Nerve growth factor attenuates oxidant-induced ß-amyloid neurotoxicity in sporadic Alzheimer's disease cybrids.

Although mitochondrial dysfunction has been linked to Alzheimer's disease (AD), it is not fully understood how this dysfunction may induce neuronal death. In this study, we show that transmitochondrial hybrid cells (cybrids) expressing mitochondrial genes from patients with sporadic AD (SAD) have substantial alterations in basal upstream tyrosine kinase signaling and downstream serine-threonine kinase signaling that are mediated by intracellular free radicals. This is associated with reduced tropomyocin receptor kinase (TrkA) and p75 neurotrophin receptor receptor expression that profoundly alters nerve growth factor signaling, increases generation of Aß and decreases viability. Many of these observed effects in SAD cybrids would be predicted to increase risk of premature neuronal death and reduce resistance to stressors and add further support for the pathogenic role of mtDNA expression in the pathogenesis of SAD.

Origin and characterization of retrograde labeled neurons supplying the rat urethra using fiberoptic confocal fluorescent microscopy in vivo and immunohistochemistry.

PURPOSE: Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. MATERIALS AND METHODS: We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. RESULTS: Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. CONCLUSIONS: Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may increase urethral resistance or regulate the reflex mechanisms controlling continence.

Fiberoptic imaging for urologic surgery.

Laparoscopic and robot-assisted surgery is likely to be improved with the development of real-time, intraoperative imaging for diagnosis, margin determination, and anatomical definition. A significant goal of much of this effort has been focused upon providing better outcomes after radical prostatectomy. The feasibility of fluorescent imaging of labeled cavernosal nerves in the operative field has been demonstrated in vivo in animals. Other applications of the technology and capability will certainly be developed over time. This article reviews and assesses the potential and capabilities of the different imaging modes currently in use or development.

Tadalafil increases Akt and extracellular signal-regulated kinase 1/2 activation, and prevents apoptotic cell death in the penis following denervation.

PURPOSE: Despite techniques to preserve the cavernous nerves during radical prostatectomy erectile dysfunction remains a complication. We determined whether bilateral cavernous nerve resection induces apoptosis in the penis. We also determined whether treatment with the phosphodiesterase-5 inhibitor tadalafil prevents apoptosis as well as the specific mechanisms involved. MATERIALS AND METHODS: Mice were subjected to cavernous nerve resection or sham surgery. Penises were processed for the identification of apoptotic cells, changes in phosphorylation of several protein kinases and immunolocalization of specific kinases. Mice were also placed on tadalafil or vehicle after cavernous nerve resection and the penises were processed as described. Statistical analysis was performed with the Mann-Whitney U test for comparisons among groups or Student's t test. RESULTS: An increase in apoptotic cavernous smooth muscle and endothelial cells was evident by 2 weeks, which further increased 4 and 6 weeks after cavernous nerve resection. Apoptosis coincided with an increase in the phosphorylation of c-jun N-terminal kinase and p38 mitogen activated protein kinase. Phospho-c-jun N-terminal kinase was immunolocalized to endothelial and smooth muscle cells. Treatment with tadalafil decreased the number of apoptotic cells and increased the phosphorylation of the 2 survival associated kinases Akt and extracellular signal-regulated kinase 1/2. CONCLUSIONS: These results provide a rationale for the early use of phosphodiesterase-5 inhibition following radical prostatectomy or extensive pelvic surgery, during which there may be injury to the cavernous nerves, to aid in the return of erectile function.

Does oxybutynin alter plaques, amyloid beta peptides and behavior in a mouse model of Alzheimer's disease?

PURPOSE: In elderly patients oxybutynin (Sigma-Aldrich) is commonly used to treat overactive bladder despite increased prevalence of Alzheimer's disease in this population. We determined whether oxybutynin altered plaque formation, amyloid beta peptide expression and behavior in a transgenic mouse model of Alzheimer's disease expressing the mutant human presenilin 1 (deltaE9) and a chimeric mouse/human amyloid precursor protein (APPswe). MATERIALS AND METHODS: Mice were treated for 30 days in an acute experiment or 5 months in a chronic experiment with oxybutynin (30 mg/kg) or vehicle. Behavioral testing was performed monthly with the elevated plus maze (Med Associates, St. Albans, Vermont) in the chronic experiment. Brains were tested for plaque burden using Hirano silver and thioflavin-S (Sigma-Aldrich) staining. Amyloid beta peptide expression was tested using enzyme-linked immunosorbent assay for amyloid beta peptides 1-40 and 1-42. RESULTS: Animals treated with chronic oxybutynin had a decreased plaque burden in the hippocampus (mean +/- SEM 2.2 +/- 0.4 vs 4.1 +/- 0.9 plaques, p <0.05) and cortex (5.8 +/- 0.7 vs 11.6 +/- 2.1, p <0.05) compared to animals treated with vehicle. Oxybutynin treated animals also had decreased expression of amyloid beta 1-42 (82.8 +/- 9.0 etag/ml vs 105.6 +/- 5.5 etag/ml, p = 0.05) compared to animals treated with vehicle. Female Alzheimer's disease mice treated with oxybutynin but not males showed improved behavior with a greater percent of time spent in the closed arm or elevated plus maze (95.9% +/- 1.6% vs 35.6% +/- 18.9%, p <0.05). The greatest difference was noted at 3 months of treatment compared to vehicle. CONCLUSIONS: These results suggest that oxybutynin may slow the progression of Alzheimer's disease in this model.

Fiberoptic imaging of cavernous nerves in vivo.

PURPOSE: A critical intraoperative variable for the return of tumescence following radical prostatectomy is preservation of the cavernous nerves. We developed a nontoxic technique that would allow high resolution, in vivo real-time imaging specifically of the cavernous nerves. MATERIALS AND METHODS: The cavernous nerves were labeled by injecting a fluorescent retrograde nerve tracer into the corpus cavernosum of male rats. Nerves were subsequently imaged in vivo using fiberoptic confocal fluorescent microscopy. Initial screening trials were performed to decide on a nerve tracer capable of axonal labeling, optimize injection concentration and characterize retrograde transport time. Toxicity studies included intracavernous pressure monitoring following electrical nerve stimulation, apoptotic staining of injected cavernous tissue and measurement of lipid peroxidation in nerves exposed to laser emissions during imaging. RESULTS: In vivo real-time video sequences of fluorescently labeled cavernous nerves were recorded. The screening trial indicated that the B subunit of cholera toxin conjugated to AlexaFluor 488 (Invitrogen) provided optimal imaging after 9 days of retrograde transport. Toxicity studies showed that maximal intracavernous pressure responses did not differ between labeled and unlabeled nerves (p = 0.9671). Tracer injection did not increase apoptosis in cavernous tissue and laser exposure did not increase lipid peroxidation in nerves. CONCLUSIONS: In vivo real-time imaging of the cavernous nerves is possible with no measurable toxicity, allowing the maintenance of erection. This novel imaging modality may allow urologists to identify cavernous nerves during pelvic surgery.

The role of corticotropin releasing factor and its antagonist, astressin, on micturition in the rat.

The purpose of this investigation was to evaluate the role of corticotropin releasing factor (CRF) on micturition. CRF is involved in the endocrine and central nervous system responses to stress and is also expressed in sites responsible for the control of micturition. In this investigation, cystometric experiments were performed in awake and unrestrained Wistar rats and on Spontaneous Hypertensive Rats, which are used as a rodent model of detrusor overactivity and anxiety. In vitro effects of CRF were evaluated using strips of detrusor muscle in an organ bath preparation. CRF (6.0 microg) administered via intrathecal and intraperitoneal routes, but not intracerebroventricularly, lowered the micturition threshold. CRF reduced the intercontraction interval by 28% and 26% after intrathecal or intraperitoneal administration, respectively, and reduced micturition volume by 34.7% and 30.2%, respectively. In Wistar-Kyoto rats, 6.0 microg intrathecal CRF significantly reduced intercontraction interval (423 +/- 79 vs. 669 +/- 59 s) and micturition volume (0.30 +/- 0.04 vs. 0.69 +/- 0.07 ml) compared to controls that received saline vehicle. These effects were blocked by pretreatment with 6.0 mug intrathecal astressin, a potent CRF antagonist, demonstrating that the effects are CRF receptor mediated. In Spontaneous Hypertensive Rats, 6.0 mug intrathecal CRF was found to have minimal stimulatory effects on the bladder, whereas astressin reduced baseline detrusor overactivity. CRF had no direct contractile effects on detrusor muscle strips. These results demonstrate that in the absence of detrusor overactivity, CRF stimulates micturition when administered via the intrathecal or intraperitoneal routes. Further studies are needed to explore the possibility whether CRF antagonists are effective for detrusor overactivity and the overactive bladder syndrome.

Methylpyridinium (MPP(+))- and nerve growth factor-induced changes in pro- and anti-apoptotic signaling pathways in SH-SY5Y neuroblastoma cells.

The parkinsonian neurotoxin methylpyridinium (MPP(+)) mimics the neuropathology of Parkinson's disease (PD) and likely kills neurons by inhibiting complex I of the electron transport chain and increasing oxidative stress. We examined the time course of activation/inactivation of multiple pro- and anti-apoptotic signaling pathways in MPP(+)-induced apoptotic death of SH-SY5Y neuroblastoma cells. We found an early increase and later decrease of transcriptional activity of the generally anti-apoptotic nuclear factor kappa-beta (NF-kappa B) and early increases in activating phosphorylation of the anti-apoptotic upstream kinase protein kinase B (PKB, also known as AKT). Sequestration-inducing phosphorylation of pro-apoptotic BAD protein increased early then declined. A small biphasic increase in the generally pro-apoptotic p38 kinase activity paralleled the biphasic rise in NF-kappa B-mediated transcription. Inhibition of p38 kinase with 5 micro M SB203540, inhibition of MEK-ERK with 50 micro M U0126, or inhibition of phosphatidylinositol-3-kinase (PI3K) with 10 micro M LY294002 reduced cell viability by 4, 18 or 37%, respectively, after 24 h. All three kinase inhibitors increased cell death in response to 24 h of MPP(+), with the greatest effect shown by LY294002. Nerve growth factor (NGF) caused an early increase in activating phosphorylation of PKB/AKT and MEK-ERK and increased cell survival during MPP(+) exposure. We found that acute MPP(+) exposure activates multiple interacting death- and survival-promoting pathways. Survival-promoting MEK-ERK and PI3K pathways contribute to viability during MPP(+) exposure, both are activated by NGF, and loss of PI3K-mediated signaling and NF-kappa B-mediated transcription may commit cells irreversibly to apoptosis in this model. It remains unknown to what extent these signaling pathways modulate dopamine neuronal death in PD.

Mechanisms of Disease: the role of nerve growth factor in the pathophysiology of bladder disorders.

The case is compelling for the involvement of nerve growth factor (NGF) in the pathogenesis of lower urinary tract disease, especially in conditions with altered neural function. Remodeling of the micturition pathways occurs following experimental bladder-outlet obstruction, denervation, spinal cord injury, cystitis, and diabetes mellitus. Clinically, NGF levels are elevated in the bladders of men with benign prostatic hyperplasia, women with interstitial cystitis and in patients with idiopathic overactive bladder. Blockade of NGF, using either an endogenous antibody or an antibody against the NGF receptor, prevents neural plasticity and bladder overactivity in experimental models of these conditions. The ability of NGF to trigger bladder overactivity might rely on altering the properties of sodium or potassium channels (or their expression) in bladder afferent fibers. Therapies based on altered NGF levels, or changes in channel properties in afferent nerves, represent an intriguing avenue of investigation for the management of detrusor overactivity or diabetic cystopathy.

Increased expression of heat shock protein 20 and decreased contractile stress in obstructed rat bladder.

PURPOSE: Bladder outlet obstruction induces detrusor hypertrophy and it can eventually lead to decreased bladder smooth muscle contractility. Heat shock protein 20 is the proposed mediator of force suppression in vascular smooth muscle. We investigated whether heat shock protein 20 could also mediate the decreased contractility observed in partially obstructed rat bladders. MATERIALS AND METHODS: Female Wistar rats (Harlan Laboratories, Indianapolis, Indiana) were randomized to partial urethral ligation or sham ligation. After 3 weeks the rats were sacrificed, and the bladders were harvested, frozen, homogenized and analyzed for heat shock protein 20 content by Western blot immunoreactivity. The content of myosin regulatory light chain, a constitutively expressed protein, was determined as a control. Bladder smooth muscle strips were dissected from some rats and mounted for force generation measurement. RESULTS: At cystectomy obstructed bladders were significantly heavier and had more residual urine compared to sham operated bladders. Heat shock protein 20 immunoreactivity was significantly increased a mean +/- 1 SEM of 1.9 +/- 0.3-fold in obstructed vs sham operated bladders. Control protein myosin regulatory light chain immunoreactivity did not significantly differ in obstructed and sham operated bladders. Maximal stress, that is force per cross-sectional area, was significantly decreased in obstructed vs sham operated bladders. Human bladder was found to express immunoreactive heat shock protein 20. CONCLUSIONS: We noted that partially obstructed rat bladders 1) express higher levels of heat shock protein 20 and 2) generate less stress than sham operated bladders. These data suggest the possibility that heat shock protein 20 over expression could at least partially mediate the decreased contractile activity observed with partial bladder outlet obstruction. The mechanism for increased heat shock protein 20 expression is unknown but it may involve increased mechanical stress or hypoxia from urethral obstruction. Human bladder expressed immunoreactive heat shock protein 20, suggesting that a similar mechanism could potentially occur in humans. If confirmed in humans, patients with clinical conditions that result in detrusor hypocontractility could potentially benefit from pharmacological interventions aimed at inhibiting heat shock protein 20.

Altered intracellular signaling and reduced viability of Alzheimer's disease neuronal cybrids is reproduced by beta-amyloid peptide acting through receptor for advanced glycation end products (RAGE).

Activation of apoptosis by increased production of amyloid beta peptides (Abeta) has been implicated in neuronal cell death of Alzheimer's disease (AD). We used mitochondrial transgenic cybrid models of sporadic AD (SAD), which overproduce Abeta compared to control (CTL) cybrids, to investigate the effects of endogenously generated Abeta on intracellular signaling pathways and viability. Reducing SAD Abeta production with gamma-secretase inhibition altered the total phosphorylation profile of SAD cybrid to one similar to CTL cybrids and enhanced viability to approximately CTL cybrid levels. Treating CTL cybrids with exogenous Abeta or conditioned media (CM) from SAD cybrids activated the signaling pathways active in SAD cybrids under basal condition and decreased viability. Antibodies against receptor for advanced glycation end products (RAGE) blocked Abeta-induced activation of the p38, JNK pathways, and NF-kappaB in CTL cybrids and offered protection against the neurotoxic effects of Abeta. Expression of SAD mitochondrial genes in cybrids activates stress-related signaling pathways and reduces viability. This SAD phenotype is produced by endogenously generated Abeta and can be replicated by exogenous Abeta acting through RAGE.

Endogenous oxidative stress in sporadic Alzheimer's disease neuronal cybrids reduces viability by increasing apoptosis through pro-death signaling pathways and is mimicked by oxidant exposure of control cybrids.

Although oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative diseases such as Alzheimer's disease (AD), it is not fully understood how mitochondrial oxidative stress may induce neuronal death. We used mitochondrial transgenic neuronal cell cybrid models of sporadic AD (SAD) to investigate the effects of endogenously generated reactive oxygen species (ROS) on viability and cell death mechanisms. Compared to control (CTL) cybrids, SAD cybrids have increased accumulation of oxidative stress markers and increased apoptosis that is blocked by N-acetylcysteine (NAC) and zVAD.fmk. SAD cybrids also have increased basal activation of the MAPKs, Akt, and NF-kappa B. NF-kappa B activation and cybrid viability are enhanced by NAC. Inhibiting the activity of the PI3K pathway or NF-kappa B aggravates neuronal death. Exposure of CTL cybrids to H2O2 decreased viability and activated in a NAC-sensitive manner, the same intracellular signaling pathways active under basal conditions in SAD cybrids.

Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death.

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling.

Activation of p38 and N-acetylcysteine-sensitive c-Jun NH2-terminal kinase signaling cascades is required for induction of apoptosis in Parkinson's disease cybrids.

Cytoplasmic hybrid cells (cybrids) are created by selective amplification of mitochondrial genes against constant nuclear genetic and environmental backgrounds. Cybrids from patients with sporadic Parkinson's disease (PD) recapitulate disease features such as decreased complex I activity, increased oxidative stress, elevated activation of NF-kappaB, and production of Lewy body inclusions. We examined the activation of signaling pathways and NF-kappaB in PD cybrids after exposure to MAPK inhibitors and/or the antioxidant N-acetylcysteine (NAC). Under basal replicating conditions, PD cybrids have decreased viability that is associated with increased DNA condensation and poly-ADP ribose polymerase (PARP) cleavage as well as elevated p38 and JNK activity. Pharmacological inhibition of oxidative stress diminished the elevated p38, JNK activity and PARP cleavage, and enhanced PD cybrid viability. PD mitochondrial genes expressed in cybrids stimulate pro-apoptotic cell signaling and biochemistry through oxidative stress. These results support development of antioxidative therapeutics for PD.

Alterations in voiding frequency and cystometry in the clomipramine induced model of endogenous depression and reversal with fluoxetine.

PURPOSE: Because serotonin (5-HT) in the central nervous system may inhibit bladder activity, we postulated that depression associated with altered 5-HT function might be associated with overactive bladder (OAB). We examined a rat model of endogenous depression caused by lowering 5-HT for effects on voiding frequency (VF) and awake cystometry (CMG), and examined the effect of reversal using the selective serotonin reuptake inhibitor fluoxetine. MATERIALS AND METHODS: Wistar rat pups were divided into 2 groups, namely clomipramine treated (CL) and saline control (SC). From postnatal days 8 to 21 each pup was injected with CL hydrochloride (22.5 mg/kg body weight) or an equal volume of saline. VF was assessed at 10 and 15 weeks. Behavioral correlates of depression were assessed using the forced swim test. At age 15 weeks CMG was performed. Fluoxetine (20 mg/kg daily) was administrated to a subset of SC/CL rats, and VF and CMG were repeated. RESULTS: In CL rats immobility was increased when rats were submitted to the forced swim test, indicating depression. CL rats voided more frequently than the SC group at 10 and 15 weeks but the difference was significant only in females. CMG of female CL group showed decreased bladder capacity, micturition volume and intermicturition contractions compared with the SC group. Treatment with fluoxetine reversed these changes, as in SC rats. CONCLUSIONS: These results support the hypothesis that OAB in a subgroup of depressed individuals may be associated with altered 5-HT function. It may explain reports of an association between depression and OAB.