Examining the effects of cigarette smoke on mouse lens through a multi OMIC approach.

Here, we report a multi OMIC (transcriptome, proteome, and metabolome) approach to investigate molecular changes in lens fiber cells (FC) of mice exposed to cigarette smoke (CS). Pregnant mice were placed in a whole-body smoke chamber and a few days later pups were born, which were exposed to CS for 5 hours/day, 5 days/week for a total of 3½ months. We examined the mice exposed to CS for CS-related cataractogenesis after completion of the CS exposure but no cataracts were observed. Lenses of CS-exposed and age-matched, untreated control mice were extracted and lens FC were subjected to multi OMIC profiling. We identified 348 genes, 130 proteins, and 14 metabolites exhibiting significant (p < 0.05) differential levels in lens FC of mice exposed to CS, corresponding to 3.6%, 4.3%, and 5.0% of the total genes, protein, and metabolites, respectively identified in this study. Our multi OMIC approach confirmed that only a small fraction of the transcriptome, the proteome, and the metabolome was perturbed in the lens FC of mice exposed to CS, which suggests that exposure of CS had a minimal effect on the mouse lens. It is worth noting that while our results confirm that CS exposure does not have a substantial impact on the molecular landscape of the mouse lens FC, we cannot rule out that CS exposure for longer durations and/or in combination with other morbidities or environmental factors would have a more robust effect and/or result in cataractogenesis.

Blood-brain barrier development: Systems modeling and predictive toxicology.

The blood-brain barrier (BBB) serves as a gateway for passage of drugs, chemicals, nutrients, metabolites, and hormones between vascular and neural compartments in the brain. Here, we review BBB development with regard to the microphysiology of the neurovascular unit (NVU) and the impact of BBB disruption on brain development. Our focus is on modeling these complex systems. Extant in silico models are available as tools to predict the probability of drug/chemical passage across the BBB; in vitro platforms for high-throughput screening and high-content imaging provide novel data streams for profiling chemical-biological interactions; and engineered human cell-based microphysiological systems provide empirical models with which to investigate the dynamics of NVU function. Computational models are needed that bring together kinetic and dynamic aspects of NVU function across gestation and under various physiological and toxicological scenarios. This integration will inform adverse outcome pathways to reduce uncertainty in translating in vitro data and in silico models for use in risk assessments that aim to protect neurodevelopmental health.

Decreased Sirtuin Deacetylase Activity in LRRK2 G2019S iPSC-Derived Dopaminergic Neurons.

Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD.

Neurite Aggregation and Calcium Dysfunction in iPSC-Derived Sensory Neurons with Parkinson's Disease-Related LRRK2 G2019S Mutation.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most-common genetic determinants of Parkinson's disease (PD). The G2019S mutation is detected most frequently and is associated with increased kinase activity. Whereas G2019S mutant dopamine neurons exhibit neurite elongation deficits, the effect of G2019S on other neuronal subtypes is unknown. As PD patients also suffer from non-motor symptoms that may be unrelated to dopamine neuron loss, we used induced pluripotent stem cells (iPSCs) to assess morphological and functional properties of peripheral sensory neurons. LRRK2 G2019S iPSC-derived sensory neurons exhibited normal neurite length but had large microtubule-containing neurite aggregations. Additionally, LRRK2 G2019S iPSC-derived sensory neurons displayed altered calcium dynamics. Treatment with LRRK2 kinase inhibitors resulted in significant, but not complete, morphological and functional rescue. These data indicate a role for LRRK2 kinase activity in sensory neuron structure and function, which when disrupted, may lead to sensory neuron deficits in PD.

Alterations in late endocytic trafficking related to the pathobiology of LRRK2-linked Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene comprise the most common cause of familial Parkinson's disease (PD), and variants increase the risk for sporadic PD. LRRK2 displays kinase and GTPase activity, and altered catalytic activity correlates with neurotoxicity, making LRRK2 a promising therapeutic target. Despite the importance of LRRK2 for disease pathogenesis, its normal cellular function, and the mechanism(s) by which pathogenic mutations cause neurodegeneration remain unclear. LRRK2 seems to regulate a variety of intracellular vesicular trafficking events to and from the late endosome in a manner dependent on various Rab proteins. At least some of those events are further regulated by LRRK2 in a manner dependent on two-pore channels (TPCs). TPCs are ionic channels localized to distinct endosomal structures and can cause localized calcium release from those acidic stores, with downstream effects on vesicular trafficking. Here, we review current knowledge about the link between LRRK2, TPC- and Rab-mediated vesicular trafficking to and from the late endosome, highlighting a possible cross-talk between endolysosomal calcium stores and Rab proteins underlying pathomechanism(s) in LRRK2-related PD.

Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.