AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/-SMA model mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/- SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

Human Cytomegalovirus Disruption of Calcium Signaling in Neural Progenitor Cells and Organoids.

The herpesvirus human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Infection can result in infants born with a variety of symptoms, including hepatosplenomegaly, microcephaly, and developmental disabilities. Microcephaly is associated with disruptions in the neural progenitor cell (NPC) population. Here, we defined the impact of HCMV infection on neural tissue development and calcium regulation, a critical activity in neural development. Regulation of intracellular calcium involves purinergic receptors and voltage-gated calcium channels (VGCC). HCMV infection compromised the ability of both pathways in NPCs as well as fibroblasts to respond to stimulation. We observed significant drops in basal calcium levels in infected NPCs which were accompanied by loss in VGCC activity and purinergic receptor responses. However, uninfected cells in the population retained responsiveness. Addition of the HCMV inhibitor maribavir reduced viral spread but failed to restore activity in infected cells. To study neural development, we infected three-dimensional cortical organoids with HCMV. Infection spread to a subset of cells over time and disrupted organoid structure, with alterations in developmental and neural layering markers. Organoid-derived infected neurons and astrocytes were unable to respond to stimulation whereas uninfected cells retained nearly normal responses. Maribavir partially restored structural features, including neural rosette formation, and dampened the impact of infection on neural cellular function. Using a tissue model system, we have demonstrated that HCMV alters cortical neural layering and disrupts calcium regulation in infected cells.IMPORTANCE Human cytomegalovirus (HCMV) replicates in several cell types throughout the body, causing disease in the absence of an effective immune response. Studies on HCMV require cultured human cells and tissues due to species specificity. In these studies, we investigated the impact of infection on developing three-dimensional cortical organoid tissues, with specific emphasis on cell-type-dependent calcium signaling. Calcium signaling is an essential function during neural differentiation and cortical development. We observed that HCMV infects and spreads within these tissues, ultimately disrupting cortical structure. Infected cells exhibited depleted calcium stores and loss of ATP- and KCl-stimulated calcium signaling while uninfected cells in the population maintained nearly normal responses. Some protection was provided by the viral inhibitor maribavir. Overall, our studies provide new insights into the impact of HCMV on cortical tissue development and function.

Studying Human Neurological Disorders Using Induced Pluripotent Stem Cells: From 2D Monolayer to 3D Organoid and Blood Brain Barrier Models.

Neurological disorders have emerged as a predominant healthcare concern in recent years due to their severe consequences on quality of life and prevalence throughout the world. Understanding the underlying mechanisms of these diseases and the interactions between different brain cell types is essential for the development of new therapeutics. Induced pluripotent stem cells (iPSCs) are invaluable tools for neurological disease modeling, as they have unlimited self-renewal and differentiation capacity. Mounting evidence shows: (i) various brain cells can be generated from iPSCs in two-dimensional (2D) monolayer cultures; and (ii) further advances in 3D culture systems have led to the differentiation of iPSCs into organoids with multiple brain cell types and specific brain regions. These 3D organoids have gained widespread attention as in vitro tools to recapitulate complex features of the brain, and (iii) complex interactions between iPSC-derived brain cell types can recapitulate physiological and pathological conditions of blood-brain barrier (BBB). As iPSCs can be generated from diverse patient populations, researchers have effectively applied 2D, 3D, and BBB models to recapitulate genetically complex neurological disorders and reveal novel insights into molecular and genetic mechanisms of neurological disorders. In this review, we describe recent progress in the generation of 2D, 3D, and BBB models from iPSCs and further discuss their limitations, advantages, and future ventures. This review also covers the current status of applications of 2D, 3D, and BBB models in drug screening, precision medicine, and modeling a wide range of neurological diseases (e.g., neurodegenerative diseases, neurodevelopmental disorders, brain injury, and neuropsychiatric disorders). © 2019 American Physiological Society. Compr Physiol 9:565-611, 2019.

SRCP1 Conveys Resistance to Polyglutamine Aggregation.

The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.

Modeling Protein Aggregation and the Heat Shock Response in ALS iPSC-Derived Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by the selective loss of the upper and lower motor neurons. Only 10% of all cases are caused by a mutation in one of the two dozen different identified genes, while the remaining 90% are likely caused by a combination of as yet unidentified genetic and environmental factors. Mutations in C9orf72, SOD1, or TDP-43 are the most common causes of familial ALS, together responsible for at least 60% of these cases. Remarkably, despite the large degree of heterogeneity, all cases of ALS have protein aggregates in the brain and spinal cord that are immunopositive for SOD1, TDP-43, OPTN, and/or p62. These inclusions are normally prevented and cleared by heat shock proteins (Hsps), suggesting that ALS motor neurons have an impaired ability to induce the heat shock response (HSR). Accordingly, there is evidence of decreased induction of Hsps in ALS mouse models and in human post-mortem samples compared to unaffected controls. However, the role of Hsps in protein accumulation in human motor neurons has not been fully elucidated. Here, we generated motor neuron cultures from human induced pluripotent stem cell (iPSC) lines carrying mutations in SOD1, TDP-43, or C9orf72. In this study, we provide evidence that despite a lack of overt motor neuron loss, there is an accumulation of insoluble, aggregation-prone proteins in iPSC-derived motor neuron cultures but that content and levels vary with genetic background. Additionally, although iPSC-derived motor neurons are generally capable of inducing the HSR when exposed to a heat stress, protein aggregation itself is not sufficient to induce the HSR or stress granule formation. We therefore conclude that ALS iPSC-derived motor neurons recapitulate key early pathological features of the disease and fail to endogenously upregulate the HSR in response to increased protein burden.

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.

Astrocyte-produced miR-146a as a mediator of motor neuron loss in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by the loss of the survival motor neuron-1 (SMN1) gene, which leads to motor neuron loss, muscle atrophy, respiratory distress, and death. Motor neurons exhibit the most profound loss, but the mechanisms underlying disease pathogenesis are not fully understood. Recent evidence suggests that motor neuron extrinsic influences, such as those arising from astrocytes, contribute to motor neuron malfunction and loss. Here we investigated both loss-of-function and toxic gain-of-function astrocyte mechanisms that could play a role in SMA pathology. We had previously found that glial derived neurotrophic factor (GDNF) is reduced in SMA astrocytes. However, reduced GDNF expression does not play a major role in SMA pathology as viral-mediated GDNF re-expression did not improve astrocyte function or motor neuron loss. In contrast, we found that SMA astrocytes increased microRNA (miR) production and secretion compared to control astrocytes, suggesting potential toxic gain-of-function properties. Specifically, we found that miR-146a was significantly upregulated in SMA induced pluripotent stem cell (iPSC)-derived astrocytes and SMNΔ7 mouse spinal cord. Moreover, increased miR-146a was sufficient to induce motor neuron loss in vitro, whereas miR-146a inhibition prevented SMA astrocyte-induced motor neuron loss. Together, these data indicate that altered astrocyte production of miR-146a may be a contributing factor in astrocyte-mediated SMA pathology.

BAR-SH3 sorting nexins are conserved interacting proteins of Nervous wreck that organize synapses and promote neurotransmission.

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.