Biotherapies for neurodegenerative diseases
While neurodegenerative diseases are mostly prevalent in the elderly, they may also arise early in life. In infants and children neurodegeneration causes severe mental retardation and premature death with devastating consequences on families and high costs for the society. Inborn errors of the metabolism, in particular lysosomal storage diseases (LSDs), are the most frequent causes of neurodegeneration in pre-adulthood. Whereas the primary event that causes neurodegeneration in adults is often unknown, knowledge accumulated on LSDs over the last decades provides detailed understanding of the genetic and biochemical bases of these disorders. Previous identification of the causative event and causative agents makes investigations of LSDs well appropriated to decipher cascades of events leading to neurodegeneration.
Our investigations in animal models over the last 10 years led to the definition of treatments based on gene therapy of the central nervous system for mucopolysaccharidoses, a family of LSDs in which saccharides accumulate. Phase I/II clinical trials have been prepared for two of these diseases (Sanfilippo syndromes type A and B). The first patients were treated in 2011 and the study will be completed in 2012-2013. Treatment consists in 12 to 16 deposits of adeno-associated vectors encoding the missing enzyme in the brain parenchyma. Although treatment will likely halt neurodegeneration, efficacy with regards to mental retardation is uncertain. It will depend on damages caused by accumulating saccharides during the post-natal period preceding treatment. Clinical manifestations in children suggest that they primarily concern cortical maturation, possibly affecting functional wiring in the frontal cortex.
Our recent results support this hypothesis. They were performed in affected mouse neurons, in neurons derived from patient’s induced pluripotent stem cells (iPSc) and in HeLa cell, in which a tetracycline inducible depletion of NAGLU, the missing enzyme in Sanflillipo type B syndrome, allows time-course investigations of the cellular toxicity of heapran sulfate oligosaccharides. Observations made in these various cell models provided evidence for expansion and disorganisation of the Golgi apparatus (Fig 1), cell polarity defects, cell migration defects, cell adhesion defects and defective neuritogenesis. Since deficient cells release high amounts of undigested saccharides in their environment, a likely cause of these defects is the alteration of cell-matrix interactions.
Fig. 1: HeLa cells were depleted of NAGLU, the missing enzyme in Sanfilippo syndrome type B, for 7 days, using tetracycline-inducible specific shRNAs. Immunostaining of the cis-Golgi marker GMA30 and the trans-Golgi marker p230 visualises Cis-Golgi expansion in depleted cells. Electron microscopy reveals elongation and circularization of Golgi ribbons forming vesicular structures that are characteristic of the cell pathology observed in lysosomal storage diseases.
Our ongoing and future studies will bring knowledge on the control of GM130 functions and its alterations when saccharides accumulate. This protein is a component of a multi-molecular highly dynamic complex, which controls the size, the position and the organization of the Golgi, the organisation of the centrosomes, and the nucleation and expansion of microtubules at the Golgi surface. Our results documenting alterations of GM130 functions in cells accumulating saccharides suggest control by extra-cellular cues, especially soluble heparan sulfate fragments. We showed that these molecules affect integrin clustering, downstream RhoGTPase activation and cell orientation. However, links between these pathways and GM130 control have not been previously investigated. Our projects will help understanding the impact of extra-cellular sugars on the intra-cellular organization and will potentially open possibilities to investigate relationships between sulfation of sugars in the matrix and impact on cell functions, providing insights into the “saccharide code”.
Acute Lateral Sclerosis (ALS) appears as a spectrum of neurodegenerative diseases preferentially affecting motoneurons in adults. It is highly devastating, untreatable and etiology is not known. We set up methods to differentiate and purify motoneurons from patient-derived iPSc with the aim to identify invariant phenotypic features that would found a new classification of the various forms of ALS. We expect that motoneurons from patients who develop ALS due to genetic predisposition will express phenotypic alterations. Identification of pathological molecular events would provide disease markers useful for diagnosis and follow-up, assessing therapies and developing new drugs. Absence of phenotypic alteration of motoneurons will in contrast suggest that the disease is not related to genetic predisposition but rather due to environmental determinants affecting the epigenome. The establishment of cohorts of patients based on this distinction will facilitate the detection of so far unknown genetic alterations and help assessing treatment efficacy.
In collaboration with a clinical research team at the Institut du Cerveau et de la Moelle Epinière (ICM, Pr. R. Meininger), we collected skin fibroblasts from series of patients with genetic and sporadic forms of ALS, from which iPSc were generated. We set up rapid and efficient methods to induce iPSc differentiation in motoneurons and GABAergic neurons (Fig. 2). However, cellular heterogeneity represents a major limitation for accurate phenotypic analyses and comparisons between cultures from different origins. Using lentivirus encoding reporter proteins under the control of cell specific promoters and novel flowcytometry techniques performed in collaboration with G. Haase (CHU Nord, Marseille), we have now access to pure populations of iPSc-derived motorneurons. Experiments are in progress to characterise these cells using markers of generic motorneurons, class-specific motoneurons or pool-specific motoneurons, and to search for phenotypic abnormalities. These investigations include the study of cell survival in various culture conditions, neurite outgrowth, protein aggregates, Golgi alterations, and miRNA expression patterns.
Fig. 2: Human neuron differentiated from iPSc. Fibroblasts cultured from a skin biopsy performed in a patient with a familial form of ALS associated with Alsin mutation were used to derived iPSc. Pluripotent stem cells were differentiated in neurons. Sorting by flowcytometry using a fluorescent marker protein expressed under the control of a neuron-specific promoters isolated pure populations of mature neurons