Sviluppo di una terapia enzimatica sostitutiva guidata da nanoparticelle per la malattia di Krabbe

Krabbe disease (KD), also known as globoid cell leukodystrophy, is a rare (one in 100,000 births) autosomal recessive neurodegenerative disorder. KD is classified as a lysosomal storage disease (LSD), a family of over 70 conditions characterized by perturbation in lysosomal homeostasis. KD is caused by mutations in the gene encoding for the lysosomal enzyme β-galactosylceramidase (or galactocerebrosidase; GALC). GALC plays a pivotal role in the physiological turnover of myelin components and the removal of toxic compounds. In detail, GALC deficiency leads to impaired degradation of its substrates, galactosylceramide (GalCer) and galactosylsphingosine (psychosine, PSY). GalCer can be degraded by another lysosomal enzyme, but PSY can be degraded only by GALC. Consequently, PSY accumulation results in the nearly total loss of myelin forming both in the oligodendrocytes and in the Schwann cells. This condition leads to severe demyelination and consequent neurodegeneration of both the central nervous system and peripheral nervous system. Despite this enormous effort, there is no cure for KD, and the current standard of care is mostly supportive only. Nowadays, only the pre-symptomatic transplantation of hematopoietic stem cells (HSCT) finds clinical applications. Over the last few years, the medical and the scientific communities have focused their efforts on alternative therapies: i. the substrate reduction therapy, or the reduction of the PSY accumulation by blocking its synthesis, ii. pharmacological therapies with anti-inflammatory and antioxidant drugs, iii. the gene therapy to replace the afunctional GALC gene, and iv. the enzyme replacement therapy (ERT). The ERT has gained broad interest thanks to the effective results achieved in other diseases. However, applications of such therapy to KD still do not exist due to one major issue to overcome: the blood-brain barrier (BBB). This physiological barrier strictly regulates the passage of molecules, ions, and cells from the blood to the brain, also hampering the penetration of therapeutics. The aim of this thesis is to investigate an innovative ERT for KD based on brain-targeted polymeric degradable nanoparticles, loaded with GALC aggregates, and functionalized with a brain targeting peptide (Angiopep-2), to restore GALC activity in the brain of a natural murine model called Twitcher (TWI). This thesis is part of the research project nanoERT – Nanoparticle based Enzyme Replacement Therapy for the treatment of Krabbe disease: a pre-clinical study in the Twitcher Mouse (ELA 2019-008I2), funded by the European Leukodystrophy Association (ELA). This study reports the development of a set of nanoparticles (NPs) formulations, composed of the copolymer poly (D, L-lactide-co-glycolide) (PLGA), and loaded with increasing concentrations of GALC cross-linked enzyme aggregates (CLEAs), from 3 to 300 ug/mg of PLGA. The targeting unit Angiopep-2 is previously conjugated to PLGA, and a small percentage of Angiopep-2-conjugated copolymer is used in NPs preparation. First, CLEAs, NPs, and CLEAs-loaded NPs are formulated to find the best preparation procedures, allowing high repeatability, maximization of enzyme loading, and suitable NPs size. Then, CLEAs-loaded NPs are characterized to study the stability up to 30 days in storage conditions (4°C, diluted in physiological solution) and their shelf life. Specifically, we investigated the enzyme activity and cargo loading, and the morphological stability of formulations with time, identifying the optimal one. Enzyme activity is measured by a specific enzymatic assay based on the cleavage of the substrate 4-methylumbelliferyl β-D-glucopyranoside, while the quantification of the protein cargo is performed by means of the ninhydrin assay. Size changes are evaluated by dynamic light scattering (DLS) analysis. To identify the optimal formulation, we quantified the leakage of CLEAs from NPs in storage conditions by means of enzyme and protein assays. After the development and the characterization of the system, postnatal day (PND) 20 TWI mice were treated intraperitoneally with CLEAs-loaded NPs and were sacrificed 4 hours (h), 24h, 72h, and 7 days after treatment. Then, GALC enzymatic assay was performed in the brain and sciatic nerve (representing CNS and PNS, respectively) and in two typical accumulation organs, liver and kidneys. From the obtained results, we identified the best recipe for the synthesis of CLEAs and for the preparation of NPs. We found that the amount of CLEAs used during the preparation affects enzymatic activity (from 0,08 to 12,8 U/mg of NPs), the size of CLEAs-loaded NPs (mean diameter: 150-250 nm), and the protein encapsulation (from 4,5 to 125,9 µg/g of NPs). Thus, we found a formulation suited to our aim in terms of CLEas activity, diameter, encapsulation, and release over time. We found that formulations are stable under storage conditions for up to 30 days (no loss of activity and significant degradation of PLGA); these data contribute to establishing which are the best storage conditions for the formulation and to a better understanding of CLEAs-loaded NPs. The brain of the TWI mice treated with CLEAs-loaded NPs showed an enzymatic activity (E.A) that is significantly higher than the E.A. of the untreated TWI from 4 to 72 hours after the NPs administration. Overall, these data demonstrate that the formulation gives TWI brain an E.A. that is maintained to a level that could potentially be of clinical interest up to 72 h from the treatment. An increase in E.A.is also observed in the sciatic nerve compared to untreated TWI. Regarding the liver, we found a high E.A. after 24 hours from the treatment, confirming the capability of our formulation to deliver enzymatically active GALC into mice tissues. Regarding the kidneys, we didn’t find an increase in E.A. compared to untreated TWI. In conclusion, our results are really encouraging at this stage, and we believe that the developed formulation could be of potential interest for the treatment of KD. Some additional aspects related to the fate of NPs, such as the potential accumulation of PLGA, and the effectiveness of released GALC in the brain, still need to be clarified, as well as the verification of the impaired functions restoration. All these aspects will be considered in the next experiments, with repeated administrations of CLEAs-loaded NPs and behavioural tests

Data from: Chronic Rapamycin administration via drinking water mitigates the pathological phenotype in a Krabbe disease mouse model through autophagy activation.

<p>ABSTRACT </p><p>Krabbe disease (KD) is a rare disorder caused by a deficiency of the lysosomal enzyme galactosylceramidase (GALC), resulting in the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration. Despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, and there is currently no cure available.</p><p>Previous studies from our lab revealed the presence of autophagy dysfunctions in KD pathogenesis, as evidenced by the presence of p62-tagged protein aggregates in the brains of KD mice and increased p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially restore the wild-type (WT) phenotype in KD primary cells by reducing the number of p62 aggregates.</p><p>In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the motor performance of the mice through grip strength and rotarod tests, along with various biochemical parameters related to KD pathogenesis (i.e. autophagy markers expression, myelination, astrogliosis, and PSY accumulation).</p><p>Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Using western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, which corroborates our earlier in-vitro results. Furthermore, RAPA treatment partially reduces PSY levels in the spinal cord.</p><p>In conclusion, our results support the consideration of RAPA as a supportive therapy for KD. Importantly, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be promptly evaluated in clinical trials.</p><p>Twitcher heterozygous (HET) mice (TWI+/− C57BL6 mice; the Jackson Laboratory, Bar Harbor, ME) were used as breeding pairs to produce homozygous TWI mice (TWI−/−, elsewhere in the Dataset abbreviated as TWI for simplicity). This breeding was carried out at the Center for Experimental Biomedicine of CNR in Pisa. The animals were housed under standard conditions and all procedures were performed in accordance with the protocols and ethical guidelines approved by the Ministry of Health (permit no. 535/2018-PR; official starting date: July 9, 2018).</p><p>Starting from post-natal days (PND) 21-23, mice received chronic Rapamycin (RAPA) dissolved in their drinking water at a concentration of 15 mg/L. The treatment continued until they reached a body weight loss ≥ 25%, which was the predetermined ethical endpoint. At that stage, they were euthanized. Wild type (WT) mice were euthanized alongside the last sacrificed TWI mouse in the specific experiment. Behavioral tests were conducted at three time points (TP): TP 1 = PND 24-25; TP 2 = PND 29-30; TP 3 = PND 35-37. All procedures were carried out with the utmost care to minimize any potential suffering of the mice. Tissues (total brain, cerebrum, cerebellum, spinal cord, sciatic nerves, liver and kidneys) were extracted from each mouse and used to investigate various biochemical parameters related to KD pathogenesis (i.e. autophagy markers expression, myelination, astrogliosis and PSY accumulation) throught western blot, immunohistochemistry and Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS).</p><p>Data files are uploaded as Plain text or as .xlsx files. The .xlsx files can be opened with Microsoft Excel or Google Sheets.</p&gt

The influence of illness-related variables, personal resources and context-related factors on real-life functioning of people with schizophrenia

In people suffering from schizophrenia, major areas of everyday life are impaired, including independent living, productive activities and social relationships. Enhanced understanding of factors that hinder real-life functioning is vital for treatments to translate into more positive outcomes. The goal of the present study was to identify predictors of real-life functioning in people with schizophrenia, and to assess their relative contribution. Based on previous literature and clinical experience, several factors were selected and grouped into three categories: illness-related variables, personal resources and context-related factors. Some of these variables were never investigated before in relationship with real-life functioning. In 921 patients with schizophrenia living in the community, we found that variables relevant to the disease, personal resources and social context explain 53.8% of real-life functioning variance in a structural equation model. Neurocognition exhibited the strongest, though indirect, association with real-life functioning. Positive symptoms and disorganization, as well as avolition, proved to have significant direct and indirect effects, while depression had no significant association and poor emotional expression was only indirectly and weakly related to real-life functioning. Availability of a disability pension and access to social and family incentives also showed a significant direct association with functioning. Social cognition, functional capacity, resilience, internalized stigma and engagement with mental health services served as mediators. The observed complex associations among investigated predictors, mediators and real-life functioning strongly suggest that integrated and personalized programs should be provided as standard treatment to people with schizophrenia

The complex relationship between self-reported 'personal recovery' and clinical recovery in schizophrenia

Self-reported 'personal recovery' and clinical recovery in schizophrenia (SRPR and CR, respectively) reflect different perspectives in schizophrenia outcome, not necessarily concordant with each other and usually representing the consumer's or the therapist's point of view.By means of a cluster analysis on SRPR-related variables, we identified three clusters. The first and third cluster included subjects with the best and the poorest clinical outcome respectively. The second cluster was characterized by better insight, higher levels of depression and stigma, lowest self-esteem and personal strength, and highest emotional coping. The first cluster showed positive features of recovery, while the third cluster showed negative features. The second cluster, with the most positive insight, showed a more complex pattern, a somewhat 'paradoxical' mixture of positive and negative personal and clinical features of recovery.The present results suggest the need for a characterization of persons with schizophrenia along SRPR and CR dimensions to design individualized and integrated treatment programs aimed to improve insight and coping strategies, reduce stigma, and shape recovery styles