Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure 4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure 4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figure panels 4A and 4E for the following reasons: Figure panel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figure panel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.

Protein aggregation and autophagy dysfunction: new lessons from mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases with strong neurological involvement. MPSs are caused by defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs), which consequently accumulate into the lysosomes as primary storage. Macroautophagy/autophagy impairment is well known to drive neurodegeneration in MPSs, however, mechanisms underlying such dysfunction are still poorly understood. Recently, by studying a mouse model for MPS-III (Sanfilippo syndrome) we have shown that the progressive aggregation of amyloid proteins in neuronal cell bodies occurs downstream of the GAG storage and, in turn, impairs the autophagy pathway by affecting lysosomal-dependent autophagosome clearance.

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

In vivo Gene Therapy to the Liver and Nervous System: Promises and Challenges.

In vivo genetic engineering has recently shown remarkable potential as a novel effective treatment for an ever-growing number of diseases, as also witnessed by the recent marketing authorization of several in vivo gene therapy products. In vivo genetic engineering comprises both viral vector-mediated gene transfer and the more recently developed genome/epigenome editing strategies, as long as they are directly administered to patients. Here we first review the most advanced in vivo gene therapies that are commercially available or in clinical development. We then highlight the major challenges to be overcome to fully and broadly exploit in vivo gene therapies as novel medicines, discussing some of the approaches that are being taken to address them, with a focus on the nervous system and liver taken as paradigmatic examples.

Retinal Degeneration in MPS-IIIA Mouse Model.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

Protein Aggregation and Dysfunction of Autophagy-Lysosomal Pathway: A Vicious Cycle in Lysosomal Storage Diseases.

Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades.

The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ß progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.

Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.

Gene therapy for mucopolysaccharidoses: in vivo and ex vivo approaches.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency in lysosomal enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs). The current therapeutic strategies of enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation have been reported to reduce patient morbidity and to improve their quality of life, but they are associated with persistence of residual disease burden, in particular at the neurocognitive and musculoskeletal levels. This indicates the need for more efficacious treatments capable of effective and rapid enzyme delivery to the affected organs, especially the brain and the skeleton. Gene therapy (GT) strategies aimed at correcting the genetic defect in patient cells could represent a significant improvement for the treatment of MPS when compared with conventional approaches. While in-vivo GT strategies foresee the administration of viral vector particles directly to patients with the aim of providing normal complementary DNA to the affected cells, ex-vivo GT approaches are based on the ex-vivo transduction of patient cells that are subsequently infused back. This review provides insights into the state-of-art accomplishments made with in vivo and ex vivo GT-based approaches in MPS and provide a vision for the future in the medical community.

Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs.

Brain Targeting in MPS-IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a childhood metabolic neuropathology caused by the inherited deficiency of the lysosomal enzyme sulfamidase and is characterized by the accumulation of undegraded glycosaminoglycans in the lysosomes of cells and tissues of affected patients. MPS-IIIA represents one of the most common forms of lysosomal storage disorders (LSDs) and to date there is no cure. Since neurodegeneration is the most relevant pathological feature in MPS-IIIA patients, the treatment of the central nervous system (CNS) lesions represents the goal of any effective therapy for this devastating disorder. During the last years many advances have been made in developing and testing new therapies for brain involvement in MPS-IIIA. These studies have been possible because of the availability of mouse and dog models that recapitulate the MPS-IIIA neuropathological features. Some of these approaches are based on direct CNS administration routes through which the therapeutic molecules access the CNS via the parenchyma (intracerebral injections) or via the cerebrospinal fluid (intraventricular/intrathecal injections). These approaches are highly invasive and poorly suited for clinical use. Minimally invasive approaches are based on systemic injections into the blood stream of therapeutics capable of crossing the blood-brain barrier (BBB). This review will present the background of the clinic and pathology aspects of MPS-IIIA and will describe the current MPS-IIIA preclinical and clinical studies focusing on how a systemic therapeutic strategy based on crossing the BBB has been successfully used to treat CNS pathology and behavioral abnormalities in a mouse model of MPS-IIIA. Future clinical applications of this approach to MPS-IIIA patients will be also discussed together with the possibility of using similar strategies in other LSDs with neurological involvement.

Brain Disorders Due to Lysosomal Dysfunction.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

Transdermal spinal catheter placement in piglets: Description and validation of the technique.

BACKGROUND: The swine species represents a perfect model for translational medicine due to its physiological and anatomical resemblance to humans. The development of techniques for spinal catheter insertion in swine is significantly useful but, at the moment, the only technique described requires laminectomy as a surgical approach. NEW METHOD: The proposed techniques represent a transdermal approach for catheter placement in piglets. The study was divided into Phase I (anatomical study on 8 cadavers) and Phase II (in vivo application of the technique in 20 anaesthetised 30-day old piglets). A spinal needle was introduced between the L2 and L3 spinous processes with a ventro-cranial orientation until cerebro-spinal fluid leakage. It was then replaced with a Tuohy needle, used to introduce the catheter into the intrathecal space. Before inserting the catheter, the approximate length from the insertion point to the external projection of the Cisterna Magna was measured using the gradation markings on the device. RESULTS: The technique described allowed spinal catheter placement in all piglets. In Phase I, the correct placement was confirmed using fluoroscopy while, in Phase II, cerebrospinal fluid leakage from the needle was relied on. No clinical alterations were detected either during the procedure or during the following days. COMPARISON WITH EXISTING METHOD: This technique is easy and requires less skilled operators when compared to the other existing method which involves a surgical approach. Moreover, being less invasive, it potentially leads to fewer complications. CONCLUSIONS: In conclusion, the technique can be performed safely in piglets, and provides an easier and less invasive approach for spinal catheter insertion.

Access to cerebrospinal fluid in piglets via the cisterna magna: optimization and description of the technique.

The collection of cerebrospinal fluid is necessary in order to determine its composition. It can then be used to diagnose various diseases. The aim of the study was to develop and optimize a technique for performing safe centesis for the collection of cerebrospinal fluid in piglets and its injection through the cisterna magna. The study was divided into three phases: (1) anatomical study of cadavers, (2) in vivo application of the technique and (3) observation of recovery time. The proposed technique resulted in a safe puncture of the cisterna magna. The authors identified and confirmed the correspondence of the crista occipitalis and the wings of the atlas with the external landmarks on the cadaver by means of direct radiological visualization. The punctures were performed successfully at the first attempt in 11 out of 12 anaesthetized piglets. The technique herein described provides a reproducible safe and easy route for approaching the cisterna magna for cerebrospinal fluid collection, drug administration and gene delivery.

Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with mucopolysaccharidosis type IIIA disease: results of a phase I/II trial.

Mucopolysaccharidosis type IIIA is a severe degenerative disease caused by an autosomal recessive defect of a gene encoding a lysosomal heparan-N-sulfamidase, the N-sulfoglycosamine sulfohydrolase (SGSH), the catalytic site of which is activated by a sulfatase-modifying factor (SUMF1). Four children (Patients 1-3, aged between 5.5 and 6 years; Patient 4 aged 2 years 8 months) received intracerebral injections of an adeno-associated viral vector serotype rh.10-SGSH-IRES-SUMF1 vector in a phase I/II clinical trial. All children were able to walk, but their cognitive abilities were abnormal and had declined (Patients 1-3). Patients 1-3 presented with brain atrophy. The therapeutic vector was delivered in a frameless stereotaxic device, at a dose of 7.2×10(11) viral genomes/patient simultaneously via 12 needles as deposits of 60 µl over a period of 2 hr. The vector was delivered bilaterally to the white matter anterior, medial, and posterior to the basal ganglia. Immunosuppressive treatment (mycophenolate mofetil and tacrolimus) was initiated 15 days before surgery and maintained for 8 weeks (mycophenolate mofetil) or throughout follow-up (tacrolimus, with progressive dose reduction) to prevent elimination of transduced cells. Safety data collected from inclusion, during the neurosurgery period and over the year of follow-up, showed good tolerance, absence of adverse events related to the injected product, no increase in the number of infectious events, and no biological sign of toxicity related to immunosuppressive drugs. Efficacy analysis was necessarily preliminary in this phase I/II trial on four children, in the absence of validated surrogate markers. Brain atrophy evaluated by magnetic resonance imaging seemed to be stable in Patients 1 and 3 but tended to increase in Patients 2 and 4. Neuropsychological evaluations suggested a possible although moderate improvement in behavior, attention, and sleep in Patients 1-3. The youngest patient was the most likely to display neurocognitive benefit.

Phosphorylation-regulated degradation of the tumor-suppressor form of PED by chaperone-mediated autophagy in lung cancer cells.

PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)-which, among other processes, is involved in chaperone-mediated autophagy (CMA)-as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.