Early dysfunction of central 5-HT system in a murine model of bovine spongiform encephalopathy.

The hypothesis of an early vulnerability of the serotonergic system to prion infection was investigated in a murine model of bovine spongiform encephalopathy (BSE). Behavioral tests targeted to 5-HT functions were performed in the course of infection to evaluate circadian activity, anxiety-like behavior, pain sensitivity and the 5-HT syndrome. The first behavioral change was a decrease in nocturnal activity detected at 30% of incubation time. Further behavioral alterations including nocturnal hyperactivity, reduced anxiety, hyperalgesia and exaggerated 5-HT syndrome were observed at 60%-70% of incubation time, before the onset of clinical signs. The same tests performed in 5-HT-depleted mice and in prion protein-deficient mice revealed behavioral abnormalities similar in many aspects to those of BSE-infected mice. Histological and biochemical analysis showed alterations of the serotonergic system in BSE-infected and prion protein-deficient mice. These results indicate that BSE infection affects the homeostasis of serotonergic neurons and suggest that the disruption of prion protein normal function contributes to the early pathological changes in our mouse model of BSE. A similar process may occur in the human variant Creutzfeldt-Jacob disease, as suggested by the early symptoms of alterations in mood, sleep and pain sensitivity.

Alzheimer's disease proteins in cerebellar and hippocampal synapses during postnatal development and aging of the rat.

Alzheimer's dementia may be considered a synaptic disease of central neurons: the loss of synapses, reflected by early cognitive impairments, precedes the appearance of extra cellular focal deposits of beta-amyloid peptide in the brain of patients. Distinct immunocytochemical patterns of amyloid precursor proteins (APPs) have previously been demonstrated in the synapses by ultrastructural analysis in the cerebellum and hippocampus of adult rats and mice. Now we show that during postnatal development and during aging in these structures, the immunocytochemical expression of APPs increases in the synapses in parallel with the known up-regulation of total APPs brain levels. Interestingly, as shown previously in the adult rodents, the presenilins (PSs) 1 and 2, which intervene in APPs metabolism, exhibit a synaptic distribution pattern similar to that of APPs with parallel quantitative changes throughout life. In the brain tissue, single and double immunocytochemistry at the ultrastructural level shows co-localisation of APPs and PSs in axonal and dendritic synaptic compartments during postnatal synaptogenesis, adulthood and aging. In addition, double-labelling immunocytofluorescence detects these proteins close to synaptophysin at the growth cones of developing cultured neurons. Thusly, the brain expression of APPs and PSs appears to be regulated synchronously during lifespan in the synaptic compartments where the proteins are colocated. This suggests that PS-dependent processing of important synaptic proteins such as APPs could intervene in age-induced adjustments of synaptic relationships between specific types of neurons.

Synaptic prion protein immuno-reactivity in the rodent cerebellum.

The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Hemisynaptic distribution patterns of presenilins and beta-APP isoforms in the rodent cerebellum and hippocampus.

Healthy brain neurons co-express Alzheimer's disease (AD) related proteins presenilins (PS) and beta-amyloid precursor protein (beta-APP). Deposition of beta-amyloid and PS in the senile plaques of AD brain and their ability to interact in vitro suggest that AD pathology could arise from a defect in the physiological interactions between beta-APP and PS within and/or between neurons. The present study compares the immunocytochemical distribution of PS (1 and 2) and beta-APP major isoforms (695 and 751/770) in the synapses of the cerebellum and hippocampus of the adult rat and mouse. In the cerebellar cortex of both species, the four molecules are immunodetected in the presynaptic or the postsynaptic compartments of synapses, suggesting that they are involved in interneuronal relationships. In contrast, PS and beta-APP are postsynaptic in almost all the immunoreactive synapses of the hippocampus. The different distribution patterns of these proteins in cerebellar and hippocampal synapses may reflect specific physiological differences, responsible for differential vulnerability of neurons to AD synaptic pathology. Defective interactions between beta-APP and PS at the synapses could impede the synaptic functions of beta-APP, inducing the selective loss of synapses that accounts for cognitive impairment in AD.

Detection and characterization of a distinct type of beet necrotic yellow vein virus RNA 5 in a sugarbeet growing area in Europe.

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26 K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25 K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

Detection and Characterization of a Distinct Type of Beet Necrotic Yellow Vein Virus RNA 5 in a Sugarbeet Growing Area in Europe.

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

Detection of beet necrotic yellow vein virus strains, variants and mixed infections by examining single-strand conformation polymorphisms of immunocapture RT-PCR products.

Single-strand conformation polymorphism analysis was found to be a powerful tool for rapidly assigning large numbers of beet necrotic yellow vein virus (BNYVV) isolates to a known strain group as well as for detecting mixed infections, minor variants or new strain groups. The prevalence of the B-type in Germany and France and the A-type in most other countries was confirmed. Minor variants with a very restricted distribution were detected occasionally. New rhizomania outbreaks in Great Britain were caused either by the A- or B-type or mixtures of both suggesting introduction of BNYVV from several sites abroad. An entirely different BNYVV type (P-type) was identified in a small area in France. Evidence for further strain groups in China was also obtained.

In situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus RNA 3.

The in situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus (BNYVV) RNA 3 and of the BNYVV coat protein (CP) was studied by immunoelectron microscopy in infected leaf and root cells of Chenopodium murale and C. quinoa. The CP was detected in the cytoplasm of all cell types except xylem, sieve elements, and companion cells. P25 was detected in the cytoplasm and nuclei of the same cell types. The intensity of CP labelling varied depending upon the stage of infection of the cell, whereas the P25 labelling intensity was similar in newly infected cells and in cels at later stages of infection. These results suggest that P25 may be synthesized at an earlier stage of infection than CP. Its presence in the nuclei of newly infected cells may be related to the reported effect of P25 on leaf symptom development.

Detection by immunogold labelling of P75 readthrough protein near an extremity of beet necrotic yellow vein virus particles.

RNA 2 of beet necrotic yellow vein virus carries the cistron for the 21 kd coat protein at its 5'-extremity. During translation, the coat protein cistron termination codon is suppressed about 10% of the time so that translation continues into the adjacent open reading frame to produce a 75 kd species, known as P75, which contains the coat protein sequence at its N-terminus. Immunoblotting experiments with a P75-specific antiserum showed that P75 is present in only trace amounts in purified virus preparations. Electron microscopic visualization of immunogold-labelled virions in crude tissue extracts has provided evidence for an association between P75 and at least a fraction of the BNYVV particles, with P75 being predominantly located near one end of the rod-shaped virions. This finding is discussed in the context of the current model for the role of P75 in virus assembly and vector transmission.