A new analytical bench assay for the determination of arylsulfatase a activity toward galactosyl-3-sulfate ceramide: implication for metachromatic leukodystrophy diagnosis.

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Expression of cathepsins S and D signals a distinctive biochemical trait in CD34+ hematopoietic stem cells of relapsing-remitting multiple sclerosis patients.

BACKGROUND: The elucidation of mechanistic aspects of relapsing-remitting multiple sclerosis (RRMS) pathogenesis may offer valuable insights into diagnostic decisions and medical treatment. RESULTS: Two lysosomal proteases, cathepsins S and D (CatS and CatD), display an exclusive pattern of expression in CD34(+) hematopoietic stem cells (HSCs) from peripheral blood of acute MS (A-MS) patients (n = 20). While both enzymes normally exist as precursor forms in the HSCs of healthy individuals (n = 30), the same cells from A-MS patients consistently exhibit mature enzymes. Further, mature cathepsins are expressed at lower rates in stable MS subjects (S-MS, n = 15) and revert to precursor proteins after interferon-ß1a treatment (n = 5). Mature CatD and CatS were induced in HSCs of healthy donors that were either co-cultured with PBMCs of A-MS patients or exposed to their plasma, suggesting a functional involvement of soluble agents. Following HSC exposure to several cytokines known to be implicated in MS, and based on relative cytokine levels displayed in A-MS, S-MS and control individuals, we identified IL-16 as a specific cell signaling factor associated with cathepsin processing. CONCLUSIONS: These data point to an evident correlation between CatS and CatD expression and MS clinical stage, and define a biochemical trait in HSCs with functional, medical, and diagnostic relevance.

Neural stem cell gene therapy ameliorates pathology and function in a mouse model of globoid cell leukodystrophy.

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of ß-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit.

Development of a New Tool for 3D Modeling for Regenerative Medicine.

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the ß-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume.

Coordinated involvement of cathepsins S, D and cystatin C in the commitment of hematopoietic stem cells to dendritic cells.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

Leukodystrophies are rare diseases caused by defects in the genes coding for lysosomal enzymes that degrade several glycosphingolipids. Gene therapy for leukodystrophies requires efficient distribution of the missing enzymes in CNS tissues to prevent demyelination and neurodegeneration. In this work, we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal protein distribution from a single injection site. We used bidirectional (bd) lentiviral vectors (LV) (bdLV) to ensure coordinate expression of a therapeutic gene (beta-galactocerebrosidase, GALC; arylsulfatase A, ARSA) and of a reporter gene, thus monitoring simultaneously transgene distribution and enzyme reconstitution. A single EC injection of bdLV.GALC in early symptomatic twitcher mice (a murine model of globoid cell leukodystrophy) resulted in rapid and robust expression of a functional GALC protein in the telencephalon, cerebellum, brainstem and spinal cord. This led to global rescue of enzymatic activity, significant reduction of tissue storage and decrease of activated astroglia and microglia. Widespread protein distribution and complete metabolic correction were also observed after EC injection of bdLV.ARSA in a mouse model of metachromatic leukodystrophy. Our data indicated axonal transport, distribution through cerebrospinal fluid flow and cross-correction as the mechanisms contributing to widespread bioavailability of GALC and ARSA proteins in CNS tissues. LV-mediated gene delivery of lysosomal enzymes by targeting highly interconnected CNS regions is a potentially effective strategy that, combined with a treatment able to target the PNS and peripheral organs, may provide significant therapeutic benefit to patients affected by leukodystrophies.

MicroRNA implications across neurodevelopment and neuropathology.

MicroRNAs (miRNAs) have rapidly emerged as biologically important mediators of posttranscriptional and epigenetic regulation in both plants and animals. miRNAs function through a variety of mechanisms including mRNA degradation and translational repression; additionally, miRNAs may guide gene expression by serving as transcription factors. miRNAs are highly expressed in human brain. Tissue and cell type-specific enrichments of certain miRNAs within the nervous system argue for a biological significance during neurodevelopmental stages. On the other hand, a large number of studies have reported links between alterations of miRNA homeostasis and pathologic conditions such as cancer, heart diseases, and neurodegeneration. Thus, profiles of distinct or aberrant miRNA signatures have most recently surged as one of the most fascinating interests in current biology. Here, the most recent insights into the involvement of miRNAs in the biology of the nervous system and the occurrence of neuropathological disorders are reviewed and discussed.

Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells.

RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34(+) hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34(+) cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34(+) cells, suggesting a potential for applications in related preclinical models.

Neural precursor cell cultures from GM2 gangliosidosis animal models recapitulate the biochemical and molecular hallmarks of the brain pathology.

In this work we showed that genotype-related patterns of hexosaminidase activity, isoenzyme composition, gene expression and ganglioside metabolism observed during embryonic and postnatal brain development are recapitulated during the progressive stages of neural precursor cell (NPC) differentiation to mature glia and neurons in vitro. Further, by comparing NPCs and their differentiated progeny established from Tay-Sachs (TS) and Sandhoff (SD) animal models with the wild-type counterparts, we studied the events linking the accumulation of undegraded substrates to hexosaminidase activity. We showed that similarly to what observed in brain tissues in TS NPCs and progeny, the stored GM2 was partially converted by sialidase to GA2, which can be then degraded in the lysosomes to its components. The latter can be used in a salvage pathway for the formation of GM3. Interestingly, results obtained from ganglioside feeding assays and from measurement of lysosomal sialidase activity suggest that a similar pathway might work also in the SD model.