[Show abstract][Hide abstract] ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.
For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal
muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the
lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies,
surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic
for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous
pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model
resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional
restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional
activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent
model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous
proteostasis mechanism and an attractive target for therapy.
No preview · Article · Oct 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Because of their prevalence, severity and lack of effective treatments, inborn errors of metabolism need novel and more effective
therapeutic approaches. The opportunity for an early treatment coming from expanded newborn screening has made this need even
more urgent. To meet this demand, a growing number of novel treatments are entering in the phase of clinical development.
Strategies to overcome the detrimental consequences of the enzyme deficiencies responsible for inborn errors of metabolism
have been focused on multiple fronts at the levels of the gene, RNA, protein and whole cell. These strategies have been accomplished
using a wide spectrum of approaches ranging from small molecules to enzyme replacement therapy, cell and gene therapy. The
applications of new technologies in the field of inborn errors of metabolism, such as genome editing, RNA interference and
cell reprogramming, along with progress in pre-existing strategies, such as gene therapy or cell transplantation, have tremendous
potential for clinical translation.
No preview · Article · Oct 2015 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Copy number variations are a common cause of intellectual disability (ID). Determining the contribution of copy number variants (CNVs), particularly gains, to disease remains challenging. Here, we report four males with ID with sub-microscopic duplications at Xp11.2 and review the few cases with overlapping duplications reported to date. We established the extent of the duplicated regions in each case encompassing a minimum of three known disease genes TSPYL2, KDM5C and IQSEC2 with one case also duplicating the known disease gene HUWE1. Patients with a duplication encompassing TSPYL2, KDM5C and IQSEC2 without gains of nearby SMC1A and HUWE1 genes have not been reported thus far. All cases presented with ID and significant deficits of speech development. Some patients also manifested behavioral disturbances such as hyperactivity and attention-deficit/hyperactivity disorder. Lymphoblastic cell lines from patients show markedly elevated levels of TSPYL2, KDM5C and SMC1A, transcripts consistent with the extent of their CNVs. The duplicated region in our patients contains several genes known to escape X-inactivation, including KDM5C, IQSEC2 and SMC1A. In silico analysis of expression data in selected gene expression omnibus series indicates that dosage of these genes, especially IQSEC2, is similar in males and females despite the fact they escape from X-inactivation in females. Taken together, the data suggest that gains in Xp11.22 including IQSEC2 cause ID and are associated with hyperactivity and attention-deficit/hyperactivity disorder, and are likely to be dosage-sensitive in males.European Journal of Human Genetics advance online publication, 10 June 2015; doi:10.1038/ejhg.2015.123.
No preview · Article · Jun 2015 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Gene therapy is entering the stage of initial clinical development to treat a growing number of inherited metabolic diseases. This review outlines the development of liver-directed gene therapy for diseases caused by deficiencies of enzymes that are primarily expressed in the liver and discusses the disorders that appear most promising for clinical translation.
Full-text · Article · Apr 2015 · Human Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Pre-existing immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for Mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder due to arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in twenty-four patients out of 34 (71%) was predicted by the type of mutations. Pre-existing Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.
Full-text · Article · Feb 2015 · Human Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity.
Electronic supplementary material
The online version of this article (doi:10.1007/s10545-014-9808-2) contains supplementary material, which is available to authorized users.
Full-text · Article · Jan 2015 · Journal of Inherited Metabolic Disease
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I), respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on KCs and LSECs, respectively. Compared with saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7-and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to greater hepatocyte transduction, compared with control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6, the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5kb) genes. Viral vectors derived from Adenovirus (Ad), Lentivirus (LV) and Herpesvirus (HV) can package large DNA sequences but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed.
Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes, and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium (RPE). We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8.
[Show abstract][Hide abstract] ABSTRACT: Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.
Full-text · Article · Jun 2014 · Developmental Cell
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.
[Show abstract][Hide abstract] ABSTRACT: Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery due to a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared to intravenous delivery liver and multi-year transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. Following vector delivery into the surgically-isolated liver, we showed phenotypic correction at the low and clinically relevant vector dose of 1x1011 vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative compared to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.
Full-text · Article · Mar 2014 · Human Gene Therapy Methods
[Show abstract][Hide abstract] ABSTRACT: Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor β (TGF-β) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-β signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-β antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.European Journal of Human Genetics advance online publication, 8 January 2014; doi:10.1038/ejhg.2013.283.
Full-text · Article · Jan 2014 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Rhabdomyolysis may result from various factors, namely trauma, exercise, medications, infections, endocrine disorders, congenital myopathies, and metabolic diseases.(1) Among the latter, mitochondrial fatty acid β-oxidation (FAO) defects frequently cause recurrent rhabdomyolysis. FAO disorders are recessively inherited and have a combined incidence of 1:9,300, estimated after implementation of newborn screening programs by tandem mass spectrometry (MS/MS).(2) Clinical manifestations of these disorders range from sudden infant death to Reye-like syndrome, nonketotic hypoglycemia, skeletal myopathy, peripheral neuropathy, and progressive cardiomyopathy. Here, we describe an 18-month-old child presenting with episodes of recurrent rhabdomyolysis related to mitochondrial trifunctional protein deficiency (MTPD), without additional manifestations of FAO defects. We discuss the diagnosis of MTPD and review the prognosis and treatments.