Shu G. Chen

LRRK2 regulates mitochondrial dynamics and function through direct interaction with DLP1

The leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of autosomal-dominant Parkinson disease (PD). Mitochondrial dysfunction represents a critical event in the pathogenesis of PD. We demonstrated that wild-type (WT) LRRK2 expression caused mitochondrial fragmentation along with increased mitochondrial dynamin-like protein (DLP1, also known as DRP1), a fission protein, which was further exacerbated by expression of PD-associated mutants (R1441C or G2019S) in both SH-SY5Y and differentiated primary cortical neurons. We also found that LRRK2 interacted with DLP1, and LRRK2-DLP1 interaction was enhanced by PD-associated mutations that probably results in increased mitochondrial DLP1 levels. Co-expression of dominant-negative DLP1 K38A or WT Mfn2 blocked LRRK2-induced mitochondrial fragmentation, mitochondrial dysfunction and neuronal toxicity. Importantly, mitochondrial fragmentation and dysfunction were not observed in cells expressing either GTP-binding deficient mutant LRRK2 K1347A or kinase-dead mutant D1994A which has minimal interaction with DLP1 and did not increase the mitochondrial DLP1 level. We concluded that LRRK2 regulates mitochondrial dynamics by increasing mitochondrial DLP1 through its direct interaction with DLP1, and LRRK2 kinase activity plays a critical role in this process.

Molecular assessment of the potential transmissibilities of BSE and scrapie to humans

More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt–Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK,. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis. Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here we show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, we used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. We found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrPSc) and BSE (PrPBSE). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions. Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.

Chronic Wasting Disease of Elk: Transmissibility to Humans Examined by Transgenic Mouse Models

Chronic wasting disease (CWD), a prion disease affecting free-ranging and captive cervids (deer and elk), is widespread in the United States and parts of Canada. The large cervid population, the popularity of venison consumption, and the apparent spread of the CWD epidemic are likely resulting in increased human exposure to CWD in the United States. Whether CWD is transmissible to humans, as has been shown for bovine spongiform encephalopathy (the prion disease of cattle), is unknown. We generated transgenic mice expressing the elk or human prion protein (PrP) in a PrP-null background. After intracerebral inoculation with elk CWD prion, two lines of “humanized” transgenic mice that are susceptible to human prions failed to develop the hallmarks of prion diseases after >657 and >756 d, respectively, whereas the “cervidized” transgenic mice became infected after 118–142 d. These data indicate that there is a substantial species barrier for transmission of elk CWD to humans.

Fatal Familial Insomnia and Familial Creutzfeldt‐Jakob Disease: Clinical, Pathological and Molecular Features

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt‐Jakob disease (CJD178) are two prion diseases that have different clinical and pathological features, the same aspartic acid to asparagine mutation (D178N) at codon 178 of the prion protein (PrP) gene, but distinct genotypes generated by the methionine‐valine polymorphism at codon 129 (129M or 129V) in the mutant allele of the PrP gene. The D178N, 129M allele segregates with FFI while the D178N, 129V allele segregates with CJD178. The proteinase K resistant PrP (PrPres) isoforms present in FFI and CJD178 differ in degree of glycosylation and size. Thus, the amino acid, methionine or valine, at position 129 of the mutant allele, in conjunction with D178N mutation results in significant alterations of PrPres in FFI and CJD178. The 129 polymorphic site also exerts influence through the normal allele: the course of the disease is shorter in the patients homozygous at codon 129 and other minor but consistent phenotypic differences occur between homozygous and heterozygous FFI patients. The comparative study of PrPres distribution in FFI homozygotes and heterozygotes at codon 129 has lead to the conclusion that the phenotypic differences observed between these two FFI patient populations may be the result of different rates of conversion of normal PrP into PrPres, at least in some brain regions.

The Roc domain of leucine-rich repeat kinase 2 is sufficient for interaction with microtubules.

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinsons disease (PD). Although this multidomain protein has been shown to have both GTPase and kinase activities through the Roc and MAPKKK domains, respectively, the protein–protein interactions and pathways involved in LRRK2‐mediated signaling remain elusive. Utilizing a combination of protein pull‐down assays, mass spectrometry, Western blotting, and immunofluorescence microscopy, this study identifies and describes the interaction between LRRK2 and microtubules. The Roc or GTPase‐like domain of LRRK2 is sufficient for interaction with α/β‐tubulin heterodimers. This interaction occurs in a guanine nucleotide‐independent manner, suggesting that tubulin might not be an effector of the LRRK2 GTPase domain. The R1441C pathogenic mutation, located within the Roc domain, retains interaction with α/β‐tubulin heterodimers, suggesting that disruption of this interaction likely is not the mechanism whereby the R1441C mutation leads to disease. At a subcellular level, endogenous LRRK2 protein was found to colocalize with α/β‐tubulin in primary hippocampal neurons. These findings are significant in that they link LRRK2 with microtubules, a structural component of the cell that is critically involved in the pathogenesis of several neurodegenerative diseases, including PD.

Identification of Novel Proteinase K-resistant C-terminal Fragments of PrP in Creutzfeldt-Jakob Disease

The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of α-helical domains accompanied by the increase of β structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of ∼19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation.

Protease-Resistant Human Prion Protein and Ferritin Are Cotransported across Caco-2 Epithelial Cells: Implications for Species Barrier in Prion Uptake from the Intestine

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrPSc), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrPSc core of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrPSc-protein complex that is avidly transcytosed in vesicular structures across an in vitro model of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrPC expression. Unexpectedly, PrPSc is cotransported with ferritin, a prominent component of the DE-treated PrPSc-protein complex. The transport of PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrPSc-associated proteins, in particular ferritin, may facilitate PrPSc uptake in the intestine from distant species, leading to a carrier state in humans.

Neuronal mitochondrial amelioration by feeding acetyl-L-carnitine and lipoic acid to aged rats

Brain function declines with age and is associated with diminishing mitochondrial integrity. The neuronal mitochondrial ultrastructural changes of young (4 months) and old (21 months) F344 rats supplemented with two mitochondrial metabolites, acetyl‐L‐carnitine (ALCAR, 0.2%[wt/vol] in the drinking water) and R‐α‐lipoic acid (LA, 0.1%[wt/wt] in the chow), were analysed using qualitative and quantitative electron microscopy techniques. Two independent morphologists blinded to sample identity examined and scored all electron micrographs. Mitochondria were examined in each micrograph, and each structure was scored according to the degree of injury. Controls displayed an age‐associated significant decrease in the number of intact mitochondria (P = 0.026) as well as an increase in mitochondria with broken cristae (P < 0.001) in the hippocampus as demonstrated by electron microscopic observations. Neuronal mitochondrial damage was associated with damage in vessel wall cells, especially vascular endothelial cells. Dietary supplementation of young and aged animals increased the proliferation of intact mitochondria and reduced the density of mitochondria associated with vacuoles and lipofuscin. Feeding old rats ALCAR and LA significantly reduced the number of severely damaged mitochondria (P = 0.02) and increased the number of intact mitochondria (P < 0.001) in the hippocampus. These results suggest that feeding ALCAR with LA may ameliorate age‐associated mitochondrial ultrastructural decay and are consistent with previous studies showing improved brain function.

Prion Protein Aggregation Reverted by Low Temperature in Transfected Cells Carrying a Prion Protein Gene Mutation

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.

Leucine-rich repeat kinase 2 (LRRK2): a key player in the pathogenesis of Parkinson's disease.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder, with a prevalence of more than 1% after the age of 65 years. Mutations in the gene encoding leucine‐rich repeat kinase‐2 (LRRK2) have recently been linked to autosomal dominant, late‐onset PD that is clinically indistinguishable from typical, idiopathic disease. LRRK2 is a multidomain protein containing several protein interaction motifs as well as dual enzymatic domains of GTPase and protein kinase activities. Disease‐associated mutations are found throughout the multidomain structure of the protein. LRRK2, however, is unique among the PD‐causing genes, because a missense mutation, G2019S, is a frequent determinant of not only familial but also sporadic PD. Thus, LRRK2 has emerged as a promising therapeutic target for combating PD. In this Mini‐Review, we look at the current state of knowledge regarding the domain structure, amino acid substitutions, and potential functional roles of LRRK2.