Huntington’s Disease

Hunter KelleyBCMB 403: Neurogenetics Lab

Dept. of Biochemistry, Cellular & Molecular Biology, Univ. of Tennessee, Knoxville

37996

Brief History of Huntington’s Disease

• Named after George Huntington1 – East Hampton, Long Island (1872)– “Huntington’s Chorea”

• Juvenile form

• 1983: Mapping of chromosomal localization of HD mutation1

• 1993: Identification of gene mutation1

• Autosominal Dominant, meaning only one copy is required for the disease– Size of CAG repeat increases as the altered gene is passed down from

generations (anticipation)2

Symptoms• Pre-manifest HD1

– Characterized by subtle motor, cognitive and behavioral changes– Alteration in executive function, decreased work performance, increase in

twitching– Behavioral changes: Increased irritability, minimal chorea present, small cognitive

declines

• Manifest HD1

– Twitches become more pronounced CHOREA• Common in early onset of HD and worsens in progression of disease• Difficulty in walking, speaking and eating• Weight loss• Increased saccadic eye movement

• Onset of Symptoms1

– Usually 30-40s but can occur in early 20s• “Westphal Variant”

Which Gene is affected?• The Huntington’s Disease Collaborative Research

Group2 – Defect in HTT gene encoding the protein product, Huntingtin

• IT15; HD• 350 kDa protein of unknown function that consists of 3,144 amino

acids

– Mutation located on short arm of chromosome 4 at position 16.3 4p16.3

– N-terminal region of huntingtin characterized by elongated CAG triplet repeat coding for Glutamine on exon 1

Exon Structure – IT15 gene

Figure 1: Schematic diagram representing the exon structure of HD gene. Vertical lines below the IT15 cDNA represent exon junctions.2

Chromosome Localization of Mutant htt

Figure2: Mutation site of IT15 gene encoding for the protein, huntingtin on short-arm of chromosome 4 at position 16.3 (NCBI Homepage: https://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=4&query=uid(-1257768356)&QSTR=3064%5Bgene%5Fid%5D&maps=gene_set&cmd=focus)3

Effect of CAG Triplet Length

Figure 3: Indirect correlation of repeat length of CAG triplet repeat coding for glutamine with age onset of disease. The greater the (CAG)n repeat, the earlier age onset of HD.1

Putative Drosophila Homolog of Huntington’s Disease

• Genomic Structure4

– Drosophila HD gene covers a smaller genome compared to human HD– Drosophila HD cDNA

• 29 exons compared to 67 as seen in vertebrate genomic sequences5

• Larger exon length (82-1151 bp) compared to 48-341 bp in human HD4

– Intron size of Drosophila HD are larger compared to intron size in other genes4

• Three introns <80 base pairs• Ten introns >1000 base pairs

• Differences in Amino Acid Sequence4

– Drosophila HD cDNA encodes protein consisting of 3583 amino acids ; 394 kDa MW– Glutamine only coded at most once compared to Human HD

– Absence of proline stretch following glutamines

Putative Drosophila Homolog of Huntington’s Disease

Figure 4: Schematic map showing Drosophila HD genomic DNA and cDNA. A) Intron-Exon structure representation of Drosophila HD. Exon are solid black boxes, and the introns are characterized as the solid lines between the exons. B) cDNA representation of Drosophila HD. Coding region is shaded solid black, whereas the 5’ and 3’ UTRs are characterized by the lines tailing off the end. Reverse-Transcription PCR products characterized by the individual boxes.4

Sequence Alignment of Drosophila HD

Figure 5: Schematic diagram representing the level of similarity between Drosophila HD (dhtt) and human huntingtin (htt).4

Effect of mutant htt on CRE Pathway

• Normal Huntingtin (htt)6

– CREB, a cellular transcription factor binds to DNA elements known as cAMP response elements (CRE) located in the promoter region

– Phosphorylation of CREB by Protein Kinase A (PKA) initiates recruitment of CREB binding protein (CBP)

– CBP has histone acetyltransferase activity, allowing chromatin to be restructured into an open form• CREB recruits TAFII130, a subunit of TAFIID followed by subsequent recruitment of

TFAIIA (B, D, E, F, H) and TATA-binding protein• Phosphorylation of RNA Polymerase II in CBD domain by TFIIH initiates

transcription• Mutant Huntingtin (mhtt)6

– CBP and TAFII130 prevented from binding to cAMP-response elements (CRE) in the promoter region

– Discrepancy in the apparatus of general transcription factors in addition to RNA Polymerase II leads to impairments in transcriptional regulation

Effect of mutant htt on NRSE-mediated transcription• Normal Huntingtin (htt)6

– Controls the expression of of Brain-Derived Neurotrophic Factor (BDNF)– Transcription factor REST-NRSF (repressor‐element‐1 transcription factor–

neuron‐restrictive silencer factor) binds to DNA elements NRSEs in promoter region to produce BDNF gene, a protein responsible for neuronal growth and survival

– Normal htt binds to REST-NRSF in the cytoplasm, preventing it from entering the nucleus and binding to NRSE site, allowing BDNF transcription

• Abnormal Huntingtin (mhtt)6

– Inhibited binding to REST-NRSF in cytoplasm, which allows increased levels of REST-NRSF in the nucleus

– REST-NRSF can now bind to NRSE sites, promoting the subsequent recruitment of Sin3A-histone-deacytlase complexes (HDACs) Remodeling of chromatin into closed structure, which in turn suppresses the transcription of BDNF

Figure 6: Transcriptional-mediation of cAMP-response element (CRE) and NRSE pathways effect of normal htt compared to mutant htt6

Mutant Huntingtin Induces Apoptosis

• Apoptosis in Transfected PC12 cells : 150Q-E vs. 20Q-E– Immunostaining with antibody, EM48, localized the majority of the 20Q-E protein to the

cytoplasm, whereas 150Q-E protein is highly concentrated in the nucleus7

– Electrophoresis of genomic DNAs showed increased DNA fragmentation in 150Q-E P12 cells compared to the control group7

• Caspase Activation– Caspase-8 and caspase-3 involved in induction of apoptosis8

– Radiolabeled with [35S]Methionine and incubated with respective cell extracts• Cleavage of caspase-3 present in 150Q-E transfected cell extract compared to control• Further experiment with cleavage of PARP showed same result of higher caspase-3 activity

in 150Q-E transfected PC12 cells compared to control7

– RT-PCR measured Induced caspase-1 expression in transfected cells with higher CAG triplet repeat7

• Release of Cytochrome C– 150Q-E cells showed increased level of cytochrome C compared to control group Increased

mitochondrial oxidative stress7,9

– Manganese superoxide dismutase (SOD2) levels were measured and showed highest levels in 150Q-E PC12 cells7,10

Caspase-1 vs. Caspase-3 Activity in Transfected PC12 cells

• Huntingtin transfected PC12 cells measured at 1,2,& 3 days after culturing to determine level of caspase activity– Higher caspase evident in 150Q-E transfected cells compared to

normal glutamine repeated cells, 20Q-E.7

– Increase in caspase-1 activity at day 2 compared to delayed increase in caspase-3 activity at day 37,11

• Increase in this activity leads to mitochrondrial permeability making the cell further susceptible to Cytochrome C release7

• Caspase 1 acts as initiator for caspase-3 activity11

Symptom Onset Due to Mutant htt

• Direct Pathway– Nerve cells in the striatum of the basal ganglia (caudate nucleus and

putamen) begin to die due to apoptosis from activation of caspase-1 and caspase-3, respectively12

– Leads to the internal globus pallidus receiving less neurotransmitter, exhibiting a form of inhibition. This form of of inhibition causes the IGP to release more neurotransmitter than usual12

– In return, the thalamus is inhibited resulting in a decreased amount of neurotransmitter release to the motor cortex, which generates decreased motor activity characterized by patients with Huntington’s Disease12

Symptom Onset due to Mutant htt• Indirect Pathway

– Death of neurons in the basal ganglia causes External Globus Pallidus (EGP) to receive less neurotransmitter. This inhibition causes the EGP to release more neurotransmitter12

– Subthalamic nuclei receives increased NT release from EGP, resulting in more pronounced inhibition Reduced release of NT12

– The thalamus receives less NT release from Internal Globus Pallidus, perceiving it as less inhibition12

– The thalamus then releases more neurotransmitter to the motor cortex, causing its over-stimulation, which is characterized by the involuntary, jerky limb movements known as chorea12

Transgenic Mice Study• Transgenic HD mice consisting of 115-150 CAG repeat overexpressed in mice

showed a neurological phenotype similar to the symptoms exhibited in HD patients13

– Involuntary Movement– Increased Tremors– Seizures– Weight loss

• Pathology reports showed presence of intranuclear inclusions identified by ubiquitin antibodies, constituting to the aggregates characterized by Huntington’s Disease13

• Another study examining mice homozygous for the gene resulted in the premature death of mice during gastrulation proving that the normal form of huntingtin is vital for embryonic development14

Treatment• Currently, there is no present cure for this disease• Insertion of the antibody Happ1 led to suppressed production of the mutant htt,

and their symptoms improved15

• Injection of small-interfering RNAs (siRNAs) into the striatum blocks transcription of mutant htt16

– Decrease in size of inclusion bodies of striatal neurons– Increased life of striatal neurons– Reduction in motor deficits

• Transgenic HD mice sustained a prolonged life expectancy when given a caspase inhibitor, such as minocycline, suppressing the activation of cell apoptosis7

• Risperidone and Olanzepine, neuroleptic agents, help control chorea and behavioral problems such as agitation1

• Physiotherapists, occupational therapists, & speech therapists1

Citations

• 1: Sturrock, A., & Leavitt, B. R. (2010). The clinical and genetic features of Huntington disease. Journal of Geriatric Psychiatry and Neurology, 0891988710383573.

• 2: MacDonald, M. E., Ambrose, C. M., Duyao, M. P., Myers, R. H., Lin, C., Srinidhi, L., ... & MacFarlane, H. (1993). A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell, 72(6), 971-983.

• 3: https://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=4&query=uid(-1257768356)&QSTR=3064%5Bgene%5Fid%5D&maps=gene_set&cmd=focus

• 4: Li, Z., Karlovich, C. A., Fish, M. P., Scott, M. P., & Myers, R. M. (1999). A putative Drosophila homolog of the Huntington's disease gene. Human molecular genetics, 8(9), 1807-1815.

• 5: Baxendale, S., Abdulla, S., Elgar, G., Buck, D., Berks, M., Micklem, G., ... & Lehrach, H. (1995). Comparative sequence analysis of the human and pufferfish Huntington's disease genes. Nature genetics, 10(1), 67-76.

• 6: Landles, C., & Bates, G. P. (2004). Huntingtin and the molecular pathogenesis of Huntington's disease. EMBO reports, 5(10), 958-963.

• 7: Li, S. H., Lam, S., Cheng, A. L., & Li, X. J. (2000). Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis. Human molecular genetics, 9(19), 2859-2867.

Citations• 8: Sanchez, I., Xu, C. J., Juo, P., Kakizaka, A., Blenis, J., & Yuan, J. (1999). Caspase-8 is required for cell

death induced by expanded polyglutamine repeats. Neuron, 22(3), 623-633.• 9: Fujimura, M., Morita-Fujimura, Y., Kawase, M., Copin, J. C., Calagui, B., Epstein, C. J., & Chan, P. H.

(1999). Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. The Journal of

neuroscience, 19(9), 3414-3422.• 10: Esposito, L. A., Melov, S., Panov, A., Cottrell, B. A., & Wallace, D. C. (1999). Mitochondrial disease

in mouse results in increased oxidative stress. Proceedings of the National Academy of Sciences, 96(9), 4820-4825.

• 11: Chen, M., Ona, V. O., Li, M., Ferrante, R. J., Fink, K. B., Zhu, S., ... & Hobbs, W. (2000). Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nature medicine, 6(7), 797-801.

• 12: Liou, S. (2010). The Basic Neurology of Huntington’s Disease. Standford University• 13: Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., ... & Bates, G. P.

(1996). Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87(3), 493-506.

• 14: Nasir, J., Floresco, S. B., O'Kusky, J. R., Diewert, V. M., Richman, J. M., Zeisler, J., ... & Hayden, M. R. (1995). Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell, 81(5), 811-823.

Citations

• 15: Southwell, A. L., Ko, J., & Patterson, P. H. (2009). Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease. The Journal of Neuroscience, 29(43), 13589-13602.

• 16: DiFiglia, M., Sena-Esteves, M., Chase, K., Sapp, E., Pfister, E., Sass, M., ... & Manoharan, M. (2007). Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proceedings

of the National Academy of Sciences, 104(43), 17204-17209.