This series of investigations aimed to evaluate in dogs the interaction between MK-467, a peripheral α2-adrenoceptor antagonist with poor penetration into the central nervous system, and dexmedetomidine, a selective α2-adrenoceptor agonist commonly employed in small animal clinical practice due its potent sedative effects. The objective of this study was to find an optimal dose-ratio of dexmedetomidine and the antagonist that could attenuate or prevent major cardiovascular changes without any significant effect on the sedation induced by the agonist. The effects on blood flow in abdominal organs and on plasma concentrations of glucose, insulin, non-esterified free fatty acids, lactate and cortisol of this optimal dose were then evaluated. The sedative effects were assessed subjectively by means of a composite sedation score. Simultaneously, hypnosis was evaluated through the bispectral index. Haemodynamic parameters that were evaluated comprised cardiac output, arterial blood pressure, heart rate, central venous pressure and systemic vascular resistance. Time-intensity parameters derived from contrast-enhanced ultrasound imaging were used to assess blood flow in selected abdominal organs. Three doses of MK-467 (250, 500 and 750 µg/kg) were tested against dexmedetomidine alone (10 µg/kg). All treatments were administered IV. Sedation was significantly lower and BIS significantly higher with the medium and highest doses of MK-467 than with dexmedetomidine. However, bioequivalence between dexmedetomidine and the combination was reached with all treatments for the two parameters analysed. Early cardiovascular effects of dexmedetomidine were not completely prevented with the lowest dose of MK-467, and the highest dose reduced mean arterial pressure. The middle dose of MK-467 (500 µg/kg) provided the best cardiovascular stability. Addition of the peripheral antagonist attenuated dexmedetomidine-induced changes in organ blood flow evaluated by the CEUS. An increase in plasma glucose was observed in dexmedetomidine-treated dogs, but not when MK-467 was added. Inversely, plasma insulin concentration was reduced with dexmedetomidine, but not when dexmedetomidine was combined with MK-467. Plasma non-esterified free fatty acids concentration decreased transiently with the combination, while with dexmedetomidine alone the reduction persisted throughout the observation period. Plasma lactate concentration increased with dexmedetomidine, but not with the combination. In conclusion, the addition of MK-467 attenuated or prevented the early cardiovascular effects of dexmedetomidine, not having clinically relevant effects on the sedation induced by the latter. Some metabolic changes induced by dexmedetomidine were halted by MK-467.

Patients with malignant peripheral nerve sheath tumor (MPNST), a rare soft tissue cancer associated with loss of the tumor suppressor neurofibromin (NF1), have poor prognosis and typically respond poorly to adjuvant therapy. We evaluated the effect of 299 clinical and investigational compounds on seven MPNST cell lines, two primary cultures of human Schwann cells, and five normal bone marrow aspirates, to identify potent drugs for MPNST treatment with few side effects. Top hits included Polo-like kinase 1 (PLK1) inhibitors (volasertib and BI2536) and the fluoronucleoside gemcitabine, which were validated in orthogonal assays measuring viability, cytotoxicity, and apoptosis. DNA copy number, gene expression, and protein expression were determined for the cell lines to assess pharmacogenomic relationships. MPNST cells were more sensitive to BI2536 and gemcitabine compared to a reference set of 94 cancer cell lines. PLK1, RRM1, and RRM2 mRNA levels were increased in MPNST compared to benign neurofibroma tissue, and the protein level of PLK1 was increased in the MPNST cell lines compared to normal Schwann cells, indicating an increased dependence on these drug targets in malignant cells. Furthermore, we observed an association between increased mRNA expression of PLK1, RRM1, and RRM2 in patient samples and worse disease outcome, suggesting a selective benefit from inhibition of these genes in the most aggressive tumors.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons and propagating Lewy body pathology. Dopamine depletion in the striatum gives rise to the cardinal motor symptoms of PD. Current PD medications are based on replenishing striatal dopamine and provide symptomatic relief to the motor deficits. However, troublesome adverse effects and diminished efficacy complicate their long-term use. There is a great unmet medical need for a therapy that could slow or halt the progression of the disease. Neurotrophic factors (NTFs) are secreted proteins that promote neuronal growth, differentiation and survival. They are able to prevent the progression of neurodegeneration and restore aberrant neuronal function in a variety of preclinical models of PD. Nonetheless, outcomes from clinical trials have been disappointing. The purpose of this work was to characterize the effects of cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and novel small molecule receptor tyrosine kinase RET agonists (BT13 and BT44) on nigrostriatal dopamine system and support their preclinical development as potential neurotrophic therapies of PD. To further clarify the functional effects of glial cell line-derived neurotrophic factor (GDNF), CDNF and MANF in the normal rat brain, microdialysis measurements were performed after a bolus injection of NTFs into the striatum. We saw augmented stimulus-evoked dopamine release and elevated dopamine turnover in the striatum of MANF-injected rats. GDNF injection increased in vivo tyrosine hydroxylase (TH) and catechol-O-methyltransferase activity and decreased monoamine oxidase A activity. These data are relevant when considering exogenously administered NTFs as a potential therapeutic approach for PD, since they have to be compatible with the existing dopaminergic medications of the patients. We also investigated the distribution properties of 125I-labeled and unlabeled CDNF after a nigral injection to intact rats. CDNF readily diffused into the brain areas surrounding the injection site and colocalized with TH-immunoreactive neurons in the substantia nigra. We did not detect active transportation of CDNF to distal brain areas. This characterization provides valuable insights into the selection of optimal delivery site and protocol for CDNF therapy. Our in vitro assays showed that RET agonists BT13 and BT44 were able to induce RET phosphorylation and activate downstream pro-survival signaling cascades Akt and ERK. They also supported the survival of cultured midbrain dopamine neurons from wild-type, but not from RET knockout, mice. The functional effects of BT13 and BT44 were evaluated in a unilateral 6-hydroxydopamine rat model of PD, where both compounds alleviated amphetamine-induced turning behavior. BT44 also showed potential to restore striatal TH-immunoreactive fibers. As blood-brain barrier penetrating compounds, BT13 and BT44 serve as promising leads that can be further developed into a disease-modifying therapy for PD.

Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.

Background-Small molecule kinase inhibitors (KIs) are a class of agents currently used for treatment of various cancers. Unfortunately, treatment of cancer patients with some of the KIs is associated with cardiotoxicity, and there is an unmet need for methods to predict their cardiotoxicity. Here, we utilized a novel computational method to identify protein kinases crucial for cardiomyocyte viability. Methods and Results-One hundred forty KIs were screened for their toxicity in cultured neonatal cardiomyocytes. The kinase targets of KIs were determined based on integrated data from binding assays. The key kinases mediating the toxicity of KIs to cardiomyocytes were identified by using a novel machine learning method for target deconvolution that combines the information from the toxicity screen and from the kinase profiling assays. The top kinases identified by the model were phosphoinositide 3-kinase catalytic subunit alpha, mammalian target of rapamycin, and insulin-like growth factor 1 receptor. Knockdown of the individual kinases in cardiomyocytes confirmed their role in regulating cardiomyocyte viability. Conclusions-Combining the data from analysis of KI toxicity on cardiomyocytes and KI target profiling provides a novel method to predict cardiomyocyte toxicity of KIs.

The aim of this study was to explore the functions of T-type calcium channels, and their possible role in neuronal stem cells migration. The role of T-type calcium channel in mature brain is known to be in producing electroencephalographic oscillations. This action in turn is the key factor in some neuronal physiological and pathophysiological functions, like non-REM sleep, memory, learning and absence epilepsy. In addition, T-type calcium channels have peripheral actions, but this study concerns on its neuronal functions. This low-voltage activated channels functions in neurogenesis is less known than its role in mature brain. It is known to promote neuronal proliferation and differentiation, but what comes to its possible actions in neuronal migration, is poorly studied. This study shows some evidence of T-type calcium channel taking part in neuronal migration in mice embryonic subventricular zones progenitor cells. Selective T-type antagonists, ethosuximide, nickelchloride and a scorpion peptide toxin kurtoxin, decreased the rate of migration in differentiating progenitor cells. This study consists of a literature review and an experimental part. Another aim of this study is to consider an alternative approach to stem cell therapies based on invasive transplantation of the cells. This other attempt is non-invasive manipulating of endogenous stem cells to proliferate and migrate to the injured or depleted area in the brain, differentiate into a desired phenotype and stop their division after they have completed their mission. Non-invasive altering of the stem cells is awaiting pharmacological solutions to resolve the problems being faced in this effort. There are some non-invasive therapies already being used successfully to cure pathological conditions such as spinal cord injury. These methods could be used as well in stem cell based therapies in the treatment of neurodegenerative diseases and brain injuries. These methods are still in the beginning of their way and lacking the full understanding of the key factors that affect neuronal development. These factors include some important endogenous inducing and inhibiting substances. One of the most important inducing substances is calcium ion regulating a variety of events in neurogenesis. T-type calcium channel, as being widely expressed during early brain development, and decaying by neuronal maturation, might have a pivotal role in conducting progenitor cells.

Neurodegenerative diseases are characterized by the dysfunction and death of specific neuronal populations. Parkinson’s disease (PD) is caused by the progressive loss of dopamine neurons in the substantia nigra, whereas motor neurons (MNs) in the motor cortex, brain stem, and spinal cord degenerate and die in amyotrophic lateral sclerosis (ALS). Accumulation of misfolded proteins and endoplasmic reticulum (ER) stress are some common hallmarks in the pathophysiology of neurodegenerative diseases. ER stress triggers the unfolded protein response (UPR), a physiological response that aims at restoring the ER homeostasis by degrading misfolded proteins, attenuating protein translation, and increasing the expression of ER chaperones important for protein folding. Initially the UPR is protective, but, upon prolonged ER stress, the UPR switches from an adaptive to a pro-apoptotic response. Cerebral dopamine neurotrophic factor (CDNF) is an ER resident protein with neurotrophic properties that is protective and restorative in preclinical models of PD. The mechanism underlying CDNF’s action is still unclear, but experimental data suggest a possible involvement of CDNF in the ER homeostasis. The aim of this thesis work was to study the therapeutic potential of CDNF in PD and ALS rodent models and investigate CDNF mode of action, with a special focus on the ER stress response. Herein, we report that co-administration of CDNF and glial cell line-derived neurotrophic factor (GDNF) showed an additive neurorestorative effect in the unilateral 6-hydroxydopamine rat model of PD, suggesting a different mechanism of action for these two proteins. We found that GDNF activated the pro-survival MAPK/ERK and PI3K/AKT pathways in the striatal dopamine neurons within 1 hour from protein administration. In contrast, CDNF activated only the PI3K/AKT pathway and at 4 hours upon treatment. Furthermore, CDNF, but not GDNF, reduced the expression of UPR markers ATF6, p-eIF2α, and GRP78. Therefore, the ability of CDNF to regulate ER stress was thoroughly investigated in three rodent models of ALS with different genetic etiology and disease progression. We showed that CDNF decreased the ER stress response specifically in MNs, by attenuating all three branches of the UPR, initiated by transducers inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6). CDNF treatment was effective in all three models, indicating that CDNF’s therapeutic effect was independent of disease etiology. CDNF rescued MNs from ER-stressed induce cell death, halting the progression of the disease and ameliorating the motor deficit in the SOD1-G93A mouse model and in the TDP43-M337V rat model. Finally, we identified that depleting endogenous CDNF from the SOD1-G93A model worsened the motor symptoms in the mice, but did not affect their lifespan. The ER stress response in the Cdnf -/- SOD1-G93A mice was especially exacerbated in the skeletal muscle, where CDNF is normally highly expressed, and an overexpression of homologous protein mesencephalic astrocyte-derived neurotrophic factor (MANF) was detected in the same tissue. We observed a reduction in the number of lumbar MNs in Cdnf -/- SOD1-G93A compared to classical SOD1-G93A mice, which would explain the aggravated motor impairment. At this point, however, we could not determine whether the increase in MNs loss was caused by CDNF depletion in MNs, or rather a consequence of CDNF-deficiency in the degenerating muscle cells, targets of MNs. It was previously reported that, in mice, endogenous CDNF is important for the development and maintenance of enteric submucosal neurons, as well as for the regulation of gastrointestinal transit. Remarkably, we found that Cdnf -/- mice had less lumbar MNs at 4 months, compared to WT littermates, although this decrease did not result in any motor deficit. These findings suggest that CDNF may also have a role in the development and/or survival of MNs. Altogether, these studies indicate that ER stress is an important therapeutic target for neurodegenerative diseases, such as PD and ALS, and that CDNF is a promising drug candidate, due to its ability to attenuate all three pathways of UPR.

Dopamine (DA) neurons of the ventral tegmental area (VTA) are critical for decision-making and motivation and have also been implicated in the development of addictive behaviors. The activity of these neurons and the subsequent changes in DA concentrations in the target regions of the VTA are strictly regulated by both excitatory and inhibitory inputs. Among those inhibitory inputs, GABAergic transmission is mediated by phasic and tonic currents generated through different GABAA receptor subtypes. Although the phasic currents arising through the activation of synaptic GABAA receptors have been well described, much less is known about extrasynaptic GABAA receptors mediating tonic currents and modulating neuronal activity in the VTA. Here, pharmacologically selective receptor modulators, transgenic mouse models and brain slice electrophysiology were all exploited to probe the role of extrasynaptic GABAA receptors in mediating neuroplasticity in VTA DA neurons. Even though they possess distinct molecular sites of action, gaboxadol (THIP) and ganaxalone (GAN) enhanced tonic inhibition by selective targeting of the extrasynaptic δ subunit-containing GABAA receptors located on VTA GABA neurons. The tonic inhibition induced in these neurons appeared to be sufficient to disinhibit DA neurons and induce persistent neuroplasticity in the glutamate synapses on VTA DA neurons, which resulted from insertion of new GluA2 subunit-lacking AMPA receptors into the synapses. Screening of reward-related behaviors associated with VTA DA activity revealed that THIP failed to induce any reinforcement during self-administration either in mice or baboons. Moreover, both THIP and GAN produced conditioned place aversion in mice. The study performed in δ subunit-knockout mice further supported the proposal that tonic inhibition of the VTA GABA neurons contributes to conditioned aversive behavior and THIP- and GAN-induced neuroplasticity. The c-Fos mapping of brain regions, which could take part in THIP-induced aversive behavioral effects and/or neuroplasticity on VTA DA neurons, revealed the bed nucleus of stria terminalis (BNST), a part of the so-called extended amygdala circuitry, as a possible participant in mediating the aforementioned THIP-induced aversive effects. In summary, these studies demonstrate that tonic inhibition mediated by δ subunit-containing GABAA receptors appears to be a significant component of the inhibition in the VTA, and thus important for the control of motivated behavior.