Deciphering the Enigma of Human Poly(Q) Disorders: Contribution of Drosophila melanogaster

M Dhruba Singh, Soram Idiyasan Chanu, Surajit Sarkar

M Dhruba Singh, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA
Soram Idiyasan Chanu, Surajit Sarkar, Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110 021, India

Correspondence to: Surajit Sarkar, Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110 021, India.
Email: sarkar@south.du.ac.in
Telephone: +91-11-24157331
Fax: + 91-11-24112761
Received: August 27, 2015
Revised: December 6, 2015
Accepted: December 11, 2015
Published online: March 1, 2016


Poly(Q) disorders includes neurodegenerative disorders associated with irreversible loss of specific neurons in adult brain. The causative factor has been identified as the abnormal expansion of CAG repeats in protein coding region of the gene which leads to formation of protein Inclusion Bodies (IBs) and altered cellular physiology. The molecular pathogenesis of poly(Q) disease involves protein mis-folding, aggregate formation, blockage of axonal transport, mitochondrial dysfunction and global transcriptional dysregulation. Formation of inclusion bodies is a hallmark and common characteristic feature of all poly(Q) disorders. In view of limitation attached with human genetics, Drosophila has emerged as an excellent system to model the human neurodegenerative disorders due to availability of flexible yet powerful genetic tools and owing to the similarity between some of the fundamental cellular processes with humans. Drosophila system has been extensively utilized not only to decipher the mechanistic detail of diseases pathogenesis but also to screen several genetic and chemical modifiers which could potentially help in designing novel therapeutic strategies. The Drosophila melanogaster, thus, expected to contribute meaningfully towards novel discoveries to design novel therapeutic strategies to combat the devastating human neurodegenerative disorders.

© 2016 ACT. All rights reserved.

Key words: Polyglutamine diseases; Inclusion bodies; Drosophila

Singh MD, Chanu SI, Sarkar S. Deciphering the Enigma of Human Poly(Q) Disorders: Contribution of Drosophila melanogaster. International Journal of Neurology Research 2016; 2(1): 216-223 Available from: URL: http://www.ghrnet.org/index.php/jnr/article/view/1361


Polyglutamine [poly(Q)] diseases represent a group of neurodegenerative disorder which exhibit some common clinical symptoms and share comparable mechanism of molecular pathogenesis. Some of the common symptoms of poly(Q) disorders include loss of body coordination, memory, cognitive thinking and difficulty in speech[1]. There are nine of the neurological diseases: Spinal and bulbar muscular atrophy (SBMA), Huntington’s disease (HD), six of the Spinocerebellar ataxias (SCA1, 2, 3, 6, 7 and 17) and Dentatorubral pallidoluysian atrophy (DRPLA) which are commonly grouped as poly(Q) diseases[2,3,4]. In general, poly(Q) disorders are late onset, progressive in nature and cause degeneration of specific population of neurons in the brain as per the characteristics of each disease type[2]. For instance, in HD basal ganglia and cortical neurons are highly affected whereas purkinje cells in cerebellum are mostly affected in case of SCA3. Most forms of the poly(Q) disorders are dominantly inherited except spinal and bulbar muscular atrophy which shows X-linked recessive inheritance pattern[5].

Molecular mechanism underlying the pathogenesis of poly(Q) disorders

The first case of poly(Q) disease was reported by George Huntington in 1872 and named “chorea” due to dance like movement of patient suffering from this neurodegenerative condition[6]. For several years the underlying mechanism and cause of disease remain obscure but the advancement in biological sciences particularly in later part of twentieth century paved way to the understanding of predominant basis of the onset of disease. It was found that expansion of CAG repeat in the coding sequence of androgen receptor was associated with X-linked neuromuscular disorder, commonly known as Spinal and Bulbar Muscular Atrophy (SBMA) or Kennedy’s disease[7]. Subsequently, genetics of Huntington’s disease (HD) was also deciphered and expansion of CAG repeat was established as the primary cause[8]. Thereafter, subsequent investigations in this area led to identification of 7 more of diseases caused by expansion triplet repeat other than CAG. For instance, fragile X syndrome (FRAXA) and Fragile X-associated tremor/ataxia syndrome (FXTAS) were identified to be associated with expansion of CGG repeats on the X chromosome[9,10]. On other hand Fragile XE mental retardation (FRAXE) is caused by expansion of GCC on X chromosome[11]. Myotonic dystrophy (DM) and Spinocerebellar ataxia type 8 (SCA8) develop due to abnormal expansion of CTG repeats on the respective genes, whereas FRDA (Friedreich’s ataxia) is caused by expansion of GAA repeat in the frataxin gene located on X chromosome[12]. Interestingly, Spinocerebellar ataxia type 12 (SCA12) is also caused by the expansion of CAG repeats in the untranslated region of the gene, and therefore, it is not considered as a poly(Q) diseases[13].

Formation of protein inclusion bodies (IB) and impairment of transcriptional machinery

Expansion of CAG repeats leads to major changes in cellular physiology such as formation of protein Inclusion Bodies (IBs), defects in protein folding machinery, impairment of transcriptional machinery, defect in axonal transport system, cellular apoptosis, mitochondrial defects and dysregulation of intracellular Ca2+ homeostasis (Figure 1). As depicted in Figure 1, expansion of CAG repeats leads to elongation of glutamine tract of the protein and alters its native conformation. The mis-folded protein interacts with other proteins comprising glutamine repeats and interrupts the molecular function(s) of the target proteins. Subsequently, such interaction results into formation of microscopic molecular aggregates commonly known as inclusion bodies[14,15]. The kinetics of the polymerization of poly(Q) proteins involve prolong lag period which facilitates formation of the nucleus of protein aggregate followed by extension period that allows rapid incorporation of monomers to form larger aggregates[16,17]. The propensity of the mutant proteins to form aggregate arises from their ability of self-interaction which is achieved above certain pathological threshold limit[18-20]. Mutated huntingtin protein (Htt) form β-pleated sheet conformations in-vitro similar to amyloid with a fibrillar structure[18]. Above the threshold limit, the poly(Q) protein are suggested to form polar zipper and associate with each other by hydrogen bonds[21]. Intriguingly, proteins with endogenous poly(Q) repeats confers instability in the conformation after acquiring additional poly(Q) in the existing stretch[19].

In neuronal cell culture, the transfection of mutant Htt resulted in formation of IBs depending upon the length of poly(Q) repeats; with longer repeats forming more number of inclusion bodies[22,23]. Transfection studies suggest that truncated mutant Htt translocate into the nucleus rapidly and exhibit more tendencies to form aggregates than the full length of the protein[24,25]. The overexpression of both, full length and truncated form of ataxin-3 protein results in aggregate formation, cellular toxicity and neurodegeneration[26-29]. In rat striatal neurons, addition of expanded poly(Q) repeats with an inert protein such as poly(Q)-green fluorescent protein (GFP) fusion construct resulted in formation of inclusion bodies demonstrating that the toxic moiety lies in the elongated poly(Q) tract[30]. Interestingly, in all the cases of murine model of Huntington’s disease, the N-terminal fragment of the mutated Htt was always found to be associated with protein aggregates. Subsequent studies have demonstrated that co-expression of the N-terminal of Htt protein along with its full length counterpart further accelerates the formation of inclusion bodies suggesting a clear role of N-terminal fragments in formation of protein aggregate[31-33].

Interestingly, similar kinds of protein aggregates have been observed in the brain tissues of SCA1 patient and cell lines expressing mutant ataxin-1[34]. Likewise, transgenic mice expressing mutant ataxin-1 in the cerebellar region develop characteristics of ataxia phenotype with the formation of inclusion bodies[35]. In majority of poly(Q) disorders, the mis-folded proteins present in nuclear inclusion bodies are highly ubiquitinated[27,36]. Occurrence of the ubiquitinated form of mutant ataxin-3 aggregates have also been reported in neurons of SCA3 patients[37]. Similarly, ubiquitinated inclusions are also found in brain tissues of SCA2 and SCA7 patients[38,39]. In SCA17, the nuclear inclusions associated with ubiquitin and TATA binding proteins (TBP) are found in putamen and frontal cortex[40]. In contrast, inclusion bodies in SCA6 patients were not ubiquitinated and were relatively cytoplasmic[41]. In spite of that, degeneration of Purkinje cells are quite prevalence in SCA6 patients and mutant CACNA1A transfected cell lines developed perinuclear inclusions resulting in apoptosis[41].

The poly(Q) repeats alters the function of calcium channel which could be one of the major factors driving the cellular degeneration[42]. Analysis of the brain cells of patients suffering from poly(Q) disorder revealed that in addition to the respective disease protein(s) the IBs were also comprised of several essential proteins/factors[43]. It appears that sequestration of essential proteins/ factors in IBs from their respective sites leads to altered homeostasis and cellular toxicity. The IBs gradually increase in size and grow as high molecular weight protein body and subsequently get transported into the nucleus by an unknown mechanism[43]. After reaching into the nuclear compartment it sequesters several transcription factors and results in alteration of transcriptional activity of the cell[43,44,45].

Formation of inclusion bodies is a hallmark and common characteristic feature of all poly(Q) disorders[46,28]. Interestingly, in most of the poly(Q) diseases the flanking DNA sequence which surround the CAG repeat domain largely contribute on the dynamics of inclusion body formation[47,48]. In Huntington’s disease, first exonic sequence which flanks the CAG repeats is crucial for the formation and regulation of inclusion bodies[49]. Similarly, in SCA3, relative position of poly(Q) stretch and a highly conserved domain, Josephin determines the kinetics of inclusion body formation[47].

Poly(Q) proteins exhibit intriguing characteristics of the ability to associate with other proteins harboring short non-pathogenic glutamine repeats by homotypic glutamate interactions[16,50]. Moreover, proteins with expanded poly(Q) repeats show tendency to accumulate in nucleus where presence of highly concentrated solutes allows it to interact with many transcription factors and cofactors[51,52]. In affected individual, the IBs grow in size with the progression of age and translocate themselves into nucleus. Inside the nucleus they bind to many more of glutamine rich transcription factors such as cAMP response element binding protein binding protein (CBP), TBP (TATA binding protein), TAFII130 and Specificity protein 1(sp1)[53-58]. The binding and sequestration of transcription factors make a significant negative impact on the rate of cellular transcription. One of the major factors that caught great attention is CBP which is a key regulator of transcription[53-58].

CBP (CREB binding protein) is one of the key transcription factors which interacts with several other factors and regulate transcription of crucial genes[59-61]. CBP core protein has histone acetyltransferase (HAT) activity which facilitates histone acetylation and opening of the histone chain around the DNA to make the DNA readily available for RNA polymerase[57,62]. In Huntington’s disease, the mutant Htt protein sequesters CBP into the inclusion bodies[53-55]. Moreover, CBP protein has also been found to localise with the inclusion bodies in post-mortem tissues, cell culture and poly(Q) disease models[63]. The sequestration of CBP by inclusion bodies leads to hypoacetylation and undermines the transcription activity depriving cell of the survival factors and ultimately cell is destined for apoptosis. The role of defective histone acetylation on poly(Q) pathogenesis is substantially documented by the fact that inhibitors of Histone deacetylase such as Suberoylanilide hydroxamic acid (SAHA), Trichostatin A (TSA) and Sodium butyrate alleviates the neurodegeneration in animal models and cell lines[53,64].

In addition to the compromised function of several transcription factors as discussed above, mice model of SCA1 shows reduced transcriptional efficiency of six additional neuronal genes which are involved in signal transduction and calcium homeostasis[65]. The down regulations of these genes are evident in Purkinje cells much before the detectable phenotype of SCA1[65]. In case of SCA3, several inflammatory response genes show up-regulation while expressions of several cell-surface receptor genes are repressed[66]. It appears that the loss of normal function of ataxin-3 protein together with the toxic gain of functions by mutant ataxin-3 contribute to the disease pathogenesis[66].

Modelling of human poly(Q) disorders in Drosophila melanogaster

Drosophila has emerged as an excellent system to model the human neurodegenerative disorders owing to the similarity between some of the fundamental cellular processes with humans and the fact that flies and humans share many structurally and functionally related gene families. It has been found that ~75% of the human disease causing genes have functional Drosophila homologues showing overall identity ranging from 40% between homologues to as much as 80-90% between conserved functional domains[67,68]. Presence of extensive yet easily analyzable form of nervous system also makes it suitable for disease modelling. Several added advantages that Drosophila offers over other conventional model systems such as yeast, C. elegans and human cell cultures include its genetic tractability due to a short generation time and life-span, ease of maintaining large population within the confines of a laboratory, high fecundity, presence of balancer chromosomes, a well worked out developmental processes and anatomy, absence of meiotic recombination in males, relatively small genome contained on four completely sequenced and annotated chromosomes and ability to perform large-scale genetic screens to identify potential modifiers of known disease phenotypes[69]. In addition, availability of a large number of various mutant lines available at several Drosophila stock centres also makes it a popular model organism. The most advantageous aspect of poly(Q) disease modelling in Drosophila lies in the easy screening of genetic and chemical modifiers which subsequently help in designing novel therapeutic strategies.

Adult flies with their sophisticated brain and nervous system organized into specialized neural centres are capable of exhibiting complex behaviours such as learning and memory, much like the human brain. Interference in such well-coordinated motor behaviours serves as a highly sensitive readout of neuronal dysfunction and subsequent genetic analysis. Moreover, fly models are extremely useful while carrying out pharmacological screens for identifying novel therapeutic drug targets as they lack a blood brain barrier that can prevent the access of the drug to the central nervous system tissues[70]. Moreover, the response towards many drugs that act within the CNS is similar to the effects observed in mammalian systems[70]. The life cycle of Drosophila consist of four stages: embryonic, larval, pupal and adult stage. The larval stage can be further subdivided into 3 stages namely 1st instar stage, second instar stage and third instar stage. The neurogenesis in Drosophila starts around 5 hours after egg laying[71,72]. The Central nervous system of Drosophila is composed of neurons and glial cells. The neurons are tightly associated with glial cells by a structure known as commissure[73]. In Drosophila various adult organs and anatomical structure develops from 10 pairs of imaginal discs and the genital disc.

One of the most valuable tools that Drosophila bestows for neurodegenerative disease modelling is the use of its compound eyes for easy and direct evaluation of neurodegeneration. The adult Drosophila eye is developed from the rudimentary organ called as eye imaginal disc[74,75]. The eyes are not indispensable for the survival and presence of regular hexagonal lattice of each ommmatidium gives easily recognizable phenotype even with the naked eye[76]. Expression of pathogenic poly(Q) protein containing 108 CAG repeat in eye perfectly mimics the degeneration of neuronal tissues as found in post-mortem brain of HD patient[72]. Interestingly, the development of disease could be traced from the third instar larval eye imaginal disc. The eye disc corresponds to the posterior region of a common imaginal disc which develops into adult eye and the anterior part develops to antennal structure. The morphogenetic furrow is the area where intense cell division and neuronal differentiation takes place[74,75].

The compound eye of Drosophila is composed of individual unit called ommatidium. Each ommatidium is composed of different units of cells i.e. 2 primary cells, 6 secondary cells, 3 tertiary cells, 8 photoreceptors cells and 4 cone cells. The 7th photoreceptors cells lie above the 8th photoreceptor cells, and therefore, not visible at the same visual focal plane[77]. In addition to eye tissues, Drosophila brain is also used as model organ in many of the neurodegenerative disorders. Drosophila adult brain is consists of about 10,0000 neurons, organised into regions with specialized functions such as learning, olfaction, memory and vision[78,79]. The mushroom body is highly advanced information processing centre composed of about 2,500 neurons with densely packed cell bodies know as Kenyon cells[80]. Mushroom body is homologous to the hippocampus of human brain and involved in olfactory memory and learning[80].

Several approaches have been exploited so far to study neurodegeneration in Drosophila. Initially, the classical forward genetic approach was utilized wherein random mutations were created to select a phenotype showing brain degeneration which was then analysed with the aim of identifying the gene responsible for the mutant phenotype[81-83]. However, such screens either remain incomplete due to ignorance of redundant loci and epigenetic effects or difficult to undertake due to non-measurable phenotypes. Additionally a classical genetic approach takes significantly longer. Therefore, alternative reverse genetics-based approaches have been undertaken to elucidate pathogenic pathways and remedial strategies of neurodegenerative disorders. Moreover, the dominant nature of most of the mutant alleles of poly(Q) disorders suggested that perhaps these diseases could be modelled in Drosophila by introducing the mutant allele into the fly genome and producing transgenics. A binary system was used for this purpose which benefits from the property of the yeast transcriptional activator GAL4 protein to bind to a small upstream activator sequence (UAS) of genes to drive transcription. Foreign genes can be cloned into transposable P-element vectors under the control of a UAS sequence and crossed with transgenic flies expressing the yeast Gal4 gene in a tissue-specific manner[84]. In this context it is important to note that the transgene remains silent until crossed with a desired Gal4 line. Most commonly used promoters in case of neurodegenerative disorders include pan-neuronal elav-Gal4 driver[85,86] and eye specific glass multiple reporter element (gmr) driver[87,88]. Neurodegeneration can be monitored either by measuring the loss of visible photoreceptors neurons in the eyes or by evaluating motor function by the climbing assay or by checking for the lethality of the organism. There are many advantages of using the UAS-Gal4 system for transgenic expression: first, the conditional nature of expression which prevents deleterious effects of constitutive expression of transgene; second, a single UAS transgenic line can be ectopically expressed in many different tissue types, depending upon the GAL4 line being used; third, the system is capable of yielding much higher levels of expression of the gene of interest as compared to direct promoter-fused transgenes.

Expression of mutant form of AR, ataxin-1, ataxin-3 and Htt produces neuropathological condition reminiscent of human form[28,89,90]. The severity of poly(Q) diseases was also found to be dependent upon the length of poly(Q) repeat as found in human disease conditions. Moreover the progressive nature was also established in flies as the defects are observed only in mature larvae, pupal stage or in adult stages. The earliest SCA1 models generated in Drosophila were SCA1Q82 and SCA1Q30 transgenic lines[89]. The overexpression of SCA1Q82 produced a strong phenotype with complete degeneration of retinal structures and neuronal cell masses. Comparatively, expression of SCA1Q30 produces milder phenotype with partial degeneration of retinal structures. Interestingly, temperature mediated increased expression of SCA1Q30 was found to enhance the degenerative phenotypes[89]. Moreover, overexpression of wild type ataxin-1 was also found to induce neurodegeneration[89].

Targeted expression of truncated form of SCA3 containing 78 poly(Q) repeats induces neuronal degeneration in Drosophila eye[28]. However, expression of normal length of 27 poly(Q) repeats did not produce any degenerative phenotype[28,91]. As depicted in Figure 2, formation of inclusion bodies are equally evident in targeted Drosophila tissues as found in affected human brain. Occurrence of such inclusion bodies in Drosophila tissues was accompanied by enhanced cell death indicating implication of inclusion bodies in progression of neurodegeneration. The designing of Drosophila HD model was done by using truncated form of htt gene containing 1st exon with the expanded form of CAG repeats[92]. The expression of HttQ93 produces progressive loss of photoreceptors with subsequent aging[92]. The transgenic stocks containing Q0 and Q128 repeats were generated using the N-terminal fragments of Htt gene with first 588 amino acids along with the caspase cleavage site. Targeted expression of Q0 in Drosophila eye did not produce any morphological defect, whereas the expression of Q128 reduces the photoreceptors depolarisation and loss of synaptic transmission[93]. The aggregates were also formed along with the axons which carry the synaptic stimulus to the CNS[93].

Modelling of human poly(Q) and other neurological disorders in model organisms provides an ultimate tool for devising novel therapeutic strategies to suppress progression or manifestation of disease phenotypes[94,95,96]. Moreover, analysis of behavioural changes and neuropathological features could be studied in greater detail as the cellular processes of disease pathogenesis are completely recapitulated in most of the model system. It also facilitates in identification of modifier genes and pathways which could be potentially utilized as drug targets. The Drosophila model system is one of the oldest as well as one of the widely used model system for devising the testing paradigms such as genetic screens and chemical screens of poly(Q) disorders. Several genetic and chemical modifiers of human poly(Q) disorders have been identified utilizing Drosophila disease models. Moreover, some of such modifiers are being investigated and potential drug targets and to design novel therapeutic approaches.

Concluding remarks

In brief, Drosophila provides a powerful genetic tool to investigate various aspects of human neurodegenerative poly(Q) disorders. However, given an exceptional model system and the varieties of tools available to study modified phenotypes, the precise mechanism which leads poly(Q) induced toxicity still stands as a debatable issue. Therefore, efforts should be given to unravel the molecular nature of the neurotoxic species arises in each disease type, and to decipher the key neuronal functions which get altered due to accumulation of toxic protein aggregates. Moreover, parallel comprehensive studies should be planed for not only to decipher the mechanistic details of disease pathogenicity but also to perform large scale screening for the designated molecules and genetic modifiers to assess their potential as the modifier of disease phenotypes, which could be verified further for their reproducibility in higher model systems. The Drosophila melanogaster has, thus, proved its worth in the field of neurobiology and will continue to contribute meaningfully towards novel discoveries to combat the devastating human neurological disorders.


Research work in laboratory is supported by grants from the Department of Biotechnology (DBT), Government of India, New Delhi; DU/DST-PURSE scheme and Delhi University R & D fund.


The authors have no conflicts of interest to declare.


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