Sabrina Ceccariglia

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca2+‐binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca2+ overload. The present study was aimed at determining whether intracellular Ca2+ homeostasis dysregulation is involved in the TMT‐induced neurodegeneration and if intracellular Ca2+‐buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time‐ and concentration‐dependent [Ca2+]i increases that were primarily due to Ca2+ release from intracellular stores although Ca2+ entry through Cav1 channels also contributed to [Ca2+]i increases in the early phase of TMT action. Cell pre‐treatment with the Ca2+ chelator, 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis(acetoxymethyl ester) (2 μM) significantly reduced the TMT‐induced neuronal death. Moreover, CR+ neurons responded to TMT with smaller [Ca2+]i increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca2+ homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR+ neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca2+ overload.

Canine cognitive dysfunction and the cerebellum: Acetylcholinesterase reduction, neuronal and glial changes

The specific functional and pathological alterations observed in Alzheimers disease are less severe in the cerebellum than in other brain areas, particularly the entorhinal cortex and hippocampus. Since dense core amyloid-beta plaque formation has been associated with an acetylcholinesterase heterogeneous nucleator action, we examined if an acetylcholinesterase imbalance was involved in cerebellum plaque deposition. By using the canine counterpart of senile dementia of the Alzheimers type, a promising model of human brain aging and early phases of Alzheimers disease, we investigated how cerebellar pathology and acetylcholinesterase density could be related with cognitive dysfunction. As in Alzheimers disease, the late affectation of the cerebellum was evidenced by its lack of amyloid-beta plaque and the presence of diffuse deposition throughout all cortical grey matter layers. The highest acetylcholinesterase optic density corresponded to cerebellar islands of the granular layer and was predominantly associated with synaptic glomeruli and the somata of Golgi cells. Its reduction correlated with aging and loss of granule cells, whereas cognitive deficit only correlated with loss of Purkinje cells. The observed Bergmann glia alterations may correspond to a reactive response to the loss and damage of the Purkinje cells, their specific neuronal partner. Regarding the role of acetylcholinesterase mediation in amyloid-beta deposition, our data argue against an interaction between these two proteins because acetylcholinesterase reduction correlates with aging but not with cognitive deficit. Finally, our data support the use of companion dogs of all breeds to study aging and early phases of Alzheimers disease.

Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process.

Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA(1)/CA(3) subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca(2+) cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca(2+) chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA(3) neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.

Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration

Trimethyltin chloride (TMT) is a neurotoxicant producing neuronal degeneration and reactive astrogliosis in the mammalian central nervous system, especially the hippocampus. A previous magnetic resonance imaging investigation in TMT-treated rats evidenced dilation of lateral ventricles, also suggesting alterations in blood-brain barrier permeability and brain edema. Aquaporin 4 (AQP4), a glial water channel protein expressed mainly in the nervous system, is considered a specific marker of vascular permeability and thought to play an important role in brain edema (conditions). We studied AQP4 expression in the hippocampus and cerebral cortex of TMT-treated rats in order to explore the molecular mechanisms involved in brain edema occurring in these experimental conditions. Real-time PCR and western blotting data showed significant up-regulation of both AQP4 mRNA and protein levels starting 14 days after TMT treatment in the hippocampus and cortex. Parallel immunofluorescence studies indicated intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting 14 days after TMT intoxication. In order to study the effects of TMT on vascular integrity, double-label immunofluorescence experiments for rat immunoglobulin G (IgG) and rat endothelial cell antigen-1 (RECA-1) or neuronal nuclei (NeuN) (endothelial and neuronal markers respectively) were performed. The results indicated, at 21 and 35 days after treatment, the presence of rat IgG in paravasal parenchyma and in some neuronal cells of the hippocampus and cortex. The extravasated IgG staining was temporally correlated with over-expression of neuronal vascular endothelial growth factor (VEGF) and the active phosphorylated form of its neuronal receptor (VEGFR-2P), suggesting that these factors may cooperate in mediating vascular leakage.

Aquaporin 4 expression increases in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration

Trimethyltin (TMT) is a neurotoxicant know to produce significant and selective neuronal degeneration in the rodent CNS (for review, 1). Magnetic resonance imaging (MRI) investigation in TMT-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability .In order to explore the molecular mechanisms involved in the phenomenon we have investigated in the hippocampus and cortex of TMT-treated rats the expression of aquaporin 4 (AQP4), a glial water channel protein believed to play a role in brain oedematous conditions. AQP4 expression was tested both by real-time PCR and western blotting analysis in hippocampus and cortex homogenates. To confirm molecular results and visualize the AQP4 cell distribution double-label immunofluorescence for AQP4 and GFAP was performed. Real-time PCR and western blotting data show a significant upregulation of AQP4 starting from 14 days of TMT treatment both in the hippocampus and the cortex. Accordingly, the immunofluorescence shows an intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. In particular, AQP4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels. The study of the Rhodamine B fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of TMT-treated rats, indicating a vascular involvement in the TMT-induced neurodegenerative processes.. AQP4 over-expression and astrogliosis occurring in the brain of TMT-treated rats might putatively play a role in alterations of vascular permeability and brain oedema formation evidenced by MRI studies.

Aquaporin 4 (AQP4) expression and blood brain barrier damage in an experimental model of neurodegeneration induced by trimethyltin

Trimethyltin (TMT) is well known to produce a distinct pattern of selective neuronal degeneration in the rodent CNS. TMT intoxication is also an important factor linked to induction of brain edema. Aquaporin-4 (AQP4), a water transporting protein, is thought to be the primary route through which water moves in and out astrocytes, is over-expressed in some pathological conditions, and is considered a marker of vascular permeability. The aim of our study was to investigate the integrity of the blood brain barrier (BBB), and the expression of AQP4 in astrocytes, after TMT intoxication. The brains of adult female Wistar rats, treated and untreated with TMT, were isolated and consecutive coronal sections obtained with a vibratome were incubated with primary fluorescent antibodies AQP4-Ig goat and GFAP-Ig rabbit. Imaging of fluorescence was performed on a confocal laser scanning microscope (Zeiss). In addition, dissected hippocampi and cortex were homogenized and the proteins were separated by SDS-PAGE. Although data in literature document absence of alterations of the BBB in TMT treated rats, our preliminary published data of the MRI investigation with Gd-DTPA suggested the presence of such alterations that concur with the passage of contrast into the damaged tissue. We have analyzed the AQP4 expression 7, 14, 21 and 35 days after TMT exposure. Immunofluorescence and Western Blotting analysis have showed an upregulation of the AQP4 in astrocytes after TMT intoxication., the expression levels of which progressively increased after TMT exposure, both in hippocampus and in brain cortex. All the data suggest that the astrocytes and their AQP4 protein are involved in the brain edema formation and in the possible alteration of the vascular permeability, in this TMT model of neurodegeneration.