Epilepsy-related CDKL5 deficiency slows synaptic vesicle endocytosis in central nerve terminals

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from Cdkl5 knockout rat males. Using a genetically encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD. SIGNIFICANCE STATEMENT Loss of cyclin-dependent kinase like 5 (CDKL5) function is a leading cause of monogenic childhood epileptic encephalopathy. However, information regarding its biological role is scarce. In this study, we reveal a selective presynaptic role for CDKL5 in synaptic vesicle endocytosis and that its protein kinase activity is both necessary and sufficient for this role. The isolated protein kinase domain is sufficient to correct this loss of function, which may facilitate future gene therapy strategies if presynaptic dysfunction is proven to be central to the disorder. It also reveals that a CDKL5-specific substrate is located at the presynapse, the phosphorylation of which is required for optimal SV endocytosis. </p

PROCEEDINGS AND PHOTO ALBUM FROM THE OFFICIAL PRESENTATION OF THE RELIGIOUS STUDIES LABORATORY (RELIGLAB) OF THE NATIONAL AND KAPODISTRIAN UNIVERSITY OF ATHENS

A special issue by Theophany Journal (Proceedings 1)A special issue by Theophany Journal (Proceedings 1

High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments

High glucose concentrations have been shown to activate endothelial cells and promote angiogenesis. In the present study, it was investigated whether high glucose concentrations could improve the vascularisation capacity of adipose-tissue-derived microvascular fragments (ad-MVF). Ad-MVF were isolated from the epididymal fat pads of donor mice and cultivated for 24 h in University of Wisconsin (UW) solution supplemented with vehicle or 30 mM glucose. Protein expression, morphology, viability and proliferation of the cultivated ad-MVF were analysed by means of proteome profiler mouse angiogenesis array, scanning electron microscopy and immunohistochemistry. Additional cultivated ad-MVF were seeded on to collagen-glycosaminoglycan scaffolds to study their in vivo vascularisation capacity in the dorsal skinfold chamber model by intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, high glucose exposure changed the protein expression pattern of ad-MVF with endoglin, interleukin (IL)-1β and monocyte chemoattractant protein (MCP)-1 as the most up-regulated pro-angiogenic factors. Moreover, high glucose exposure induced the formation of nanopores in the ad-MVF wall. In addition, ad-MVF contained significantly larger numbers of proliferating endothelial and perivascular cells while exhibiting a comparable number of apoptotic cells when compared to vehicle-treated controls. In vivo, scaffolds seeded with high-glucose-exposed ad-MVF exhibited an improved vascularisation and tissue incorporation. These findings demonstrated that the exposure of cultivated ad-MVF to high glucose concentrations is a promising approach to improve their in vivo performance as vascularisation units for tissue engineering and regenerative medicine

Insulin-like growth factor 1 stimulates the angiogenic activity of adipose tissue-derived microvascular fragments

Angiogenesis in adipose tissue is promoted by insulin-like growth factor 1 signaling. We analyzed whether this regulatory mechanism also improves the angiogenic activity of adipose tissue-derived microvascular fragments. Murine adipose tissue-derived microvascular fragments were cultivated for 24 h in the University of Wisconsin (UW) solution supplemented with vehicle, insulin-like growth factor 1, or a combination of insulin-like growth factor 1 and insulin-like growth factor-binding protein 4. Subsequently, we assessed their cellular composition, viability, proliferation, and growth factor expression. Moreover, cultivated adipose tissue-derived microvascular fragments were seeded onto collagen-glycosaminoglycan scaffolds, which were implanted into dorsal skinfold chambers to study their vascularization and incorporation. Insulin-like growth factor 1 increased the viability and growth factor expression of adipose tissue-derived microvascular fragments without affecting their cellular composition and proliferation. Accordingly, scaffolds containing insulin-like growth factor 1-stimulated adipose tissue-derived microvascular fragments exhibited an enhanced in vivo vascularization and incorporation. These positive insulin-like growth factor 1 effects were reversed by additional exposure of adipose tissue-derived microvascular fragments to insulin-like growth factor-binding protein 4. Our findings indicate that insulin-like growth factor 1 stimulation of adipose tissue-derived microvascular fragments is suitable to improve their vascularization capacity

Variation at the TRIM11 Locus Modifies Progressive Supranuclear Palsy Phenotype

Objective The basis for clinical variation related to underlying progressive supranuclear palsy (PSP) pathology is unknown. We performed a genome‐wide association study (GWAS) to identify genetic determinants of PSP phenotype. Methods Two independent pathological and clinically diagnosed PSP cohorts were genotyped and phenotyped to create Richardson syndrome (RS) and non‐RS groups. We carried out separate logistic regression GWASs to compare RS and non‐RS groups and then combined datasets to carry out a whole cohort analysis (RS = 367, non‐RS = 130). We validated our findings in a third cohort by referring to data from 100 deeply phenotyped cases from a recent GWAS. We assessed the expression/coexpression patterns of our identified genes and used our data to carry out gene‐based association testing. Results Our lead single nucleotide polymorphism (SNP), rs564309, showed an association signal in both cohorts, reaching genome‐wide significance in our whole cohort analysis (odds ratio = 5.5, 95% confidence interval = 3.2–10.0, p = 1.7 × 10−9). rs564309 is an intronic variant of the tripartite motif‐containing protein 11 (TRIM11) gene, a component of the ubiquitin proteasome system (UPS). In our third cohort, minor allele frequencies of surrogate SNPs in high linkage disequilibrium with rs564309 replicated our findings. Gene‐based association testing confirmed an association signal at TRIM11. We found that TRIM11 is predominantly expressed neuronally, in the cerebellum and basal ganglia. Interpretation Our study suggests that the TRIM11 locus is a genetic modifier of PSP phenotype and potentially adds further evidence for the UPS having a key role in tau pathology, therefore representing a target for disease‐modifying therapies

Molecular Mechanism of Ochratoxin A Transport in the Kidney

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity

Delay Analysis in Marshalling Yards : The case study of Malmö

The management of terminal yards requires quite complex day-to-day operations when hundreds of trains could be entered and/or exited marshalling yards every day. More specifically, multi-dimensional decisions are necessary to be taken in daily basis for management of operations. Manual solutions might cause inefficiency in the yard’s operation and consequently to the network. Nowadays, many freight trains in Sweden fail to follow their scheduled plan. In particular, a small portion of trains are following the scheduled arrival and departure time while the majority of trains run ahead of schedule.This master thesis aims to conduct evaluation of internal procedures within a marshalling yard in terms of time, examine the magnitude of delays from the scheduled departure time, and to identify the key reasons causing the delay during departure and their main implications. Furthermore, the master this aims to investigate ways of optimizing operations to increase system punctuality. The Malmo’s marshalling yard was used as a case study.The methods are used for this master thesis are a qualitative as well as a quantitative assessment. A literature review has been conducted using journal papers, conference papers and technical reports from other relevant projects as well as on-site visit and interviews. In order to manipulate the data for the research, the software Planimate was used and a simulation model is built based on operations in Malmo’s marshalling yard. Three scenarios are performed. The first one is considered without any usage of automation. The second one, automation is applied in the case of the arrival yard and the third one, automation is applied in the arrival and the departure yard.The findings from the qualitative research show that there are several factors cause delays, either network failures such as the late arrival of trains in the yard or internal factors as the reduced railway capacity. Also, as the simulation model has been demonstrated, any application of automated processes within the marshalling yard’s operations will be beneficial because will speed up the internal processes. For instance, the third scenario turns out the best scenario among the others because time is reduced almost to half time compared to the first scenario

Techniques and methodologies for railway capacity analysis: comparative studies and integration perspectives

The article provides an analysis of methods and techniques for the qualification of the railway capacity, classified by reference to factors having a direct relationship with the obtained results. Synthetic, analytical and analogical methods are compared and developed

Presynaptic dysfunction in CDKL5 deficiency disorder

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a monogenic developmental and epileptic encephalopathy with onset in early infancy that is caused by mutations in the CDKL5 gene. CDD patients often exhibit profound neurodevelopmental delay, visual and motor deficits, and autistic-like manifestations, whereas epileptic seizures typically appear as early as the third week after birth. CDKL5 is a neuron-specific serine/threonine kinase that has been implicated in different cellular processes including neurite outgrowth, microtubule remodelling, and synaptogenesis. Animal models of CDKL5 deficiency have revealed phenotypes associated with defective neurotransmission. However, the potential role of CDKL5 in presynaptic processes and synaptic vesicle (SV) membrane trafficking remains unknown. In this project, we used a novel CDKL5 KO rat model to detect potential phenotypes that are linked to loss of CDKL5 function. Using a genetically encoded fluorescent reporter, we revealed that absence of CDKL5 results in defective SV recycling in an activity-dependent manner in primary hippocampal neurons. Using a molecular replacement strategy, we showed that the kinase domain of CDKL5 was able to restore the speed of SV endocytosis indicating that the catalytic activity of CDKL5 is essential for its role in SV recycling. In agreement, we revealed that CDKL5 mutants either lacking the kinase domain or containing kinase-inactive mutations reported in CDD patients were unable to rescue this impairment suggesting that defective presynaptic processes may contribute to the CDD onset. Since the kinase activity is critical for CDKL5-mediated SV recycling, we also explored whether the phosphorylation levels of its in vitro presynaptic substrate, amphiphysin 1 (Amph1), were altered in CDKL5 KO neurons. We revealed that CDKL5 does not exert its presynaptic role by phosphorylating Amph1 at S293. At the same time, this work showed that Amph1-mediated complexes are important for SV endocytosis in presynaptic terminals. Furthermore, we mapped the Amph1 motif that interacts with a different endocytosis protein, endophilin A1, and we demonstrated that the Amph1-endophilin A1 complex is essential for SV regeneration. Finally, the phosphorylation dynamics at Amph1-S293 dictates both Amph1-mediated interactions with endophilin A1 and SV endocytosis, indicating that phosphorylation-dependent Amph1-endophilin A1 interaction is essential for optimal SV endocytosis. Overall, this study offers the first evidence of a presynaptic role of CDKL5 that is mediated through its kinase activity and creates the basis for future research on presynaptic CDKL5 that could lead to potential treatments for CDD patients