Paternal Western diet causes transgenerational increase in food consumption in Drosophila with parallel alterations in the offspring brain proteome and microRNAs.

Several lines of evidence indicate that ancestral diet might play an important role in determining offspring's metabolic traits. However, it is not yet clear whether ancestral diet can affect offspring's food choices and feeding behavior. In the current study, taking advantage of Drosophila model system, we demonstrate that paternal Western diet (WD) increases offspring food consumption up to the fourth generation. Paternal WD also induced alterations in F1 offspring brain proteome. Using enrichment analyses of pathways for upregulated and downregulated proteins, we found that upregulated proteins had significant enrichments in terms related to translation and translation factors, whereas downregulated proteins displayed enrichments in small molecule metabolic processes, TCA cycles, and electron transport chain (ETC). Using MIENTURNET miRNA prediction tool, dme-miR-10-3p was identified as the top conserved miRNA predicted to target proteins regulated by ancestral diet. RNAi-based knockdown of miR-10 in the brain significantly increased food consumption, implicating miR-10 as a potential factor in programming feeding behavior. Together, these findings suggest that ancestral nutrition may influence offspring feeding behavior through alterations in miRNAs.

In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons.

Background: Diabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown. Aim: In this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM. Methods: Major pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45 mM) for 24 or 48 hours and compared to time-matched controls (25 mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50 mg/kg, n = 4), and 5 weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100. Outcomes: Length, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture. Results: The total number of neurons and the length and number of branches were significantly decreased after 24 and 48 hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05). Clinical Translation: Glucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply. Strengths and Limitations: Exposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM. Conclusion: Culturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.

Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival.

BACKGROUND: Prostatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn't been explored. AIM: To determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT. METHODS: Major pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling. OUTCOMES: We measured neurite length, branching, specific neuron populations and apoptosis. RESULTS: Ex vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss. CLINICAL TRANSLATION: These findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT. STRENGTHS AND LIMITATIONS: This is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo. CONCLUSION: Unirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions. Randolph JT, Pak ES, McMains JC, et al. Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival. J Sex Med 2022;19:1333-1342.

High-fat diet induces obesity in adult mice but fails to develop pre-penile and penile vascular dysfunction.

Obesity can lead to cardiovascular disease, diabetes, and erectile dysfunction (ED), which decreases overall quality of life. Mechanisms responsible for obesity-induced ED are unknown. Current mouse models of high-fat diet (HFD)-induced obesity yield conflicting results. Genetic variants among common "wild type" strains may explain contradictory data. Adult male C57BL/6N and 6J mice were fed a 45% HFD for 12 weeks. Weekly food intake, weight gain, and body-fat percentage were measured. After 12 weeks, ex vivo vascular reactivity was measured in aortas, internal pudendal arteries, and penises. We assessed smooth muscle contractility, endothelial-dependent and -independent relaxation, and penile neurotransmitter-mediated relaxation. C57BL/6N mice developed greater obesity and glucose sensitivity compared to C57BL/6J mice. Aortas from both strains that fed a HFD had decreased contraction, yet contraction was unchanged in HFD pudendal arteries and penises. Interestingly, endothelial-dependent and -independent relaxation was unchanged in both systemic and penile vasculature. Likewise, HFD did not impair penile neurotransmitter-mediated relaxation. Both strains fed 12 weeks of HFD-developed obese phenotypes. However, HFD did not impair pre-penile or penile smooth muscle vasoreactivity as demonstrated in previous studies, suggesting that this preclinical model does not accurately represent the clinical phenotype of obesity-induced ED.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning.

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Ex vivo Akt inhibition reverses castration induced internal pudendal artery and penile endothelial dysfunction.

AIMS: Androgen deprivation therapy is a common prostate cancer treatment which causes men to have castrate levels of testosterone. Unfortunately, most testosterone deficient patients will suffer severe erectile dysfunction (ED) and have no effective ED treatment options. Testosterone deficiency causes endothelial dysfunction and impairs penile vasodilation necessary to maintain an erection. Recent evidence demonstrates testosterone activates androgen receptors (AR) and generates nitric oxide (NO) through the Akt-endothelial NO synthase (eNOS) pathway; however, it remains unknown how castration impacts this signaling pathway. MATERIALS AND METHODS: In this study, we used a surgically castrated rat model to determine how castration impacts ex vivo internal pudendal artery (IPA) and penile relaxation through the Akt-eNOS pathway. KEY FINDINGS: Unlike systemic vasculature, castration causes significant IPA and penis endothelial dysfunction associated with a 50% AR reduction. Though testosterone and acetylcholine (ACh) both phosphorylate Akt and eNOS, castration did not affect testosterone-mediated IPA and penile Akt or eNOS phosphorylation. Surprisingly, castration increases ACh-mediated Akt and eNOS phosphorylation but reduces the eNOS dimer to monomer ratio. Akt inhibition using 10DEBC preserves IPA eNOS dimers. Functionally, 10DEBC reverses castration induced ex vivo IPA and penile endothelial dysfunction. SIGNIFICANCE: These data demonstrate how castration uncouples eNOS and provide a novel strategy for improving endothelial-dependent relaxation necessary for an erection. Further studies are needed to determine if Akt inhibition may treat or even prevent ED in testosterone deficient prostate cancer survivors.

Impact of prostatic radiation therapy on bladder contractility and innervation.

AIMS: To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats. METHODS: Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat. RESULTS: At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression. CONCLUSION: In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.

Preference and detrimental effects of high fat, sugar, and salt diet in wild-caught Drosophila simulans are reversed by flight exercise.

High saturated fat, sugar, and salt contents are a staple of a Western diet (WD), contributing to obesity, metabolic syndrome, and a plethora of other health risks. However, the combinatorial effects of these ingredients have not been fully evaluated. Here, using the wild-caught Drosophila simulans, we show that a diet enriched with saturated fat, sugar, and salt is more detrimental than each ingredient separately, resulting in a significantly decreased lifespan, locomotor activity, sleep, reproductive function, and mitochondrial function. These detrimental effects were more pronounced in female than in male flies. Adding regular flight exercise to flies on the WD markedly negated the adverse effects of a WD. At the molecular level, the WD significantly increased levels of triglycerides and caused mitochondrial dysfunction, while exercise counterbalanced these effects. Interestingly, fruit flies developed a preference for the WD after pre-exposure, which was averted by flight exercise. The results demonstrate that regular aerobic exercise can mitigate adverse dietary effects on fly mitochondrial function, physiology, and feeding behavior. Our data establish Drosophila simulans as a novel model of diet-exercise interaction that bears a strong similarity to the pathophysiology of obesity and eating disorders in humans.

Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons.

BACKGROUND: Prostatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. AIM: To determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. METHODS: MPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. OUTCOMES: After 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. RESULTS: RT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P < .001) and whole MPG culture via upregulation of caspase 3 gene expression (P < .05). Nitrergic neurons were markedly decreased in irradiated dissociated culture (P < .05), while nNOS gene expression was upregulated in irradiated whole organ culture (P < .05). The proportion of dissociated sympathetic neurons and whole organ TH gene expression remained unchanged after RT. Interestingly, RT dissociated neurites were 22% shorter than controls, while RT whole organ neurites were 15% longer than controls (P < .01). MPG Schwann cells markers (Sox10, Krox20) were elevated after RT in whole organ culture. CLINICAL TRANSLATION: Prostatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. STRENGTHS & LIMITATIONS: The advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. CONCLUSION: The 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT. Randolph JT, Pak ES, Koontz BF, et al. Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons. J Sex Med 2020;17:1423-1433.

Chronic high-fat diet decreased detrusor mitochondrial respiration and increased nerve-mediated contractions.

AIMS: To assess the impact of chronic high-fat diet (HFD) on behavioral voiding patterns, detrusor contractility, and smooth muscle mitochondrial function in male mice. MATERIALS AND METHODS: Male C57BL/6J mice (6 weeks) were fed a control or HFD for 20 weeks. Bladder function was assessed by void spot assays. Bladders were collected and detrusor contractility to carbachol (10-9 -10-5 M), and electrical field stimulation (EFS, 0.5-32 Hz) in the presence and absence of atropine was measured. Homogenized detrusor samples were placed in oxygraphs to assess the rate of oxygen consumption of the mitochondria within the detrusor in the presence of different substrates. Mitochondrial hydrogen peroxide (H2 O2 ) emission was measured fluorometrically. Detrusor citrate synthase activity was measured via enzyme activity kit and Western blots assessed the electron transport chain (ETC) protein content. RESULTS: HFD significantly increased body weight, adiposity, and blood glucose levels. HFD mice demonstrated increased voiding frequency and increased EFS-induced detrusor contractility. There were no changes in detrusor relaxation or cholinergic-medicated contraction. Mitochondrial respiration was decreased with HFD and H2 O 2 emission was increased. The relative amount of mitochondria in the detrusor was similar between groups. However, ETC complexes V and III were increased following HFD. CONCLUSIONS: Chronic HFD increased adiposity, lead to more frequent voiding, and enhanced EFS-mediated detrusor contractions. Mitochondrial respiration was decreased and H2 O 2 emission increased following HFD. Further research is required to determine if alterations in mitochondrial function could play a role in the development of HFD-induced bladder dysfunction.

Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth.

BACKGROUND: Erectile dysfunction (ED) is common following radiation therapy (RT) for prostate cancer. Although the cause of RT-induced ED is unknown, damage to both the neuronal and vascular components supporting erections are often implicated. AIM: To determine the effects of prostatic RT on erections, penile vascular physiology, and major pelvic ganglia (MPG) neuron growth and survival in a rat model. METHODS: Male rats underwent 0 Gy or 22 Gy single fraction of prostate-confined, conformal RT. At 2 weeks or 10 weeks post-RT (n = 10/group), cavernous nerve stimulation was performed and erections were assessed. Tissue bath experiments were performed to assess both penile artery and internal pudendal artery (IPA) function. MPGs were dissociated and neurons grown in culture for 72 hours. Immunofluorescence staining was done to quantify neuron survival (terminal deoxynucleotidyl transferase nick-end labeling), outgrowth (beta-tubulin III), type (nitric oxide synthase [nNOS] and tyrosine hydroxylase [TH]), and nerve injury markers (small GTPase Rac1 and ninjurin-1 [Ninj-1]). Whole MPG real-time quantitative polymerase chain reaction (qPCR) was performed to measure expression of genes related to nerve type, neuron injury, repair, and myelination, such as Ninj-1, Rac1, ATF3, GAP43, GFAP, SOX10, and KROX20. OUTCOMES: Intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio, smooth muscle contractility and relaxation, gene expression, neuritogenesis, and apoptosis. RESULTS: Following RT, ICP/MAP was unchanged at 2 weeks or 10 weeks. Nerve-mediated penile contraction was increased at 2 weeks, whereas adrenergic contraction was reduced at 10 weeks. Penile relaxation and IPA vasoreactivity were unchanged. Neuronal apoptosis was more than doubled both early and late post-RT. RT caused a progressive decrease in neurite branching but an early increase and then late decrease in neurite lengthening. RT reduced the numbers of nNOS-positive neurons both early and late and also decreased MPG nitrergic gene expression. TH neurons and gene expression were unchanged at 2 weeks; however, both were decreased after 10 weeks. Although most markers of gene injury and repair were unaffected early post-RT, MPG expression of Ninj1 and GFAP increased. After 10 weeks, Ninj1 and GFAP remained elevated while markers of neuron injury (ATF3), outgrowth (GAP43 and Rac1), and myelin regulation (SOX10) were decreased. CLINICAL TRANSLATION: RT-induced ED may result from damage to the ganglia controlling erections. STRENGTHS & LIMITATIONS: This study used a clinically relevant, prostate-confined model to examine neurovascular structures not accessible in human studies. Unfortunately, rats did not exhibit ED at this time point. CONCLUSION: This is the first study to demonstrate impaired health and regeneration potential of dissociated MPG neurons following RT. Neuronal injury was apparent early post-RT and persisted or increased over time but was insufficient to cause ED at the time points examined. Powers SA, Odom MR, Pak ES, et al. Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth. J Sex Med 2019;16:27-41.

Clarifying the Relative Impacts of Vascular and Nerve Injury That Culminate in Erectile Dysfunction in a Pilot Study Using a Rat Model of Prostate Irradiation and a Thrombopoietin Mimetic.

PURPOSE: Radiation therapy (RT) offers an important and curative approach to treating prostate cancer, but it is associated with a high incidence of erectile dysfunction (ED). It is not clear whether the etiology of radiation-induced ED (RI-ED) is driven by RT-mediated injury to the vasculature, the nerves, or both. This pilot study sought to distinguish the effects of vascular and nerve injury in RI-ED by applying a vascular radioprotectant in a rat model of prostate RT. METHODS: A single dose of the thrombopoietin mimetic (TPOm; RWJ-800088), previously shown to mitigate radiation-induced vascular injury, was administered 10 minutes after single-fraction conformal prostate RT. Nine weeks after RT, rats were assessed for erectile and arterial function. Nerve markers were quantified with reverse transcriptase polymerase chain reaction. Immunofluorescent microscopy further characterized vascular effects of RT and TPOm. RESULTS: Sham animals and animals that received RT and TPOm showed significant arterial vasodilation in response to systemic hydralazine (24.1% ± 7.3% increase; P = .03 in paired t test). However, animals that received RT and vehicle were unable to mount a vasodilatory response (-7.4% ± 9.9% increase; P = .44 in paired t test). TPOm prevented RT-induced change in the penile artery cross-sectional area (P = .036), but it did not ameliorate cavernous nerve injury as evaluated by gene expression of neuronal injury markers. Despite significant structural and functional vascular protective effects and some trends for differences in nerve injury/recovery markers, TPOm did not prevent RI-ED at 9 weeks, as assessed by intracavernous pressure monitoring after cavernous nerve stimulation. CONCLUSIONS: These data suggest that vascular protection alone is not sufficient to prevent RI-ED and that cavernous nerve injury plays a key role in RI-ED. Further research is required to delineate the multifactorial nature of RI-ED and to determine if TPOm with modified dosing regimens can mitigate against nerve injury either through direct or vascular protective effects.

miRNA-431 Prevents Amyloid-ß-Induced Synapse Loss in Neuronal Cell Culture Model of Alzheimer's Disease by Silencing Kremen1.

Synapse loss is well regarded as the underlying cause for the progressive decline of memory function over the course of Alzheimer's disease (AD) development. Recent observations suggest that the accumulation of the Wnt antagonist Dickkopf-1 (Dkk1) in the AD brain plays a critical role in triggering synaptic degeneration. Mechanistically, Dkk1 cooperates with Kremen1 (Krm1), its transmembrane receptor, to block the Wnt/ß-catenin signaling pathway. Here, we show that silencing Krm1 with miR-431 prevents amyloid-ß-mediated synapse loss in cortico-hippocampal cultures isolated from triple transgenic 3xTg-AD mice. Exposure to AßDDL (an amyloid-ß derived diffusive ligand) or Dkk1 reduced the number of pre- and post-synaptic puncta in primary neuronal cultures, while treatment with miR-431 prevented synapse loss. In addition, treatment with miR-431 also prevented neurite degeneration. Our findings demonstrate that miR-431 protects synapses and neurites from Aß-toxicity in an AD cell culture model and may be a promising therapeutic target.

NLRP3/IL-1ß mediates denervation during bladder outlet obstruction in rats.

AIMS: Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3-mediated inflammation on bladder denervation during BOO. METHODS: There were five groups of rats: (i) Control (no surgery); (ii) Sham-operated; (iii) BOO rats given vehicle; (iv) BOO rats given the NLRP3 inhibitor glyburide; and (v) BOO rats given the IL-1 receptor antagonist anakinra. BOO was constructed by ligating the urethra over a 1 mm catheter and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Bladder sections were stained for PGP9.5, a pan-neuronal marker. Whole transverse sections were used to identify and count nerves while assessing cross-sectional area. For in vitro studies, pelvic ganglion neurons were isolated and treated with IL-1ß. After a 48 h incubation apoptosis, neurite length and branching were assessed. RESULTS: In obstructed bladders, the number of nerves decreased while total area increased, indicating a loss of cell number and/or branching. The decrease in nerve density was blocked by glyburide or anakinra, clearly implicating the NLRP3 pathway in denervation. In vitro analysis demonstrated that IL-1ß, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1ß triggered apoptosis. CONCLUSIONS: The NLRP3/IL-1ß-mediated inflammation pathway plays a significant role in denervation during BOO.

Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries.

BACKGROUND: The internal pudendal arteries (IPAs) supply blood to the penis and are highly susceptible to vascular remodeling in rodent models of diabetes, hypertension, aging, and chronic kidney disease, thus contributing to erectile dysfunction. Interestingly, vascular remodeling primarily occurs in the distal and not in the proximal IPA, suggesting distinct local physiologic signaling differences within the IPA. AIM: To examine the role of purinergic signaling and neurotransmitter release by electrical field stimulation (EFS) in the regulation of proximal and distal IPA vascular tone. METHODS: Proximal and distal IPAs were mounted in wire myographs and vascular responses to phenylephrine, acetylcholine, and 2-(N,N-diethylamino)-diazenolate-2-oxide, diethyl-ammonium salt (DEA NONOate) were measured. EFS-mediated contraction and non-adrenergic non-cholinergic (NANC) relaxation were evaluated in the absence and presence of a nitric oxide synthase antagonist. Purinergic agonist and NANC relaxation responses were assessed in the presence and absence of P2X1 and P2Y1 antagonists. Protein expression of P2X1 and P2Y1 receptors was measured by western blot. MAIN OUTCOME MEASURES: Proximal and distal IPA contraction and relaxation were measured during increasing agonist administration and EFS in the presence and absence of antagonists. RESULTS: Proximal and distal IPA concentration response curves to phenylephrine, acetylcholine, and DEA NONOate did no differ. Interestingly, distal IPA exhibited greater EFS-mediated contraction and NANC relaxation compared with proximal IPA. Nitric oxide synthase inhibition completely inhibited distal IPA NANC relaxation but did not affect proximal IPA relaxation. P2X1 or P2Y1 receptor antagonism during NANC relaxation increased distal IPA relaxation but decreased proximal IPA relaxation. Combined P2X1 and P2Y1 receptor antagonism had no effect on proximal IPA relaxation but significantly increased distal IPA NANC relaxation. CLINICAL TRANSLATION: Understanding neurovascular regulation of IPA vascular tone through nitrergic and purinergic mechanisms could yield new therapeutic targets to improve IPA blood flow and treat vasculogenic erectile dysfunction. STRENGTHS AND LIMITATIONS: This study is the first to illustrate the differences in mechanisms responsible for regulating vascular tone in the proximal and distal IPAs. All presented findings are currently limited to ex vivo vascular function. CONCLUSION: The regulation of vascular tone differs regionally in the IPA. The distal IPA is controlled through neurotransmitter-mediated NO-dependent mechanisms and increased sensitivity to purinergic P2X1 and P2Y1 receptor inhibition. Odom MR, Pak ES, Brown DA, Hannan JL. Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries. J Sex Med 2017;14:1285-1296.

Paternal long-term exercise programs offspring for low energy expenditure and increased risk for obesity in mice.

Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin-resistant phenotype. Although diet-induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long-term voluntary wheel-running in C57BL/6J male mice on their offspring's predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel-running for 12 wk produced offspring that were more susceptible to the adverse effects of a high-fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long-term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro-RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA-induced modification of sperm.

Neurogenic potential of spinal cord organotypic culture.

There are several neurogenic niches in the adult mammalian central nervous system. In the central nervous system, neural stem cells (NSC) localize not only to the periventricular area, but are also diffusely distributed in the parenchyma. Here, we assessed neurogenic potential of organotypic cultures prepared from adult mouse spinal cord. Slices were placed on Millipore inserts for organotypic culture and incubated in neurobasal media supplemented with B27 and N2 for up to 9 weeks. After 3-4 weeks, the cell's aggregates formed in the slices. The aggregate's cells were BrdU-uptake, nestin and alkaline phosphatase positive. At the later stage of incubation, we observed Oct3/4 in the inner mass of the neurospheres as well as expression of Dppa1, which is an Oct-4 downstream target gene and a marker for pluripotency. To check differentiation, the formed neurospheres were isolated and cultured for several days in differentiation media. The obtained data demonstrated the cells from isolated neurospheres differentiate into astrocytes and MAP2-positive neurons. Immunostaining for HB9 and Lim2 revealed subsequent differentiation of MAP2-positive cells into motor neurons and interneurons, respectively. We hypothesized neuronal loss and/or long-term culturing of spinal cord slices may trigger a reset of the internal cell program and promote proliferation and further differentiation of NSC.

Multi-walled carbon nanotubes inhibit regenerative axon growth of dorsal root ganglia neurons of mice.

Recent observations have demonstrated that nanomaterials may be toxic to human tissue. While the ability of nano-scaled particulate matter is known to cause a range of problems in respiratory system, recent observations suggest that the nervous system may be vulnerable as well. In the current paper we asked whether exposure of primary neuronal cell cultures to nanoparticles might compromise regenerative axon growth. Regenerative response was triggered by performing a conditioning lesion of sciatic nerve five days prior to collection of dorsal root ganglia (DRG). DRG neurons were plated at a low density and incubated with multi-walled carbon nanotubes (MWCNTs) (0.1-10 µg/ml in 10% of surfactant in saline) overnight. The experiments showed that exposure of DRG cultures to MWCNT significantly impaired regenerative axonogenesis without concomitant cell death. These results indicate that MWNCTs may have detrimental effect on nerve regeneration and may potentially trigger axonal pathology.

Embryonic stem cells inhibit expression of erythropoietin in the injured spinal cord.

Recent observations have demonstrated neuroprotective role of erythropoietin (Epo) and Epo receptor in the central nervous system. Here we examined Epo function in the murine spinal cord after transplantation of pluripotent mouse embryonic stem (ES) cells pre-differentiated towards neuronal type following spinal cord injury. Expression of Epo was measured at both mRNA and protein levels in the ES cells as well as in the spinal cords after 1 and 7 days. Our data demonstrated that expression of Epo mRNA, as well as its protein content, in ES cells was significantly decreased after differentiation procedure. In the spinal cords, analysis showed that Epo mRNA level was significantly decreased after 1 day of ES cell injections in comparison to media-injected control. Epo protein level detected by Western blot was diminished as well. Examination of Epo production in the injured spinal cords after media or ES cells injections by indirect immunofluorescence showed increased Epo-immunopositive staining after media injections 1 day after injection. In contrast, ES cell transplantation did not induce Epo expression. Seven days after ES cell injections, Epo-immunopositive cells' distribution in the ipsilateral side was not changed, while the intensity of immunostaining on the contralateral side was increased, approaching levels in control media-injected tissues. Our data let us to presume that previously described immediate positive effects of ES cells injected into the injured zone of spinal cord are not based on Epo, but on other factors or hormones, which should be elucidated further.

Pre-differentiated embryonic stem cells promote neuronal regeneration by cross-coupling of BDNF and IL-6 signaling pathways in the host tissue.

The mechanism of embryonic stem (ES) cell therapeutic action remains far from being elucidated. Our recent report has shown that transplantation of ES cells, predifferentiated into neuronal progenitors, prevented appearance of chronic pain behaviors in mice after experimentally induced spinal cord injury. In the current study, we tested the hypothesis that this beneficial effect is mediated by antiapoptotic and regenerative signaling pathways activated in the host tissue by transplanted ES cells. Spinal cord injury was induced by unilateral microinjections of quisqualic acid at spinal levels T12-L2. At 1 week after injury, the pre-differentiated towards neuronal phenotype ES cells were transplanted into the site of injury. Here we show that transplantation of pre-differentiated ES cells activate both brain-derived neurotrophic factor (BDNF) and interleukin-6 (IL-6) signaling pathways in the host tissue, leading to activation of cAMP/PKA, phosporylation of cofilin and synapsin I, and promoting regenerative growth and neuronal survival.