions, including pulmonary hypertension and cancer, but its role in AD/ADRD remains unclear. The present study builds on our previous findings, demonstrating that inhibition of ERK and PKC leads to a dose-dependent dilation of the middle cerebral artery and penetrating arteriole, with a more pronounced effect in Dusp5 KO rats. Both ERK and PKC inhibitors resulted in a significant reduction of myogenic tone in vessels from Dusp5 KO rats. Dusp5 KO rats exhibited stronger autoregulation of the surface but not deep cortical cerebral blood flow. Inhibition of ERK and PKC significantly enhanced the contractile capacity of vascular smooth muscle cells from both strains. Finally, a significant improvement in learning and memory was observed in Dusp5 KO rats 24 hours after initial training. Our results suggest that altered vascular reactivity in Dusp5 KO rats may involve distinct mechanisms for different vascular beds, and DUSP5 deletion could be a potential therapeutic target for AD/ADRD. Further investigations are necessary to determine the effects of DUSP5 inhibition on capillary stalling, blood-brain barrier permeability, and neurodegeneration in aging and disease models.

Peripheral sensory neurons and non-neuronal cells express functional Piezo1 channels.

Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ∼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.

Longitudinal characterization of cerebral hemodynamics in the TgF344-AD rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a global healthcare crisis. The TgF344-AD rat is an AD model exhibiting age-dependent AD pathological hallmarks. We confirmed that AD rats developed cognitive deficits at 6 months without alteration of any other major biophysical parameters. We longitudinally characterized cerebral hemodynamics in AD rats at 3, 4, 6, and 14 months. The myogenic responses of the cerebral arteries and arterioles were impaired at 4 months of age in the AD rats. Consistent with the ex vivo results, the AD rat exhibited poor autoregulation of surface and deep cortical cerebral blood flow 2 months preceding cognitive decline. The dysfunction of cerebral hemodynamics in AD is exacerbated with age associated with reduced cerebral perfusion. Further, abolished cell contractility contributes to cerebral hemodynamics imbalance in AD. This may be attributed to enhanced ROS production, reduced mitochondrial respiration and ATP production, and disrupted actin cytoskeleton in cerebral vascular contractile cells.

Keratinocyte Piezo1 drives paclitaxel-induced mechanical hypersensitivity.

Recent work demonstrates that epidermal keratinocytes are critical for normal touch sensation. However, it is unknown if keratinocytes contribute to touch evoked pain and hypersensitivity following tissue injury. Here, we used inhibitory optogenetic and chemogenetic techniques to determine the extent to which keratinocyte activity contributes to the severe neuropathic pain that accompanies chemotherapeutic treatment. We found that keratinocyte inhibition largely alleviates paclitaxel-induced mechanical hypersensitivity. Furthermore, we found that paclitaxel exposure sensitizes mouse and human keratinocytes to mechanical stimulation through the keratinocyte mechanotransducer Piezo1. These findings demonstrate the contribution of non-neuronal cutaneous cells to neuropathic pain and pave the way for the development of new pain-relief strategies that target epidermal keratinocytes and Piezo1.

Selective block of sensory neuronal T-type/Cav3.2 activity mitigates neuropathic pain behavior in a rat model of osteoarthritis pain.

BACKGROUND: Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (CaV3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/CaV3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/CaV3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/CaV3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA. METHODS: MIA model of knee OA was induced in male and female rats by a single injection of 2 mg MIA into intra-knee articular cavity. Two weeks after induction of knee MIA-OA pain, recombinant adeno-associated viruses (AAV)-encoding potent CaV3.2 inhibitory peptide aptamer 2 (CaV3.2iPA2) that have been characterized in our previous study were delivered into the ipsilateral lumbar 4/5 DRG. Effectiveness of DRG-CaV3.2iPA2 treatment on evoked (mechanical and thermal) and spontaneous (conditioned place preference) pain behavior, as well as weight-bearing asymmetry measured by Incapacitance tester, in the arthritic limbs of MIA rats were evaluated. AAV-mediated transgene expression in DRG was determined by immunohistochemistry. RESULTS: AAV-mediated expression of CaV3.2iPA2 selective in the DRG-PSNs produced significant and comparable mitigations of evoked and spontaneous pain behavior, as well as normalization of weight-bearing asymmetry in both male and female MIA-OA rats. Analgesia of DRG-AAV-CaV3.2iPA1, another potent CaV3.2 inhibitory peptide, was also observed. Whole-cell current-clamp recordings showed that AAV-mediated CaV3.2iPA2 expression normalized hyperexcitability of the PSNs dissociated from the DRG of MIA animals, suggesting that CaV3.2iPA2 attenuated pain behavior by reversing MIA-induced neuronal hyperexcitability. CONCLUSIONS: Together, our results add therapeutic support that T-type/CaV3.2 in primary sensory pathways contributes to MIA-OA pain pathogenesis and that CaV3.2iPAs are promising analgesic leads that, combined with AAV-targeted delivery in anatomically segmental sensory ganglia, have the potential for further development as a peripheral selective T-type/CaV3.2-targeting strategy in mitigating chronic MIA-OA pain behavior. Validation of the therapeutic potential of this strategy in other OA models may be valuable in future study.

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus-mediated peripheral analgesia.

ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.

S-SCAM inhibits Axin-dependent synaptic function of GSK3ß in a sex-dependent manner.

S-SCAM/MAGI-2 gene duplication is associated with schizophrenia (SCZ). S-SCAM overexpression in the forebrain induces SCZ-like phenotypes in a transgenic (Tg) mouse model. Interestingly, S-SCAM Tg mice show male-specific impairments in synaptic plasticity and working memory. However, mechanisms underlying the sex-specific deficits remain unknown. Here we report that S-SCAM Tg mice have male-specific deficits in synaptic GSK3ß functions, as shown by reduced synaptic protein levels and increased inhibitory phosphorylation of GSK3ß. This GSK3ß hyper-phosphorylation was associated with increased CaMKII activities. Notably, synaptic levels of Axin1, to which GSK3ß binds in competition with S-SCAM, were also reduced in male S-SCAM Tg mice. We demonstrated that Axin-binding is required for the S-SCAM overexpression-induced synaptic GSK3ß reduction. Axin stabilization using XAV939 rescued the GSK3ß deficits and restored the temporal activation of GSK3ß during long-term depression in S-SCAM overexpressing neurons. Interestingly, synaptic Axin2 levels were increased in female S-SCAM Tg mice. Female sex hormone 17ß-estradiol increased Axin2 expression and increased synaptic GSK3ß levels in S-SCAM overexpressing neurons. These results reveal the role of S-SCAM in controlling Axin-dependent synaptic localization of GSK3ß. Moreover, our studies point out the pathological relevance of GSK3ß hypofunction found in humans and contribute to understanding the molecular underpinnings of sex differences in SCZ.

Sigma-1 receptor activity in primary sensory neurons is a critical driver of neuropathic pain.

The Sigma-1 receptor (σ1R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ1R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ1R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ1R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ1R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ1R suppressed neuronal excitability, suggesting that σ1R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ1R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ1R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ1R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.

Combination Therapy of the Active KRAS-Targeting Antibody inRas37 and a PI3K Inhibitor in Pancreatic Cancer.

KRAS activating mutations, which are present in more than 90% of pancreatic cancers, drive tumor dependency on the RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Therefore, combined targeting of RAS/MAPK and PI3K/AKT signaling pathways may be required for optimal therapeutic effect in pancreatic cancer. However, the therapeutic efficacy of combined MAPK and PI3K/AKT signaling target inhibitors is unsatisfactory in pancreatic cancer treatment, because it is often accompanied by MAPK pathway reactivation by PI3K/AKT inhibitor. Therefore, we developed an inRas37 antibody, which directly targets the intra-cellularly activated GTP-bound form of oncogenic RAS mutation and investigated its synergistic effect in the presence of the PI3K inhibitor BEZ-235 in pancreatic cancer. In this study, inRas37 remarkably increased the drug response of BEZ-235 to pancreatic cancer cells by inhibiting MAPK reactivation. Moreover, the co-treatment synergistically inhibited cell proliferation, migration, and invasion and exhibited synergistic anticancer activity by inhibiting the MAPK and PI3K pathways. The combined administration of inRas37and BEZ-235 significantly inhibited tumor growth in mouse models. Our results demonstrated that inRas37 synergistically increased the antitumor activity of BEZ-235 by inhibiting MAPK reactivation, suggesting that inRas37 and BEZ-235 co-treatment could be a potential treatment approach for pancreatic cancer patients with KRAS mutations.

Piezo2 mechanosensitive ion channel is located to sensory neurons and nonneuronal cells in rat peripheral sensory pathway: implications in pain.

ABSTRACT: Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naïve rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.

One-Step Etching Characteristics of ITO/Ag/ITO Multilayered Electrode in High-Density and High-Electron-Temperature Plasma.

This paper presents the dry etching characteristics of indium tin oxide (ITO)/Ag/ITO multilayered thin film, used as a pixel electrode in a high-resolution active-matrix organic light-emitting diode (AMOLED) device. Dry etching was performed using a combination of H2 and HCl gases in a reactive ion etching system with a remote electron cyclotron resonance (ECR) plasma source, in order to achieve high electron temperature. The effect of the gas ratio (H2/HCl) was closely observed, in order to achieve an optimal etch profile and an effective etch process, while other parameters-such as the radio frequency (RF) power, ECR power, chamber pressure, and temperature-were fixed. The optimized process, with an appropriate gas ratio, constitutes a one-step serial dry etch solution for ITO and Ag multilayered thin films.

Intracellular KRAS-specific antibody enhances the anti-tumor efficacy of gemcitabine in pancreatic cancer by inducing endosomal escape.

KRAS mutation is associated with the progression and growth of pancreatic cancer and contributes to chemo-resistance, which poses a significant clinical challenge in pancreatic cancer. Here, we developed a RT22-ep59 antibody (Ab) that directly targets the intracellularly activated GTP-bound form of oncogenic KRAS mutants after it is internalized into cytosol by endocytosis through tumor-associated receptor of extracellular epithelial cell adhesion molecule (EpCAM) and investigated its synergistic anticancer effects in the presence of gemcitabine in pancreatic cancer. We first observed that RT22-ep59 specifically recognized tumor-associated EpCAM and reached the cytosol by endosomal escape. In addition, the anticancer effect of RT22-ep59 was observed in the high-EpCAM-expressing pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells, but it had little effect on the low-EpCAM-expressing pancreatic cancer cells. Additionally, co-treatment with RT22-ep59 and gemcitabine synergistically inhibited cell viability, migration, and invasion in 3D-cultures and exhibited synergistic anticancer activity by inhibiting the RAF/ERK or PI3K/AKT pathways in cells with high-EpCAM expression. In an orthotopic mouse model, combined administration of RT22-ep59 and gemcitabine significantly inhibited tumor growth. Furthermore, the co-treatment suppressed cancer metastasis by blocking EMT signaling in vitro and in vivo. Our results demonstrated that RT22-ep59 synergistically increased the antitumor activity of gemcitabine by inhibiting RAS signaling by specifically targeting KRAS. This indicates that co-treatment with RT22-ep59 and gemcitabine might be considered a potential therapeutic strategy for pancreatic cancer patients harboring KRAS mutation.

Enhanced T-type calcium channel 3.2 activity in sensory neurons contributes to neuropathic-like pain of monosodium iodoacetate-induced knee osteoarthritis.

The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.

Satellite glial cells in sensory ganglia express functional transient receptor potential ankyrin 1 that is sensitized in neuropathic and inflammatory pain.

Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund's adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.

Direct targeting of oncogenic RAS mutants with a tumor-specific cytosol-penetrating antibody inhibits RAS mutant-driven tumor growth.

Oncogenic RAS mutant (RASMUT) proteins have been considered undruggable via conventional antibody regimens owing to the intracellular location restricting conventional-antibody accessibility. Here, we report a pan-RAS-targeting IgG antibody, inRas37, which directly targets the intracellularly activated form of various RASMUT subtypes after tumor cell-specific internalization into the cytosol to block the interactions with effector proteins, thereby suppressing the downstream signaling. Systemic administration of inRas37 exerted a potent antitumor activity in a subset of RASMUT tumor xenografts in mice, but little efficacy in RASMUT tumors with concurrent downstream PI3K mutations, which were overcome by combination with a PI3K inhibitor. The YAP1 protein was up-regulated as an adaptive resistance-inducing response to inRas37 in RASMUT-dependent colorectal tumors; accordingly, a combination of inRas37 with a YAP1 inhibitor manifested synergistic antitumor effects in vitro and in vivo. Our study offers a promising pan-RAS-targeting antibody and the corresponding therapeutic strategy against RASMUT tumors.

Aberrant expression of S-SCAM causes the loss of GABAergic synapses in hippocampal neurons.

The duplication and deletion mutations of the S-SCAM/MAGI-2 gene are associated with schizophrenia and infantile spasms, respectively. S-SCAM is a unique synaptic scaffolding protein that localizes to both excitatory and GABAergic synapses. However, consequences of aberrant S-SCAM expression on GABAergic synapses is little studied. Here we report the effect of S-SCAM knockdown and overexpression on GABAergic synapses. S-SCAM knockdown in cultured hippocampal neurons caused a drastic loss of both pre- and post-synaptic components of GABAergic synapses, indicating its essential role in GABAergic synapse formation and maintenance. Surprisingly, S-SCAM overexpression also attenuated GABAergic synapses, but the effect is mediated by the loss of postsynaptic GABAA receptors, gephyrin, and neuroligin 2 and does not involve presynaptic component vesicular GABA transporters. Overexpression studies using S-SCAM mutants with various domain deletions indicated that GABAergic synapse loss correlates with their ability to increase excitatory synaptic function. Consistently, AMPA receptor antagonist CNQX or calcineurin inhibitor FK506 abolished the S-SCAM overexpression-induced loss of GABAA receptors, supporting that GABAergic synapse loss by S-SCAM overexpression is due to the activity-induced dispersal of synaptic GABAA receptors. These results suggest that abnormal S-SCAM protein levels disrupt excitation/inhibition balance in neurons, which may explain the pathogenic nature of S-SCAM copy number variations.