Brain-targeted enzyme loaded nanoparticles: a breach through the blood brain barrier for enzyme replacement therapy in Krabbe disease

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement

Incorporación de materiales ecológicos de los envases de botellas de agua en la empresa Nestlé en la actualidad

Problema: Este trabajo de investigación se elaboró para comprender la manera de incorporación de materiales ecológicos para la reducción de la contaminación ambiental. Objetivos: La incorporación de materiales ecológicos, y analizar la implementación del reciclaje del plástico PET en los envases de agua mineral de la marca Nestlé. Metodología: El estudio comprendió la descripción del fenómeno central mediante la recolección de datos cualitativos obtenidos de unidades en artículos científicos. Se trató de una investigación mixta (cualitativa y cuantitativa). Para la etapa cualitativa se usó la técnica guía de observación, y para la etapa cuantitativa se usó la técnica del cuestionario. Para el análisis de los datos cualitativos se usó estadística descriptiva por medio del software Microsoft Excel. Resultados: La mayoría de los encuestados está de acuerdo en la incorporación del plástico PET. Conclusión: La mayoría de los usuarios encuestados son conscientes del daño que ocasiona el plástico en el medio ambient

Autophagy as a pathogenic mechanism and a therapeutic target in Krabbe disease

Globoid Cell Leukodystrophy (GLD), also known as Krabbe disease, is a rare hereditary neurodegenerative condition. This disorder affects about 1 in 100000 live births and the early-infantile form represents approximately 85-90% of all cases. GLD is rapidly progressive and results in neurological degeneration and death within the first 1-2 years from birth. The cause of this disorder has been attributed to the deficiency of a lysosomal enzyme called galactosyl-ceramidase (GALC). Normally, GALC degrades galactosyl-ceramide (GLC) and other sphingolipids, among which galactosylsphingosine (psychosine, PSY). GALC loss of function leads to PSY accumulation in the central and peripheral nervous system, which causes massive oligodendrocyte and Schwann cell death, with resulting demyelination. The accumulation of PSY afflicts myelinating cells and neurons as well, leading to a series of cellular and tissutal modifications. Unfortunately, the systemic administration of GALC is not an effective therapy, since the enzyme can not cross the blood brain barrier (BBB). Many therapeutic approaches have been attempted but, except for symptomatic and life-support treatments, there is no cure currently available. Owing to the poor general understanding of GLD physio-pathology, novel approaches to investigate the underlying mechanisms prove necessary. Lysosomal disorders are typically characterized by an abnormal accumulation of substances within lysosomes. Lysosomes are intracytoplasmic organelles endowed with the enzymes necessary to degrade all principal classes of biological molecules and capable of eliminating entire organelles, thus playing a fundamental role in metabolizing endogenous substances whose accumulation within the cell is potentially toxic. The process by which lysosomal enzymes make contact with the substances to be degraded is known as autophagy. The accumulation of un-degraded substrates interferes with different cellular functions and autophagic mechanisms in particular result frequently altered. As in other lysosomal storage disorders, autophagy might have a significant role also in GLD, but this process has never been investigated for this pathology. The aim of this thesis is to study autophagy as a pathogenic mechanism and a therapeutic target. Given that oligodendrocyte degeneration is among the major characteristics of Krabbe disease, a human oligodendrocytes hybrid cell line (MO3.13) has been used as a cellular model. MO3.13 have been treated with exogenous PSY alone or in conjunction with lithium acetate, an autophagy modulator, and the reaction of cells to this drug has been assessed by determining cellular modifications such as viability, different expression of autophagy-involved factors and Reactive Oxygen Species (ROS). Flow cytometry analysis of cells stained with both Annexin V FITC (AV), a marker for cell apoptosis, and Propidium Iodide (PI), an indicator for cell necrosis, revealed an increased mortality rate in cells treated with PSY, while the additional administration of lithium incremented cell viability, suggesting some sort of lithium-mediated betterment in oligodendrocytes disturbed by the presence of PSY. To confirm the hypothesis that lithium-induced autophagy may be the cause of such improvement, cells were treated with exogenous PSY alone, or else in conjunction with lithium, and then tested for LC3 and p62 as autophagic activity markers. LC3 localization within the cell was investigated through confocal fluorescence microscopy, whereas levels of LC3-II and p62 were examined through western blot. To further inspect whether the cause of the improved viability obtained with lithium could be a lithium-induced autophagy, in addition to the usual treatment, the cells were administered with prolyl endopeptidase inhibitor 2 (PEI), an inhibitor of prolyl oligopeptidase activity, which nullifies the effects of lithium. Here as well, cell viability was checked by staining the cells with AV-PI and examining the emitted fluorescence with a cell sorter. Moreover, to investigate whether the supposed lithium-induced autophagy may decrease PSY levels, cells treated with the usual treatments were lysed, lipids were extracted and levels of PSY were quantified by HPLC and mass spectrometry. Finally, since PSY-induced cell death is associated with ROS production, variation in ROS levels was examined by administering the cells with the usual treatment, staining them with a fluorogenic marker for ROS and detecting the emitted fluorescence with a cell sorter

Cross linked enzyme aggregates as versatile tool for enzyme delivery: application to polymeric nanoparticle.

ABSTRACT: Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly(lactide-co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher’s disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs

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

Chronic lithium administration in a mouse model for Krabbe disease

none12: Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro, laying the foundation for their in-vivo pre-clinical testing. Here, we test lithium carbonate in-vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the β-catenin-dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the β-catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.noneDel Grosso, Ambra; Parlanti, Gabriele; Angella, Lucia; Giordano, Nadia; Tonazzini, Ilaria; Ottalagana, Elisa; Carpi, Sara; Pellegrino, Roberto Maria; Alabed, Husam B R; Emiliani, Carla; Caleo, Matteo; Cecchini, MarcoDel Grosso, Ambra; Parlanti, Gabriele; Angella, Lucia; Giordano, Nadia; Tonazzini, Ilaria; Ottalagana, Elisa; Carpi, Sara; Pellegrino, Roberto Maria; Alabed, Husam B R; Emiliani, Carla; Caleo, Matteo; Cecchini, Marc

Cross-Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery: Application to Polymeric Nanoparticles

Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly­(lactide-<i>co</i>-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher’s disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs

Altered Subpopulations of Red Blood Cells and Post-treatment Anemia in Malaria

International audienceIn acute malaria, the bulk of erythrocyte loss occurs after therapy, with a nadir of hemoglobin generally observed 3–7 days after treatment. The fine mechanisms leading to this early post-treatment anemia are still elusive. We explored pathological changes in RBC subpopulations by quantifying biochemical and mechanical alterations during severe malaria treated with artemisinin derivatives, a drug family that induce “pitting” in the spleen. In this study, the hemoglobin concentration dropped by 1.93 G/dl during therapy. During the same period, iRBC accounting for 6.12% of all RBC before therapy (BT) were replaced by pitted-RBC, accounting for 5.33% of RBC after therapy (AT). RBC loss was thus of 15.9%, of which only a minor part was due to the loss of iRBC or pitted-RBC. When comparing RBC BT and AT to normal controls, lipidomics revealed an increase in the cholesterol/phosphatidylethanolamine ratio (0.17 versus 0.24, p &lt; 0.001) and cholesterol/phosphatidylinositol ratio (0.36 versus 0.67, p = 0.001). Using ektacytometry, we observed a reduced deformability of circulating RBC, similar BT and AT, compared to health control donors. The mean Elongation Index at 1.69Pa was 0.24 BT and 0.23 AT vs. 0.28 in controls ( p &lt; 0.0001). At 30Pa EI was 0.56 BT and 0.56 AT vs. 0.60 in controls ( p &lt; 0.001). The retention rate (rr) of RBC subpopulations in spleen-mimetic microsphere layers was higher for iRBC (rr = 20% p = 0.0033) and pitted-RBC (rr = 19%, p = 0.0031) than for healthy RBC (0.12%). Somewhat surprisingly, the post-treatment anemia in malaria results from the elimination of RBC that were never infected

Natural History of Drusenoid Pigment Epithelial Detachment Associated with Age-Related Macular Degeneration Age-Related Eye Disease Study 2 Report No. 17

PurposeTo investigate the natural history and genetic associations of drusenoid pigment epithelial detachment (DPED) associated with age-related macular degeneration (AMD).DesignRetrospective analysis of a prospective cohort study.ParticipantsOf the 4203 Age-Related Eye Disease Study 2 (AREDS2) participants, 391 eyes (325 participants) had DPED without late AMD at the time of DPED detection. Genetic analyses included 120 white AREDS2 participants and 145 Age-Related Eye Disease Study (AREDS) participants with DPED.MethodsBaseline and annual stereoscopic fundus photographs were graded centrally to detect DPED, a well-defined yellow elevated mound of confluent drusen ≥433 μm in diameter, and to evaluate progression rates to late AMD: geographic atrophy (GA) and neovascular (NV)-AMD. Five single nucleotide polymorphisms (CFH [rs10611670], C3 [rs2230199], CFI [rs10033900], C2/CFB [rs114254831], ARMS2 [rs10490924]) and genetic risk score (GRS) group were investigated for association with DPED development. Kaplan-Meier analyses and multivariable proportional hazard regressions were performed.Main outcome measuresProgression rates to late AMD and decrease of ≥3 lines in visual acuity (VA) from the time of DPED detection; association of rate of DPED development with genotype.ResultsMean (standard deviation [SD]) follow-up time from DPED detection was 4.7 (0.9) years. DPED was associated with increased risk of progression to late AMD (hazard ratio [HR], 2.36; 95% confidence interval [CI], 1.98-2.82; P &lt; 0.001); 67% of eyes progressed to late AMD 5 years after DPED detection. Drusenoid pigment epithelial detachment was associated with increased risk of ≥3 lines of VA loss (HR, 3.08; CI, 2.41-3.93; P &lt; 0.001) with 46% of eyes experiencing vision loss at 5 years (with or without progression to late AMD). ARMS2 risk alleles (1 vs. 0: HR, 2.72, CI, 1.58-4.70; 2 vs. 0: HR, 3.16, CI, 1.60-6.21, P &lt; 0.001) and increasing GRS group (4 vs. 1) (HR, 12.17, CI, 3.66-40.45, P &lt; 0.001) were significantly associated with DPED development in AREDS. There were no significant genetic results in AREDS2.ConclusionsThis study replicates the results of previous natural history studies of eyes with DPED including the high rates of progression to late AMD and vision loss (regardless of progression to late AMD). The genetic associations are consistent with genes associated with AMD progression

Progression of Geographic Atrophy in Age-related Macular Degeneration AREDS2 Report Number 16

PurposeTo analyze the prevalence, incidence, and clinical characteristics of eyes with geographic atrophy (GA) in age-related macular degeneration (AMD), including clinical and genetic factors affecting enlargement.DesignProspective cohort study within a controlled clinical trial.ParticipantsAge-Related Eye Disease Study 2 (AREDS2) participants, aged 50-85 years.MethodsBaseline and annual stereoscopic color fundus photographs were evaluated for GA presence and area. Analyses included GA prevalence and incidence rates, Kaplan-Meier rates, mixed-model regression, and multivariable analysis of the square root of GA, area adjusted for covariates, including clinical/imaging characteristics and genotype.Main outcome measures(1) Presence or development of GA; (2) change in the square root of GA area over time.ResultsAt baseline, 517 eyes (6.2%) of 411 participants (9.8%) had pre-existing GA (without neovascular AMD), with the following characteristics: 33% central, 67% noncentral; and the following configurations: 36% small, 26% solid/unifocal, 24% multifocal, 9% horseshoe/ring, and 6% indeterminate. Of the remaining 6530 eyes at risk, 1099 eyes (17.3%) of 883 participants developed incident GA without prior neovascular disease during mean follow-up of 4.4 years. The Kaplan-Meier rate of incident GA was 19% of eyes at 5 years. In eyes with incident GA, 4-year risk of subsequent neovascular AMD was 29%. In eyes with incident noncentral GA, 4-year risk of central involvement was 57%. GA enlargement rate (following square root transformation) was similar in eyes with pre-existing GA (0.29 mm/year; 95% confidence interval 0.27-0.30) and incident GA (0.28 mm/year; 0.27-0.30). In the combined group, GA enlargement was significantly faster with noncentrality, multifocality, intermediate baseline size, and bilateral GA (P &lt; 0.0001 for interaction in each case) but not with AREDS2 treatment assignment (P&nbsp;= 0.33) or smoking status (P&nbsp;= 0.05). Enlargement was significantly faster with ARMS2 risk (P &lt; 0.0001), C3 non-risk (P&nbsp;= 0.0002), and APOE non-risk (P&nbsp;= 0.001) genotypes.ConclusionsAnalyses of AREDS2 data on natural history of GA provide representative data on GA evolution and enlargement. GA enlargement, which was influenced by lesion features, was relentless, resulting in rapid central vision loss. The genetic variants associated with faster enlargement were partially distinct from those associated with risk of incident GA. These findings are relevant to further investigations of GA pathogenesis and clinical trial planning