A Computational Approach: The Functional Effects of Thyroid Peroxidase Variants in Thyroid Cancer and Genetic Disorders.

PURPOSE: Thyroid peroxidase (TPO) is essential for the synthesis of thyroid hormones. However, specific mutations render TPO antigenic and prone to autoimmune attacks leading to thyroid cancer, TPO deficiency, and congenital hypothyroidism (CH). Despite technological advancement, most experimental procedures cannot quickly identify the genetic causes of CH nor detect thyroid cancer in the early stages. METHODS: We performed saturated computational mutagenesis to calculate the folding energy changes (∆∆G) caused by missense mutations and analyzed the mutations involved in post-translational modifications (PTMs). RESULTS: Our results showed that the functional important missense mutations occurred in the heme peroxidase domain. Through computational saturation mutagenesis, we identified the TPO mutations in G393 and G348 affecting protein stability and PTMs. Our folding energy calculations revealed that seven of nine somatic thyroid cancer mutations destabilized TPO. CONCLUSION: These findings highlight the impact of these specific mutations on TPO stability, linking them to thyroid cancer and other genetic thyroid-related disorders. Our results show that computational mutagenesis of proteins provides a quick insight into rare mutations causing Mendelian disorders and cancers in humans.

Recombinant immunotoxin induces tumor intrinsic STING signaling against head and neck squamous cell carcinoma.

The innate immune stimulator of interferon genes (STING) pathway is known to activate type I interferons (IFN-I) and participate in generating antitumor immunity. We previously produced hDT806, a recombinant diphtheria immunotoxin, and demonstrated its efficacy against head and neck squamous cell carcinoma (HNSCC). However, it's unknown whether the tumor-intrinsic STING plays a role in the anti-HNSCC effects of hDT806. In this study, we investigated the innate immune modulation of hDT806 on HNSCC. hDT806 significantly upregulated the level of STING and the ratio of p-TBK1/TBK1 in the HNSCC cells. Moreover, intratumoral hDT806 treatment increased the expression of STING-IFN-I signaling proteins including IFNA1, IFNB, CXCL10 and MX1, a marker of IFN-I receptor activity, in the HNSCC xenografts. Overexpression of STING mimicked the hDT806-induced upregulation of the STING-IFN-I signaling and induced apoptosis in the HNSCC cells. In the mouse xenograft models of HNSCC with STING overexpression, we observed a significant suppression of tumor growth and reduced tumor weight with increased apoptosis compared to their control xenograft counterparts without STING overexpression. Collectively, our data revealed that hDT806 may act as a stimulator of tumor-intrinsic STING-IFN-I signaling to inhibit tumor growth in HNSCC.

Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma.

Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10-30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.

Converged DNA Damage Response Renders Human Hepatocellular Carcinoma Sensitive to CDK7 Inhibition.

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality. The inhibition of cyclin-dependent kinase 7 (CDK7) activity has shown therapeutic efficacy in HCC. However, the underlying molecular mechanisms remain elusive. Here, we show that three HCC lines, HepG2, Hep3B, and SK-Hep-1, were highly susceptible to the CDK7 inhibitor THZ1. In mouse models, THZ1 effectively reduced HepG2 tumor growth and tumor weight. THZ1 arrested cell cycle and triggered MYC-related apoptosis in HepG2. To evaluate how MYC protein levels affected THZ1-induced apoptotic cell death, we overexpressed MYC in HepG2 and found that exogenously overexpressed MYC promoted cell cycle progression and increased cells in the S phase. THZ1 drastically engendered the apoptosis of MYC-overexpressing HepG2 cells in the S and G2/M phases. Importantly, transcription-inhibition-induced apoptosis is associated with DNA damage, and exogenous MYC expression further enhanced the THZ1-induced DNA damage response in MYC-overexpressing HepG2 cells. Consistently, in the HepG2 xenografts, THZ1 treatment was associated with DNA-damage-induced cell death. Together, our data indicate that the converged effect of MYC-promoted cell cycle progression and CDK7 inhibition by THZ1 confers the hypersensitivity of HCC to DNA-damage-induced cell death. Our findings may suggest a new therapeutic strategy of THZ1 against HCC.

PTEN promoter methylation predicts 10-year prognosis in hormone receptor-positive early breast cancer patients who received adjuvant tamoxifen endocrine therapy.

PURPOSE: There remain a lack of biomarkers for endocrine therapy resistance in patients with breast cancer (BC), which is proving to be a great challenge. In vitro experiments have shown that downregulation of PTEN expression leads to resistance to tamoxifen (TAM) in BC cells. We aimed to investigate the predictive role of tumor PTEN promoter methylation and PTEN expression in long-term survival after TAM adjuvant therapy in patients with early-stage BC. METHODS: From 2001 to 2013, 105 patients with stage I-III BC who were treated with standardized adjuvant TAM for 5 years or until relapse in West China Hospital (WCH) were enrolled in this study. PTEN expression and DNA methylation of three specified sequences from the PTEN promoter in primary tumors were measured using immunohistochemistry and pyrosequencing. A cohort of 159 hormone receptor-positive patients receiving TAM treatment from The Cancer Genome Atlas (TCGA) database was used for verification. RESULTS: Median follow-up time for the WCH cohort was 141.7 months. The low, moderate, and high PTEN expression groups had differing 10-year disease-free survival (DFS) (42.3%, 55%, 81%, respectively, P = 0.027) and overall survival (OS) rates (65%, 84.2%, 90.5%, respectively, P = 0.027). Higher methylation levels of the second sequence (- 819 to - 787 bp), rather than the first (- 1143 to - 1107 bp) or third sequence (- 663 to - 593 bp), independently increased the risk of disease recurrence (hazard ratio = 2.60) and death (hazard ratio = 3.79) in the WCH cohort, according to multivariate Cox regression analysis. Importantly, out of the five CpG islands located within this sequence, only high methylation of the - 796 CpG island predicted shorter DFS and OS. In TCGA validation cohort, there was also a trend of higher methylation of the - 796 CpG island correlating with shorter disease-free intervals, with borderline significance (P = 0.057). CONCLUSION: Low PTEN expression and high methylation of its promoter (sequence - 819 to - 787 bp) in tissue predict poor DFS and OS in hormone receptor-positive early BC patients who received adjuvant TAM.

Mitogen-activated protein kinase inhibition-induced modulation of epidermal growth factor receptor signaling in human head and neck squamous cell carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is one of the most notable characteristics in head and neck squamous cell carcinoma (HNSCC). The MAPK kinase (MEK) inhibitor trametinib has shown efficacy to treat HNSCC; however, the molecular mechanism remains unclear. METHODS: HNSCC lines, mouse models, Western blot, and flow cytometry were employed to analyze the anticancer effects of trametinib. RESULTS: The JHU-011, JHU-022, and JHU-029 HNSCC cells with different genetic alterations were highly susceptible to trametinib. Trametinib effectively reduced EGFR expression, which was accompanied by the reduction of pro-survival protein MYC, and the increased expression of a MYC-targeted cyclin-dependent kinase inhibitor p27kip1 and pro-apoptotic protein BIM. Trametinib resulted in G1 arrest of the cells, markedly reduced cell numbers in S phase, and significantly increased apoptosis. In mouse models, trametinib strongly inhibited tumors growth. CONCLUSIONS: The MAPK-ERK signaling inhibition by trametinib may target EGFR and the downstream proteins against HNSCC.

Inhibition of MEK suppresses hepatocellular carcinoma growth through independent MYC and BIM regulation.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignancy. In HCC, mitogen-activated protein kinase (MAPK) signaling is overactivated. The MAPK kinase (MEK) inhibitor trametinib has been approved to treat several types of advanced cancers with a BRAF mutation. Herein, we examined whether trametinib has efficacy against HCC. METHODS: The effects of trametinib on cell viability, proliferation and tumor growth were assessed in HCC-derived cell lines and mouse xenograft models. Western blot analysis and immunohistochemistry were used to identify key regulators critical for HHC cell proliferation and tumor growth. RESULTS: We found that trametinib dose-dependently inhibited the viability and proliferation of HCC cells. We also found that a strong suppression of MEK by trametinib downregulated the pro-survival protein MYC, but upregulated the pro-apoptotic protein BIM. This dual differential regulation of MYC and BIM was found to be accompanied by upregulation of a MYC-targeted cyclin dependent kinase inhibitor, p27kip1 (p27), and an apoptosis marker, cleaved poly (ADP ribose) polymerase 1 (PARP), indicating a concurrent modulation of cell cycle- and apoptosis-related pathways. Importantly, we found that MYC overexpression did not block increased BIM in trametinib-treated HCC cells, indicating that MAPK signaling independently regulates MYC and BIM. Finally, we found that trametinib in vivo inhibited HepG2 xenograft tumor growth and attenuated tumor invasion into surrounding tissues. Consistent with the in vitro findings, MYC expression was found to be reduced, while p27 expression was found to be elevated, and BIM expression and cleaved PARP levels were found to be increased in trametinib-treated xenograft tumors. CONCLUSIONS: Collectively, our data indicate that trametinib exhibits efficacy in treating HCC cells via distinct regulation of the MYC and BIM pathways. As such, targeting MEK to block MAPK signaling with trametinib may provide novel treatment opportunities for HCC.

Antitumor properties of Salvianolic acid B against triple-negative and hormone receptor-positive breast cancer cells via ceramide-mediated apoptosis.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options. It is urgent to develop new therapeutics against this disease. Salvinolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bunge, a well-known Chinese medicine for treating various diseases without appreciable adverse effects. To understand the antitumor properties of Sal-B against TNBC, we analyzed its effects on the cell viability, cell cycle and apoptosis of triple-negative MDA-MB-231 cells with the hormone receptor-positive MCF-7 cells as the control. The in vitro analysis showed that Sal-B could significantly reduce the cell viability and suppress the proliferation of both MDA-MB-231 and MCF-7 cells with decreased cyclin B1 expression, but with no noticeable cell cycle phase change. In mouse models, Sal-B markedly inhibited the growth, decreased the PCNA expression, and increased the cell apoptosis of MDA-MB-231 tumor xenografts. To understand the antitumor mechanisms, we analyzed the expression levels of ceramides, and anti-apoptotic (Bcl-xL and survivin) and pro-apoptotic (caspase-3 and caspase-8) proteins. We found that Sal-B enhanced the ceramide accumulation and inhibited the anti-apoptotic protein expression. Interestingly, the ceramide accumulation was accompanied by decreased expression of glucosylceramide and GM3 synthases, two key enzymes regulating ceramide metabolism. These findings indicate that Sal-B exerts its antitumor effects at least partially by inducing the ceramide accumulation and ceramide-mediated apoptosis via inhibiting the expression of glucosylceramide and GM3 synthases, which was independent of estrogen receptor α. Sal-B appears to be a promising therapeutic agent against TNBC.

Simple quantitative PCR analysis for allelic Pten loss in tumor progression.

We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR. As a method to detect both mutation and allele-specific gene loss, our approach is less subject to the variability of sample amount that are often very limited in clinical analysis. Since conventional PCR is easy to be carried out, our method simplifies the workflow in any laboratory and would provide significant advantages for simplicity to quantify allele-specific gene loss.

Ethanol drives aversive conditioning through dopamine 1 receptor and glutamate receptor-mediated activation of lateral habenula neurons.

There has been increasing interest in the lateral habenula (LHb) given its potent regulatory role in many aversion-related behaviors. Interestingly, ethanol can be rewarding as well as aversive; we therefore investigated whether ethanol exposure alters pacemaker firing or glutamate receptor signaling in LHb neurons in vitro and also whether LHb activity in vivo might contribute to the acquisition of conditioned place aversion to ethanol. Surprisingly, in epithalamic slices, low doses of ethanol (1.4 mM) strongly accelerated LHb neuron firing (by ~60%), and ethanol's effects were much reduced by blocking glutamate receptors. Ethanol increased presynaptic glutamate release, and about half of this effect was mediated by dopamine subtype 1 receptors (D1Rs) and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In agreement with these findings, c-Fos immunoreactivity in LHb regions was enhanced after a single administration of a low dose of ethanol (0.25 g/kg i.p.). Importantly, the same dose of ethanol in vivo also produced strong conditioned place aversion, and this was prevented by inhibiting D1Rs or neuronal activity within the LHb. By contrast, a higher dose (2 g/kg) led to ethanol conditioned place preference, which was enhanced by inhibiting neuronal activity or D1Rs within the LHb and suppressed by infusing aminomethylphosphonic acid or the D1R agonist SKF38393 within the LHb. Our in vitro and in vivo observations show, for the first time, that ethanol increases LHb excitation, mediated by D1R and glutamate receptors, and may underlie a LHb aversive signal that contributes to ethanol-related aversion.

Downregulation of miR-221-3p and upregulation of its target gene PARP1 are prognostic biomarkers for triple negative breast cancer patients and associated with poor prognosis.

The purpose of this study was to identify microRNAs (miRNAs) closely associated with the prognosis of triple-negative breast cancer (TNBC) and their possible targets. This study recruited 125 early-stage TNBC patients, including 40 cases in the experimental group (20 cases with poor prognoses vs. 20 cases with good prognoses) and 85 cases in the validation group (27 cases with poor prognoses vs. 58 cases with good prognoses). In the experimental group, miRNA microarray showed 34 differentially expressed miRNAs in patients with different prognoses. We selected 5 miRNAs for validation. The differential expression of miR-221-3p was further verified in the experimental and validation groups using real-time polymerase chain reaction (PCR). High miR-221-3p expression was associated with better 5-year disease-free survival (DFS) (HR = 0.480; 95% CI, 0.263-0.879; p = 0.017) of TNBC patients. High expression of its target gene PARP1 predicted poorer 5-year DFS (HR = 2.236, 95% CI, 1.209-4.136, p = 0.010). MiR-221-3p down-regulated PARP1 by targeting its 3'-untranslated region. In conclusion, low miR-221-3p expression may contribute to the poor outcome of TNBC patients through regulating PARP1. MiR-221-3p likely plays a role as a PARP1 inhibitor by directly regulating PARP1 expression, thereby affecting the prognoses of TNBC patients.

MiR-4653-3p and its target gene FRS2 are prognostic biomarkers for hormone receptor positive breast cancer patients receiving tamoxifen as adjuvant endocrine therapy.

Long-term tamoxifen treatment significantly improves the survival of hormone receptor-positive (HR+) breast cancer (BC) patients. However, tamoxifen resistance remains a challenge. We aimed to identify prognostic biomarkers for tamoxifen resistance and reveal the underlying mechanism. From March 2001 to September 2013, 400 HR+ BC women (stage I~III) were treated with adjuvant tamoxifen for 5 years or until relapse in West China Hospital. We included a discovery set of 6 patients who were refractory to tamoxifen, and a validation cohort of 88 patients including 35 cases with relapse. In the discovery set, microRNA microarray showed that miR-4653-3p decreased in recurrent/metastatic lesions compared to the matched primary lesions. In the validation cohort, real-time RT-PCR demonstrated that, following tamoxifen treatment, miR-4653-3p overexpression in the primary tumors decreased the risk of relapse (adjusted hazard ratio [HR] = 0.17, 95% confidence interval [CI] = 0.05~0.57, P = 0.004). Conversely, high expression of FRS2, the key adaptor protein required by FGFR signaling, predicted poor disease-free survival (DFS) (adjusted HR = 2.70, 95% CI = 1.11~6.56, P = 0.03). MiR-4653-3p down regulated FRS2 by binding to its 3' untranslated region. Either overexpressing miR-4653-3p or attenuating FRS2 expression could restore TAM sensitivity in two tamoxifen-resistant BC cell lines. In conclusion, high miR-4653-3p level was the potential predictor for favorable DFS, while FRS2 overexpression was potential high-risk factor for relapse in HR+ BC patients receiving TAM adjuvant therapy. FGFR/FRS2 signaling might be a promising target for reversing tamoxifen resistance.

High Frequency Electrical Stimulation of Lateral Habenula Reduces Voluntary Ethanol Consumption in Rats.

BACKGROUND: Development of new strategies that can effectively prevent and/or treat alcohol use disorders is of paramount importance, because the currently available treatments are inadequate. Increasing evidence indicates that the lateral habenula (LHb) plays an important role in aversion, drug abuse, and depression. In light of the success of high-frequency stimulation (HFS) of the LHb in improving helplessness behavior in rodents, we assessed the effects of LHb HFS on ethanol-drinking behavior in rats. METHODS: We trained rats to drink ethanol under an intermittent access two-bottle choice procedure. We used c-Fos immunohistochemistry and electrophysiological approaches to examine LHb activity. We applied a HFS protocol that has proven effective for reducing helplessness behavior in rats via a bipolar electrode implanted into the LHb. RESULTS: c-Fos protein expression and the frequency of both spontaneous action potential firings and spontaneous excitatory postsynaptic currents were higher in LHb neurons of ethanol-withdrawn rats compared to their ethanol-naïve counterparts. HFS to the LHb produced long-term reduction of intake and preference for ethanol, without altering locomotor activity. Conversely, low-frequency electrical stimulation to the LHb or HFS applied to the nearby nucleus did not affect drinking behavior. CONCLUSIONS: Our results suggest that withdrawal from chronic ethanol exposure increases glutamate release and the activity of LHb neurons, and that functional inhibition of the LHb via HFS reduces ethanol consumption. Thus, LHb HFS could be a potential new therapeutic option for alcoholics.

Serotonin modulates glutamatergic transmission to neurons in the lateral habenula.

The lateral habenula (LHb) is bilaterally connected with serotoninergic raphe nuclei, and expresses high density of serotonin receptors. However, actions of serotonin on the excitatory synaptic transmission to LHb neurons have not been thoroughly investigated. The LHb contains two anatomically and functionally distinct regions: lateral (LHbl) and medial (LHbm) divisions. We compared serotonin's effects on glutamatergic transmission across the LHb in rat brains. Serotonin bi-directionally and differentially modulated glutamatergic transmission. Serotonin inhibited glutamatergic transmission in higher percentage of LHbl neurons but potentiated in higher percentage of LHbm neurons. Magnitude of potentiation was greater in LHbm than in LHbl. Type 2 and 3 serotonin receptor antagonists attenuated serotonin's potentiation. The serotonin reuptake blocker, and the type 2 and 3 receptor agonists facilitated glutamatergic transmission in both LHbl and LHbm neurons. Thus, serotonin via activating its type 2, 3 receptors, increased glutamate release at nerve terminals in some LHb neurons. Our data demonstrated that serotonin affects both LHbm and LHbl. Serotonin might play an important role in processing information between the LHb and its downstream-targeted structures during decision-making. It may also contribute to a homeostatic balance underlying the neural circuitry between the LHb and raphe nuclei.

Probe-free allele-specific copy number detection and analysis of tumors.

Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping.

Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I((5-HTi))) and accelerated spontaneous firing in ∼80% of LHb neurons in rat brain slices. I((5-HTi)) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I((5-HTi)) was diminished by 5-HT(2/3) receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT(2/3) agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I((5-HTi)) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression.

Neural differentiation of mesenchymal stem cells influences their chemotactic responses to stromal cell-derived factor-1α.

Mesenchymal stem cells (MSCs) are proposed as a promising source for cell-based therapies in neural disease. Although increasing numbers of studies have been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic response and the differentiation status of these cells is still unclear. In the present study, we demonstrated that MSCs in varying neural differentiation states display various chemotactic responses to stromal cell-derived factor-1α (SDF-1α). The chemotactic responses of MSCs under different differentiation stages in response to SDF-1α were analyzed by Boyden chamber, and the results showed that cells of undifferentiation, 24-h preinduction, 5-h induction, and 18-h maintenance states displayed a stronger chemotactic response to SDF-1α, while 48-h maintenance did not. Further, we found that the phosphorylation levels of PI3K/Akt, ERK1/2, SAPK/JNK, and p38MAPK are closely related to the differentiation states of MSCs subjected to SDF-1α, and finally, inhibition of SAPK/JNK signaling significantly attenuates SDF-1α-stimulated transfilter migration of MSCs of undifferentiation, 24-h preinduction, 18-h maintenance, and 48-h maintenance, but not MSCs of 5-h induction. Meanwhile, interference with PI3K/Akt, p38MAPK, or ERK1/2 signaling prevents only cells at certain differentiation state from migrating in response to SDF-1α. Collectively, these results demonstrate that MSCs in varying neural differentiation states have different migratory capacities, thereby illuminating optimization of the therapeutic potential of MSCs to be used for neural regeneration after injury.

Salsolinol modulation of dopamine neurons.

Salsolinol, a tetrahydroisoquinoline present in the human and rat brains, is the condensation product of dopamine and acetaldehyde, the first metabolite of ethanol. Previous evidence obtained in vivo links salsolinol with the mesolimbic dopaminergic (DA) system: salsolinol is self-administered into the posterior of the ventral tegmental area (pVTA) of rats; intra-VTA administration of salsolinol induces a strong conditional place preference and increases dopamine release in the nucleus accumbens (NAc). However, the underlying neuronal mechanisms are unclear. Here we present an overview of some of the recent research on this topic. Electrophysiological studies reveal that DA neurons in the pVTA are a target of salsolinol. In acute brain slices from rats, salsolinol increases the excitability and accelerates the ongoing firing of dopamine neurons in the pVTA. Intriguingly, this action of salsolinol involves multiple pre- and post-synaptic mechanisms, including: (1) depolarizing dopamine neurons; (2) by activating µ opioid receptors on the GABAergic inputs to dopamine neurons - which decreases GABAergic activity - dopamine neurons are disinhibited; and (3) enhancing presynaptic glutamatergic transmission onto dopamine neurons via activation of dopamine type 1 receptors, probably situated on the glutamatergic terminals. These novel mechanisms may contribute to the rewarding/reinforcing properties of salsolinol observed in vivo.

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs.

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin ßIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke.