Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity.

Introduction: Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. Methods: To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). Results: We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. Discussion: This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

Impaired lipid metabolism in astrocytes underlies degeneration of cortical projection neurons in hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) are caused by a length-dependent axonopathy of long corticospinal neurons, but how axons of these cortical projection neurons (PNs) degenerate remains elusive. We generated isogenic human pluripotent stem cell (hPSC) lines for two ATL1 missense mutations associated with SPG3A, the most common early-onset autosomal dominant HSP. In hPSC-derived cortical PNs, ATL1 mutations resulted in reduced axonal outgrowth, impaired axonal transport, and accumulated axonal swellings, recapitulating disease-specific phenotypes. Importantly, ATL1 mutations dysregulated proteolipid gene expression, reduced lipid droplet size in astrocytes, and unexpectedly disrupted cholesterol transfer from glia to neurons, leading to cholesterol deficiency in SPG3A cortical PNs. Applying cholesterol or conditioned medium from control astrocytes, a major source of cholesterol in the brain, rescued aberrant axonal transport and swellings in SPG3A cortical PNs. Furthermore, treatment with the NR1H2 agonist GW3965 corrected lipid droplet defects in SPG3A astrocytes and promoted cholesterol efflux from astrocytes, leading to restoration of cholesterol levels and rescue of axonal degeneration in SPG3A cortical PNs. These results reveal a non-cell autonomous mechanism underlying axonal degeneration of cortical PNs mediated by impaired cholesterol homeostasis in glia.

Kinetic Heterogeneity of Cancer Cell Fractional Killing.

Lethal drugs can induce incomplete cell death in a population of cancer cells, a phenomenon referred to as fractional killing. Here, we show that high-throughput population-level time-lapse imaging can be used to quantify fractional killing in response to hundreds of different drug treatments in parallel. We find that stable intermediate levels of fractional killing are uncommon, with many drug treatments resulting in complete or near-complete eradication of all cells, if given enough time. The kinetics of fractional killing over time vary substantially as a function of drug, drug dose, and genetic background. At the molecular level, the antiapoptotic protein MCL1 is an important determinant of the kinetics of fractional killing in response to MAPK pathway inhibitors but not other lethal stimuli. These studies suggest that fractional killing is governed by diverse lethal stimulus-specific mechanisms.

Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

Application of a Substrate-Mediated Selection with c-Src Tyrosine Kinase to a DNA-Encoded Chemical Library.

As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound (SrcDEL10) showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed IC50's ranging from of 8-100 µM. NMR analysis of SrcDEL10 bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.

Impaired mitochondrial dynamics underlie axonal defects in hereditary spastic paraplegias.

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

Robustness of In Vitro Selection Assays of DNA-Encoded Peptidomimetic Ligands to CBX7 and CBX8.

The identification of protein ligands from a DNA-encoded library is commonly conducted by an affinity selection assay. These assays are often not validated for robustness, raising questions about selections that fail to identify ligands and the utility of enrichment values for ranking ligand potencies. Here, we report a method for optimizing and utilizing affinity selection assays to identify potent and selective peptidic ligands to the highly related chromodomains of CBX proteins. To optimize affinity selection parameters, statistical analyses (Z' factors) were used to define the ability of selection assay conditions to identify and differentiate ligands of varying affinity. A DNA-encoded positional scanning library of peptidomimetics was constructed around a trimethyllysine-containing parent peptide, and parallel selections against the chromodomains from CBX8 and CBX7 were conducted over three protein concentrations. Relative potencies of off-DNA hit molecules were determined through a fluorescence polarization assay and were consistent with enrichments observed by DNA sequencing of the affinity selection assays. In addition, novel peptide-based ligands were discovered with increased potency and selectivity to the chromodomain of CBX8. The results indicate low DNA tag bias and show that affinity-based in vitro selection assays are sufficiently robust for both ligand discovery and determination of quantitative structure-activity relationships.

Recapitulating and Correcting Marfan Syndrome in a Cellular Model.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in FBN1 gene, which encodes a key extracellular matrix protein FIBRILLIN-1. The haplosufficiency of FBN1 has been implicated in pathogenesis of MFS with manifestations primarily in cardiovascular, muscular, and ocular tissues. Due to limitations in animal models to study the late-onset diseases, human pluripotent stem cells (PSCs) offer a homogeneic tool for dissection of cellular and molecular pathogenic mechanism for MFS in vitro. Here, we first derived induced PSCs (iPSCs) from a MFS patient with a FBN1 mutation and corrected the mutation, thereby generating an isogenic "gain-of-function" control cells for the parental MFS iPSCs. Reversely, we knocked out FBN1 in both alleles in a wild-type (WT) human embryonic stem cell (ESC) line, which served as a loss-of-function model for MFS with the WT cells as an isogenic control. Mesenchymal stem cells derived from both FBN1-mutant iPSCs and -ESCs demonstrated reduced osteogenic differentiation and microfibril formation. We further demonstrated that vascular smooth muscle cells derived from FBN1-mutant iPSCs showed less sensitivity to carbachol as demonstrated by contractility and Ca2+ influx assay, compared to the isogenic controls cells. These findings were further supported by transcriptomic anaylsis of the cells. Therefore, this study based on both gain- and loss-of-function approaches confirmed the pathogenetic role of FBN1 mutations in these MFS-related phenotypic changes.

Modeling Axonal Defects in Hereditary Spastic Paraplegia with Human Pluripotent Stem Cells.

BACKGROUND: Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis (ALS). Since the discovery of the first HSP gene, SPAST that encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remains largely unknown. The development of induced pluripotent stem cell (iPSC) technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathologic processes including axonal defects. METHODS: In this article, we will frst review the pathology and pathways affected in the common forms of HSP subtypes by searching the PubMed database. We will then summurize the findings and insights gained from studies using iPSC-based models, and discuss the challenges and future directions. RESULTS: HSPs, a heterogeneous group of genetic neurodegenerative disorders, are characterized by lower extremity weakness and spasticity that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit disease-relevant axonal defects, such as impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport. CONCLUSION: These patient-derived neurons offer unique tools to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy.

Calcium/calmodulin-dependent protein kinase kinase ß is neuroprotective in stroke in aged mice.

Stroke is a devastating neurological disease and the leading cause of long-term disability, particularly in the elderly. Calcium/calmodulin-dependent protein kinase kinase ß (CaMKK ß) is a major kinase activated by elevated levels of intracellular calcium. Our previous findings in young mice have suggested that CaMKK ß is neuroprotective as KO mice had worse stroke outcomes. Because age is an important determinant of stroke outcome, we evaluated the functional role of CaMKK ß in stroke in aged mice. We used middle cerebral artery occlusion to induce stroke in aged wild-type (WT) and CaMKK ß KO male mice. Lentiviral vectors carrying CaMKK ß (LV-CaMKK ß) were used to overexpress CaMKK ß in the mouse brain. Baseline levels of CaMKK ß in the aged brain were significantly lower than those in young mice. LV-CaMKK ß treatment reduced infarcts and neurological deficits assessed 3 days after stroke. In chronic survival experiments, CaMKK ß KO mice showed increased tissue loss in the ipsilateral hemisphere 3 weeks after stroke. In addition, KO mice showed poorer functional recovery during the 3-week survival period, as measured by the rotarod test, corner test, locomotor activity assay, and novel object recognition test, compared with WT controls. The loss of blood-brain barrier proteins, inactivation of survival gene expression such as B-cell lymphoma 2 (Bcl-2) and an increase in inflammatory cytokines in the serum were observed after stroke with CaMKK ß inhibition. We demonstrate that CaMKK ß is neuroprotective in stroke in aged mice. Therefore, our data suggest that CaMKK ß may be a potential target for reducing long-term disability after stroke.

Nuclear translocation of histone deacetylase 4 induces neuronal death in stroke.

Mounting evidence suggests that epigenetic modifications play critical roles in the survival/death of stressed neurons. Chief among these modifications is the deacetylation of histones within the chromatin by histone deacetylases (HDACs). HDAC4 is highly expressed in neurons and is usually trapped in cytosol. However, tightly regulated signal-dependent shuttling of this molecule between cytosol and nucleus occurs. Here, we studied the intracellular trafficking of HDAC4 and regulatory mechanisms during stroke. HDAC4 translocated from the cytosol into the nucleus of neurons in response to stroke induced by middle cerebral artery occlusion (MCAO) in mice. Similar translocation was seen after oxygen-glucose deprivation (OGD) in cultured mouse neurons. Expression of nuclear-restricted HDAC4 increased neuronal death after OGD and worsened infarcts and functional deficits in mice following MCAO; however, expression of cytosolic-restricted HDAC4 did not affect outcome after ischemia. In contrast, HDAC4 knockdown with siRNA improved neuronal survival after OGD. Furthermore, expression of nuclear-restricted HDAC4 reduced the acetylation of histones 3 and 4 as well as the levels of pro-survival downstream molecules after OGD. Finally, genetic deletion of calcium/calmodulin-dependent protein kinase IV (CaMKIV) increased the nuclear accumulation of HDAC4 in MCAO model, while overexpression of CaMKIV reduced the levels of nuclear HDAC4 following OGD. When HDAC4 was inhibited, the neuroprotection provided by CaMKIV overexpression was absent during OGD. Our data demonstrate a detrimental role of the nuclear accumulation of HDAC4 following stroke and identify CaMKIV as a key regulator of neuronal intracellular HDAC4 trafficking during stroke.

Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy.

Spinal muscular atrophy (SMA), characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1) gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs) and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC) mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.

Crosslinking of DNA-linked ligands to target proteins for enrichment from DNA-encoded libraries.

Achieving sufficient enrichment of ligands from DNA-encoded libraries for detection can be difficult, particularly for low affinity ligands within highly complex libraries. To address this challenge, we present an approach for crosslinking DNA-linked ligands to target proteins using electrophilic or photoreactive groups. The approach involves the teathering of a ssDNA oligonucleotide to a DNA-encoded molecule to enable attachment of a reactive group post-synthetically via DNA hybridization. Crosslinking traps ligand-protein complexes while in solution and allows for stringent washing conditions to be applied in the subsequent purification. Five reactive groups (tosyl, NHS ester, sulfonyl fluoride, phenyl azide, and diazirine) were tested for crosslinking efficiency and specificity with three DNA-linked ligands to their target proteins. In a model selection, crosslinking resulted in improved enrichment of both high and a low affinity ligands in comparison to a selection with a solid-phase immobilized protein.

Modeling Axonal Phenotypes with Human Pluripotent Stem Cells.

Impaired axonal development and degeneration are implicated in many debilitating disorders, such as hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS), and periphery neuropathy. Human pluripotent stem cells (hPSCs) have provided researchers with an excellent resource for modeling human neuropathologic processes including axonal defects in vitro. There are a number of steps that are crucial when developing an hPSC-based model of a human disease, including generating induced pluripotent stem cells (iPSCs), differentiating those cells to affected cell types, and identifying disease-relevant phenotypes. Here, we describe these steps in detail, focusing on the neurodegenerative disorder HSP.

Pharmacologic rescue of axon growth defects in a human iPSC model of hereditary spastic paraplegia SPG3A.

Hereditary spastic paraplegias are a large, diverse group of neurological disorders (SPG1-71) with the unifying feature of prominent lower extremity spasticity, owing to a length-dependent axonopathy of corticospinal motor neurons. The most common early-onset form of pure, autosomal dominant hereditary spastic paraplegia is caused by mutation in the ATL1 gene encoding the atlastin-1 GTPase, which mediates homotypic fusion of ER tubules to form the polygonal ER network. We have identified a p.Pro342Ser mutation in a young girl with pure SPG3A. This residue is in a critical hinge region of atlastin-1 between its GTPase and assembly domains, and it is conserved in all known eukaryotic atlastin orthologs. We produced induced pluripotent stem cells from skin fibroblasts and differentiated these into forebrain neurons to generate a human neuronal model for SPG3A. Axons of these SPG3A neurons showed impaired growth, recapitulating axonal defects in atlastin-1-depleted rat cortical neurons and impaired root hair growth in loss-of-function mutants of the ATL1 ortholog rhd3 in the plant Arabidopsis. Both the microtubule cytoskeleton and tubular ER are important for mitochondrial distribution and function within cells, and SPG3A neurons showed alterations in mitochondrial motility. Even so, it is not clear whether this change is involved in disease pathogenesis. The SPG3A axon growth defects could be rescued with microtubule-binding agents, emphasizing the importance of tubular ER interactions with the microtubule cytoskeleton in hereditary spastic paraplegia pathogenesis. The prominent alterations in axon growth in SPG3A neurons may represent a particularly attractive target for suppression in screens for novel pharmacologic agents.

Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia.

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP.

Identification of a possible respiratory arsenate reductase in Denitrovibrio acetiphilus, a member of the phylum Deferribacteres.

Denitrovibrio acetiphilus N2460(T) is one of the few members of the phylum Deferribacteres with a sequenced genome. N2460(T) was capable of growing with dimethyl sulfoxide, selenate, or arsenate provided as a terminal electron acceptor, and we identified 15 genes that could possibly encode respiratory reductases for these compounds. The protein encoded by one of these genes, YP_003504839, clustered with respiratory arsenate reductases on a phylogenetic tree. Transcription of the gene for YP_003504839, Dacet_2121, was highly induced when arsenate was provided as a terminal electron acceptor. Dacet_2121 exists in a possible operon that is distinct from the previously characterized respiratory arsenate reductase operon in Shewanella sp. ANA-3.

The specification of telencephalic glutamatergic neurons from human pluripotent stem cells.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.