LIS1 and NDEL1 Regulate Axonal Trafficking of Mitochondria in Mature Neurons.

Defective mitochondrial dynamics in axons have been linked to both developmental and late-onset neurological disorders. Axonal trafficking is in large part governed by the microtubule motors kinesin-1 and cytoplasmic dynein 1 (dynein). Dynein is the primary retrograde transport motor in axons, and mutations in dynein and many of its regulators also cause neurological diseases. Depletion of LIS1, famous for linking dynein deregulation to lissencephaly (smooth brain), in adult mice leads to severe neurological phenotypes, demonstrating post-developmental roles. LIS1 stimulates retrograde transport of acidic organelles in cultured adult rat dorsal root ganglion (DRG) axons but findings on its role in mitochondrial trafficking have been inconsistent and have not been reported for adult axons. Here we report that there is an increased number of mitochondria in cross-sections of sciatic nerve axons from adult LIS1+/- mice. This is probably related to reduced dynein activity as axons from adult rat nerves exposed to the dynein inhibitor, ciliobrevin D also had increased numbers of mitochondria. Moreover, LIS1 overexpression (OE) in cultured adult rat DRG axons stimulated retrograde mitochondrial transport while LIS1 knockdown (KD) or expression of a LIS1 dynein-binding mutant (LIS1-K147A) inhibited retrograde transport, as did KD of dynein heavy chain (DHC). These findings are consistent with our report on acidic organelles. However, KD of NDEL1, a LIS1 and dynein binding protein, or expression of a LIS1 NDEL1-binding mutant (LIS1-R212A) also dramatically impacted retrograde mitochondrial transport, which was not the case for acidic organelles. Manipulations that disrupted retrograde mitochondrial transport also increased the average length of axonal mitochondria, suggesting a role for dynein in fusion or fission events. Our data point to cargo specificity in NDEL1 function and raise the possibility that defects in the LIS1/NDEL1 dynein regulatory pathway could contribute to mitochondrial diseases with axonal pathologies.

Quantitative Neurotoxicology: An Assessment of the Neurotoxic Profile of Kainic Acid in Sprague Dawley Rats.

This study consisted of a qualitative and quantitative assessment of neuropathological changes in kainic acid (KA)-treated adult male rats. Rats were administered a single 10 mg/kg intraperitoneal injection of KA or the same volume of saline and sacrificed 24 or 48 hours posttreatment. Brains were collected, sectioned coronally (∼ 81 slices), and stained with amino cupric silver to reveal degenerative changes. For qualitative assessment of neural degeneration, sectioned material was evaluated by a board-certified pathologist, and the level of degeneration was graded based upon a 4-point scale. For measurement of quantitative neural degeneration in response to KA treatment, the HALO digital image analysis software tool was used. Quantitative measurements of specific regions within the brain were obtained from silver-stained tissue sections with quantitation based on stain color and optical density. This quantitative evaluation method identified degeneration primarily in the cerebral cortex, septal nuclei, amygdala, olfactory bulb, hippocampus, thalamus, and hypothalamus. The KA-produced neuronal degeneration in the cortex was primarily in the piriform, insular, rhinal, and cingulate areas. In the hippocampus, the dentate gyrus was found to be the most affected area. Our findings indicate global neurotoxicity due to KA treatment. Certain brain structures exhibited more degeneration than others, reflecting differential sensitivity or vulnerability of neurons to KA.

Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology, US Food and Drug Administration Public Workshop-Workshop Proceedings.

PURPOSE: Next-generation sequencing (NGS) oncology panels are becoming integral in hospital and academic settings to guide patient treatment and enrollment in clinical trials. Although NGS technologies have revolutionized decision-making for cancer therapeutics, physicians may face many challenges in parsing and prioritizing NGS-based test results to determine the best course of treatment for individual patients. On January 29, 2018, the US Food and Drug Administration held a public workshop entitled, "Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology." Here, we discuss the presentations and discussion highlights across the four sessions of the workshop. METHODS: The goal of the public workshop was to engage stakeholders and solicit input from experts in precision oncology to discuss the integration of complex NGS data into patient management and regulatory innovation within the precision oncology community. The US Food and Drug Administration gathered representatives from academia, industry, patient advocacy, government, and professional organizations for a series of presentations followed by panel discussions. After the workshop, the transcript and speaker presentation slides were reviewed and summarized for manuscript preparation. RESULTS: Speakers and panelists provided diverse perspectives on the integration of NGS technology into patient care for oncology and on the complexities that surround data interpretation and sharing. Discussions highlighted the challenges with standardization for variant classification while expressing the utility of consensus recommendations among stakeholders in oncology for driving innovation in the era of precision medicine. CONCLUSION: As precision medicine advances, clear communication within the field of precision oncology will be key to creating an environment that facilitates the generation and sharing of data that have value to patients.

Engineering allosteric regulation in protein kinases.

Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed "hotspots" that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.

Hierarchical Organization Endows the Kinase Domain with Regulatory Plasticity.

The functional diversity of kinases enables specificity in cellular signal transduction. Yet how more than 500 members of the human kinome specifically receive regulatory inputs and convey information to appropriate substrates-all while using the common signaling output of phosphorylation-remains enigmatic. Here, we perform statistical co-evolution analysis, mutational scanning, and quantitative live-cell assays to reveal a hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. We find that three quasi-independent "sectors"-groups of evolutionarily coupled residues-represent functional units in the kinase domain that encode for catalytic activity, substrate specificity, and regulation. Sector positions impact both disease and pharmacology: the catalytic sector is significantly enriched for somatic cancer mutations, and residues in the regulatory sector interact with allosteric kinase inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity.

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis.

Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

A Cdk5-dependent switch regulates Lis1/Ndel1/dynein-driven organelle transport in adult axons.

Lissencephaly is a human developmental brain abnormality caused by LIS1 haploinsufficiency. This disorder is in large part attributed to altered mitosis and migration in the developing brain. LIS1 and an interacting protein, NDEL1, bind to cytoplasmic dynein, a microtubule motor protein. While the tripartite complex is clearly important for developmental events, we are intrigued by the fact that Lis1 and Ndel1 expression remain high in the adult mouse nervous system. Dynein plays a crucial role in retrograde axonal transport, a process that is used by mature neurons. Here, we monitored acidic organelles moving in axons of adult rat sensory neurons to determine whether Lis1 and Ndel1 contribute to axonal transport. Lis1 RNAi significantly reduced axon transport of these organelles. Ndel1 RNAi had little impact, but combined Lis1 and Ndel1 RNAi caused a more severe phenotype than Lis1 RNAi alone, essentially shutting down transport. Lis1 overexpression stimulated retrograde transport, while a Lis1 dynein-binding mutant severely disrupted transport. Overexpression of Ndel1 or a Lis1 Ndel1-binding mutant only mildly perturbed transport. However, expressing a mutant Ndel1 lacking key phosphorylation sites shut down transport completely, as did a dominant-negative Cdk5 construct. We propose that, in axons, unphosphorylated Ndel1 inhibits the capacity of dynein to transport acidic organelles. Phosphorylation of Ndel1 by Cdk5 not only reduces this inhibition but also allows Lis1 to further stimulate the cargo transport capacity of dynein. Our data raise the possibility that defects in a Lis1/Ndel1 regulatory switch could contribute to neurodegenerative diseases linked to axonal pathology in adults.