The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system.

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

The chaperone Clusterin in neurodegeneration-friend or foe?

Fibrillar protein aggregates are the pathological hallmark of a group of age-dependent neurodegenerative conditions, including Alzheimer's and Parkinson's disease. Aggregates of the microtubule-associated protein Tau are observed in Alzheimer's disease and primary tauopathies. Tau pathology propagates from cell to cell in a prion-like process that is likely subject to modulation by extracellular chaperones such as Clusterin. We recently reported that Clusterin delayed Tau fibril formation but enhanced the activity of Tau oligomers to seed aggregation of endogenous Tau in a cellular model. In contrast, Clusterin inhibited the propagation of α-Synuclein aggregates associated with Parkinson's disease. These findings raise the possibility of a mechanistic link between Clusterin upregulation observed in Alzheimer's disease and the progression of Tau pathology. Here we review the diverse functions of Clusterin in the pathogenesis of neurodegenerative diseases, focusing on evidence that Clusterin may act either as a suppressor or enhancer of pathology.

The extracellular chaperone Clusterin enhances Tau aggregate seeding in a cellular model.

Spreading of aggregate pathology across brain regions acts as a driver of disease progression in Tau-related neurodegeneration, including Alzheimer's disease (AD) and frontotemporal dementia. Aggregate seeds released from affected cells are internalized by naïve cells and induce the prion-like templating of soluble Tau into neurotoxic aggregates. Here we show in a cellular model system and in neurons that Clusterin, an abundant extracellular chaperone, strongly enhances Tau aggregate seeding. Upon interaction with Tau aggregates, Clusterin stabilizes highly potent, soluble seed species. Tau/Clusterin complexes enter recipient cells via endocytosis and compromise the endolysosomal compartment, allowing transfer to the cytosol where they propagate aggregation of endogenous Tau. Thus, upregulation of Clusterin, as observed in AD patients, may enhance Tau seeding and possibly accelerate the spreading of Tau pathology.

DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress.

Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT), the protein encoded by DPAGT1, is an endoplasmic reticulum (ER)-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met), c.791T>G (p.Val264Gly), c.901C>T (p.Arg301Cys), c.902G>A (p.Arg301His), c.1154T>G (p.Leu385Arg), and of the novel mutation c.329T>C (p.Phe110Ser), were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress) was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies.

Pharmacological Chaperoning: A Potential Treatment for PMM2-CDG.

The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG), the most common N-glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease-causing mutations described in patients with PMM2-CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss-of-function mutations. The present work describes the high-throughput screening, by differential scanning fluorimetry, of 10,000 low-molecular-weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof-of-concept of a possible treatment for PMM2-CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.

The Effects of PMM2-CDG-Causing Mutations on the Folding, Activity, and Stability of the PMM2 Protein.

Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones.

Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option.

PURPOSE: Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. METHODS: The disease was confirmed based on the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1. KEY FINDINGS: Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.1217_1218delAT, and c.1482-1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093-?_1620+?). Only one mutation, p.G477R (c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful. SIGNIFICANCE: The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application.

SHOX interacts with the chondrogenic transcription factors SOX5 and SOX6 to activate the aggrecan enhancer.

SHOX (short stature homeobox-containing gene) encodes a transcription factor implicated in skeletal development. SHOX haploinsufficiency has been demonstrated in Leri-Weill dyschondrosteosis (LWD), a skeletal dysplasia associated with disproportionate short stature, as well as in a variable proportion of cases with idiopathic short stature (ISS). In order to gain insight into the SHOX signalling pathways, we performed a yeast two-hybrid screen to identify SHOX-interacting proteins. Two transcription factors, SOX5 and SOX6, were identified. Co-immunoprecipitation assays confirmed the existence of the SHOX-SOX5 and SHOX-SOX6 interactions in human cells, whereas immunohistochemical studies demonstrated the coexpression of these proteins in 18- and 32-week human fetal growth plates. The SHOX homeodomain and the SOX6 HMG domain were shown to be implicated in the SHOX-SOX6 interaction. Moreover, different SHOX missense mutations, identified in LWD and ISS patients, disrupted this interaction. The physiological importance of these interactions was investigated by studying the effect of SHOX on a transcriptional target of the SOX trio, Agc1, which encodes one of the main components of cartilage, aggrecan. Our results show that SHOX cooperates with SOX5/SOX6 and SOX9 in the activation of the upstream Agc1 enhancer and that SHOX mutations affect this activation. In conclusion, we have identified SOX5 and SOX6 as the first two SHOX-interacting proteins and have shown that this interaction regulates aggrecan expression, an essential factor in chondrogenesis and skeletal development.

Identification of the first de novo PAR1 deletion downstream of SHOX in an individual diagnosed with Léri-Weill dyschondrosteosis (LWD).

Léri-Weill dyschondrosteosis (LWD, MIM 127300), is a dominantly inherited skeletal dysplasia with disproportionate short stature, mesomelic limb shortening, and the characteristic Madelung deformity. Two regions of the pseudoautosomal region 1 (PAR1) have been shown to be involved in LWD, SHOX (short-stature homeobox-containing gene) and the downstream enhancer region. We report our genetic findings of a young girl clinically diagnosed with LWD. We analyzed the proband and her family using MLPA and microsatellite analysis. We identified a deletion, 726-866 kb in size, of the downstream SHOX enhancer region in the proband. Neither parent carried the deletion. Microsatellite analysis showed that the deleted allele was of paternal origin. The mutation is more likely to have arisen from a de novo event but paternal gonadal mosaicism cannot be excluded. In conclusion, we report the clinical and molecular details of the first case of a de novo deletion of the downstream PAR1 region in an LWD individual. De novo deletions of SHOX and the downstream enhancer region must be therefore considered in cases of isolated LWD.