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CFERV 2019 Frankel

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NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in genes that encode receptor subunits are associated with several intractable neurodevelopmental disorders, including developmental and epileptic encephalopathy (DEE). To extend understanding of these intractable childhood diseases, we have begun to model these variants in laboratory mice.

Models studied so far are a constitutive knockin for GRIN2A variant p.Ser644Gly (S644G) based on one case, and a GRIN2D variant, p.Val667Ile (V667I), noted in at least three unrelated cases. Homozygous and heterozygous Grin2a S644G mice exhibit altered hippocampal morphology at 2 weeks, and homozygotes exhibit lethal tonic-clonic seizures in week 3. Heterozygotes have a normal lifespan without spontaneous seizures, but display a variety of distinct features including
resistance to electrically induced limbic seizures, as well as hyperactivity and repetitive and reduced anxiety behaviors. Multielectrode recordings of mutant neuronal networks reveal hyperexcitability and altered bursting and synchronicity of both mutant genotypes. When expressed in heterologous cells, mutant receptors exhibit enhanced NMDAR agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. Consistent with this, NMDAR-mediated synaptic currents in hippocampal slices from mutant mice show a prolonged deactivation time course. Standard antiepileptic drug monotherapy was ineffective in the patient, but combined treatment of NMDAR antagonists with antiepileptic drugs substantially reduced the seizure burden albeit without appreciable developmental improvement. Chronic treatment of homozygous mutant mouse pups with NMDAR antagonists delayed the onset of lethal seizures but did not prevent them.

Grin2d V667I knockin mice are at an earlier stage of study. In contrast to Grin2a S644G, V667I heterozygous mice are severely impaired and suffer from lethal tonic-clonic seizures with an onset from about 18 days to 3 months. The impairment precludes natural mating such that the line needs to be propagated by ovary transplantation. Young heterozygotes adults in video-EEG also exhibit very frequent interictal epileptiform spiking and spike-wave discharge activity, resembling absence seizures. Preliminary histology shows significant pyknotic nuclei appearing to evidence
cell death in the cerebral cortex. Pup developmental milestones, while not evidencing significant developmental delay, show unusual features including excessive maternal separation induced pup vocalization. Further studies are ongoing. Together these efforts illustrate the power of modelling severe neurodevelopmental seizure disorders using multiple experimental modalities and suggest their utility in identifying and evaluating new therapies.

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CFERV 2019 Frankel

  1. 1. Hammer Bldg IGM 4-5-6 floors Mouse Models of GRIN Gain-Of-Function Genetic Variants Wayne N. Frankel, Ph.D. Institute of Genomic Medicine (IGM) Department of Genetics & Development Columbia University Irving Medical Center, NY, NY © Wayne N. Frankel, Ph.D.
  2. 2. Columbia/IGM Frankel lab David Goldstein Michael Boland Jennifer Gelinas Yueqing Peng Emory University Stephen Traynelis & colleagues University of Vermont Matthew Weston The Jackson Laboratory Cat Lutz Boston Children’s/Harvard Anapurna Poduri & colleagues Nationwide Children’s/Ohio St. Scott Harper • Explore mouse models of severe childhood epileptic encephalopathy • Can we obtain relevant in vivo phenotypes when modeling the mouse version of disease? • Including and beyond seizures • Establish studies across several experimental platforms • in vitro, in vivo, ex vivo • Phenotypes & mechanisms • Intervention feasibility • Test predictable or invent new interventions • Evaluate in ≥1 platform • Test efficacy vs. ‘standard of care.’ Objectives of our research © Wayne N. Frankel, Ph.D.
  3. 3. Human genetics, genomics David Goldstein Erin Heinzen Mouse models: seizures, sleep Wayne Frankel Jennifer Gelinas Yueqing Peng Mouse models: neurobehavior Mu Yang, MNBC Fibroblasts hiPSCs O S K M Neural Networks Integration-free reprogramming Neuronal Differentiation Electrophysiology Multielectrode Array Dermal biopsy Genetic mouse models Small Molecule Screening Genome Editing (CRISPR/Cas9) Gene Expression Analyses Patient-specific mutations Seizure monitoring Seizure threshold testing Comprehensive behavior monitoring Drug Pharmacology Genetic variant detection Patients Clinical partners Neurogenetics research in the IGM - a precision medicine ‘ecosystem’ Cellular models Michael Boland © Wayne N. Frankel, Ph.D.
  4. 4. But please keep in mind: • Mice are mice, and people are people • Clinical features not expected to be identical (and they are not!) • Beyond speech deficits • Variation in type and prominence of clinical features and gene dosage impact • However, most genes and many basic neurological functions are very highly conserved • Same gene -> neuron function -> brain-wide function -> mouse version of disease = Best possible animal model of genetic disease, including neurological (despite clear differences between species, and the fact that we are only just beginning to get good at assessing phenotypes during “mouse childhood”) © Wayne N. Frankel, Ph.D.
  5. 5. A2M BHLHE22 CHD4 DIP2C FETUB HDAC4 LANCL2 MYO5A PACS2 PTPRO RXFP1 STK36 TTN AAK1 BMP2 CHIA DISP1 FLG HECW2 LCE1A MYO7B PAK6 PTPRT RYR2 STX1B TTYH1 ABCA2 BMS1 CLDN19 DNAH7 FLNA HFE LDLRAD1 MYOM3 PALLD PURA RYR3 STXBP1 TUBB2A ABCB9 C16orf62 CLIC5 DNAH9 FLNC HIPK3 LEKR1 N6AMT1 PAQR8 PWWP2A SAFB2 SVOPL UBQLN4 ACOT4 C17orf53 CNTN5 DNAJC6 FLRT1 HIST1H2BD LEMD2 NBAS PASK QRSL1 SCAF4 SYNE2 UHRF1BP1L ADAM21 C18orf25 COL4A4 DNM1 FOCAD HIST2H2BE LETM1 NBEA PCDHB13 RAB5C SCN1A SYTL5 UNC5CL ADAMTSL4 C1orf123 COL7A1 DSG2 FRAT2 HLTF LIN7A NCBP1 PDCL2 RAD54L2 SCN2A TAAR2 USP7 AGPAT3 C1orf56 COQ3 DTYMK FRMD4A HNRNPH1 LRP1 NCOR2 PDIK1L RAET1L SCN8A TAF1 UTRN AHCY C1QTNF6 CPAMD8 EDEM1 G3BP1 HNRNPU LRP4 NEDD4L PHF21A RALGAPB SCYL1 TAS2R4 VPS37A AKAP6 C3orf22 CR2 EMILIN3 GABBR2 HRG LUC7L3 NEDD9 PHIP RALGPS1 SDCBP2 TCF4 WDFY2 AKR1C4 C4orf37 CREBBP EPHB1 GABRA1 HSF2 MAML3 NETO2 PIGS RANBP17 SELRC1 TCTE3 WDR1 ALG13 C5orf22 CRTAC1 ERG GABRB1 HSPG2 MAN1A2 NFASC PIK3AP1 RANGAP1 SERPINC1 TEP1 WDR19 ALMS1 C6orf222 CSMD2 ETNK2 GABRB3 IFT172 MAP3K8 NFE2L1 PIKFYVE RARS SETX TET3 WDR45 ALS2CL CACNA1A CSNK1E ETS1 GAS2 IQSEC2 MAPK8IP1 NFRKB PITX1 RASIP1 SGK223 TEX15 WDR82 ANK3 CACNA1E CTTNBP2NL EXOSC2 GCM2 ITGAM MAST1 NIPA1 PLA1A RBM12 SKA3 THAP4 WHSC1L1 ANKRD12 CAMK4 CUBN EXPH5 GFM2 ITGB4 MCM3 NLGN2 PLCG2 RBM45 SLAMF1 THOC2 WRN ANKRD24 CANT1 CUL2 FAM102A GLB1L3 ITPR1 MCM7 NLRP11 PLXNA1 RCL1 SLC16A3 TIFA XPO1 ANKRD50 CASP14 CUX2 FAM116B GLIS3 KCNB1 MEOX2 NLRP5 PLXNB1 RD3 SLC1A2 TMPRSS5 YPEL4 AP3S2 CASP9 CXXC11 FAM133B GLUL (KCNQ2) MIOX NLRP8 PNMAL1 RET SLC25A13 TNKS2 YWHAG ARFGEF1 CASQ1 CYP2U1 FAM134A GNAO1 GNB1 (KCNQ3) MKLN1 NOLC1 PPP1R3B RFX3 SLC26A11 TNNI3K ZBTB40 ARRDC1 CCDC125 DAO FAM21C GPR108 KCNT1 MLL NOTUM PPP3CA RGS14 SLC26A8 TPTE2 ZC3H3 ARHGEF9 ASH1L CDC25B DBP FAM50A GPR128 GPR98 KDR MLL2 NPAT PPP6R2 RHOG SLC35A2 TRIM29 ZFHX3 ASXL1 CDHR2 DCX FAM63B GRAMD2 KIAA0913 MMP27 NR1H2 PRDM12 RIOK3 SLC5A10 TRIM32 ZNF248 ATAD2B CDKL5 DDX50 FAM86C1 GRIN1 KIAA1324L MRS2 NTSR2 PRDM4 RNF186 SLCO1B7 TRIM8 ZNF282 ATIC CDS2 DDX58 FARSA GRIN2A KIAA2018 MSANTD1 OR10S1 PRG3 RP1L1 SMG9 TRIO ZNF354C ATP2B4 CELA3B DECR2 FASN GRIN2B GRIN2D KLHL11 MTOR OR2F2 PRKX RRP1B SZT2 SMURF1 TRRAP ZNF572 B3GNT4 CELSR1 DHDDS FBXL4 GTF2B KMT2B MTRF1 OR52E8 PRR19 RTKN2 SNX30 TSNAXIP1 ZNF839 BCL2L13 CEP55 DHTKD1 FBXO41 HBS1L KNDC1 MVK OSBPL5 PSD3 RTN1 SORBS3 TSPYL1 ZNFX1 BCLAF1 CHD2 DIAPH3 FCGR2B HCK KRT34 MYH6 OSBPL7 PTEN RTP1 SP TTC16 ZSCAN2 BEST2 DIP2B FERMT3 HCN4 KRTAP1-3 MYO3A OXA1L PTK2B RUVBL2 SPG7 TTF1 ZSCAN21 Origins of our research – breakthroughs in identifying putative genetic variants for epilepsy Epi4k EPGP EpiGen & other Genome sequencing groups & consortia © Wayne N. Frankel, Ph.D.
  6. 6. Gene and variant Whole animal phenotypes? Histological phenotypes? Neuron culture phenotypes? Therapy efficacy studies begun? Grin2aS644G NMDA receptor/ion channel Seizure & behavior Hippocampal atrophy Yes Yes (NMDAR antagonists) Grin2dV664I NMDA receptor/ion channel Seizure (behavior NDY) Cell death NDY Yes (NMDAR antagonists) Gnb1K78R G-protein b1 subunit Seizure & behavior No Yes Yes (antiepileptics) Kcnt1Y796H “Slack” K+ ion channel, mild Seizure & behavior No Yes Excluded proposed therapy Kcnt1condR428Q “Slack” K+ ion channel, severe Seizure (behavior NDY) NDY NDY NDY Arhgef9G55A GEF & scaffold for inhibitory synapses Seizures NDY; startle Protein aggregates, selective neuronal death NDY NDY Arfgef1fs GEF and trans-Golgi protein Seizure (↓ threshold only) and pup milestones Hippocampal atrophy No NDY Iqsec2fs GEF & scaffold for excitatory synapses Seizure & behavior Hippocampal ‘swelling’ NDY NDY Ppp3cafs* Calcineurin: Ser/Thr prot. phosphatase Seizure (behavior NDY) NDY NDY NDY Stxbp1-/+ MUNC-18: synaptic vesicle exocytosis Seizure & behavior (no) Yes NDY Dnm1Ftfl/Ftfl Dynamin 1: synaptic vesicle endocytosis Seizure & behavior Cell death, dendritic morphology Yes Yes (mRNA silencing) Genetically diverse (not just ion channels) mouse models under study in our group
  7. 7. 4 mos 9 mos Various Various (incl. atyp. absence) No No NMDAR antagonists lower sz. freq. Profound Hypotonia Spastic Ocularmotor apraxia Non-verb. Non ambul. LBD M1 M3 ABD ATD M4 M2 * d GluN1/GluN2A Tetramer S644 Transmembrane Domains Agonist Binding Domain Amino Terminal Domain COOH GluN2A Out In Agonist * Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660) Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661) Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658) Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688) Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662) Val667Ile (GluN2D) Ser644G (GluN2A) * Two NMDA receptor subunit variants causing early onset epileptic encephalopathy S644G V667I Onset: Seizures: AED ther. resp.: Other drugs: Devel delay: Movement: Other: GRIN2A (GluN2A) GRIN2D (GluN2D) NMDAR = Ligand-gated (glycine, glutamate) selective cation channel (Na+, Ca2+) Anapurna Poduri et al., Boston Children’s Hospital Stephen Traynelis et al., Emory University
  8. 8. a%ent’s and muta%on’s informa%on. (a) (b) pa%ent’s informa%on. (c) Schema%c topology ents a GluN2A subunit (asterisk notes the posi%on of the S644G muta%on). (d) A model of A subunit shown as space fill built from the GluN2B crystallographic data. The red asterisk artoon indica%ng the domain arrangement of a GluN2A subunit shows the posi%on of the transmembrane domain M3 (TM3), a cri%cal domain that may influence the channel LBD M1 M3 ABD ATD M4 M2 * d GluN1/GluN2A Tetramer S644 Transmembrane Domains Agonist Binding Domain Amino Terminal Domain COOH GluN2A Out In Agonist c GRIN2A ATD S1 S2 CTD M1 M2 M3 M4 * * Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660) Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661) Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658) Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688) Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662) MGI 958 Grin2a Exon 10 WT S644G 429kb Chr. 16 GCA AGT TAC GCA GGT TAC A S Y A G Y CRISPR/Cas9 JAX GET oligo-directed HDR 221 1 1 2 221 1 1 2 221 1 2 221 1 2 1 1 1 2 221 1 1 2 221 1 1 2 221 1 2 221 1 2 1 1 1 2 1. All homozygotes have lethal seizures by PND17 Days postnatal 0 10 20 30 0 50 100 Age postnatal (days) %Survival +/+ S644G/+ S644G/S644G %Survival Grin2aS644G knockin mouse model Cat Lutz, Aamir Zuberi, JAX Center for Precision Genetics Frankel lab, Columbia Most overt features 2. Heterozygous mothers inattentive to pups 3. Heterozygous mice are hyperactive
  9. 9. HIP COR HIPHIP CA1 CA2 DG UL CA1 DL L1 VZ +/+ +/S644G S644G/S644Ga HIP CA2 DG CA1 HIP CA2 DG CA1 Grin2aS644G heterozygous and homozygous mice have hippocampal atrophy at postnatal day 14 (2-3 days before lethal seizures) JJ Teoh, Frankel lab, Columbia Heterozygous adults resistant to electrically induced limbic seizure +/+ S644G/+ 0 5 10 15 20 iRMSCurrent(mA) **** iRMScurrent(±1SD) © Wayne N. Frankel, Ph.D.
  10. 10. +/ + Baseline 80 90 100 110 120 0 100 200 300 400 Startleresponse(Vmax) +/+ S644G/+ Stimulus (dB) * *** *** ***N=25 N=22 * circling repetititve exploring resting 0 20 40 60 80 100 Percent(%) +/+ S644G/+ N=12 N=11 *** ** Stimulus (dB) WT Mutant 0 10 20 30 40 50 60 70 80 90 Threshold 0 20 40 80 60 ABRthreshold(dB) p<0.05 Acoustic startle Repetitive behaviors Self-grooming p<0.1 p<0.05 p<0.001 10 20 30 40 50 60 500 1000 1500 2000 Recording time (min) Distancetraveled(cm) +/+ female (N=9) S644G/+ female (N=13) +/+ male (N=12) S644G/+ male (N=12) 10 20 30 40 50 60 0 100 200 300 Recording time (min) Timespentincenter(s) +/+ female S644G/+ female +/+ male S644G/+ male Open field (total ambulation) Open field (center time) females males both 0 20 40 60 80 Time(s/10mins) +/+ S644G/+ * 4 4 7 9 11 13 Grin2aS644G heterozygous adults have a variety of behavioral phenotypes Ayla Kanber, Ariadna Amador, Frankel lab, Columbia
  11. 11. 11 1E-8 1E-7 1E-6 0 20 40 60 80 100 0.01 0.1 1 10 100 0 20 40 60 80 100 1E-3 0.01 0.1 1 10 0 20 40 60 80 100 S644G WT 2A Glutamate, µM MaximalResponse,% Glycine, µM S644G WT 2A MaximalResponse,% H+, M MaximalResponse,% 500 ms 0.1 normalized S644G WT 2A ▼ glutamate GluN1/GluN2A-S644G Heterologous Expression System S644G WT 2A Functional properties of receptor-channel: GRIN2A (GluN2A) -S644G enhances virtually all aspects of receptor function Yuan, Traynelis labs, Emory© Wayne N. Frankel, Ph.D.
  12. 12. 12 + / + S 6 4 4 G / + 0 6 0 1 2 0 1 8 0 tweighted(ms) N M D A R S y n a p t i c D e c a y N = 1 5 N = 9 * * * Evoked CA1 EPSC from S644G 200 ms 50 pA WT S644G +/- 0.1 0.2 100 ms S644G mutation prolongs NMDAR synaptic time course (more excitable) 200 ms 50 pA Bhattacharya, Camp - Yuan/Traynelis labs, Emory 10 100 1000 GluN2AC1/GluN2AC2 GluN2AC1-S644G/GluN2AC2 GluN2AC1-S644G/GluN2AC2-S644G glutamate 300 ms tweighted(ms) GluN2AC1/ GluN2AC2 GluN2AC1-S644G/ GluN2AC2-S644G GluN2AC1-S644G/ GluN2AC2 (14) (8) (9) GluN1/GluN2A-S644G HEK cells © Wayne N. Frankel, Ph.D.
  13. 13. Electrodes embedded in cell culture plate (48 well – 16 electrodes per well) Advantages (vs. single cell patch-clamp recordings): • Observe populations of neurons rather than single cells • Non-invasive method to record neuronal network activity • Monitor network development/establishment • Test several experimental conditions in parallel • Medium-throughput allows for small-scale compound screening/testing Neurons on MEA Multiwell MEA Network firing properties 1˚ neuron culture in Multielectrode Array (MEA) © Wayne N. Frankel, Ph.D.
  14. 14. Grin2a-S644G enhances spike and network burst firing in 1˚ cortical neuron culture – MEA Christopher Bostick, Daniel Krizay, Goldstein/Boland labs, Columbia +/+ S644G/S644G 0 20 40 60 1 16 S644G/+ 0 20 40 60 1 16 Electrodes 0 20 40 60 Time (s) 1 16 p < 0.0001 each DIV 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 0 1 0 2 0 3 0 D a y s In V itro (D IV ) Burst/min + /+ S 6 4 4 G /S 6 4 4 G S 6 4 4 G /+ P e rm p -v a lu e : < 0 .0 0 1 + /+ v s S 6 4 4 G /+ a n d S 6 4 4 G /S 6 4 4 G , 0 .2 7 9 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 0 .0 0 .1 0 .2 0 .3 D a y s In V itro (D IV ) MeanFrequencyinBurst + /+ S 6 4 4 G /S 6 4 4 G S 6 4 4 G /+ P e rm p -v a lu e : < 0 .0 0 1 + /+ v s S 6 4 4 G /+ a n d S 6 4 4 G /S 6 4 4 G , 0 .6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G p < 0.0001 each DIV 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 0 5 1 0 1 5 D a y s In V itro (D IV ) MFR(Hz/AE) + /+ S 6 4 4 G /S 6 4 4 G S 6 4 4 G /+ P e rm p -va lu e : < 0 .0 0 1 + /+ vs S 6 4 4 G /+ a n d S 6 4 4 G /S 6 4 4 G , 0 .6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G , p < 0.0001 each DIV 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 D a y s In V itro (D IV ) + /+ S 6 4 4 G /S 6 4 4 G S 6 4 4 G /+ : < 0 .0 0 1 + /+ vs S 6 4 4 G /+ a n d .6 4 S 6 4 4 G /+ v s S 6 4 4 G /S 6 4 4 G , Mean firing rate # Burst per minute Mutual information Mutual Information © Wayne N. Frankel, Ph.D.
  15. 15. Memantine Dextromethorphan Topiramate Levetiracetam Valproic acid Zonisamide Max dose No.seizurespermonth 0 50 100 150 200 250 Mar-15 May-15 Jul-15 Sep-15 Nov-15 Jan-16 Mar-16 May-16 Jul-16 Sep-16 Nov-16 Jan-17 Mar-17 May-17 Jul-17 Sep-17 Monthly Seizure Count GRIN2A S644G patient seizure freq. lowered by NMDAR antagonist & AED combined therapy (“comorbid” behaviors unimproved) Anapurna Poduri, Jurrian Peters, Heather Olson, Harvard Univ/Boston Children’s Hospital © Wayne N. Frankel, Ph.D.
  16. 16. MFR(Hz)%Baseline -7 -6 -5 -4 -3 0 2 0 4 0 6 0 8 0 1 0 0 G lu N 2 A C1 /G lu N 2 A C2 G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -S 6 4 4 G L o g [M e m a n tin e ] Response(%Control) -7 -6 -5 -4 -3 0 2 0 4 0 6 0 8 0 1 0 0 G lu N 2 A C1 /G lu N 2 A C2 G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -W T G lu N 2 A C1 -S 6 4 4 G /G lu N 2 A C2 -S 6 4 4 G L o g [D e x tro m e th o rp h a n ] Response(%Control) MFR(Hz)%Baseline Memantine (µM) Dextromethorphan (µM) %survival Log [Memantine] (µM) Log [Dextromethorphan] (µM) Response(%ofcontrol) Response(%ofcontrol) Age (days postnatal) 0 10 20 30 0 50 100 Age (days postnatal) Percentsurvival Vehicle (N=24) Radiprodil (N=15) Nuedexta (N=17) Dextromethorphan (N=16) Quinidine (N=11) 0.01 0.1 1 10 100 0 50 100 150 Memantine (µM) MFR(Hz)%Untreated +/+ S644G/+ Non-treated S644G/S644G 0.1 1 10 0 50 100 150 Dextromethorphan (µM) MFR(Hz)%Untreated +/+ S644G/+ S644G/S644G Non-treated 0.01 0.1 1 10 100 0 50 100 150 Memantine (µM) MFR(Hz)%Untreated +/+ S644G/+ Non-treated S644G/S644G Response to FDA-approved NMDAR antagonists in vitro, ex vivo and in vivo Weiting Tang, Traynelis/Myers/Yuan labs, Emory Christopher Bostick, Goldstein/Boland labs, Columbia Ariadna Amador, Ayla Kanber, Frankel lab, Columbia Nuedexta prolongs survival by 30% © Wayne N. Frankel, Ph.D.
  17. 17. Summary: Grin2a S644G model Strong in vitro, in vivo and ex vivo phenotypes Seizures Hyperactive, hypoanxious, repetitive behaviors, poor maternal care Mostly consistent with each other Some unusual (e.g. resistance to limbic seizures) NMDAR targeted drug studies mimic partial rescue seen in human Does not mitigate developmental delay (treated mice still very small) © Wayne N. Frankel, Ph.D.
  18. 18. 4 mos 9 mos Various Various (incl. atyp. absence) No No NMDAR antagonists lower sz. freq. Profound Hypotonia Spastic Ocularmotor apraxia Non-verb. Non ambul. LBD M1 M3 ABD ATD M4 M2 * d GluN1/GluN2A Tetramer S644 Transmembrane Domains Agonist Binding Domain Amino Terminal Domain COOH GluN2A Out In Agonist * Human GluN2A (620) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEFVD (660) Human GluN2B (621) QNPKGTTSKIMVSVWAFFAVIFLASYTANLAAFMIQEEYVD (661) Human GluN2C (618) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEQYID (658) Human GluN2D (648) ENPRGTTSKIMVLVWAFFAVIFLASYTANLAAFMIQEEYVD (688) Human GluN1 (622) GAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEE (662) Val667Ile (GluN2D) Ser644G (GluN2A) * Two NMDA receptor subunit variants causing early onset epileptic encephalopathy S644G V667I Onset: Seizures: AED ther. resp.: Other drugs: Devel delay: Movement: Other: GRIN2A (GluN2A) GRIN2D (GluN2D) NMDAR = Ligand-gated (glycine, glutamate) selective cation channel (Na+, Ca2+) Anapurna Poduri et al., Boston Children’s Hospital Stephen Traynelis et al., Emory University 3 4 children have the same variant
  19. 19. Grin2dV667I/+ heterozygotes experience lethal seizures b/w PND22-50 JAX: Heroic approach to line maintenance: ovary transplantation Sabrina Petri, Frankel lab, Columbia Cat Lutz lab, JAX In our hands, memantine ad lib in the drinking water does not significantly mitigate lethal seizures – but its difficult to control dosage or confirm degree of brain exposure. © Wayne N. Frankel, Ph.D.
  20. 20. WT32 WT33 WT34 Het35 Het36 Het40 Het39 F F F F M F M Seizure phenotypes of Grin2dV667I/+ mice seen as early as 17 days of age 1. Continual spike-wave discharge-like seizure activity in Grin2d V667I mice 2. Single, lethal tonic-clonic seizure Sabrina Petri, Frankel lab, Columbia © Wayne N. Frankel, Ph.D.
  21. 21. Grin2D V667I – abnormal motor/coordination Wildtype hindlimbs splayed Heterozygote hindlimbs clasped © Wayne N. Frankel, Ph.D.
  22. 22. Het/Hom weigh less than wt as they grow No difference Different at P7 No difference No difference Different at P6 Grin2d V667I abnormal pup behaviors at a very early age postnatal JJ Teoh, Frankel lab, Columbia Postnatal day 3! © Wayne N. Frankel, Ph.D.
  23. 23. Summary and plans: Grin2aV667I model Early days in our studies Challenge to overcome breeding obstacle (due to lethal seizures of otherwise fertile adults) Continue with ovarian transplantation, but conditional model (“floxxed”) – underway at JAX Seizure phenotypes – striking Determine age-of-onset and progression of continual spike-wave seizures Histopathology Determine age-of-onset of cell death, cell types Cellular physiology Whole-cell recordings in neuron culture (Columbia) & acute slices (Emory) Multielectrode array mass culture recordings Cellular etiology (is defect in interneurons rate-limiting for disease?) Ideally want conditional mutation for this Treatment Continue to explore memantine + (chronic treatment challenges in mice) Genetic therapy (e.g. AAV9-mediated mutation-specific RNA elimination?) © Wayne N. Frankel, Ph.D.
  24. 24. Novel gene therapy approach: Dnm1Fitful mouse model for Dnm1 epileptic encephalopathy GTPase Middle PHD GED PRD 1 314 499 631 746 864 G43S S45N T65N G139V Q148R A177P K206N K206G R237W S238I I289F G346V G359A G359R G373K H396D G397D N363_R364insLP K535E A408T Ftfl Mutant mouse discovered spontaneously at Jackson Lab ca. 2008 Virginia Aimiuwu, Ph.D. candidate• Dnm1Ftfl/Ftfl homozygotes have severe seizures, usually lethal by 3rd week • Modeling EE • Various neurodevelopmental and behavioral deficits We previously showed seizures due to inhibitory neuron mutant expression, but neurobehavioral comorbidities due to excitatory neuron mutant expression © Wayne N. Frankel, Ph.D.
  25. 25. DNM1 codes for a protein involved in endocytosis and synaptic vesicle recycling Schmid and Frolov 2011 • Dnm1Ftfl molecules interfere with DNM1 multimolecular assembly © Wayne N. Frankel, Ph.D.
  26. 26. Dnm1a specific miRNA construct Scott Harper, Nationwide Children’s Hospital Center for Gene Therapy Novel gene therapy approach: inactivate (degrade) mutant mRNA © Wayne N. Frankel, Ph.D.
  27. 27. miDnm1A-4 shows the highest efficacy of Dnm1a knockdown in vitro • miDnm1A-1: 84% • miDnm1A-2: 64% • miDnm1A-3: 80% • miDnm1A-4: 95% Scott Harper, Nationwide Children’s Hospital Center for Gene Therapy © Wayne N. Frankel, Ph.D.
  28. 28. Package and deliver miRNA construct in adeno-associated virus, AAV9 (neurotropic subtype) Schultz and Chamberlain., 2009 © Wayne N. Frankel, Ph.D.
  29. 29. Experimental design scAAV9-miDnm1a icv injection E10 E15 E20 P2 P4 P6 P8 P10 P12 P14 P16 P18 P20P0 Neurogenesis Synaptogenesis growth delay Ataxia Wobbly gait Seizures Hypotonia Lethal seizures D nm 1a nm 1b D nm 1 0 2 4 6 ΔCTofDnm1relativemRNA +/+ eGFP +/+ miDnm1a Virginia Aimiuwu, Frankel lab, Columbia © Wayne N. Frankel, Ph.D.
  30. 30. 0 10 20 30 0 50 100 PND Percentsurvival Survival Ftfl/Ftfl Ftfl/Ftfl: 1x1010 vg miDnm1a Ftfl/Ftfl: 1.85x1011 vg miDnm1a Ftfl/Ftfl: 3.25x1011 vg miDnm1a +/Ftfl untreated +/+ 0 10 20 30 0 5 10 15 20 25 PND Weight(g) Growth Ftfl/Ftfl Ftfl/Ftfl: 1.85x1011 vg miDnm1a Ftfl/Ftfl: 3.25x1011 vg miDnm1a +/Ftfl untreated +/+ 0 10 20 30 0 PND Ftfl/Ftfl Ftfl/Ftfl: 1x1010 vg miDnm1a Ftfl/Ftfl: 1.85x1011 vg miDnm1a Ftfl/Ftfl: 3.25x1011 vg miDnm1a +/Ftfl untreated +/+ 0 10 20 30 0 50 100 PND Percentsurvival F2 Hybrid Survival +/+ +/+: 5.2x1011 vg miDnm1a Ftfl/Ftfl: 5.2x1011 vg eGFP Ftfl/Ftfl: 5.20x1011 vg miDnm1a 0 10 20 30 0 5 10 15 20 25 PND Weight(g) F2 Hybrid Growth +/+ +/+: 5.2x1011 vg miDnm1a Ftfl/Ftfl: 5.2x1011 vg eGFP mRNA silencing therapy in Dnm1Ftfl/Ftfl neonates significantly mitigates lethal seizures AND developmental delay (and on two different strain backgrounds)(B6JxFVB)F2 hybrid B6Jinbred Growth Survival Virginia Aimiuwu, Frankel lab, Columbia Rescues features previously shown to depend on gene defect in excitatory neurons ……inhibitory neurons © Wayne N. Frankel, Ph.D.
  31. 31. 2 4 6 8 10 12 0 2 4 6 8 PND weight(g) Growth Ftfl/Ftfl Treated: 3.25x1011 vg Ftfl/Ftfl +/+ 3 5 7 9 11 13 0 10 20 30 40 PND Time(S) Negative geotaxis 90o Ftfl/Ftfl: 3.25x1011 vg miDnm1a Ftfl/Ftfl +/+ 3 5 7 9 11 13 0 10 20 30 40 PND Time(S) Negative geotaxis 180o Ftfl/Ftfl: 3.25x1011 vg miDnm1a Ftffl/Ftfl +/+ It also rescues deficiency in some developmental performance tasks Virginia Aimiuwu, Frankel lab, Columbia © Wayne N. Frankel, Ph.D.
  32. 32. Promising, but why isn’t rescue complete? treatment E10 E15 E20 P2 P4 P6 P8 P10 P12 P14 P16 P18 P20P0 Neurogenesis Synaptogenesis growth delay Ataxia Wobbly gait Seizures Hypotonia Lethal seizures Human window of opportunity should be longer Quality of life improvement expected, regardless Disease engages before miDnm1a fully expressed? Dose not high enough? Transduction inefficient?
  33. 33. David Goldstein & Michael Boland Lab Sophie Colombo, PhD Chris Bostick, PhD Sarah Dugger Daniel Krizay Wayne Frankel Lab Megha Sah, PhD *JJ Teoh, PhD *Ariadna Amador, PhD *Sabrina Petri *Ayla Kanber Chana Rosenthal-Weiss *Virginia Aimiuwu Devin Jones Wanqi Wang Mouse NeuroBehavior Core Mu Yang, PhD, Director Elizabeth Rafikian IGM Electrophysiology Damian Williams, PhD Acknowledgements NIH R37 NS031348 NIH JCPG U54 OD020351Columbia Columbia Univ. Precision Medicine The Jackson Laboratory Cat Lutz University of Vermont Amy Shore & Matt Weston Emory University Stephen Traynelis/Hongjie Yuan, Scott Myers &labs Nationwide Children’s Hospital Scott Harper © Wayne N. Frankel, Ph.D.

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