Sex-specific pleiotropic changes in emotional behavior and alcohol consumption in human α-synuclein A53T transgenic mice during early adulthood.

Point mutations in the α-synuclein coding gene may lead to the development of Parkinson's disease (PD). PD is often accompanied by other psychiatric conditions, such as anxiety, depression, and drug use disorders, which typically emerge in adulthood. Some of these point mutations, such as SNCA and A30T, have been linked to behavioral effects that are not commonly associated with PD, especially regarding alcohol consumption patterns. In this study, we investigated whether the familial PD point mutation A53T is associated with changes in alcohol consumption behavior and emotional states at ages not yet characterized by α-synuclein accumulation. The affective and alcohol-drinking phenotypes remained unaltered in female PDGF-hA53T-synuclein-transgenic (A53T) mice during both early and late adulthood. Brain region-specific activation of ceramide-producing enzymes, acid sphingomyelinase (ASM), and neutral sphingomyelinase (NSM), known for their neuroprotective properties, was observed during early adulthood but not in late adulthood. In males, the A53T mutation was linked to a reduction in alcohol consumption in both early and late adulthood. However, male A53T mice displayed increased anxiety- and depression-like behaviors during both early and late adulthood. Enhanced ASM activity in the dorsal mesencephalon and ventral hippocampus may potentially contribute to these adverse behavioral effects of the mutation in males during late adulthood. In summary, the A53T gene mutation was associated with diverse changes in emotional states and alcohol consumption behavior long before the onset of PD, and these effects varied by sex. These alterations in behavior may be linked to changes in brain ceramide metabolism.

Adult alcohol drinking and emotional tone are mediated by neutral sphingomyelinase during development in males.

Alcohol use, abuse, and addiction, and resulting health hazards are highly sex-dependent with unknown mechanisms. Previously, strong links between the SMPD3 gene and its coded protein neutral sphingomyelinase 2 (NSM) and alcohol abuse, emotional behavior, and bone defects were discovered and multiple mechanisms were identified for females. Here we report strong sex-dimorphisms for central, but not for peripheral mechanisms of NSM action in mouse models. Reduced NSM activity resulted in enhanced alcohol consumption in males, but delayed conditioned rewarding effects. It enhanced the acute dopamine response to alcohol, but decreased monoaminergic systems adaptations to chronic alcohol. Reduced NSM activity increased depression- and anxiety-like behavior, but was not involved in alcohol use for the self-management of the emotional state. Constitutively reduced NSM activity impaired structural development in the brain and enhanced lipidomic sensitivity to chronic alcohol. While the central effects were mostly opposite to NSM function in females, similar roles in bone-mediated osteocalcin release and its effects on alcohol drinking and emotional behavior were observed. These findings support the view that the NSM and multiple downstream mechanism may be a source of the sex-differences in alcohol use and emotional behavior.

Toll-like Receptor 2-Melatonin Feedback Loop Regulates the Activation of Spinal NLRP3 Inflammasome in Morphine-Tolerant Rats.

BACKGROUND AND PURPOSE: Morphine is amongst the most effective analgesics available for the management of severe pain. However, prolonged morphine treatment leads to analgesic tolerance which limits its clinical usage. Previous studies have demonstrated that melatonin ameliorates morphine tolerance by reducing neuroinflammation. However, little is known about the relationship between Toll like receptor 2 (TLR2) and neuroinflammation in morphine tolerance. The aim of this study was to explore the role of TLR2 in morphine tolerance and its connections with melatonin and Nod-like receptor protein 3 (NLRP3) inflammasome. METHODS: Sprague-Dawley rats were treated with morphine for 7 days and tail-flick latency test was performed to identify the induction of analgesic tolerance. The roles of TLR2 in microglia activation and morphine tolerance were assessed pharmacologically, and the possible interactions between melatonin, TLR2 and NLRP3 inflammasome were investigated. KEY RESULTS: Morphine tolerance was accompanied by increased TLR2 expression and NLRP3 inflammasome activation in spinal cord. whereas melatonin level was down-regulated. Chronic melatonin administration resulted in a reduced TLR2 expression and NLRP3 inflammasome activation. Moreover, the analgesic effect of morphine was partially restored. Inhibition of TLR2 suppressed the microglia and NLRP3 inflammasome activation, as well as restored the spinal melatonin level while attenuated the development of morphine tolerance. Furthermore, the inhibition of microglia activation ameliorated morphine tolerance via inhibiting TLR2-NLRP3 inflammasome signaling in spinal cord. CONCLUSION: In this study, we directly demonstrate a TLR2-melatonin negative feedback loop regulating microglia and NLRP3 inflammasome activation during the development of morphine tolerance.

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects.

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone-brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental-physical co-morbidity trias of alcohol abuse-depression/anxiety-bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental-physical co-morbidity trias.

Synergistic Control of Transmitter Turnover at Glycinergic Synapses by GlyT1, GlyT2, and ASC-1.

In addition to being involved in protein biosynthesis and metabolism, the amino acid glycine is the most important inhibitory neurotransmitter in caudal regions of the brain. These functions require a tight regulation of glycine concentration not only in the synaptic cleft, but also in various intracellular and extracellular compartments. This is achieved not only by confining the synthesis and degradation of glycine predominantly to the mitochondria, but also by the action of high-affinity large-capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminals or glial cells surrounding the synapses. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their synergistic functional interaction is only poorly understood. In this review, we summarize our current knowledge of the two high-affinity transporters for glycine, the sodium-dependent glycine transporters 1 (GlyT1; SLC6A9) and 2 (GlyT2; SLC6A5) and the alanine-serine-cysteine-1 transporter (Asc-1; SLC7A10).

Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo.

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4-5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

The Cortical Neuroimmune Regulator TANK Affects Emotional Processing and Enhances Alcohol Drinking: A Translational Study.

Alcohol abuse is a major public health problem worldwide. Understanding the molecular mechanisms that control regular drinking may help to reduce hazards of alcohol consumption. While immunological mechanisms have been related to alcohol drinking, most studies reported changes in immune function that are secondary to alcohol use. In this report, we analyse how the gene "TRAF family member-associated NF-κB activator" (TANK) affects alcohol drinking behavior. Based on our recent discovery in a large GWAS dataset that suggested an association of TANK, SNP rs197273, with alcohol drinking, we report that SNP rs197273 in TANK is associated both with gene expression (P = 1.16 × 10-19) and regional methylation (P = 5.90 × 10-25). A tank knock out mouse model suggests a role of TANK in alcohol drinking, anxiety-related behavior, as well as alcohol exposure induced activation of insular cortex NF-κB. Functional and structural neuroimaging studies among up to 1896 adolescents reveal that TANK is involved in the control of brain activity in areas of aversive interoceptive processing, including the insular cortex, but not in areas related to reinforcement, reward processing or impulsiveness. Our findings suggest that the cortical neuroimmune regulator TANK is associated with enhanced aversive emotional processing that better protects from the establishment of alcohol drinking behavior.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.

Prenatal androgen receptor activation determines adult alcohol and water drinking in a sex-specific way.

Alcohol use disorders are major psychiatric disorders. Correlational studies in humans suggested organizational hormonal effects during embryonic development as a risk factor for adult alcohol dependence. Permanent changes can be induced by the activity of sex hormones, like testosterone. Here, we demonstrate a relationship between prenatal androgen receptor (AR)-activation and adult alcohol as well as water drinking in mice in a sex-dependent fashion. Prenatal AR inhibition using the antagonist flutamide decreased adult male alcohol consumption. In contrast, prenatal AR activation by dihydrotestosterone (DHT) led to an increase in adult alcohol consumption in females. These effects were different in adult water drinking, flutamide increased water consumption in females and DHT increased water consumption in males. Prenatal flutamide reduced locomotion and anxiety in adult males but was ineffective in females. We found that prenatal AR activation controls adult levels of monoaminergic modulatory transmitters in the brain and blood hormone levels in a sex-specific way. RNA-Seq analysis confirmed a prenatal AR mediated control of adult expression of alcohol drinking-related genes like Bdnf and Per2. These findings demonstrate that prenatal androgen activity is a risk factor for the establishment of alcohol consumption in adults by its organizational effects.

Deficit in acoustic signal-in-noise detection in glycine receptor α3 subunit knockout mice.

Hearing is an essential sense for communication in animals and humans. Normal function of the cochlea of higher vertebrates relies on a fine-tuned interplay of afferent and efferent innervation of both inner and outer hair cells. Efferent inhibition is controlled via olivocochlear feedback loops, mediated mainly by acetylcholine, γ-aminobutyric acid (GABA) and glycine, and is one of the first sites affected by synapto- and neuropathy in the development of hearing loss. While the functions of acetylcholine, GABA and other inhibitory transmitters within these feedback loops are at least partially understood, especially the function of glycine still remains elusive. To address this question, we investigated hearing in glycine receptor (GlyR) α3 knockout (KO) and wildtype (WT) mice. We found no differences in pure tone hearing thresholds at 11.3 and 16 kHz between the two groups as assessed by auditory brainstem response (ABR) measurements. Detailed analysis of the ABR waves at 11.3 kHz, however, revealed a latency decrease of wave III and an amplitude increase of wave IV in KO compared to WT animals. GlyRα3 KO animals showed significantly impaired prepulse inhibition of the auditory startle response in a noisy environment, indicating that GlyRα3-mediated glycinergic inhibition is important for signal-in-noise detection.

Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis.

Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking-but not forced alcohol exposure-reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism.

Three-Step Test System for the Identification of Novel GABAA Receptor Modulating Food Plants.

Potentiation of γ-amino butyric acid (GABA)-induced GABAA receptor (GABAAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1ß2-GABAAR expressed in Xenopus laevis oocytes. Positively tested food plants were then subjected to quantification of GABA content by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to exclude test foods, which evoke a TEVC-response by endogenous GABA. In the third step, specificity of GABAA-modulating activity was assessed by TEVC analysis of Xenopus laevis oocytes expressing the homologous glycine receptor (GlyR). The three-component test was then applied to screen 10 aqueous extracts of food plants for their GABAAR activity. Thus, hop cones (Humulus lupulus) and Sideritis sipylea were identified as the most potent specific GABAAR modulators eliciting significant potentiation of the current by 182 ± 27 and 172 ± 19 %, respectively, at the lowest concentration of 0.5 µg/mL. The extracts can now be further evaluated by in vivo studies and by structural evaluation of the active components.

Long-term application of glycine transporter inhibitors acts antineuropathic and modulates spinal N-methyl-D-aspartate receptor subunit NR-1 expression in rats.

BACKGROUND: Dysfunction of spinal glycinergic neurotransmission is a major pathogenetic factor in neuropathic pain. The synaptic glycine concentration is controlled by the two glycine transporters (GlyT) 1 and 2. GlyT inhibitors act antinociceptive in various animal pain models when applied as bolus. Yet, in some studies, severe neuromotor side effects were reported. The aim of the current study was to elucidate whether continuous inhibition of GlyT ameliorates neuropathic pain without side effects and whether protein expression of GlyT1, GlyT2, or N-methyl-D-aspartate receptor subunit NR-1 in the spinal cord is affected. METHODS: In the chronic constriction injury model of neuropathic pain, male Wistar rats received specific GlyT1 and GlyT2 inhibitors (ALX5407 and ALX1393; Sigma-Aldrich, St. Louis, MO) or vehicle for 14 days via subcutaneous osmotic infusion pumps (n = 6). Mechanical allodynia and thermal hyperalgesia were assessed before, after chronic constriction injury, and every 2 days during substance application. At the end of behavioral assessment, the expression of GlyT1, GlyT2, and NR-1 in the spinal cord was determined by Western blot analysis. RESULTS: Both ALX5407 and ALX1393 ameliorated thermal hyperalgesia and mechanical allodynia in a time- and dose-dependent manner. Respiratory or neuromotor side effects were not observed. NR-1 expression in the ipsilateral spinal cord was significantly reduced by ALX5407, but not by ALX1393. The expression of GlyT1 and GlyT2 remained unchanged. CONCLUSIONS: Continuous systemic inhibition of GlyT significantly ameliorates neuropathic pain in rats. Thus, GlyT represent promising targets in pain research. Modulation of N-methyl-D-aspartate receptor expression might represent a novel mechanism for the antinociceptive action of GyT1 inhibitors.

A retroelement modifies pre-mRNA splicing: the murine Glrb(spa) allele is a splicing signal polymorphism amplified by long interspersed nuclear element insertion.

The glycine receptor-deficient mutant mouse spastic carries a full-length long interspersed nuclear element (LINE1) retrotransposon in intron 6 of the glycine receptor ß subunit gene, Glrb(spa). The mutation arose in the C57BL/6J strain and is associated with skipping of exon 6 or a combination of the exons 5 and 6, thus resulting in a translational frameshift within the coding regions of the GlyR ß subunit. The effect of the Glrb(spa) LINE1 insertion on pre-mRNA splicing was studied using a minigene approach. Sequence comparison as well as motif prediction and mutational analysis revealed that in addition to the LINE1 insertion the inactivation of an exonic splicing enhancer (ESE) within exon 6 is required for skipping of exon 6. Reconstitution of the ESE by substitution of a single residue was sufficient to prevent exon skipping. In addition to the ESE, two regions within the 5' and 3' UTR of the LINE1 were shown to be critical determinants for exon skipping, indicating that LINE1 acts as efficient modifier of subtle endogenous splicing phenotypes. Thus, the spastic allele of the murine glycine receptor ß subunit gene is a two-hit mutation, where the hypomorphic alteration in an ESE is amplified by the insertion of a LINE1 element in the adjacent intron. Conversely, the LINE1 effect on splicing may be modulated by individual polymorphisms, depending on the insertional environment within the host genome.

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord.

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.

Lidocaine metabolites inhibit glycine transporter 1: a novel mechanism for the analgesic action of systemic lidocaine?

BACKGROUND: Lidocaine exerts antinociceptive effects when applied systemically. The mechanisms are not fully understood but glycinergic mechanisms might be involved. The synaptic glycine concentration is controlled by glycine transporters. Whereas neurons express two types of glycine transporters, astrocytes specifically express glycine transporter 1 (GlyT1). This study focuses on effects of lidocaine and its major metabolites on GlyT1 function. METHODS: The effects of lidocaine and its metabolites monoethylglycinexylidide (MEGX), glycinexylidide, and N-ethylglycine on GlyT1 function were investigated in uptake experiments with [¹4C]-labeled glycine in primary rat astrocytes. Furthermore, the effect of lidocaine and its metabolites on glycine-induced currents were investigated in GlyT1-expressing Xenopus laevis oocytes. RESULTS: Lidocaine reduced glycine uptake only at toxic concentrations. The metabolites MEGX, glycinexylidide, and N-ethylglycine, however, significantly reduced glycine uptake (P < 0.05). Inhibition of glycine uptake by a combination of lidocaine with its metabolites at a clinically relevant concentration was diminished with increasing extracellular glycine concentrations. Detailed analysis revealed that MEGX inhibits GlyT1 function (P < 0.05), whereas N-ethylglycine was identified as an alternative GlyT1 substrate (EC50 = 55 µM). CONCLUSIONS: Although lidocaine does not function directly on GlyT1, its metabolites MEGX and N-ethylglycine [corrected] were shown to inhibit GlyT1-mediated glycine uptake by at least two different mechanisms. Whereas N-ethylglycine [corrected] was demonstrated to be an alternative GlyT1 substrate, MEGX was shown to inhibit GlyT1 activity in both primary astrocytes and in GlyT1-expressing Xenopuslaevis oocytes at clinically relevant concentrations. These findings provide new insights into the possible mechanisms for the antinociceptive effect of systemic lidocaine.

Evaluation of a Luminometric Cell Counting System in Context of Antimicrobial Photodynamic Inactivation.

Antimicrobial resistance belongs to the most demanding medical challenges, and antimicrobial photodynamic inactivation (aPDI) is considered a promising alternative to classical antibiotics. However, the pharmacologic characterization of novel compounds suitable for aPDI is a tedious and time-consuming task that usually requires preparation of bacterial cultures and counting of bacterial colonies. In this study, we established and utilized a luminescence-based microbial cell viability assay to analyze the aPDI effects of two porphyrin-based photosensitizers (TMPyP and THPTS) on several bacterial strains with antimicrobial resistance. We demonstrate that after adaptation of the protocol and initial calibration to every specific bacterial strain and photosensitizer, the luminometric method can be used to reliably quantify aPDI effects in most of the analyzed bacterial strains. The interference of photosensitizers with the luminometric readout and the bioluminescence of some bacterial strains were identified as possible confounders. Using this method, we could confirm the susceptibility of several bacterial strains to photodynamic treatment, including extensively drug-resistant pathogens (XDR). In contrast to the conventional culture-based determination of bacterial density, the luminometric assay allowed for a much more time-effective analysis of various treatment conditions. We recommend this luminometric method for high-throughput tasks requiring measurements of bacterial viability in the context of photodynamic treatment approaches.

Nociception in the Glycine Receptor Deficient Mutant Mouse Spastic.

Glycine receptors (GlyRs) are the primary mediators of fast inhibitory transmission in the mammalian spinal cord, where they modulate sensory and motor signaling. Mutations in GlyR genes as well as some other genes underlie the hereditary disorder hyperekplexia, characterized by episodic muscle stiffness and exaggerated startle responses. Here, we have investigated pain-related behavior and GlyR expression in the spinal cord of the GlyR deficient mutant mouse spastic (spa). In spastic mice, the GlyR number is reduced due to a ß subunit gene (Glrb) mutation resulting in aberrant splicing of GlyRß transcripts. Via direct physical interaction with the GlyR anchoring protein gephyrin, this subunit is crucially involved in the postsynaptic clustering of heteromeric GlyRs. We show that the mutation differentially affects aspects of the pain-related behavior of homozygous Glrbspa/Glrbspa mice. While response latencies to noxious heat were unchanged, chemically induced pain-related behavior revealed a reduction of the licking time and an increase in flinching in spastic homozygotes during both phases of the formalin test. Mechanically induced nocifensive behavior was reduced in spastic mice, although hind paw inflammation (by zymosan) resulted in allodynia comparable to wild-type mice. Immunohistochemical staining of the spinal cord revealed a massive reduction of dotted GlyRα subunit immunoreactivity in both ventral and dorsal horns, suggesting a reduction of clustered receptors at synaptic sites. Transcripts for all GlyRα subunit variants, however, were not reduced throughout the dorsal horn of spastic mice. These findings suggest that the loss of functional GlyRß subunits and hence synaptically localized GlyRs compromises sensory processing differentially, depending on stimulus modality.

The Meta-Substituted Isomer of TMPyP Enables More Effective Photodynamic Bacterial Inactivation than Para-TMPyP In Vitro.

Porphyrinoid-based photodynamic inactivation (PDI) provides a promising approach to treating multidrug-resistant infections. However, available agents for PDI still have optimization potential with regard to effectiveness, toxicology, chemical stability, and solubility. The currently available photosensitizer TMPyP is provided with a para substitution pattern (para-TMPyP) of the pyridinium groups and has been demonstrated to be effective for PDI of multidrug-resistant bacteria. To further improve its properties, we synthetized a structural variant of TMPyP with an isomeric substitution pattern in a meta configuration (meta-TMPyP), confirmed the correct structure by crystallographic analysis and performed a characterization with NMR-, UV/Vis-, and IR spectroscopy, photostability, and singlet oxygen generation assay. Meta-TMPyP had a hypochromic shift in absorbance (4 nm) with a 55% higher extinction coefficient and slightly improved photostability (+6.9%) compared to para-TMPyP. Despite these superior molecular properties, singlet oxygen generation was increased by only 5.4%. In contrast, PDI, based on meta-TMPyP, reduced the density of extended spectrum ß-lactamase-producing and fluoroquinolone-resistant Escherichia coli by several orders of magnitude, whereby a sterilizing effect was observed after 48 min of illumination, while para-TMPyP was less effective (p < 0.01). These findings demonstrate that structural modification with meta substitution increases antibacterial properties of TMPyP in PDI.

Photodynamic Inactivation of SARS-CoV-2 Infectivity and Antiviral Treatment Effects In Vitro.

Despite available vaccines, antibodies and antiviral agents, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic still continues to cause severe disease and death. Current treatment options are limited, and emerging new mutations are a challenge. Thus, novel treatments and measures for prevention of viral infections are urgently required. Photodynamic inactivation (PDI) is a potential treatment for infections by a broad variety of critical pathogens, including viruses. We explored the infectiousness of clinical SARS-CoV-2 isolates in Vero cell cultures after PDI-treatment, using the photosensitizer Tetrahydroporphyrin-tetratosylate (THPTS) and near-infrared light. Replication of viral RNA (qPCR), viral cytopathic effects (microscopy) and mitochondrial activity were assessed. PDI of virus suspension with 1 µM THPTS before infection resulted in a reduction of detectable viral RNA by 3 log levels at day 3 and 6 after infection to similar levels as in previously heat-inactivated virions (<99.9%; p < 0.05). Mitochondrial activity, which was significantly reduced by viral infection, was markedly increased by PDI to levels similar to uninfected cell cultures. When applying THPTS-based PDI after infection, a single treatment had a virus load-reducing effect only at a higher concentration (3 µM) and reduced cell viability in terms of PDI-induced toxicity. Repeated PDI with 0.3 µM THPTS every 4 h for 3 d after infection reduced the viral load by more than 99.9% (p < 0.05), while cell viability was maintained. Our data demonstrate that THPTS-based antiviral PDI might constitute a promising approach for inactivation of SARS-CoV-2. Further testing will demonstrate if THPTS is also suitable to reduce the viral load in vivo.