Abnormalities of mitochondrial dynamics and bioenergetics in neuronal cells from CDKL5 deficiency disorder.

CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by pathogenic variants in the Cyclin-dependent kinase-like 5 (CDKL5) gene, resulting in dysfunctional CDKL5 protein. It predominantly affects females and causes seizures in the first few months of life, ultimately resulting in severe intellectual disability. In the absence of targeted therapies, treatment is currently only symptomatic. CDKL5 is a serine/threonine kinase that is highly expressed in the brain, with a critical role in neuronal development. Evidence of mitochondrial dysfunction in CDD is gathering, but has not been studied extensively. We used human patient-derived induced pluripotent stem cells with a pathogenic truncating mutation (p.Arg59*) and CRISPR/Cas9 gene-corrected isogenic controls, differentiated into neurons, to investigate the impact of CDKL5 mutation on cellular function. Quantitative proteomics indicated mitochondrial defects in CDKL5 p.Arg59* neurons, and mitochondrial bioenergetics analysis confirmed decreased activity of mitochondrial respiratory chain complexes. Additionally, mitochondrial trafficking velocity was significantly impaired, and there was a higher percentage of stationary mitochondria. We propose mitochondrial dysfunction is contributing to CDD pathology, and should be a focus for development of targeted treatments for CDD.

NXY-059, a Failed Stroke Neuroprotectant, Offers No Protection to Stem Cell-Derived Human Neurons.

BACKGROUND: Developing new medicines is a complex process where understanding the reasons for both failure and success takes us forward. One gap in our understanding of most candidate stroke drugs before clinical trial is whether they have a protective effect on human tissues. NXY-059 is a spin-trap reagent hypothesized to have activity against the damaging oxidative biology which accompanies ischemic stroke. Re-examination of the preclinical in vivo dataset for this agent in the wake of the failed SAINT-II RCT highlighted the presence of a range of biases leading to overestimation of the magnitude of NXY-059's effects in laboratory animals. Therefore, NXY-059 seemed an ideal candidate to evaluate in human neural tissues to determine whether human tissue testing might improve screening efficiency. MATERIALS AND METHODS: The aim of this randomized and blinded study was to assess the effects of NXY-059 on human stem cell-derived neurons in the presence of ischemia-like injury induced by oxygen glucose deprivation or oxidative stress induced by hydrogen peroxide or sodium nitroprusside. RESULTS: In MTT assays of cell survival, lactate dehydrogenase assays of total cell death and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic-like cell death, NXY-059 at concentrations ranging from 1 µm to 1 mm was completely without activity. Conversely an antioxidant cocktail comprising 100 µm each of ascorbate, reduced glutathione, and dithiothreitol used as a positive control provided marked neuronal protection in these assays. CONCLUSION: These findings support our hypothesis that stroke drug screening in human neural tissues will be of value and provides an explanation for the failure of NXY-059 as a human stroke drug.

Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells.

In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.