A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, β-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti–glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver–Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.Facultad de Ciencias Veterinaria

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, β-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti–glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver–Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.Facultad de Ciencias Veterinaria

A novel form of deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, β-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti–glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver–Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.Facultad de Ciencias Veterinaria

Implicancias de la proteína miosina Va en los procesos de generación de asimetrías y polarización celular

Orientador: José R. Sotel

Reserpine-induced depression is associated in female, but not in male, adolescent rats with heightened, fluoxetine-sensitive, ethanol consumption

Depression usually emerges during adolescence, is significantly more frequent in women, and exhibits comorbidity with alcohol (ethanol) use disorders. Most of the pre-clinical studies assessing the link between depression and ethanol intake, however, have employed only males or relied on stress-induced depression, or induced the experimentally-induced, depressive-like phenotype, during adolescence yet measured ethanol intake at adulthood. This study assessed, in Wistar male and female adolescent rats, the effects of inducing experimental depression (via administration of 1.0 mg/kg reserpine [RES], a monoamine depleting drug, between postnatal day [PD] 30 to PD33) on the acquisition of voluntary ethanol drinking during PD38 to PD42), and the modulation of these effects by fluoxetine (FLUOX, 10.0 mg/kg) on PDs 34–37. RES-treated rats exhibited a significant reduction of dopamine levels at the insula, no significant changes in circulating levels of thyroxine T4, and reduced distance travelled in an open field. Repeated treatment with RES heightened ethanol intake in female, but not in male, rats; and effect that was inhibited by FLUOX. Similarly, RES significantly increased, and FLUOX reversed, risk-taking behaviors in a concentric square field (CSF) test. FLUOX significantly increased shelter-seeking in the CSF and reduced insular dopamine levels. These results indicate that depression, in females, can kindle the initiation of voluntary ethanol drinking in adolescence (one of the most reliable predictors of being diagnosed with an AUD), and pinpoint alterations in risk-taking as potential mechanisms underlying this effect. Adolescent women afflicted by mood disorders should be specifically targeted for interventions directed towards delaying initiation of alcohol consumption.Fil: Ruiz, Paul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad de la República; UruguayFil: Calliari, Aldo. Universidad de la República; UruguayFil: Pautassi, Ricardo Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Alcohol consumption in adolescent rats treated with reserpine and fluoxetine

Si bien se ha estudiado el vínculo entre los trastornos de estado de ánimo y el consumo de alcohol, tanto en humanos como en animales, todavía no es del todo claro cómo se da esta relación, y menos aún durante la adolescencia. La administración de reserpina, un depletor de monoaminas, es un modelo ampliamente utilizado en roedores adultos para inducir comportamientos asociados a depresión, pero se desconoce su utilidad en otras etapas del desarrollo. En este trabajo validamos este modelo en ratas adolescentes y posteriormente estudiamos el consumo de alcohol en estos animales, así como su modulación por antidepresivos. En el experimento 1 se administró reserpina (0.0 o 1.0 mg/kg, durante 4 días, IP) a ratas Wistar machos de 30 días. El consumo de alcohol fue evaluado luego de observar comportamientos asociados a depresión e indagar indicadores neuroendocrinos de esta patología. En el experimento 2 los animales recibieron reserpina, seguida por el antidepresivo fluoxetina (0.0 o 10.0 mg/kg, durante 4 días, IG), y luego se evaluó el consumo de alcohol. Los resultados mostraron que la reserpina aumentó significativamente los comportamientos asociados a depresión y alteró los niveles de dopamina en la corteza insular y de hormonas tiroideas en sangre. En la prueba de consumo de alcohol los animales deprimidos, pero no los controles, mostraron un incremento en el consumo a través de los días. El segundo experimento replicó parcialmente este perfil, y no se observó un efecto significativo del antidepresivo en el consumo de alcohol. Los resultados indican que la reserpina es útil para modelar en ratas adolescentes comportamientos asociados a depresión. Se encontró una relación entre este estado y el consumo de alcohol, que no se pudo revertir con antidepresivos.The relationship between mood disorders and alcohol consumption has been studied in humans and animals, although it is still not fully clear how this relationship unfolds, much less during adolescence. The administration of reserpine — a monoamine depletor — is an approach traditionally used in adult rodents to induce depression-associated behaviours, but its usefulness in other developmental stages is still unknown. In this study, this model was evaluated in adolescent rats in order to study alcohol consumption, as well as its modulation by antidepressants in these animals. In Experiment 1, 30 day-old male Wistar rats were treated with reserpine (0.0 or 1.0 mg/kg, for 4 days, IP). Alcohol consumption was tested after observing depression-associated behaviours and assessing neuroendocrine indicators of this pathology. In Experiment 2, the rats were administered reserpine followed by an antidepressant (fluoxetine, 0.0 or 10.0 mg/kg, for 4 days, IG). Alcohol consumption was then tested. The results showed that reserpine significantly increased depression-associated behaviours and altered insular dopamine and thyroid hormone levels. Alcohol consumption tests showed that reserpine-treated animals — but not control animals — increased their consumption throughout the days. The second experiment partially replicated this profile, and no significant effect of antidepressants was observed in alcohol consumption. The results show that reserpine is instrumental in modelling depression-associated behaviours in adolescent rats. A relationship was found between this condition and alcohol intake, which could not be reversed by antidepressants.Fil: Ruiz, Paul. Universidad de la República; UruguayFil: Calliari, Aldo. Universidad de la República; UruguayFil: Pautassi, Ricardo Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

La memoria espacial, y los niveles de BDNF en el hipocampo, disminuyen en ratas adolescentes deprimidas farmacológicamente con reserpina

Depression is associated with significant deficits in spatial memory. The present study assessed spatial memory in an animal model of depression. Wistar rats (n=28), males and females, were administered reserpine (0.0 or 1.0 mg/kg/every 2 days for 4 days). At termination of the treatment the rats were tested for motor function (bar test) and for anxiety response (elevated plus maze). The animals were then trained for a week (daily sessions of 5 min) in a multiple T-maze, which assesses spatial memory. The elevated plus maze test was then repeated and subsequently the animals were sacrificed and the hippocampus dissected to measure BDNF levels through a Western blot. In the bar test, the reserpine-treated rats exhibited similar grasping, yet reduced mobility, when compared to vehicle-treated peers. Reserpine-treated rats exhibited, in both elevated plus maze tests and when compared to control counterparts, significantly less time spent in the open arms, and significantly greater latency to enter, for the first time, in the closed arms. They also spent significantly more time to complete the multiple T-maze and made more performance errors than controls in this test, a profile that was associated with lower levels of BDNF at the hippocampus. These results suggest that reserpine induced a depression-like phenotype, without gross motor alterations, which was associated with spatial memory deficits.Los cuadros depresivos en humanos cursan con significativas alteraciones en la memoria espacial. Este trabajo estudió esta función en un modelo animal de depresión. Ratas Wistar (n=28), machos y hembras, fueron administradas con reserpina (0.0 o 1.0 mg/kg/cada 2 días por cuatro días). Luego del tratamiento se evaluó la función motora a través de la prueba de la barra y la respuesta de ansiedad a través de un laberinto elevado en cruz. Posteriormente, los animales fueron entrenados durante una semana (en sesiones diarias de 5 minutos) en un laberinto de múltiples T que indaga la memoria espacial. Luego se repitió la evaluación en el laberinto elevado en cruz y los animales fueron sacrificados para disecar el hipocampo y medir los niveles de BDNF a través de western blot. En la prueba de la barra los animales tratados con reserpina tuvieron una respuesta motora con igual agarre, pero menor movilidad, que los controles. En ambas tomas del laberinto elevado en cruz los animales tratados con reserpina pasaron significativamente menos tiempo en los brazos abiertos y exhibieron una latencia más elevada para entrar por primera vez en los brazos cerrados. Los animales tratados con reserpina tardaron significativamente más tiempo en resolver el laberinto de múltiples T que los controles, y cometieron más errores, efecto que estuvo asociado a niveles menores de BDNF en el hipocampo. Estos resultados sugieren que la reserpina indujo una depresión experimental, sin compromiso motor, que estuvo asociada a déficits en la adqui-sición de información espacial. Dépression chez l’homme présentent des altérations importantes de la mémoire spatiale. Ce travail a étudié cette fonction dans un modèle animal de dépression. Des rats Wistar (n = 28), mâles et femelles, ont été administrés avec de la réserpine (0.0 ou 1.0 mg / kg / tous les 2 jours pendant quatre jours). Après le traitement, la fonction motrice a été évaluée par le test des barres et la réponse anxieuse par un labyrinthe croisé surélevé. Par la suite, les animaux ont été entraînés pendant une semaine (par sessions quotidiennes de 5 minutes) dans un labyrinthe multi-T qui étudie la mémoire spatiale. Ensuite, l’évaluation a été répétée dans le labyrinthe en croix et les animaux ont été sacrifiés pour disséquer l’hippocampe et mesurer les taux de BDNF par Western blot. Lors du test des barres, les animaux traités à la réserpine présentaient une réponse motrice avec la même adhérence, mais avec une mobilité moindre, que les témoins. Dans les deux prises du labyrinthe surélevé dans la croix, les animaux traités à la réserpine ont passé beaucoup moins de temps dans les bras ouverts et ont montré une plus grande latence pour entrer dans les bras fermés pour la première fois. Les animaux traités avec de la réserpine ont mis beaucoup plus de temps à résoudre le labyrinthe multi-T que les témoins et ont commis plus d’erreurs, un effet associé à des niveaux inférieurs de BDNF dans l’hippocampe. Ces résultats suggèrent que la réserpine a induit une dépression expérimentale, sans compromis moteur, associée à des déficits dans l’acquisition d’informations spatiales.Depressão em humanos apresenta depressão alterações significativas na memória espacial. Este trabalho estudou essa função em um modelo animal de depressão. Ratos Wistar (n = 28), machos e fêmeas, foram administrados com reserpina (0.0 ou 1.0 mg / kg / a cada 2 dias por quatro dias). Após o tratamento, a função motora foi avaliada através do teste de bar e da resposta de ansiedade através de um labirinto cruzado elevado. Posteriormente, os animais foram treinados por uma semana (em sessões diárias de 5 minutos) em um labirinto multi-T que investiga a memória espacial. Na avaliação foi repetida no labirinto elevado em cruz e os animais foram sacrificados para dissecar o hipocampo e medir os níveis de BDNF através de western blot. No teste de barra, os animais tratados com reserpina tiveram uma resposta motora com a mesma aderência, mas menor mobilidade, que os controles. Em ambas as entradas do labirinto elevado na cruz, os animais tratados com reserpina gastaram significa-tivamente menos tempo nos braços abertos e exibiram uma maior latência para entrar nos braços fechados pela primeira vez. Os animais tratados com reserpina levaram significativamente mais tempo para resolver o labirinto multi-T do que os controles, e cometeram mais erros, um efeito que foi associado com níveis mais baixos de BDNF no hipocampo. Esses resultados sugerem que a reserpina induziu uma depressão experimental, sem comprometimento motor, associada a déficits na aquisição de informações espaciais