Natural harmaline acts as novel fluorescent probe for hypochlorous acid and promising therapeutic candidate for rheumatoid arthritis.

Endogenous hypochlorous acid (HOCl) is one of the most important reactive oxygen species (ROS) and acts as a distinct biomarker that is involved in various inflammatory responses including rheumatoid arthritis (RA). Therefore, it's crucial to develop an efficient method for the tracking and analysis of HOCl levels in vivo. Natural products continue to be compounds of interest, because they not only offer diverse and specific molecular scaffolds but also provide invaluable sources for new drug discovery. Herein, we firstly demonstrated harmaline (HML), a natural alkaloid mainly found in Peganum harmala L, could be acted as a novel fluorescent probe for HOCl with exceptional precision and responsiveness. Remarkably, this probe not only specifically tracked HOCl levels in cells and inflammatory RA mouse models, but also exhibited effective anti-inflammatory effects on RAW264.7 cells and anti-proliferative effects on fibroblast-like synoviocytes. Furthermore, HML has the potential to alleviate LPS-induced inflammation by inhibiting the NF-κB signaling pathway. This study represents the first example of a natural product that can simultaneously act as a fluorescent probe for specific ROS and a promising therapeutic candidate for a specific disease, which will undoubtedly extend the application of fluorophore-rich natural products.

Phytochemical Profile, Antioxidant, Anti-Alzheimer, And α-Glucosidase Inhibitory Effect Of Algerian Peganum harmala Seeds Extract.

Peganum harmala seeds crude hydro-methanolic extract and their fractions (obtained with ethyl acetate and butan-1-ol) were analyzed and compared for their chemical profiles of alkaloids and polyphenols content. Moreover, their antioxidant, a-glucosidase, acetylcholinesterase, and butyrylcholinesterase inhibitory activities were evaluated. The butan-1-ol fraction revealed the highest total phenolic content and exhibited the highest antioxidant capacity. From the inhibitory enzyme evaluations, it should be highlighted the butan-1-ol fraction inhibitory potential of ɑ-glucosidase (the IC50= 141.18±4µg/mL), which was better than the acarbose inhibitory effect (IC50= 203.41±1.07 µg/mL). The extracts' chemical profile analysis revealed several compounds, in which quercetin dimethyl ether, harmine and harmaline emerged as the major compounds. The different solvents used impacted Peganum harmala seed contents and biological responses. Statistical analysis showed a significant correlation between bioactive compounds and biological activities. Thus, Peganum harmala seeds could be a promising natural source of bioactive compounds at the crossroads of many human diseases, and its cultivation may be encouraged.

Repurposing harmaline as a novel approach to reverse tmexCD1-toprJ1-mediated tigecycline resistance against klebsiella pneumoniae infections.

BACKGROUND: A novel plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 in Klebsiella pneumoniae tremendously threatens the use of convenient therapeutic options in the post-antibiotic era, including the "last-resort" antibiotic tigecycline. RESULTS: In this work, the natural alkaloid harmaline was found to potentiate tigecycline efficacy (4- to 32-fold) against tmexCD1-toprJ1-positive K. pneumoniae, which also thwarted the evolution of tigecycline resistance. Galleria mellonella and mouse infection models in vivo further revealed that harmaline is a promising candidate to reverse tigecycline resistance. Inspiringly, harmaline works synergistically with tigecycline by undermining tmexCD1-toprJ1-mediated multidrug resistance efflux pump function via interactions with TMexCD1-TOprJ1 active residues and dissipation of the proton motive force (PMF), and triggers a vicious cycle of disrupting cell membrane integrity and metabolic homeostasis imbalance. CONCLUSION: These results reveal the potential of harmaline as a novel tigecycline adjuvant to combat hypervirulent K. pneumoniae infections.

Resveratrol and 1,25-dihydroxyvitamin D decrease Lingo-1 levels, and improve behavior in harmaline-induced Essential tremor, suggesting potential therapeutic benefits.

Essential tremor (ET) is a neurological disease that impairs motor and cognitive functioning. A variant of the Lingo-1 genetic locus is associated with a heightened ET risk, and increased expression of cerebellar Lingo-1. Lingo-1 has been associated with neurodegenerative processes; however, neuroprotection from ET-associated degeneration can be conferred by the protein Sirt1. Sirt1 activity can be promoted by Resveratrol (Res) and 1,25-dihydroxyvitamin D3 (VitD3), and thus these factors may exert neuroprotective properties through a Sirt1 mechanism. As Res and VitD3 are linked to Sirt1, enhancing Sirt1 could counteract the negative effects of increased Lingo-1. Therefore, we hypothesized that a combination of Res-VitD3 in a harmaline injection model of ET would modulate Sirt1 and Lingo-1 levels. As expected, harmaline exposure (10 mg/kg/every other day; i.p.) impaired motor coordination, enhanced tremors, rearing, and cognitive dysfunction. When Res (5 mg/kg/day; i.p.) and VitD3 (0.1 mg/kg/day; i.p.) were given to adult rats (n = 8 per group) an hour before harmaline, tremor severity, rearing, and memory impairment were reduced. Individual treatment with Res and VitD3 decreased Lingo-1 gene expression levels in qPCR assays. Co-treatment with Res and VitD3 increased and decreased Sirt1 and Lingo-1 gene expression levels, respectively, and in some cases, beneficial effects on behavior were noted, which were not seen when Res or VitD3 were individually applied. Taken together, our study found that Res and VitD3 improved locomotor and cognitive deficits, modulated Sirt1 and Lingo-1. Therefore, we would recommend co-treatment of VitD3 and Res to leverage complementary effects for the management of ET symptoms.

A Role for GABAA Receptor ß3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Background: Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6ß3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the ß3 subunit for tremor suppression. Methods: We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact ß3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Results: Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed ß3 cre- mice, but had no effect on tremor in floxed ß3 cre+ littermates that have ß3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed ß3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. Discussion: As α6ß3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express ß3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6ß3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. Highlights: We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that ß3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6ß3δ extra-synaptic GABAA receptors.

Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of ß-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.

In vitro pro-apoptotic and anti-metastatic potentials of harmaline-silver containing folate-linked chitosan nano-drug delivery system.

Objectives: Harmaline and green-synthesized silver nanoparticles were encapsulated by folate-linked chitosan molecules as a receptor-mediated drug delivery system to evaluate its pro-apoptotic and anti-metastatic potentials on human ovarian (A2780) and epithelioid (PANC) cancer cells. Materials and Methods: The Ag nanoparticles (AgNP) were synthesized utilizing an herbal bio-platform (Bistorta officinalis) and embedded with harmalin. The Harmaline-ag containing folate-linked chitosan nanoparticles (HA-fCNP) were synthesized utilizing the ionic gelation method. Both the AgNP and HA-fCNP nanoparticles were characterized by DLS, FESEM, and Zeta potential analysis. Moreover, the chemical properties of HA-fCNP and the crystallinity of AgNPs were determined by applying FTIR and XRD methods, respectively. The HA-fCNP cytotoxicity was analyzed on A2780, PANC, and HFF cell lines. Moreover, pro-apoptotic and anti-metastatic potentials of HA-fCNP were studied by analyzing the BAX-BCL2 and MMP2-MMP9 gene expression profiles, respectively. The A2780 cellular death was determined by AO/PI and flow cytometry methods. Results: The HA-fCNP significantly exhibited a selective cytotoxic impact on A2780 and PANC cancerous cell lines compared with normal human foreskin fibroblast (HFF) cells. The increased SubG1-arrested A2780 cells and up-regulated BAX gene expression following the increased treatment concentrations of hA-fCNP indicated its selective pro-apoptotic activity on A2780 cells. Also, the notable down-regulated expressions of MMP2 and MMP9 metastatic genes following the increasing doses of HA-fCNP treatment on A2780 cells confirmed its anti-metastatic activity. Conclusion: The cancer-selective cytotoxicity, apoptotic, and anti-metastatic properties of HA-fCNP are considered the appropriate properties of an anticancer compound.

The Neuroanatomical Basis of the 5-HT Syndrome and Harmalineinduced Tremor.

The 5-HT syndrome in rats is composed of head weaving, body shaking, forepaw treading, flat body posture, hindlimb abduction, and Straub tail. The importance of the brainstem and spinal cord for the syndrome is underlined by findings of 5,7-dihydroxytryptamine (5,7-DHT)-induced denervation supersensitivity in response to 5-HT-stimulant drugs. For head weaving and Straub tail, supersensitivity occurred when the neurotoxin was injected into the cisterna magna or spinal cord, for forepaw treading in cisterna magna, and for hindlimb abduction in the spinal cord. Although 5,7- DHT-related body shaking increased in the spinal cord, the sign decreased when injected into the striatum, indicating the modulatory influence of the basal ganglia. Further details on body shaking are provided by its reduced response to harmaline after 5-HT depletion caused by intraventricular 5,7-DHT, electrolytic lesions of the medial or dorsal raphe, and lesions of the inferior olive caused by systemic injection of 3-acetylpyridine along with those found in Agtpbp1pcd or nr cerebellar mouse mutants. Yet the influence of the climbing fiber pathway on other signs of the 5-HT syndrome remains to be determined.

Evaluation of therapeutic role of harmaline: in vitro cytotoxicity targeting nucleic acids.

Use of small molecules as valuable drugs against diseases is still an indefinable purpose due to the lack of in-detail knowledge regarding proper bio-target identification, specificity aspects, mode-mechanism of binding and proper in vitro study. Harmaline, an important beta-carboline alkaloid, shows effective anti-proliferative action against different types of human cancers and is also found to be a nucleic acid targeting natural molecule. This review sought to address the different signal pathways of apoptosis by harmaline in different cancer cell lines and simultaneously to characterize the structure activity aspects of the alkaloid with different motifs of nucleic acid to show its preference, biological efficacy and genotoxicity. The results open up new insights for the design and development of small molecule-based nucleic acid therapeutic agents.

Search for Novel Therapies for Essential Tremor Based on Positive Modulation of α6-Containing GABAA Receptors.

Background: Prior work using GABAA receptor subunit knockouts and the harmaline model has indicated that low-dose alcohol, gaboxadol, and ganaxolone suppress tremor via α6ßδ GABAA receptors. This suggests that drugs specifically enhancing the action of α6ßδ or α6ßγ2 GABAA receptors, both predominantly expressed on cerebellar granule cells, would be effective against tremor. We thus examined three drugs described by in vitro studies as selective α6ßδ (ketamine) or α6ßγ2 (Compound 6, flumazenil) receptor modulators. Methods: In the first step of evaluation, the maximal dose was sought at which 6/6 mice pass straight wire testing, a sensitive test for psychomotor impairment. Only non-impairing doses were used to evaluate for anti-tremor efficacy in the harmaline model, which was assessed in wildtype and α6 subunit knockout littermates. Results: Ketamine, in maximally tolerated doses of 2.0 and 3.5 mg/kg had minimal effect on harmaline tremor in both genotypes. Compound 6, at well-tolerated doses of 1-10 mg/kg, effectively suppressed tremor in both genotypes. Flumazenil suppressed tremor in wildtype mice at doses (0.015-0.05 mg/kg) far lower than those causing straight wire impairment, and did not suppress tremor in α6 knockout mice. Discussion: Modulators of α6ßδ and α6ßγ2 GABAA receptors warrant attention for novel therapies as they are anticipated to be effective and well-tolerated. Ketamine likely failed to attain α6ßδ-active levels. Compound 6 is an attractive candidate, but further study is needed to clarify its mechanism of action. The flumazenil results provide proof of principle that targeting α6ßγ2 receptors represents a worthy strategy for developing essential tremor therapies. Highlights: We tested for harmaline tremor suppression drugs previously described as in vitro α6ßδ or α6ßγ2 GABAA receptor-selective modulators. Well-tolerated flumazenil doses suppressed tremor in α6-wildtype but not α6-knockout mice. Compound 6 and ketamine failed to display this profile, likely from off-target effects. Selective α6 modulators hold promise as tremor therapy.

Harmaline toxicity on dorsal striatal neurons and its role in tremor.

Harmaline is one of the ß-carboline derivative compounds that is widely distributed in the food chain and human tissues. Harmine, a dehydrogenated form of harmaline, appeared to have a higher concentration in the brain, and appeared to be elevated in essential tremor (ET) and Parkinson's disease. Exogenous harmaline exposure in high concentration has myriad consequences, including inducing tremor, and causing neurodegeneration of Purkinje cells in the cerebellum. Harmaline-induced tremor is an established animal model for human ET, but its underlying mechanism is still controversial. One hypothesis posits that the inferior olive-cerebellum pathway is involved, and CaV3.1 T-type Ca2+ channel is a critical target of action. However, accumulating evidence indicates that tremor can be generated without disturbing T-type channels. This implies that additional neural circuits or molecular targets are involved. Using in vitro slice Ca2+-imaging and patch clamping, we demonstrated that harmaline reduced intracellular Ca2+ and suppressed depolarization-induced spiking activity of medium spiny striatal neurons (MSN), and this effect of harmaline can be partially attenuated by sulpiride (5 µM). In addition, the frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on MSNs were also significantly attenuated. Furthermore, the induced tremor in C57BL/6 J mice by harmaline injections (i.p. 12.5-18 mg/kg) was also shown to be attenuated by sulpiride (20 mg/kg). This series of experiments suggests that the dorsal striatum is a site of harmaline toxic action and might contribute to tremor generation. The findings also provide evidence that D2 signaling might be a part of the mechanism underlying essential tremor.

Mapping Pharmacologically Evoked Neurovascular Activation and Its Suppression in a Rat Model of Tremor Using Functional Ultrasound: A Feasibility Study.

Functional ultrasound (fUS), an emerging hemodynamic-based functional neuroimaging technique, is especially suited to probe brain activity and primarily used in animal models. Increasing use of pharmacological models for essential tremor extends new research to the utilization of fUS imaging in such models. Harmaline-induced tremor is an easily provoked model for the development of new therapies for essential tremor (ET). Furthermore, harmaline-induced tremor can be suppressed by the same classic medications used for essential tremor, which leads to the utilization of this model for preclinical testing. However, changes in local cerebral activities under the effect of tremorgenic doses of harmaline have not been completely investigated. In this study, we explored the feasibility of fUS imaging for visualization of cerebral activation and deactivation associated with harmaline-induced tremor and tremor-suppressing effects of propranolol. The spatial resolution of fUS using a high frame rate imaging enabled us to visualize time-locked and site-specific changes in cerebral blood flow associated with harmaline-evoked tremor. Intraperitoneal administration of harmaline generated significant neural activity changes in the primary motor cortex and ventrolateral thalamus (VL Thal) regions during tremor and then gradually returned to baseline level as tremor subsided with time. To the best of our knowledge, this is the first functional ultrasound study to show the neurovascular activation of harmaline-induced tremor and the therapeutic suppression in a rat model. Thus, fUS can be considered a noninvasive imaging method for studying neuronal activities involved in the ET model and its treatment.

Alcohol and Ganaxolone Suppress Tremor via Extra-Synaptic GABAA Receptors in the Harmaline Model of Essential Tremor.

Background: A long-standing question is why essential tremor often responds to non-intoxicating amounts of alcohol. Blood flow imaging and high-density electroencephalography have indicated that alcohol acts on tremor within the cerebellum. As extra-synaptic δ-subunit-containing GABAA receptors are sensitive to low alcohol levels, we wondered whether these receptors mediate alcohol's anti-tremor effect and, moreover, whether the δ-associated GABAA receptor α6 subunit, found abundantly in the cerebellum, is required. Methods: We tested the hypotheses that low-dose alcohol will suppress harmaline-induced tremor in wild-type mice, but not in littermates lacking GABAA receptor δ subunits, nor in littermates lacking α6 subunits. As the neurosteroid ganaxolone also activates extra-synaptic GABAA receptors, we similarly assessed this compound. The harmaline mouse model of essential tremor was utilized to generate tremor, measured as a percentage of motion power in the tremor bandwidth (9-16 Hz) divided by background motion power at 0.25-32 Hz. Results: Ethanol, 0.500 and 0.575 g/kg, and ganaxolone, 7 and 10 mg/kg, doses that do not impair performance in a sensitive psychomotor task, reduced harmaline tremor compared to vehicle-treated controls in wild-type mice but failed to suppress tremor in littermates lacking the δ or the α6 GABAA receptor subunit. Discussion: As cerebellar granule cells are the predominant brain site intensely expressing GABAA receptors containing both α6 and δ subunits, these findings suggest that this is where alcohol acts to suppress tremor. It is anticipated that medications designed specifically to target α6ßδ-containing GABAA receptors may be effective and well-tolerated for treating essential tremor. Highlights: How does alcohol temporarily ameliorate essential tremor? This study with a mouse model found that two specific kinds of GABA receptor subunits were needed for alcohol to work. As receptors with both these subunits are found mainly in cerebellum, this work suggests this is where alcohol acts to suppress tremor.

Chemical suppression of harmaline-induced body tremor yields recovery of pairwise neuronal coherence in cerebellar nuclei neurons.

Neuronal oscillations occur in health and disease; however, their characteristics can differ across conditions. During voluntary movement in freely moving rats, cerebellar nuclei (CN) neurons display intermittent but coherent oscillations in the theta frequency band (4-12 Hz). However, in the rat harmaline model of essential tremor, a disorder attributed to cerebellar malfunction, CN neurons display aberrant oscillations concomitantly with the emergence of body tremor. To identify the oscillation features that may underlie the emergence of body tremor, we analyzed neuronal activity recorded chronically from the rat CN under three conditions: in freely behaving animals, in harmaline-treated animals, and during chemical suppression of the harmaline-induced body tremor. Suppression of body tremor did not restore single neuron firing characteristics such as firing rate, the global and local coefficients of variation, the likelihood of a neuron to fire in bursts or their tendency to oscillate at a variety of dominant frequencies. Similarly, the fraction of simultaneously recorded neuronal pairs oscillating at a similar dominant frequency (<1 Hz deviation) and the mean frequency deviation within pairs remained similar to the harmaline condition. Moreover, the likelihood that pairs of CN neurons would co-oscillate was not only significantly lower than that measured in freely moving animals, but was significantly worse than chance. By contrast, the chemical suppression of body tremor fully restored pairwise neuronal coherence; that is, unlike in the harmaline condition, pairs of neurons that oscillated at the same time and frequency displayed high coherence, as in the controls. We suggest that oscillation coherence in CN neurons is essential for the execution of smooth movement and its loss likely underlies the emergence of body tremor.

Dapagliflozin modulates neuronal injury via instigation of LKB1/p-AMPK/GABAB R2 signaling pathway and suppression of the inflammatory cascade in an essential tremor rat model.

BACKGROUND: However, disturbances in cellular energy demarcate neuronal hyperexcitability in essential tremor (ET); nevertheless, no available data relates energy sensors and GABAergic neurotransmission in ET. Noteworthy, reports have asserted dapagliflozin's (DAPA) role in enhancing autophagic sensors in other disorders. Herein, this study aims to investigate DAPA's impact on the GABAB receptor subunit (GABAB R2), notwithstanding the GABA A involvement, in an ET model. METHODS: ET was induced by a single dose of harmaline (30 mg/kg; i.p.), while DAPA (1 mg/kg/day; p.o.) was given for 5 days before ET induction. The autophagic sensors were examined by injecting a single dose of dorsomorphin (DORSO) AMPK inhibitor (0.2 mg/kg; i.p.) on the 5th day before ET induction. RESULTS: DAPA decreased the HAR-induced tremor score and alleviated motor disabilities observed in the open field, rotarod, wire grip strength, and gait kinematics confirmed by reduced electrical activity in electroencephalogram. In the cerebella, DAPA curbed HAR-evoked inflammatory cytokines, apoptotic markers, and glutamate while restoring the disturbed GABA, BDNF, LKB1, p-AMPK, and GABAB R2 levels. DAPA's effect was mostly obliterated by DORSO. CONCLUSION: DAPA offers a potential neuroprotective effect in ET by augmenting the neuronal inhibitory machinery via suppressing the inflammatory and excitotoxicity systems through LKB1/p-AMPK/GABAB R2 signaling.

Harmaline exerts potentially anti-cancer effects on U-87 human malignant glioblastoma cells in vitro.

BACKGROUND: Harmaline is a ß-carboline alkaloid that can be extracted from the seeds of Peganum harmala. Harmaline has been shown to exhibit a potent cytotoxic effect against tumor cells. In this study, the anti-glioblastoma activity of harmaline was investigated in vitro. METHODS AND RESULTS: Cell viability, apoptosis, and cell cycle arrest were assessed in U-87 cells treated with harmaline at different doses. Reactive oxygen species (ROS) generation and the mRNA expression of apoptosis-associated genes were assessed. The anti-metastatic effect of harmaline on U-87 cells was evaluated by gelatin zymography assay where matrix metalloproteinase [MMP]-2/-9 enzymatic activity was measured, and the scratch assay was used to assess migratory responses. Flow cytometry demonstrated that harmaline could suppress the proliferation and induce sub-G1 cell cycle arrest and apoptotic cell death in glioblastoma cells. Harmaline treatment was also associated with an upregulation of the cell cycle-related genes, p21 and p53, and pro-apoptotic Bax, as well as the induction of ROS. The zymography assay indicated that the essential steps of metastasis were potently suppressed by harmaline through inhibiting the expression of MMP-2 and - 9. In addition, the migration of U-87 cells was significantly reduced after harmaline treatment. CONCLUSION: Our data suggest a basis for further research of harmaline which has potential cytotoxic activities in glioblastoma cells; inducing cell cycle arrest and apoptosis, repression of migration, possibly invasion, and metastasis.

Cerebellar α6GABAA Receptors as a Therapeutic Target for Essential Tremor: Proof-of-Concept Study with Ethanol and Pyrazoloquinolinones.

Ethanol has been shown to suppress essential tremor (ET) in patients at low-to-moderate doses, but its mechanism(s) of action remain unknown. One of the ET hypotheses attributes the ET tremorgenesis to the over-activated firing of inferior olivary neurons, causing synchronic rhythmic firings of cerebellar Purkinje cells. Purkinje cells, however, also receive excitatory inputs from granule cells where the α6 subunit-containing GABAA receptors (α6GABAARs) are abundantly expressed. Since ethanol is a positive allosteric modulator (PAM) of α6GABAARs, such action may mediate its anti-tremor effect. Employing the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of ethanol and α6GABAAR-selective pyrazoloquinolinone PAMs. The burrowing activity, an indicator of well-being in rodents, was measured concurrently. Ethanol significantly and dose-dependently attenuated action tremor at non-sedative doses (0.4-2.4 g/kg, i.p.). Propranolol and α6GABAAR-selective pyrazoloquinolinones also significantly suppressed tremor activity. Neither ethanol nor propranolol, but only pyrazoloquinolinones, restored burrowing activity in harmaline-treated mice. Importantly, intra-cerebellar micro-injection of furosemide (an α6GABAAR antagonist) had a trend of blocking the effect of pyrazoloquinolinone Compound 6 or ethanol on harmaline-induced tremor. In addition, the anti-tremor effects of Compound 6 and ethanol were synergistic. These results suggest that low doses of ethanol and α6GABAAR-selective PAMs can attenuate action tremor, at least partially by modulating cerebellar α6GABAARs. Thus, α6GABAARs are potential therapeutic targets for ET, and α6GABAAR-selective PAMs may be a potential mono- or add-on therapy.

Epicranial Direct Current Stimulation Suppresses Harmaline Tremor in Rats.

INTRODUCTION: Essential tremor (ET) is the most common neurologic movement disorder worldwide. It is characterized by a postural tremor, mostly in the upper extremities, causing difficulties in daily activities that may lead to social exclusion. Some patients with ET do not respond well to or do not tolerate medication. Thus, deep brain stimulation can be offered. In a recent study, we proposed a novel neuromodulation technique called epicranial current stimulation (ECS) that works in a minimally invasive way by placing the electrodes subcutaneously under the skin and directly over the skull. In this study, we investigated the feasibility of using epicranial direct current stimulation (EDCS) to suppress tremor in a rat harmaline ET model. MATERIALS AND METHODS: In experiment 1, seven Sprague Dawley rats were implanted with ECS electrodes placed over the motor cortex (MC) and the cerebellum to investigate whether stimulating between them could suppress tremor. In experiments 2 and 3, eight rats were implanted with ECS electrodes placed over the MC, cerebellum, and the rostral skull to separate the effects on tremor caused by stimulating each target. During each experiment, the rats were injected with harmaline, which induced tremor that was quantified using an accelerometer. EDCS was then applied through the different electrode configurations to evaluate their tremor suppression effectiveness. RESULTS: Results from experiment 1 showed that MCcathode-Cerebellaranode suppressed tremor compared with stimulation-OFF but MCanode-Cerebellarcathode did not. Furthermore, experiments 2 and 3 showed that it was the cerebellar anodal electrode that was driving tremor suppression. CONCLUSION: Cerebellar EDCS suppressed harmaline tremor in rats in a polarity-dependent manner. EDCS could be a promising neuromodulation method for patients with ET.

Targeting inhibition of microtubule affinity regulating kinase 4 by Harmaline: Strategy to combat Alzheimer's disease.

Microtubule-affinity regulating kinase 4 (MARK4) is linked with the development of cancer, diabetes and neurodegenerative diseases. Due to its direct role in the hyperphosphorylation of tau protein, MARK4 is considered as an attractive target to fight Alzheimer's disease (AD) and neuroinflammation. In the present study, we have selected Harmaline (HAR), an alkaloid of Paganum harmala, to investigate its MARK4 inhibitory potential and its binding mechanism. Molecular docking and fluorescence binding studies were carried out to estimate the binding affinity of the HAR with the MARK4. We observed an excellent binding affinity of HAR to the MARK4 (K = 107 M-1), further complemented by isothermal titration calorimetric measurements. In addition, HAR significantly inhibits the kinase activity of MARK4 (IC50 value of 4.46 µM). Structural investigations suggested that HAR binds to the active site pocket and forms several non-covalent interactions with biologically important residues of MARK4. All-atom molecular dynamics simulation studies further advocated that the MARK4-HAR complex is stabilized throughout the trajectory of 200 ns and causes a little conformational change. All these findings suggest that HAR is a potential MARK4 inhibitor that can be implicated in managing MARK4-associated diseases, including AD.