Anne-Marie Haeberlé

Synaptic prion protein immuno‐reactivity in the rodent cerebellum

The cellular prion protein PrPc is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrPc is converted to infectious protease‐resistant isoforms PrPres or prions. Prions provoque predictable and distinctive patterns of PrPres accumulation and neurodegeneration depending on the prion strain and on regional cell‐specific properties modulating PrPc affinity for infectious PrPres in the host brain. Synaptolysis and synaptic accumulation of PrPres during PrP‐related diseases suggests that the synapses could be primary sites able to propagate PrPres 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 PrPc, suggesting that synapse‐specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrPc remain unknown, the concentration of PrPc 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. Microsc. Res. Tech. 50:66–75, 2000.

Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain.

Expression of the cellular prion protein (PrPc) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrPc is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrPc in the mouse brain, the immunocytochemical localization of PrPc was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrPc was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrPc antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrPc immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrPc. Transgenic mice expressing PrP‐GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases. J. Comp. Neurol. 473:244–269, 2004.

Selective Recapture of Secretory Granule Components After Full Collapse Exocytosis in Neuroendocrine Chromaffin Cells

In secretory cells, calcium‐regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo‐endocytosis, including kiss‐and‐run, cavicapture and full‐collapse fusion. During kiss‐and‐run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full‐collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain.

Fibrillar Prion Peptide (106 -126) and Scrapie Prion Protein Hamper Phagocytosis in Microglia

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106–126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106–126) is internalized and processed. However, both synthetic prion peptide PrP(106–126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106–126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106–126) did not prevent F‐actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.

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

Alzheimers 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.

Phospholipid Scramblase-1-Induced Lipid Reorganization Regulates Compensatory Endocytosis in Neuroendocrine Cells

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1−/− mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.

Scrg1 is induced in TSE and brain injuries, and associated with autophagy

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. In this study, Scrg1‐immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie‐infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non‐infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie‐infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non‐transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.

Survival of interneurons and parallel fiber synapses in a cerebellar cortex deprived of Purkinje cells: Studies in the double mutant mouse Grid2Lc/+;Bax−/−

The Lurcher mutation in the Grid2 gene causes the cell autonomous death of virtually all cerebellar Purkinje cells and the target‐related death of 90% of the granule cells and 60–75% of the olivary neurons. Inactivation of Bax, a pro‐apoptotic gene of the Bcl‐2 family, in heterozygous Lurcher mutants (Grid2Lc/+) rescues ∼60% of the granule cells, but does not rescue Purkinje or olivary neurons. Given the larger size of the cerebellar molecular layer in Grid2Lc/+;Bax−/− double mutants compared to Grid2Lc/+ mutants, we analyzed the survival of the stellate and basket interneurons as well as the synaptic connectivity of parallel fibers originating from the surviving granule cells in the absence of their Purkinje cell targets in the Grid2Lc/+;Bax−/− cerebellum. Quantification showed a significantly higher density of interneurons (∼60%) in the molecular layer of the Grid2Lc/+;Bax−/− mice compared to Grid2Lc/+, suggesting that interneurons are subject to a BAX‐dependent target‐related death in the Lurcher mutants. Furthermore, electron microscopy showed the normal ultrastructural aspect of a number of parallel fibers in the molecular layer of the Grid2Lc/+; Bax−/− double mutant mice and preserved their numerous synaptic contacts on interneurons, suggesting that interneurons could play a trophic role for axon terminals of surviving granule cells. Finally, parallel fibers varicosities in the double mutant established “pseudo‐synapses” on glia as well as displayed autophagic profiles, suggesting that the connections established by the parallel fibers in the absence of their Purkinje cell targets were subject to a high turnover involving autophagy. J. Comp. Neurol. 497:622–635, 2006.

Autophagy and Cell Death of Purkinje Cells Overexpressing Doppel in Ngsk Prnp-deficient Mice

In Ngsk prion protein (PrP)‐deficient mice (NP0/0), ectopic expression of PrP‐like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late‐onset ataxia. NP0/0 PC death is partly prevented by either knocking‐out the apoptotic factor BAX or overexpressing the anti‐apoptotic factor BCL‐2 suggesting that apoptosis is involved in Dpl‐induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie‐responsive gene 1 (Scrg1)—potentially associated with autophagy—and the autophagic markers LC3B and p62 increased in the NP0/0 PCs whereas RT‐PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP0/0 PCs. At the ultrastructural level, autophagic‐like profiles accumulated in somatodendritic and axonal compartments of NP0/0, but not wild‐type PCs. The most robust autophagy was observed in NP0/0 PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP0/0 PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP0/0 PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.

Hemisynaptic distribution patterns of presenilins and β‐APP isoforms in the rodent cerebellum and hippocampus

Healthy brain neurons co‐express Alzheimers disease (AD) related proteins presenilins (PS) and β‐amyloid precursor protein (β‐APP). Deposition of β‐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 β‐APP and PS within and/or between neurons. The present study compares the immunocytochemical distribution of PS (1 and 2) and β‐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 β‐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 β‐APP and PS at the synapses could impede the synaptic functions of β‐APP, inducing the selective loss of synapses that accounts for cognitive impairment in AD. Synapse 35:96–110, 2000.