Lack of caspase-dependent apoptosis in spinal motor neurons of the wobbler mouse.

The mechanism of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is still unclear and the post-mortem analysis of samples from ALS patients does not permit a clarification of the early events of cell death occurring in ALS. Animal models of motor neuron degeneration represent a reliable tool to investigate the type of cell death. Attention was focused on the possible role of apoptosis in a spontaneous model of cervical spinal cord motor neuron degeneration, the wobbler mouse. Firstly, the rate of motor neuron loss occurring in the cervical spinal cord region of wobbler mice during different phases of symptoms progression was quantified by CholineAcetyltransferase (ChAT) immunohistochemistry. This was followed by a series of immunohistological studies to ascertain whether apoptosis was actually involved. ChAT immunostaining confirmed the severe loss of cholinergic neurons. Since the rate of motor neuron death is maximal in the first phase of the disease (from the 3rd to the 5th postnatal week), apoptotic markers were evaluated in 4-week-old wobbler mice. This study, carried out by examining a large number of cervical spinal cord sections from 20 affected animals and 20 healthy littermates, did not show either caspase activation or DNA fragmentation. These results strongly suggest that motor neuron death occurring in the wobbler mouse is not related to a caspase-dependent apoptotic mechanism.

Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein.

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Activated p38MAPK is a novel component of the intracellular inclusions found in human amyotrophic lateral sclerosis and mutant SOD1 transgenic mice.

Cytoskeletal abnormalities with accumulation of ubiquilated inclusions in the anterior horn cells are a pathological hallmark of both familial and sporadic amyotrophic lateral sclerosis (ALS) and of mouse models for ALS. Phosphorylated neurofilaments besides ubiquitin and dorfin have been identified as one of the major components of the abnormal intracellular perikaryal aggregates. As we recently found that p38 mitogen-activated protein kinase (p38MAPK) colocalized with phosphorylated neurofilaments in spinal motor neurons of SOD1 mutant mice, a model of familial ALS, we investigated whether this kinase also contributed to the inclusions found in ALS patients and SOD1 mutant mice. Intense immunoreactivity for activated p38MAPK was observed in degenerating motor neurons and reactive astrocytes in ALS cases. The intracellular immunostaining for activated p38MAPK appeared in some neurons as filamentous skein-like and ball-like inclusions, with an immunohistochemical pattern identical to that of ubiquitin. Intracellular p38MAPK-positive aggregates containing ubiquitin and neurofilaments were also found in the spinal motor neurons of SOD1 mutant mice. Our observations indicate that activation of p38MAPK might contribute significantly to the pathology of motor neurons in ALS.

A murine model of a familial prion disease.

We have produced a mouse model of a familial prion disorder by introduction of a transgene that encodes the moPrP homolog of a nine-octapeptide insertional mutant associated with an inherited form of CJD in humans. These mice develop progressive neurologic symptoms, display neuropathologic changes, and accumulate a form of mutant PrP in their brains and peripheral tissues that displays some of the biochemical properties of PrPSc. These mice have been extremely valuable for analyzing the cellular and biochemical mechanisms involved in inherited prion disorders and correlating the appearance of the PrPSc-like form with clinical and neuropathologic findings. Because the mutant protein in the mice is highly neurotoxic but appears to lack infectivity, further analysis of its properties promises to shed new light on the molecular distinction between pathogenic and infectious forms of PrP.

EM-ISEL: a useful tool to visualize DNA damage at the ultrastructural level.

A method for the localization of DNA strand breaks at the ultrastructural level is presented. The technique involves the use of terminal deoxynucleotidyl transferase and labeled dUTP. Incorporation of labeled nucleotides is visualized through colloidal gold labeling. Cells undergoing apoptotic or necrotic cell death, as well as cells showing death-unrelated DNA damage, can be easily distinguished. The technique uses tissues routinely processed for electron microscopy. It has been successfully applied to study DNA damage and apoptosis in different pathologic conditions. The feasibility of this technique for retrospective studies on archival material is emphasized.

Expression of mutant or cytosolic PrP in transgenic mice and cells is not associated with endoplasmic reticulum stress or proteasome dysfunction.

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the Ub(G76V)-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.

The collagen chaperone HSP47 is a new interactor of APP that affects the levels of extracellular beta-amyloid peptides.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive function that represents one of the most dramatic medical challenges for the aging population. Aß peptides, generated by processing of the Amyloid Precursor Protein (APP), are thought to play a central role in the pathogenesis of AD. However, the network of physical and functional interactions that may affect their production and deposition is still poorly understood. The use of a bioinformatic approach based on human/mouse conserved coexpression allowed us to identify a group of genes that display an expression profile strongly correlated with APP. Among the most prominent candidates, we investigated whether the collagen chaperone HSP47 could be functionally correlated with APP. We found that HSP47 accumulates in amyloid deposits of two different mouse models and of some AD patients, is capable to physically interact with APP and can be relocalized by APP overexpression. Notably, we found that it is possible to reduce the levels of secreted Aß peptides by reducing the expression of HSP47 or by interfering with its activity via chemical inhibitors. Our data unveil HSP47 as a new functional interactor of APP and imply it as a potential target for preventing the formation and/or growth amyloid plaques.