Gene expression andHuntington’s disease

A hallmark of many neurodegenerativedisorders is the appearance and persistence ofaggregates in neuronal cells. Recent evidencesuggests normal interactions that occurbetween proteins and nucleic acids (RNA) inthese cells may become seeds for theformation of aggregates under prolongedstressful conditions. Such irreversibleassemblies of proteins and RNA are consideredtoxic and impair neuronal functions. Mutationsin proteins that bind RNA (RNA bindingproteins) have been implicated in neurodegen -erative disorders such as amyotrophic lateralsclerosis, spinal muscular atrophy, and fragileX syndrome.

Huntington’s disease is a heritableneurodegenerative disease caused by amutation in the Huntingtin gene, HTT. Themutation results in an increased number ofrepeats (greater than 40) of the amino acidglutamine in the Huntingtin protein (HTT). A normal HTT protein has between 7 and 35glutamines. The mutant HTT protein is toxicto cells and causes cell death over time. The HTT protein is present in all human cellsthroughout life. Despite this, neurons aremost vulnerable to death. The normal HTTprotein has been implicated in many cellularfunctions. My lab discovered new functions ofHTT in gene expression. Understanding thesefunctions and how they are altered in thecells that harbour a mutant HTT gene shallreveal pathways and targets for therapeuticintervention to prevent the death of neurons.

Genes are blueprints for making proteins

The process by which the information encodedin a gene is converted to a gene product istermed gene expression. This occurs in two

steps: transcription and translation. Duringtranscription, an enzyme makes a copy of thegene. The copy is termed messenger RNA(mRNA) or transcript. During translation, amulti-protein complex called the ribosomedecodes the mRNA and generates a stringof amino acids. The string of amino acidsfolds itself to create stable structures knownas proteins.

The program of gene expression is specific toeach cell-type. The process is highly regulatedboth temporally and spatially to preserve thespecific identity of each cell. Regulation ofgene expression occurs during transcriptionand post-transcriptional events that involveprocessing of the synthesized mRNA. Acategory of proteins called RNA bindingproteins (mentioned above) bind newly mademRNA and control its stability and locationwithin the cell. Some RNA binding proteinsdo this by interacting with one end of the mRNAmolecule, forming a stable RNA-proteincomplex. RNA binding proteins can influencegene expression through the assembly ofmultiple RNA-protein complexes thussequestering mRNAs. When cells are stressedthese complexes grow in size and numberand become visible under a microscope.They are called granules, which are reversibleaggregates of mRNA-binding and mRNA-degrading proteins that trap mRNAs in thecell. Through degradation or temporarystorage of mRNAs, these granules reduce theamount of protein that can be made at anygiven time thus affecting gene expression.

Gene expression can also be regulated post-transcriptionally through the transport ofmRNA transcript to specific locations in thecell. This is particularly important in neurons.Neurons are highly asymmetric cells with long

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branches that send signals from the tip toanother neuron nearby through a gapbetween two cells known as the synapse.The interaction between two neurons isstrengthened or weakened based on howmuch stimulating signal the synapse receivesover time. This change in strength is knownas synaptic plasticity. Synaptic plasticity iscritical to learning and forming new memories.Increasing evidence suggests transport ofmRNAs to the synapse and local translationof these mRNAs contributes to synapticplasticity. Following synaptic transmission,proteins within the branches of the neuronneed be rapidly replenished. RNA bindingproteins not only facilitate the transport ofmRNAs but also regulate the efficiency atwhich mRNAs are decoded by the ribosometo make new proteins where and when theyare needed.

HTT protein has a role in RNA transportand translation

My lab has studied transcriptional and post-transcriptional gene regulatory processes inmammalian cells for many years. Because thenormal function of HTT and the mechanismby which its mutant counterpart contributesto the pathogenesis of Huntington’s diseaseremains unclear, we began investigating therole of HTT in post-transcriptional generegulatory pathways. With advanced imagingtechniques we determined the location of thenormal HTT protein in neurons grown in aculture dish. Strikingly, we discovered thatHTT could be found near RNA granulespresent in neurons. As described above, RNAgranules are assemblies of large RNA-proteincomplexes responsible for transporting mRNAto specific locations in the cell. To determine

whether HTT influences mRNA localization,we reduced the level of HTT in neurons grownin culture and examined its effect on transportof mRNA. We found that the reduction of HTTin neurons disrupts proper mRNA localization.The result suggests that HTT contributes tothe integrity of RNA granules and therebyfacilitates mRNA transport.

To understand the cellular processes thatHTT is involved in and how they may differfor mutant HTT, we carried out experimentalprocedures to isolate normal and mutantHTT protein from cells and tissues. We nextidentified proteins that associate with eachHTT form. By identifying the functions ofthe proteins that co-isolated with HTT, weuncovered a new role for HTT. Analysis ofthe binding partners of HTT proteinsrevealed that both normal and mutant HTTinteract with proteins involved in RNAprocessing/metabolism and protein synthesis.

Naoko Tanese, PhDAssociate Dean for Biomedical Sciences

Director, Sackler Institute of GraduateBiomedical Sciences

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HTT protein associates with its ownmRNA

The identification of proteins co-isolated withthe HTT protein provided further support toHTT’s role in mRNA transport and translation.We next asked whether HTT regulates geneexpression post-transcriptionally throughinteractions with mRNAs and if so, whichmRNAs. Towards this end we extracted mRNAsthat co-isolated with the HTT protein complexand analysed by direct sequencing, an advancedtechnology that permits rapid identificationof nucleic acids. Surprisingly, we found bothHTT protein forms to associate with their ownHTT mRNA.

Further examination of the HTT protein’sassociation with its own mRNA revealed thatmutant HTT associates with a shorter versionof the normal HTT mRNA. The HTT geneencodes a long mRNA transcript. However,the presence of a much smaller mRNAtranscript encoding a short amino acidsequence from one end of the HTT proteinhas been detected in cells harbouring amutant HTT gene. The short transcript wasproduced as a result of a post-transcriptionalprocessing step termed mRNA splicing.Splicing is a commonly occurring event, whichresults in the removal of unnecessarysequences from the primary mRNA sequenceto produce mature mRNAs used to makeproteins by the ribosome. Normally mRNAscan be spliced in different ways to producemultiple transcripts from one gene. However,in the case of the short HTT transcript, theHTT mRNA has been incorrectly spliced. Thismis-splicing event produced a smaller proteinconsisting of a short amino acid sequence.

Significantly, the truncated protein containedthe expanded glutamine repeats, which isknown to be toxic to cells. The fact that mis-spliced mRNA was detected only in thepresence of mutant HTT and not normalHTT suggests the possibility that it may play arole in the disease pathogenesis. Preventingthe production of the mis-spliced mRNAwould be one way to reduce cell toxicity thatcontributes to the progression of Huntington’disease.

Silencing mutant HTT gene

Although the gene that causes Huntington’sdisease was discovered more than two decadesago, development of therapies for the diseasehas been challenging. A therapeutic approachthat has shown promise involves reducing theamount of mutant HTT mRNA and protein inaffected cells. Gene silencing is a revolutionarytechnology with broad applications that maybe used to treat or cure many diseases. In thecase of Huntington’s disease, most patientshave one normal copy and one mutant copyof the HTT gene. The normal HTT gene isrequired to maintain healthy neurons. Thus,selective silencing of the mutant HTT gene ispreferred. However, this is difficult to achievebecause normal and mutant HTT genes areidentical in their DNA sequence except for thelength of the repeat sequence. However, it hasbeen reported that partial silencing of mutantHTT (accompanied by partial silencing ofnormal HTT) might be enough to alleviate thetoxic effects of the mutant gene in a mousemodel of Huntington’s diseases. Further,successful alterations of the mutant HTT genein mice using the CRISPR gene editing system

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have been reported. These groundbreakingapproaches are being actively pursued inhopes of delaying the onset and progressionof this terrible disease.

mRNAs regulated by HTT

In addition to its own mRNA, we know thatHTT protein associates with other mRNAs. Wethink HTT associates with a subset of mRNAspresent in neuronal RNA granules. We thinkHTT regulates transport and local translationof these mRNAs in response to synapticactivity. We hypothesize that HTT may regulatethe transport and translation of mRNAs thatare vital to the survival of specific neurons.Our goal is to identify additional mRNAs thatassociate with normal HTT and mutant HTT.The involvement of the HTT protein in post-transcriptional gene regulation could explainthe specific pattern of neuronal loss andsymptoms seen in Huntington’s disease. It is possible that mutant HTT affects selectgroups of genes/mRNAs more adversely overothers. These potential changes are likelysmall to account for the normal developmentof affected individuals and delay in symptomappearance. An emerging body of evidencesuggests regulated transport and localtranslation of mRNAs in neurons play a criticalrole in establishing their connectivity. Ourfindings implicate the normal HTT in theseimportant dynamic processes in neurons. It is possible that the mutant HTT perturbsthem in some way, contributing to the diseasepathogenesis.

We have uncovered novel roles for normaland mutant Huntington’s disease protein

HTT in post-transcriptional gene regulatorypathways in neurons. These novel functionshave several implications for the developmentof Huntington’s disease. As discussed at the beginning, mutations in RNA bindingproteins have been implicated in severalneurodegenerative disorders. These RNAbinding proteins normally play a role in mRNAtransport, translation of mRNA, and formationof RNA-protein granules. The RNA bindingproteins whose mutations have been linked toneurological disorders have been localized togranules found in neurons. Our study is thefirst to report that HTT plays a similar role inmRNA transport and translation. HTT proteinis much larger than RNA binding proteinslinked to other diseases. It is possible thatHTT has a scaffolding role in RNA transportand translation. Recent studies indicate thatmutant RNA binding proteins show alteredbiophysical properties. They have increasedpropensity to interact with one another andaffect the formation and function of RNAgranules. The findings are consistent with theidea that mutant RNA binding proteins foundin these granules accumulate and becomeconverted to irreversible toxic aggregatesunder prolonged stress. We suggest that HTTaggregates may be formed in a similar manner.Development of chemical agents that preventaggregation or disrupt aggregates may serveto reverse toxicity associated with mutantproteins. Through understanding of howHTT supports neurons with these functions,we hope to reveal effective new targets fortherapeutic intervention.

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Since the discovery of the gene that causesHuntington’s disease in 1993, exponentialprogress has been made in elucidating thetrue scope of Huntington’s disease, but thereare still miles to go to truly understand theimpact of symptoms of HD families.Huntington’s disease (HD), is an autosomaldominant neurological disease caused by anexpanded CAG repeat in the Huntingtin gene.The disease is characterised by progressivefunctional decline and motor, psychiatric andcognitive symptoms, in addition to weightloss, sleep disturbances and dysregulation ofthe autonomic nervous system1. Each child ofa person who carries the gene mutation thatcauses HD has a 50% chance of inheriting thefaulty gene.

“Although we currently have no cure forthis disease, we do have the ability toallow individuals to access treatments tohelp them manage this debilitatingillness. While we work on a cure and findhope for tomorrow, we have to ensurethat families affected by HD can accessthe help they need today.”

Cognitive and behavioral symptoms aremost impactful to HD patients and families

In preparation for a Patient Focused DrugDevelopment meeting with the U.S. Food andDrug Administration, the Huntington’s

Disease Society of America (HDSA) surveyedthe HD community in the U.S. on topicsrelated to HD symptoms and treatments.Between two surveys, more than 3,600responses were collected from individualsaffected by HD, Juvenile Huntington’s disease(JHD) and caregivers for those with HD andJHD. In reviewing the data collected, cleartrends began to emerge between caregiversand HD/JHD patients alike. Caregiversresponded most frequently that chorea was the most impactful symptom of HD(30%), but in aggregate, behavioral andcognitive symptoms were reported as themost impactful to their lives by more than50% of both caregivers and HD/JHDpatients2.

Huntington’s disease has long been classifiedas a movement disorder, though prodromalfeatures encompass cognitive and behavioralsymptoms of HD3. Although classified as“prodromal”, the cognitive and behavioralsymptoms of HD are major elements of thedisease and its impact on the individual andtheir families. Cognitive and behavioralsymptoms can manifest as much as a decadebefore motor symptoms develop, and as aresult often go undiagnosed as symptoms ofHD. It is not uncommon for individuals withHD to be misdiagnosed with a variety ofpsychiatric disorders before being correctlydiagnosed with HD at the onset of motor

Jennifer Simpson of the Huntington’s Disease Society of America highlights the diseaseand how there is still a long way to go before it can be truly understood

Huntington’s disease: Understanding the impact

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symptoms. Delayed diagnosis may unfairlydisadvantage people with HD and cognitive-behavioral symptoms, especially in terms ofaccessing the kind of care and benefits peoplewith HD really need to best manage theprogression of their disease4.

For Huntington’s disease patients,treatment options are lacking

In the world of HD, treatment options are fewand far between. As of the publication of thisarticle, only two medications exist that areFDA approved for the treatment of HD, andboth treat chorea symptoms associated withthe disease. Currently, there are no disease-modifying treatments or cures. Whensurveyed on availability and efficacy ofcurrent treatments for cognitive symptoms ofHD, more than 80% of respondents notedthat they or their loved one were not takingany kind of medication for symptoms like

deterioration of memory and thinking2. Forbehavioral symptoms like anxiety, depressionand irritability, individuals responded mostfrequently that they or their loved one wasnot taking any kind of medication to treatthose symptoms2. The lack of treatmentoptions, especially for cognitive symptoms ofHD, stands in stark contrast to the impactthose symptoms have on the lives of peoplewith Huntington’s disease.

Access to care early on is critical tomanaging Huntington’s disease

As patients with HD become symptomatic, itis key that those individuals have access tocomprehensive care with doctors who areknowledgeable in HD. HD patients in early tomiddle stages of the disease need coordinatedmultidisciplinary healthcare services, includingassessment of cognitive function and counselling by (neuro) psychologists,

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rehabilitation programmes, activephysiotherapeutic interventions, speechtherapist training and occupational therapy5.Lack of access to care for families with HDmeans unmanaged or poorly managedsymptoms, higher rates of caregiver burnout,potential unnecessary hospitalisations andearly entry into long-term care facilities. Withaccess to specialists knowledgeable in HD,families can avoid unnecessary additionalemotional and financial burdens. In the U.S.,HDSA has created a clinical care modelthrough the Center of Excellence programme,awarding grants to HD clinics around thecountry to provide an all-in-one service centerfor families affected by Huntington’s disease.HDSA currently funds 41 Centers of Excellencearound the U.S.

“In the world of HD, treatment optionsare few and far between. As of thepublication of this article, only twomedications exist that are FDA approvedfor the treatment of HD, and both treatchorea symptoms associated with thedisease. Currently, there are no disease-modifying treatments or cures.”

Expanded access to governmentprogrammes can help HD families

Individuals with HD access multiple forms ofgovernmental support as their diseaseprogresses. Because HD symptom onsetcommonly occurs during prime working years,many families are devastated financially, andneed to rely on programmes like SocialSecurity Disability Income (SSDI), Medicaidand Medicare. Utilising these programmes canhelp families access professionals like

neurologists, neuropsychiatrists, speechtherapists and physical therapists. Expansionof Medicaid programmes has resulted in areduction of unmet need for mental healthservices, in addition to positive impacts on thebudgets of states that expanded Medicaid asstates no longer needed to use some of theirgeneral funds to pay for behavioral healthtreatment for the uninsured6. Individuals withHD are included amongst those who havebenefitted from the expansion ofprogrammes like Medicaid, and would furtherbenefit from expedited access to Medicarethrough the Social Security Disability Incomeprogramme. HDSA has been advocating,alongside the HD community, for a waiver ofthe two-year Medicare waiting period forindividuals who are disabled by Huntington’sdisease and utilising the SSDI programme. TheHuntington’s disease Parity act of 2017 is a bi-partisan solution to an HD shaped hole in thesocial safety net. It is one step of many to helpensure access to important behavioral healthservices and specialist neurologists who canassist families maintain quality of life for folkswith HD for as long as possible. Although wecurrently have no cure for this disease, we dohave the ability to allow individuals to accesstreatments to help them manage thisdebilitating illness. While we work on a cureand find hope for tomorrow, we have toensure that families affected by HD can accessthe help they need today. ■

1 Vinther-Jensen, Tua, Larsen, I.U., Hjermind, L.E., Budtz-

Jørgensen, E., Nielsen, T.T., Nørremølle, A., et al. A

clinical classification acknowledging neuropsychiatric

and cognitive impairment in Huntington’s disease.

Orphanet J Rare Dis. 2014;9(1):114.

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2 Simpson, Jennifer A., Lovecky, D., Kogan, J., Vetter, L.A.,

and Yohrling, G.J. “Survey of the Huntington’s Disease

Patient and Caregiver Community Reveals Most

Impactful Symptoms and Treatment Needs.” Journal of

Huntingtons Disease 5, no. 4 (2016): 395-403.

doi:10.3233/jhd-160228.

3 Stout, Jane C., Paulsen, J.S., Queller, S., Solomon, A.C.,

Whitlock, K.B., Campbell, J.C., et al. Neurocognitive

signs in prodromal Huntington disease.

Neuropsychology. 2011; 25(1):1-14.

4 Loy, Clement T., and McCusker, E.A. “Is a Motor

Criterion Essential for the Diagnosis of Clinical

Huntington Disease?” PLoS Currents, 2013.

doi:10.1371/currents.hd.f4c66bd51e8db11f55e1701af937

a419.

5 Van Walsem, Marleen R., Howe, E.I., Iversen, K., Frich,

J.C. and Andelic, N. “Unmet needs for healthcare and

social support services in patients with Huntington’s

disease: a cross-sectional population-based study.”

Orphanet Journal of Rare Diseases 10, no. 124

(September 28, 2015).

https://ojrd.biomedcentral.com/articles/10.1186/s13023

-015-0324-8 .

6 Dey, Judith, Rosenoff, E., and West, K. “Benefits of

Medicaid Expansion for Behavioral Health.” March 28,

2016. Accessed May 20, 2017.

https://aspe.hhs.gov/system/files/pdf/190506/BHMedi

caidExpansion.pdf .

Jennifer SimpsonManager of Advocacy and YouthProgrammesHuntington’s Disease Society of Americawww.hdsa.orgwww.twitter.com/HDSA

Huntington’s DiseaseSociety of America

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Naoko Tanese, PhDAssociate Dean for Biomedical Sciences

Director, Sackler Institute of Graduate Biomedical Sciences

New York University School of MedicineTel: +1 212 263 8945

tanesn01@med.nyu.edu https://med.nyu.edu/faculty/naoko-tanese

http://sackler.med.nyu.edu