Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinson's disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

The role of chaperones in Parkinson's disease and prion diseases.

The etiologies of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, polyglutamine diseases, or prion diseases may be diverse; however, aberrations in protein folding, processing, and/or degradation are common features of these entities, implying a role of quality control systems, such as molecular chaperones and the ubiquitin-proteasome pathway. There is substantial evidence for a causal role of protein misfolding in the pathogenic process coming from neuropathology, genetics, animal modeling, and biophysics. The presence of protein aggregates in all neurodegenerative diseases gave rise to the hypothesis that protein aggregates, be it intracellular or extracellular deposits, may perturb the cellular homeostasis and disintegrate neuronal function (Table 1). More recently, however, an increasing number of studies have indicated that protein aggregates are not toxic per se and might even serve a protective role by sequestering misfolded proteins. Specifically, experimental models of polyglutamine diseases, Alzheimer's disease, and Parkinson's disease revealed that the appearance of aggregates can be dissociated from neuronal toxicity, while misfolded monomers or oligomeric intermediates seem to be the toxic species. The unique features of molecular chaperones to assist in the folding of nascent proteins and to prevent stress-induced misfolding was the rationale to exploit their effects in different models of neurodegenerative diseases. This chapter concentrates on two neurodegenerative diseases, Parkinson's disease and prion diseases, with a special focus on protein misfolding and a possible role of molecular chaperones.

A sensitive filter retention assay for the detection of PrP(Sc) and the screening of anti-prion compounds.

A hallmark of prion diseases is the accumulation of an abnormally folded prion protein, denoted PrP(Sc). Here we describe a new and highly sensitive method for the detection of PrP(Sc) in brain and other tissue samples that utilizes both PrP(Sc) diagnostic criteria in combination; protease resistance and aggregation. Upon filtration of tissue extracts derived from scrapie- or bovine spongiform encephalopathy-infected animals, PrP(Sc) is retained and detected on the membranes. Laborious steps such as SDS-PAGE and Western blotting are avoided with concomitant gain in sensitivity and reliability. The new procedure also proved useful in a screen for anti-prion compounds in a scrapie-infected cell culture model.

Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.

Cationic lipopolyamines induce degradation of PrPSc in scrapie-infected mouse neuroblastoma cells.

In prion diseases the endogenous prion protein (PrPC) is converted into an abnormally folded isoform, denoted PrPSc, which represents the major component of infectious scrapie prions. The mechanism of the conversion is largely unknown, but the conversion is thought to occur after PrPC has reached the plasma membrane. Here we show that exogenous administration of the cationic lipopolyamine DOSPA interfered with the accumulation of PrPSc in scrapie-infected neuroblastoma cells. Structural analysis of the compounds tested revealed that inhibition of PrPSc was specific for lipids with a headgroup composed of the polyamine spermine and a quarternary ammonium ion between the headgroup and the lipophilic tail. The cationic lipopolyamine DOSPA induced the cellular degradation of preexisting PrPSc aggregates within 12 hours and interfered with the de novo synthesis of PrPSc. Biosynthesis of PrPC, or the assembly of sphingolipid-cholesterol microdomains (rafts) on the plasma membrane, were not affected by this inhibitor. After removal of DOSPA and replating into normal medium propagation of PrPSc commenced, although initially at a reduced rate. Incubation of ScN2a cells in free spermidine had no inhibitory effect on the accumulation of PrPSc. Our results indicate that membrane targeting of a small polyamine molecule creates a potent inhibitor of PrPSc propagation and offers the possibility to degrade preexisting PrPSc aggregates in living cells.

Human cytomegalovirus immediate-early gene 2 expression leads to JCV replication in nonpermissive cells via transcriptional activation of JCV T antigen.

Human papovavirus JCV is the causative agent of the demyelinating brain disease progressive multifocal leukoencephalopathy (PML) that typically develops as a complication of impaired immunocompetence. JCV displays a strong tropism for glial cells which is correlated by glial-specific transcriptional regulation of viral gene expression. In a previous report HCMV was shown to overcome the restricted cell specificity of JCV by inducing DNA replication of a PML-derived JCV strain in human fibroblasts which are nonpermissive for the replication of JCV alone. Here we show that productive JCV replication is induced by HCMV in human glioblastoma cells. Both in fibroblasts and in glioblastoma cells, the HCMV immediate-early transactivator 2 (IE2) is sufficient to mediate JCV replication. Furthermore, IE2 induces DNA replication of several structurally different brain- or kidney-derived JCV variants. IE2-induced JCV DNA replication is accompanied by the induction of JCV T antigen expression due to stimulation of the JCV early promoter. Our results indicate that stimulation of JCV early gene expression by HCMV-IE2 is sufficient to overcome the restricted cell specificity of JCV.

Geldanamycin restores a defective heat shock response in vivo.

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.

Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease.

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.