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BackgroundCLN2 disease (Neuronal Ceroid Lipofuscinosis Type 2) is an ultra-rare, neurodegenerative lysosomal storage disease, caused by an enzyme deficiency of tripeptidyl peptidase 1 (TPP1). Lack of disease awareness and the non-specificity of presenting symptoms often leads to delayed diagnosis. These guidelines provide robust evidence-based, expert-agreed recommendations on the risks/benefits of disease-modifying treatments and the medical interventions used to manage this condition.MethodsAn expert mapping tool process was developed ranking multidisciplinary professionals, with knowledge of CLN2 disease, diagnostic or management experience of CLN2 disease, or family support professionals. Individuals were sequentially approached to identify two chairs, ensuring that the process was transparent and unbiased. A systematic literature review of published evidence using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidance was independently and simultaneously conducted to develop key statements based upon the strength of the publications. Clinical care statements formed the basis of an international modified Delphi consensus determination process using the virtual meeting (Within3) online platform which requested experts to agree or disagree with any changes. Statements reaching the consensus mark became the guiding statements within this manuscript, which were subsequently assessed against the Appraisal of Guidelines for Research and Evaluation (AGREEII) criteria.ResultsTwenty-one international experts from 7 different specialities, including a patient advocate, were identified. Fifty-three guideline statements were developed covering 13 domains: General Description and Statements, Diagnostics, Clinical Recommendations and Management, Assessments, Interventions and Treatment, Additional Care Considerations, Social Care Considerations, Pain Management, Epilepsy / Seizures, Nutritional Care Interventions, Respiratory Health, Sleep and Rest, and End of Life Care. Consensus was reached after a single round of voting, with one exception which was revised, and agreed by 100% of the SC and achieved 80% consensus in the second voting round. The overall AGREE II assessment score obtained for the development of the guidelines was 5.7 (where 1 represents the lowest quality, and 7 represents the highest quality).ConclusionThis program provides robust evidence- and consensus-driven guidelines that can be used by all healthcare professionals involved in the management of patients with CLN2 disease and other neurodegenerative disorders. This addresses the clinical need to complement other information available.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative disorders whose molecular mechanisms remain largely unknown. Omics approaches are among the methods that generate new information on modifying factors and molecular signatures. Moreover, omics data integration can address the need to progressively expand knowledge around the disease and pinpoint specific proteins to promote as candidate biomarkers.In this work, we integrated a total of 62 proteomic and transcriptomic datasets originating from humans and mice, employing a new approach able to define dysregulated processes across species, stages and NCL forms. Moreover, we selected a pool of differentially expressed proteins and genes as species- and form-related biomarkers of disease status/progression and evaluated local and spatial differences in most affected brain regions.Our results offer promising targets for potential new therapeutic strategies and reinforce the hypothesis of a connection between NCLs and other forms of dementia, particularly Alzheimer's disease.

Abstract Background CLN2 disease (Neuronal Ceroid Lipofuscinosis Type 2) is an ultra-rare, neurodegenerative lysosomal storage disease, caused by an enzyme deficiency of tripeptidyl peptidase 1 (TPP1). Lack of disease awareness and the non-specificity of presenting symptoms often leads to delayed diagnosis. These guidelines provide robust evidence-based, expert-agreed recommendations on the risks/benefits of disease-modifying treatments and the medical interventions used to manage this condition. Methods An expert mapping tool process was developed ranking multidisciplinary professionals, with knowledge of CLN2 disease, diagnostic or management experience of CLN2 disease, or family support professionals. Individuals were sequentially approached to identify two chairs, ensuring that the process was transparent and unbiased. A systematic literature review of published evidence using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidance was independently and simultaneously conducted to develop key statements based upon the strength of the publications. Clinical care statements formed the basis of an international modified Delphi consensus determination process using the virtual meeting (Within3) online platform which requested experts to agree or disagree with any changes. Statements reaching the consensus mark became the guiding statements within this manuscript, which were subsequently assessed against the Appraisal of Guidelines for Research and Evaluation (AGREEII) criteria. Results Twenty-one international experts from 7 different specialities, including a patient advocate, were identified. Fifty-three guideline statements were developed covering 13 domains: General Description and Statements, Diagnostics, Clinical Recommendations and Management, Assessments, Interventions and Treatment, Additional Care Considerations, Social Care Considerations, Pain Management, Epilepsy / Seizures, Nutritional Care Interventions, Respiratory Health, Sleep and Rest, and End of Life Care. Consensus was reached after a single round of voting, with one exception which was revised, and agreed by 100% of the SC and achieved 80% consensus in the second voting round. The overall AGREE II assessment score obtained for the development of the guidelines was 5.7 (where 1 represents the lowest quality, and 7 represents the highest quality). Conclusion This program provides robust evidence- and consensus-driven guidelines that can be used by all healthcare professionals involved in the management of patients with CLN2 disease and other neurodegenerative disorders. This addresses the clinical need to complement other information available.

A dual chamber system was deployed in two different environments to study the potential of ambient air, that was directly injected into the chambers, to form secondary organic and inorganic aerosol. A total of 16 experiments took place during March 2022 in a polluted environment in the Po Valley, Italy which is dominated by anthropogenic emissions. Another experiments were conducted in the Pertouli forest, Greece which is dominated by biogenic emissions. In both campaigns, ambient air containing highly oxidized (average O:C 0.7-0.8) aerosol was the starting point of the experiments and its chemical evolution under the presence of OH radicals was followed. In the Po Valley SOA formation was observed in all experiments but one and the formed SOA ranged from 0.1 to 10 µg m-3. Experiments conducted under more polluted conditions (usually at night and early morning) had significantly higher SOA formation, with the concentration of the organic aerosol at the end being about four times higher than the initial. Also, production of 4-230 µg m-3 of ammonium nitrate was observed in all experiments due to the high levels of ammonia in this area. The produced SOA increased as the ambient relative humidity increased, but there was not a clear relationship between the SOA and temperature. Higher SOA production was observed when the PM1 levels in Po Valley were high. Contrary to the Po Valley, only one experiment in the Pertouli forest resulted in the formation of detectable SOA (about 1 µg m-3). This experiment was characterized by higher ambient concentrations of both monoterpenes and isoprene. In two experiments, some SOA was formed, but its concentration dropped below detection levels after 30 min. This behavior is consistent with local formation in a chamber that was not well mixed. Although both environments have OA with O:C in the range of 0.7-0.8, the atmosphere of the two sites had very different potentials of forming SOA. In the Po Valley, the system reacts rapidly forming large amounts of SOA, while in Pertouli the corresponding SOA formation chemistry appears to have been practically terminated before the beginning of most experiments, so there is little additional SOA formation potential left. [ABSTRACT FROM AUTHOR]

The main aim of this review is to summarize the current state-of-art in the field of childhood Neuronal Ceroid Lipofuscinosis (NCL), a group of rare neurodegenerative disorders. These are genetic diseases associated with the formation of toxic endo-lysosomal storage. Following a brief historical review of the evolution of NCL definition, a clinically-oriented approach is used describing how the early symptoms and signs affecting motor, visual, cognitive domains, and including seizures, may lead clinicians to a rapid molecular diagnosis, avoiding the long diagnostic odyssey commonly observed. We go on to focus on recent advances in NCL research and summarize contributions to knowledge of the pathogenic mechanisms underlying NCL. We describe the large variety of experimental models which have aided this research, as well as the most recent technological developments which have shed light on the main mechanisms involved in the cellular pathology, such as apoptosis and autophagy. The search for innovative therapies is described. Translation of experimental data into therapeutic approaches is being established for several of the NCLs, and one drug is now commercially available. Lastly, we show the importance of palliative care and symptomatic treatments which are still the main therapeutic interventions.

ATR-X syndrome is a rare X-linked congenital disorder caused by hypomorphic mutations in the ATRX gene. A typical phenotype is well defined, with cognitive impairment, characteristic facial dysmorphism, hypotonia, gastrointestinal, skeletal, urogenital, and hematological anomalies as characteristic features. With a few notable exceptions, general phenotypic differences related to specific ATRX protein domains are not well established and should not be used, at least at the present time, for prognostic purposes. The phenotypic spectrum and genotypic correlations are gradually broadening, mainly due to rapidly increasing accessibility to NGS. In this scenario, it is important to continue describing new patients, illustrating the mode and age of onset of the typical and non-typical features, the classical ones and those tentatively added more recently. This report of well-characterized and mostly unreported patients expands the ATR-X clinical spectrum and emphasizes the importance of better clinical delineation of the condition. We compare our findings to those of the largest ATR-X series reported so far, discussing possible explanations for the different drawn conclusions.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5−/− mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.

CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.

We are attempting to attribute a value to the right of access to the internal documents of EU Institutions, going beyond a simple analysis of the instrinsic structure of that right as such. We are analysing case-law of the European Court of Justice that solves conflicts in which the right of access competes with the right to privacy. We then compare our results with standard practice in the Italian Courts to check for deviations of Supranational European Standars from traditional national standards.

Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42–53).

CLN1 disease (OMIM #256730) is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1) is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1) and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2) positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs) in SH-p.wtCLN1 (as compared to empty-vector transfected cells), whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45) was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM) and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to the axonal and synaptic compartments), which can be associated with an altered gene dosage of wtCLN1. Moreover, these modules were interrelated with the pathological effects associated with loss of PPT1 function, similarly as observed in the Ppt1 knockout mice and patients with CLN1 disease.