Citicoline (CDP-choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke.

CDP-choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP-choline and placebo groups, CDP-choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t-PA. Several mechanisms have been proposed to explain the beneficial actions of CDP-choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP-choline. Fischer rats and Sirt1⁻/⁻ mice were subjected to permanent focal ischemia. CDP-choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP-choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. CDP-choline failed to reduce infarct volume in Sirt1⁻/⁻ mice. Our present results demonstrate a robust effect of CDP-choline like SIRT1 activator by up-regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke. Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1⁻/⁻ mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke.

Insulin receptor substrate 2 (IRS2)-deficient mice show sensorineural hearing loss that is delayed by concomitant protein tyrosine phosphatase 1B (PTP1B) loss of function.

The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and ß-cell failure and develop type 2-like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2⁻/⁻Ptpn1⁺/⁺, Irs2⁺/⁺Ptpn1⁻/⁻ and Irs2⁻/⁻Ptpn1⁻/⁻ mice at different postnatal ages. The results indicated that Irs2⁻/⁻Ptpn1⁺/⁺ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2⁻/⁻Ptpn1⁻/⁻ mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes.

Cortical migration defects in mice expressing A-RAF from the B-RAF locus.

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.

Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells.

Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1-/- mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal-regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

Bcl-2 determines susceptibility to induction of lung cancer by oncogenic CRaf.

The efficiency of tumor induction by oncogenes is influenced by modifier genes that determine individual susceptibility. We have used a transgenic mouse model to examine the role of a candidate susceptibility gene, bcl-2, for development of Raf oncogene-induced lung adenomas. Loss of bcl-2 greatly retarded tumor development without affecting tumor phenotype. Tumor tissues from bcl-2 positive and negative mice were compared for the fraction of S phase cells by staining for proliferating cell nuclear antigen and for the fraction of apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The data indicate that the increased tumor latency in the absence of bcl-2 results primarily from an increased apoptotic rate but also involves a decrease in tumor cell proliferation. Both effects can be rescued by breeding with H2K-bcl-2 transgenic mice demonstrating that loss of bcl-2 was the major genetic factor determining tumor resistance. These findings suggest that bcl-2 is a major susceptibility gene for development of lung cancer in mice and perhaps in humans.

Transforming growth factor ß1 inhibition protects from noise-induced hearing loss.

Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor ß (TGF-ß) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-ß as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-ß1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-ß1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-ß1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.

BAG-1 haplo-insufficiency impairs lung tumorigenesis.

BACKGROUND: BAG-1 is a multifunctional co-chaperone of heat shock proteins (Hsc70/Hsp70) that is expressed in most cells. It interacts with Bcl-2 and Raf indicating that it might connect protein folding with other signaling pathways. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial. METHODS: We have evaluated the effect of BAG-1 heterozygosity in mice in a model of non-small-cell lung tumorigenesis with histological and molecular methods. We have generated mice heterozygous for BAG-1, carrying a BAG-1 null allele, that in addition express oncogenic, constitutively active C-Raf kinase (SP-C C-Raf BxB) in type II pneumocytes. SP-C C-Raf BxB mice develop multifocal adenomas early in adulthood. RESULTS: We show that BAG-1 heterozygosity in mice impairs C-Raf oncogene-induced lung adenoma growth. Lung tumor initiation was reduced by half in BAG-1 heterozygous SP-C C-Raf BxB mice compared to their littermates. Tumor area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies. Whereas BAG-1 heterozygosity did not affect the rate of cell proliferation or signaling through the mitogenic cascade in adenoma cells, it increased the rate of apoptosis. CONCLUSION: Reduced BAG-1 expression specifically targets tumor cells to apoptosis and impairs tumorigenesis. Our data implicate BAG-1 as a key player in oncogenic transformation by Raf and identify it as a potential molecular target for cancer treatment.

Cochlear abnormalities in insulin-like growth factor-1 mouse mutants.

Insulin-like growth factor 1 (IGF-1) modulates inner ear cell proliferation, differentiation and survival in culture. Its function in human hearing was first evidenced by a report of a boy with a homozygous deletion of the Igf-1 gene, who showed severe sensorineural deafness [Woods et al., New Engl. J. Med. 335 (1996) 1363-1367]. To better understand the in vivo role of IGF-1 during inner ear differentiation and maturation, we studied the cochleae of Igf-1 gene knockout mice by performing morphometric stereological analyses, immunohistochemistry and electron microscopy on postnatal days 5 (P5), P8 and P20. At P20, but not at P5, the volumes of the cochlea and cochlear ganglion were significantly reduced in mutant mice, although the reduction was less severe than whole body dwarfism. A significant decrease in the number and average size of auditory neurons was also evident at P20. IGF-1-deficient cochlear neurons showed increased apoptosis, along with altered expression of neurofilament 200 kDa and vimentin. The eighth nerve, the cochlear ganglion and the fibers innervating the sensory cells of the organ of Corti of the P20 mouse mutants presented increased expression of vimentin, whereas the expression of neurofilament was decreased. In addition, the myelin sheath was severely affected in ganglion neurons. In conclusion, IGF-1 deficit in mice severely affects postnatal survival, differentiation and maturation of the cochlear ganglion cells.

Trophic effects of insulin-like growth factor-I (IGF-I) in the inner ear.

Insulin-like growth factors (IGFs) have a pivotal role during nervous system development and in its functional maintenance. IGF-I and its high affinity receptor (IGF1R) are expressed in the developing inner ear and in the postnatal cochlear and vestibular ganglia. We recently showed that trophic support by IGF-I is essential for the early neurogenesis of the chick cochleovestibular ganglion (CVG). In the chicken embryo otic vesicle, IGF-I regulates developmental death dynamics by regulating the activity and/or levels of key intracellular molecules, including lipid and protein kinases such as ceramide kinase, Akt and Jun N-terminal kinase (JNK). Mice lacking IGF-I lose many auditory neurons and present increased auditory thresholds at early postnatal ages. Neuronal loss associated to IGF-I deficiency is caused by apoptosis of the auditory neurons, which presented abnormally increased levels of activated caspase-3. It is worth noting that in man, homozygous deletion of the IGF-1 gene causes sensory-neural deafness. IGF-I is thus necessary for normal development and maintenance of the inner ear. The trophic actions of IGF-I in the inner ear suggest that this factor may have therapeutic potential for the treatment of hearing loss.

Drug delivery to the inner ear: strategies and their therapeutic implications for sensorineural hearing loss.

Hearing aids or cochlear implants constitute almost exclusively the treatment options currently available to patients suffering from sensorineural hearing loss and related conditions, such as noise-induced hearing loss, ototoxicity or autoimmune inner ear disease. While some systemic treatments exist, they generally exert adverse secondary effects and their efficacy is hampered by the blood-cochlear barrier that limits drug access to the inner ear. Hence, the new therapies that are being developed for hearing loss focus on strategies for direct drug delivery to the inner ear. The passive and active methods for local delivery can be categorized into two general groups: intratympanic or intracochlear. The intratympanic approach is a non-invasive method that preserves hearing and takes advantage of the permeability of the round window to gain access to the cochlea. However, this technique is limited by not knowing the dose of the drug that reaches the cochlea, (a handicap which might be overcome by the use of tagged drugs). While direct access to the inner ear by intracochlear administration avoids this problem, this method requires surgery. Currently, laboratory animals are being used to explore which therapeutic approaches are best suited to address this problem. These include cochleostomy and the insertion of devices that pump drugs into the inner ear without inducing cochlear damage. In this article, we review the different techniques under evaluation in animal models of deafness, and their potential use for drug delivery and treatment of human inner ear diseases.

Direct drug application to the round window: a comparative study of ototoxicity in rats.

OBJECTIVE: To assess the validity of inducing ototoxicity in rats by applying a sponge soaked in kanamycin and furosemide on the round window. STUDY DESIGN: Basic, randomized, nonblind experimental study. SETTING: Animal models of cochlear damage and reliable methods of local drug delivery are fundamental to study hearing loss and to design new therapies. SUBJECTS AND METHODS: Four experimental groups of six Wistar rats with different methods of drug administration were used: (1) injection of subcutaneous kanamycin (400 mg/kg) and intravenous furosemide (100 mg/kg); (2) local application of a sponge soaked in saline close to the round window; (3) animals for which the sponge was soaked in a solution containing kanamycin (200 mg/mL) and furosemide (50 mg/mL); and (4) sham-operated rats. The tympanic bulla was exposed using a ventral approach, and a bullostomy was performed to visualize the round window membrane. Cochlear function was assessed by measuring the auditory brainstem response, and hearing thresholds in response to click and tone burst stimuli were determined as peak and interpeak latencies. At the end of the study, cochlear histology was analyzed. RESULTS: Systemic administration of kanamycin and furosemide induced profound hearing loss and severe hair cell damage. Local delivery of these ototoxic drugs caused comparable damage but avoided the systemic side effects of the drug. Sham-operated and saline control animals did not experience functional alterations. CONCLUSION: Situating a sponge soaked in kanamycin and furosemide on the round window membrane through the ventral approach is a reliable method to provoke local ototoxicity in rats.