Publications by authors named "Holly J Carlisle"

13 Publications

  • Page 1 of 1

Selective Inhibitors of G2019S-LRRK2 Kinase Activity.

J Med Chem 2020 12 16;63(23):14821-14839. Epub 2020 Nov 16.

ESCAPE Bio, South San Francisco, California 94080, United States.

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01243DOI Listing
December 2020

Identification of modulators of autophagic flux in an image-based high content siRNA screen.

Autophagy 2016 ;12(4):713-26

b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.

Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.
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http://dx.doi.org/10.1080/15548627.2016.1147669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836002PMC
December 2016

Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?

F1000Res 2016 4;5:137. Epub 2016 Feb 4.

Department of Neuroscience, Amgen Inc., Thousand Oaks, CA, USA; Department of Neuroscience, Amgen Inc., Cambridge, MA, USA.

A common pathological hallmark of age-related neurodegenerative diseases is the intracellular accumulation of protein aggregates such as α-synuclein in Parkinson's disease, TDP-43 in ALS, and tau in Alzheimer's disease. Enhancing intracellular clearance of aggregation-prone proteins is a plausible strategy for slowing progression of neurodegenerative diseases and there is great interest in identifying molecular targets that control protein turnover. One of the main routes for protein degradation is through the proteasome, a multisubunit protease that degrades proteins that have been tagged with a polyubiquitin chain by ubiquitin activating and conjugating enzymes. Published data from cellular models indicate that Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), slows the degradation of tau and TDP-43 by the proteasome and that an inhibitor of USP14 increases the degradation of these substrates. We conducted similar experiments designed to evaluate tau, TDP-43, or α-synuclein levels in cells after overexpressing USP14 or knocking down endogenous expression by siRNA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792207PMC
http://dx.doi.org/10.12688/f1000research.7800.2DOI Listing
May 2016

Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.

J Biol Chem 2015 Feb 22;290(8):4908-27. Epub 2014 Dec 22.

From the Division of Biology and Biological Engineering, and

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.
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http://dx.doi.org/10.1074/jbc.M114.614420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335230PMC
February 2015

Deletion of densin-180 results in abnormal behaviors associated with mental illness and reduces mGluR5 and DISC1 in the postsynaptic density fraction.

J Neurosci 2011 Nov;31(45):16194-207

Division of Biology, California Institute of Technology, Pasadena, California 91105, USA.

Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with αCaMKII and α-actinin. To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (LRRC7). Homozygous knock-out mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum disorders. At the cellular level, loss of densin results in reduced levels of α-actinin in the brain and selective reduction in the localization of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depression, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology. These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5 and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.
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http://dx.doi.org/10.1523/JNEUROSCI.5877-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235477PMC
November 2011

Assessment of motor balance and coordination in mice using the balance beam.

J Vis Exp 2011 Mar 10(49). Epub 2011 Mar 10.

Department of Biology, California Institute of Technology, USA.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.
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http://dx.doi.org/10.3791/2376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197288PMC
March 2011

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment.

EMBO Rep 2010 Jun 14;11(6):473-8. Epub 2010 May 14.

Centre for Neuroregeneration, The University of Edinburgh, Institute of Immunology and Infection, Ashworth Buildings, Kings Buildings, Edinburgh EH9 3JT, UK.

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.
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http://dx.doi.org/10.1038/embor.2010.63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892321PMC
June 2010

SynGAP regulates steady-state and activity-dependent phosphorylation of cofilin.

J Neurosci 2008 Dec;28(50):13673-83

Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP(+/-) mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP(+/-) mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 mum NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP(+/-) mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.
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http://dx.doi.org/10.1523/JNEUROSCI.4695-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615239PMC
December 2008

Opposing effects of PSD-93 and PSD-95 on long-term potentiation and spike timing-dependent plasticity.

J Physiol 2008 Dec 20;586(24):5885-900. Epub 2008 Oct 20.

Department of Physiology, David Geffen School of Medicine at UCLA, 53-231 Center for the Health Sciences, Box 951751, Los Angeles, CA 90095-1751, USA.

The membrane-associated guanylate kinases (MAGUKs) PSD-95, PSD-93 and SAP102 are thought to have crucial roles in both AMPA receptor trafficking and formation of NMDA receptor-associated signalling complexes involved in synaptic plasticity. While PSD-95, PSD-93, and SAP102 appear to have similar roles in AMPA receptor trafficking, it is not known whether these MAGUKs also have functionally similar roles in synaptic plasticity. To explore this issue we examined several properties of basal synaptic transmission in the hippocampal CA1 region of PSD-93 and PSD-95 mutant mice and compared the ability of a number of different synaptic stimulation protocols to induce long-term potentiation (LTP) and long-term depression (LTD) in these mutants. We find that while both AMPA and NMDA receptor-mediated synaptic transmission are normal in PSD-93 mutants, PSD-95 mutant mice exhibit clear deficits in AMPA receptor-mediated transmission. Moreover, in contrast to the facilitation of LTP induction and disruption of LTD observed in PSD-95 mutant mice, PSD-93 mutant mice exhibit deficits in LTP and normal LTD. Our results suggest that PSD-95 has a unique role in AMPA receptor trafficking at excitatory synapses in the hippocampus of adult mice and indicate that PSD-93 and PSD-95 have essentially opposite roles in LTP, perhaps because these MAGUKs form distinct NMDA receptor signalling complexes that differentially regulate the induction of LTP by different patterns of synaptic activity.
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http://dx.doi.org/10.1113/jphysiol.2008.163469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655416PMC
December 2008

Integration of biochemical signalling in spines.

Nat Rev Neurosci 2005 Jun;6(6):423-34

Division of Biology 216-76, California Institute of Technology, Pasadena, California 91125, USA.

Short-term and long-term changes in the strength of synapses in neural networks underlie working memory and long-term memory storage in the brain. These changes are regulated by many biochemical signalling pathways in the postsynaptic spines of excitatory synapses. Recent findings about the roles and regulation of the small GTPases Ras, Rap and Rac in spines provide new insights into the coordination and cooperation of different pathways to effect synaptic plasticity. Here, we present an initial working representation of the interactions of five signalling cascades that are usually studied individually. We discuss their integrated function in the regulation of postsynaptic plasticity.
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http://dx.doi.org/10.1038/nrn1685DOI Listing
June 2005

Spine architecture and synaptic plasticity.

Trends Neurosci 2005 Apr;28(4):182-7

California Institute of Technology, Division of Biology 216-76, Pasadena, CA 91125, USA.

Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using live-imaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.
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http://dx.doi.org/10.1016/j.tins.2005.01.008DOI Listing
April 2005

SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor.

J Neurosci 2002 Nov;22(22):9721-32

Division of Neuroscience, University of Edinburgh, Edinburgh EH8-9JZ, United Kingdom.

At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757832PMC
November 2002

Postsynaptic induction and presynaptic expression of group 1 mGluR-dependent LTD in the hippocampal CA1 region.

J Neurophysiol 2002 Mar;87(3):1395-403

Department of Physiology, UCLA School of Medicine, 53-231 Center for the Health Sciences, Los Angeles, CA 90095, USA.

Activation of metabotropic glutamate receptors (mGluRs) with the group I mGluR selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induces a long-term depression (LTD) of excitatory synaptic transmission in the CA1 region of the hippocampus. Here we investigated the potential roles of pre- and postsynaptic processes in the DHPG-induced LTD at excitatory synapses onto hippocampal pyramidal cells in the mouse hippocampus. Activation of mGluRs with DHPG, but not ACPD, induced LTD at both Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells and at associational/commissural fiber synapses onto CA3 pyramidal cells. DHPG-induced LTD was blocked when the G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) was selectively delivered into postsynaptic CA1 pyramidal cells via an intracellular recording electrode, suggesting that DHPG depresses synaptic transmission through a postsynaptic, GTP-dependent signaling pathway. The effects of DHPG were also strongly modulated, however, by experimental manipulations that altered presynaptic calcium influx. In these experiments, we found that elevating extracellular Ca(2+) concentrations ([Ca(2+)](o)) to 6 mM almost completely blocked the effects of DHPG, whereas lowering [Ca(2+)](o) to 1 mM significantly enhanced the ability of DHPG to depress synaptic transmission. Enhancing Ca(2+) influx by prolonging action potential duration with bath applications of the K(+) channel blocker 4-aminopyridine (4-AP) also strongly reduced the effects of DHPG in the presence of normal [Ca(2+)](o) (2 mM). Although these findings indicate that alterations in Ca(2+)-dependent signaling processes strongly regulate the effects of DHPG on synaptic transmission, they do not distinguish between potential pre- versus postsynaptic sites of action. We found, however, that while inhibiting both pre- and postsynaptic K(+) channels with bath-applied 4-AP blocked the effects of DHPG; inhibition of postsynaptic K(+) channels alone with intracellular Cs(+) and TEA had no effect on the ability of DHPG to inhibit synaptic transmission. This suggests that presynaptic changes in transmitter release contribute to the depression of synaptic transmission by DHPG. Consistent with this, DHPG induced a persistent depression of both AMPA and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents in voltage-clamped pyramidal cells. Together our results suggest that activation of postsynaptic mGluRs suppresses transmission at excitatory synapses onto CA1 pyramidal cells through presynaptic effects on transmitter release.
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http://dx.doi.org/10.1152/jn.00723.2001DOI Listing
March 2002