Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) What our children teach us

Transcription

1 Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) What our children teach us IV Congreso Internacional del Síndrome X Frágil Associació Catalana SXF October 10, 2015 Paul J Hagerman MD. PhD Professor, Department of Biochemistry and Molecular Medicine, Investigator, UC Davis MIND Institute University of California, Davis, School of Medicine

2 Adult-onset problems among premutation carriers of the fragile X gene Es necesario explicar claramente su problema

3 Mothers of children with FXS: Child with fragile X syndrome

4 Mothers of children with FXS: problems with their own (carrier) fathers Frequent falls/ balance problems Difficulty writing, eating Memory loss Numbness/tingling in hands legs Loss of bladder/bowel control Child with fragile X syndrome

5 Fragile X-associated tremor/ataxia syndrome (FXTAS) Case DR: Premutation carrier with 89 CGG repeats, identified through two grandchildren with fragile X syndrome. First identified case of a carrier grandfather with progressive neurological dysfunction Core features Tremor Onset in right hand at age 54, left hand within two years; writing illegible at 58 yr; retired early as an electrician at 58 yr. Ataxia Progressive difficulty with balance and gait; frequent falls

6 Core clinical features of FXTAS Non-resting tremor Gait ataxia Associated forms of clinical involvement - Peripheral neuropathy - Cognitive decline - Autonomic dysfunction - Anxiety, mood instability - Parkinsonism

7 Core clinical features of FXTAS Non-resting tremor Gait ataxia Associated forms of clinical involvement - Peripheral neuropathy - Cognitive decline - Autonomic dysfunction - Anxiety, mood instability - Parkinsonism

8 Prior diagnoses for individuals with FXTAS Sixty-two patients FXTAS and family history of fragile X syndrome (55 men, 7 women; age of onset, 60.2 ± 7.1 years) 56 sought medical help 98 prior diagnoses Cerebrovascular 10% Miscellaneous 16% Parkinsonism 24% Dementia 13% Ataxia 17% Tremor 20% Hall et al. (2005) Neurol 65:299

9 Penetrance of FXTAS in families with known fragile X syndrome Approximately 40% of male premutation carriers (>50 yr) have combined tremor and ataxia The percent with clinical features increases with age - males: 17% (50s) 75% (80s) - far lower in females Jacquemont et al. (2004) Screening studies find the premutation in 2 5% of unexplained ataxia cases over 50 yr Jacquemont et al. (2006)

10 Women with FXTAS have less severe involvement than men FXTAS is less frequent in women: 16% vs >50% (Jacquemont et al 2004; Coffey et al 2008; Rodriquez-Revenga et al 2009) Less severe white matter disease; less severe brain atrophy; less frequent MCP sign (Adams et al 2007) Less frequent (rare) dementia (Seritan et al 2010; Schneider et al 2014) When dementia does occur in women it is associated with Alzheimer changes in brain pathology (Tassone et al 2012)

11 Medical History 146 women carriers Coffey et al., 2008 AJMG **Fisher s exact test for 2 2 contingency table analysis p<0.05

12 FXTAS: Imaging correlates Brunberg et al. (2002) AJNR 23: MRI T2-weighted image Male 75y MCP sign ~60% of cases Increased signal (T2/FLAIR) in cerebral white matter and middle cerebellar peduncles (MCP) Moderate/severe cortical atrophy/ ventricular enlargement correlates with CGG repeat length

13 What is causing FXTAS?

14 The fragile X gene is turned off in the full mutation range Typical (CGG) < 45 Full mutation (CGG) > 200 mrna FMRP Clinical Typical Fragile X syndrome

15 The fragile X gene is too active in the premutation range Typical Premutation Full mutation (CGG) (CGG) < (CGG) > 200 mrna FMRP Clinical Typical Fragile X syndrome Primary Ovarian Insufficiency (POI) FXTAS Neurodevelopmental problems The RNA itself may be the cause of the premutation-specific disorders - concept of RNA toxicity

16 FXTAS is an inclusion disorder - Ubiquitin-positive - Distinct from nucleoli in neuronal nuclei - Never observed in oligodendroglia Greco nucleolus Astrocytes inclusion Ubiquitin immunostaining of cortical neurons and astrocytes

17 Inclusions can be formed in cultured neural cells Elimination of all of the FMR1 gene except the CGG repeat region still gives rise to cellular abnormalities of FXTAS FMR1 promoter CGG FMRP coding Viral promoter CGG Reporter (GFP) coding SK neural cells 88 CGG repeats DAPI crystallin merged - Inclusions can be induced in cell culture - Induction requires expanded CGG repeat as RNA - Expanded CGG repeat as DNA does not cause inclusion formation Garcia-Arocena et al., 2005

18 FXTAS is an inclusion disorder Also in multiple cell culture systems DAPI Greco et al. (2002, 2006) Willemsen et al., 2003 Jin et al., 2003 crystallin merged SK cells 88 CGG Garcia-Arocena et al., 2005 Pure inclusion preparations has led to identification of numerous proteins of interest to pathogenic mechanism Iwahashi et al. (2006)

19 Broad distribution of intranuclear inclusions in FXTAS in brain, exclusively in nuclei of neurons and astrocytesl Also present in numerous peripheral tissues anterior and posterior pituitary thyroid dorsal root ganglia paraspinal sympathetic ganglia subepicardial autonomic ganglia of the heart ganglion cells of adrenal medulla myenteric ganglia of the stomach/intestine ovarian stromal cells testicular (Leydig) cells Greco et al., 2002 Brain; Willemsen et al., 2003 Hum Mol Genet; Greco et al., 2006 Brain Greco et al., 2007 J Urology; Brouwer et al., 2008 Psychoneuroendocrinology Godken et al., 2009 Neuropathology; Hunsaeker et al., 2011

20 The concept of RNA toxicity Myotonic dystrophy model: The expanded repeat binds to one or more proteins, thus removing them from their normal function. Myotonic dystrophy DMPK 3 UTR Increased binding of specific proteins (e.g., MBNL1) to the expanded CUG repeat reduces free protein levels AAAAAAAAAAA Loss of proteins prevents their normal function

21 The concept of RNA toxicity Sequestration model applied to FXTAS: The expanded CGG repeat binds to one or more proteins, thus removing them from their normal function. Myotonic dystrophy FXTAS DMPK 3 UTR Increased binding of specific proteins (e.g., MBNL1) to the expanded CUG repeat reduces free protein levels AAAAAAAAAAA FMR1 5 UTR Loss of proteins prevents their normal function DGCR8/DROSHA Pur α hnrnp A2/B1 CUGBP Etc.?

22 Expanded CGG repeat inhibits DGCR8/ DROSHA acovity FMR1 5 UTR DGCR8 C CGGCGGCGGCGGCGGCGGCGGCGG G G CGGCGGCGGCGGCGGCGGCGGCGG DROSHA DGCR8 DROSHA DGCR8 DROSHA? Sellier C et al. (2013) Cell Rep 3:

23 Expanded CGG repeat inhibits DGCR8/ DROSHA acovity DNA mirna sequence FMR1 5 UTR pri-mir pre-mir mature mirna X Transcription DGCR8 DROSHA decreased mirna levels DGCR8 C CGGCGGCGGCGGCGGCGGCGGCGG G G CGGCGGCGGCGGCGGCGGCGGCGG DROSHA DGCR8 DROSHA DGCR8 DROSHA? Sellier C et al. (2013) Cell Rep 3:

24 Expanded CGG repeat inhibits DGCR8/ DROSHA acovity mirna sequence DNA pri-mir pre-mir mature mirna X Transcription DGCR8 DROSHA DGCR8 C CGGCGGCGGCGGCGGCGGCGGCGG G G CGGCGGCGGCGGCGGCGGCGGCGG DROSHA DGCR8 DROSHA DGCR8 DROSHA Observed decreases in mirna levels mir-26a1 CTL FXTAS qrt-pcr mir-190 Sellier C et al. (2013) Cell Rep 3:

25 Possible mechanism(s) of FXTAS pathogenesis Post-transcriptional mechanisms FMR1 transcription Protein sequestration Inclusion Formation Evidence of cellular pathology

26 A Repeat- associated non- AUG (RAN) translaoon model for pathogenesis Todd et al. (2013) Neuron 78: RAN products (poly glycine) are detected in mouse and human inclusions. Such products are capable of eliciting neurodegenerative phenotypes in Drosophila FMRpolyG Ubiquitin Hoechst Merge Hukema et al. (2015) Human Molecular GeneOcs 24:

27 Possible mechanism(s) of FXTAS FMR1 pathogenesis transcription Post-transcriptional mechanisms Protein sequestration RAN translation RAN products FMRP Todd 2013 Inclusion Formation Inclusion Formation Evidence of cellular pathology Evidence of cellular pathology

28 FXTAS inclusions point to a role of DNA damage repair in FXTAS DNA damage and repair: - Evidence of DNA damage/repair response in the FXTAS inclusions. Presence of the phosphorylated variant of the H2A histone variant (γh2ax). Hoem et al. (2011) Intranuclear inclusion Working model: Unrepaired DNA damage at/near the CGG repeat leads to inclusion formation and neuronal cell damage.

29 A co-transcriptional (R-loop) model Newly made RNA reinvades the transcribing DNA to form an R-loop, which is known to lead to DNA damage if not quickly eliminated. Skourti-Stathaki K and Proud foot NJ (2015) Genes Devel 28: We observe R-loop formation near the start of transcription of the FMR1 gene, and this observation may provide an explanation for other features of the pathogenesis of FXTAS. Loomis E et al., (2014)

30 Pathogenesis of FXTAS DNA damage response (DDR) model C Iwahashi nucleolus inclusion inclusion

31 Pathogenesis of FXTAS (DDR model) Inclusion

32 Pathogenesis of FXTAS (DDR model) Inclusion

33 Pathogenesis of FXTAS (DDR model) Inclusion

34 Pathogenesis of FXTAS (DDR model) Inclusion

35 The DNA Damage Repair (DDR) model

36 The DNA Damage Repair (DDR) model predicts elevated p53 levels Neuronal cell death

37 Evidence of mitochondrial involvement in human FXTAS fibroblasts Several patients with FXTAS were initially diagnosed with mitochondrial myopathy, prompting us to look at mitochondrial function Ca 2+ Progressive mitochondrial dysfuncoon ROS Increased oxidative damage Decreased mito MnSOD Ross-Inta et al., 2010

38 what our children teach us FXTAS may reflect a broader (much earlier onset) disease process among children with the premutation ~8-13% with childhood seizures ~10-15% with ASD ~5% with intellectual impairment ~50% with ADHD Important for two reasons: - likely reflects common underlying mechanism(s) so common target for therapy - should allow treatment at far earlier ages, before onset of clinical symptoms

39 Reduced dendritic complexity in neonatal premutation mice Expanded CGG (premutation) neurons show reduced dendrite length and dendritic complexity - 6,13, and 20 DIV (days in vitro) Chen et al., (2010) Chen et al., (2010) Hum Mol Genet 19:

40 Neurons from neonatal premutation mice show reduced cell survival Perhaps reflecting increased susceptibility to: To environmental toxins To seizures To oxidative stress To second genetic hits Chen et al 2009 HMG

41 Mitochondrial dysfunction begins Reduced mitochondrial motility in mouse premutation hippocampal neurons in culture (4DIV) Decreased mitochondrial density in proximal neurite of premutation hippocampal neurons compared to wild type Kaplan et al 2012 early in development

42 Cultured mouse premutation neurons have increased spike/burst activity Consistent with increase seizures (8-13%) in children with the premutation Chen et al 2009

43 Reversal of spike frequency and burst activity with allopregnanolone

44 Expectations for research on FXTAS Some neurodegenerative diseases may be reversible Huntington disease mouse created with abnormal gene that can be turned off Control 18 wk 34 wk gene on Brain pathology: inclusions, loss of brain mass

45 Expectations for research on FXTAS Some neurodegenerative diseases may be reversible Huntington disease mouse created with abnormal gene that can be turned off Control 18 wk 34 wk gene on Brain pathology: inclusions, loss of brain mass Control 18 wk 34 wk gene off Reversal of brain pathology, recovery of normal behavior

46 ParOal reversibility of FXTAS inclusions in the premutaoon mouse Hukema et al, Mice engineered to have premutation CGG repeat (~100 CGG) Gene can be turned on / off with an antibiotic (doxycycline) Weeks CGG-repeat gene on (green) Start (3 wks) à 8 12 weeks gene off (yellow) Mouse brain CGG-repeat reporter gene On Off

47 Stopping expression aaer eight weeks in FXTAS mouse: substan'al reversal of inclusion size and number 8 weeks gene on 12 weeks gene off percentage Inclusion number dox wash-out * area (um) Inclusion size dox wash-out ** But no reversal seen if the gene was turned off after 12 weeks Need early diagnosis and treatment wks wks

48 Biochem Molec Med Flora Tassone Chris Iwahashi Anna Ludwig Chris Raske Gry Hoem Erick Loomis Kasia Koscielska Sean Roenspie Khaled Amiri (UAE University) Glenda Espinal Jun Yin Jun Yi Wang Biosta9s9cs Danh Nguyen Kiyoung Kim Molecular Biosciences Isaac Pessah Yucui Chen Cecilia Giulivi Zhongwei Cao Susan Hulsizer Neurosurgery Robert Berman Ryan Hunsaker Dolores Garcia- Arocena Current and Recent Collaborators UC Davis School of Medicine MIND Ins9tute Randi Hagerman Louise Gane Jennifer Cogswell Patrick Adams Michele Ono Susan Rivera Iin Wirnarni Len Abbeduto Radiology James Brunberg Rehab Med Veronica MarOnez- Cerdeno Neurology Lin Zhang Michael Rogawski John Olichney Psychiatry David Hessl Andreea Seritan Susan Rivera Noelle L Etoile Andrea Schneider Steve Noctor Chris Cunningham Stem Cell Program Jan Nolta Jeanie Liu UCHSC (Denver) Neurology Maureen Leehey Medicine James Grigsby RUSH Med Ctr (Chicago) Pediatrics Elizabeth Berry-Kravis Neurology Christopher Goetz Deborah Hall Erasmus MC (Rotterdam) Clinical Gene9cs Edwin Mientjes Rob Willemsen IGBMC (Illkirch)) Nicolas Charlet Chantal Sellier Pacific Biosciences Jackie Yen John Major David Rank John Eid Paul Peluso Luke Hickey Thang Pham LaTrobe University (Melbourne) Psychology Danuta Loesch 10/06/15