8 research outputs found
Recommended from our members
Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics.
Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder
Translating international HIV treatment guidelines into local priorities in Indonesia
Objective: International guidelines recommend countries to expand antiretroviral therapy (ART) to all HIV-infected individuals and establish local-level priorities in relation to other treatment, prevention and mitigation interventions through fair processes. However, no practical guidance is provided for such priority-setting processes. Evidence-informed deliberative processes (EDPs) fill this gap and combine stakeholder deliberation to incorporate relevant social values with rational decision-making informed by evidence on these values. This study reports on the first-time implementation and evaluation of an EDP in HIV control, organised to support the AIDS Commission in West Java province, Indonesia, in the development of its strategic plan for 2014–2018. Methods: Under the responsibility of the provincial AIDS Commission, an EDP was implemented to select priority interventions using six steps: (i) situational analysis; (ii) formation of a multistakeholder Consultation Panel; (iii) selection of criteria; (iv) identification and assessment of interventions’ performance; (v) deliberation; and (vi) selection of funding and implementing institutions. An independent researcher conducted in-depth interviews (n = 21) with panel members to evaluate the process. Results: The Consultation Pa
Cyclin-dependent-like kinase 5 is required for pain signaling in human sensory neurons and mouse models
Cyclin-dependent-like kinase 5 (Cdkl5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognised anamnestic deficiency in pain perception. Consistent with a role in nociception, we discovered that Cdkl5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in iPS-derived human nociceptors. CDKL5 deficient mice display defective epidermal innervation and conditional deletion of Cdkl5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, Cdkl5 interacts with CaMKIIα to control outgrowth as well as TRPV1-dependent signaling, which are disrupted in both Cdkl5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for Cdkl5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder
Translating international HIV treatment guidelines into local priorities in Indonesia
Contains fulltext :
189873.pdf (publisher's version ) (Open Access
Novel CDKL5 substrates and functions in neurodevelopment
Cyclin-Dependent Kinase-Like 5 (CDKL5) is a serine/threonine protein kinase important for neuronal development. Mutations in the CDKL5 gene are responsible for CDKL5 deficiency disorder, a rare neurodevelopmental disorder displaying a heterogeneous range of clinical phenotypes. CDKL5 is enriched in the brain during early postnatal development, and is known to be involved in the development of dendritic spines and synapses. However, direct downstream effectors of CDKL5 and its molecular mechanisms of action remain unknown. We have generated analogue-specific CDKL5 by mutating the gatekeeper residue in the ATP-binding pocket, and introduced a second-site mutation to rescue kinase activity. Based on the utilization of bulky ATP analogues by analogue-specific CDKL5, we used an unbiased chemical genetic screen to identify CDKL5 substrates and phosphorylation sites in mouse brain. In vitro validation of ARHGEF2, EB2 and two sites in MAP1S revealed RPxS as a common CDKL5 phosphorylation motif. We show that EB2 and MAP1S phosphorylation is strongly reduced in brains of Cdkl5 KO mice. In neurons derived from CDKL5 patient iPSCs, we observe similar reductions indicating that regulation of these phosphorylation sites is conserved in humans. By using CDKL5 substrate phosphorylation as a read-out, we have been able to show that CDKL5 activity is regulated during brain development and affected by neuronal activity. These novel CDKL5 substrates share microtubule binding properties, but only phosphorylation of MAP1S by CDKL5 is directly regulating its microtubule binding affinity. Attempts to identify a molecular function of EB2 phosphorylation did not lead to satisfying results. Both MAP1S and EB2 are known to be involved in microtubule dynamic instability, the process of constant growth and collapse of microtubule plus-ends. We show that primary cortical cultures of Cdkl5 KO mice have altered microtubule plus-end dynamics visualised by sparser, but longer EB3 comets. Knockdown of MAP1S, but not EB2, partially rescued this phenotype in Cdkl5 KO neurons, indicating a downstream function of MAP1S in the regulation of microtubule dynamics