Exp Neurobiol 2017; 26(6): 321-328
Published online December 31, 2017
© The Korean Society for Brain and Neural Sciences
Dong-Kyu Kim1, Kyu-Won Cho1, Woo Jung Ahn1, Dayana Perez-Acuña1,Hyunsu Jeong1,2, He-Jin Lee3,4 and Seung-Jae Lee1*
1Department of Medicine and Biomedical Sciences and Neuroscience Research Institute, Seoul National University College ofMedicine, Seoul 03080, 2Department of Psychology, Seoul National University, Seoul 08826, 3Department of Anatomy, School of Medicine, Konkuk University, Seoul 05029, 4IBST, Konkuk University, Seoul 05029, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-3668-7037, FAX: 82-2-447-5683
Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an
The abnormal aggregation and accumulation of specific proteins in the form of cytoplasmic inclusion is common pathological feature of most age-related neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD) and amyotrophic lateral sclerosis (ALS). In general, higher the age, greater the incidence of the disease onset. Protein aggregation is promoted with aging , probably due to increased oxidative stress [2,3] and the decline in protein degradation system [4,5].
HD is a representative autosomal dominant inherited disease. Clinically, HD manifests motor defects, including facial convulsions, tremors, and chorea, and non-motor symptoms, such as memory disorders, depression, and anxiety . Classically, this neurological disorder has been associated with the progressive degeneration of the medium spiny neurons (MSNs) in the striatum , although evidence states that several brain areas are involved [8,9].
HD has a comprehensible cause, that is, an expansion of CAG repeat in the exon 1 of huntingtin gene, which translates to the expanded polyglutamine (polyQ) tract over pathogenic threshold (Q34) in huntingtin (HTT) protein . Extension of polyQ causes aggregation of HTT protein. The huntingtin aggregates were initially found in the nucleus [11,12] and also discovered in the cytoplasm and the processes of neurons in the brains of HD patients . Although the precise mechanism of neurodegeneration in HD has not been elucidated yet, misfolding and intracellular aggregation of HTT with expanded polyQ is clearly the key element of the pathogenesis and progression of the disease, leading to a toxic gain of function and neuronal cell death .
The pathological polyQ aggregates begin to appear in specifically in the putamen and caudate nuclei in the basal ganglia region, and subsequently spread to a wide area of the cerebral cortex, including motor cortex and frontal cortex . It has been suggested that the aggregate spreading is mediated by the direct cell-to-cell transmission of polyQ aggregates, a process that involves seeded polymerization of the protein in a prion like-fashion [16,17,18].
Elucidating the mechanism of aggregation transmission is an essential step towards the understanding of the development and progression of brain diseases. Here, we generated an
All worms were maintained, grown, on nematode growth medium (NGM) plates fed with
Plasmids, including P
To make vectors expressing the human huntingtin exon 1 with a polyglutamine stretch, a sense primer containing a
The procedure for BiFC transgenic lines was performed as previously described . To analyze the effects of expanded polyglutamine on aggregate transmission, P
Single-worm PCR analyses were performed as previously described . The genomic DNA released from a gravid single worm in each line was mixed with Ex Taq™ polymerase (RR001A; Takara Shuzo Co. Ltd, Shiga, Japan) and then single-worm PCR for target genes was performed in Bio-Rad MyCycler PCR Thermal Cycler system (Bio-Rad Laboratories Inc., Hercules, CA, USA).
The procedure for western blotting was performed as previously described . Protein samples obtained from the worm pellets were loaded onto 12% SDS-PAGE gels. Monoclonal anti-polyglutamine primary antibody was used for western blotting (MAB1574; Millipore Corporation, Temecula, CA, USA). Chemiluminescence detection was performed with ECL™ prime solution (RPN2232; GE Healthcare Life Sciences, Marlborough, MA, USA), and images were obtained using the Amersham imager 600 (GE Healthcare Life Sciences, Marlborough, MA, USA) and quantified with Multi Gauge (v3.0) software (Fujifilm, Tokyo, Japan).
Worms were collected, washed with M9 buffer (22 mM KH2PO4, 22 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4), and then immobilized with 10 mM sodium azide (S2002, Sigma-Aldrich) in M9 buffer. After removing the buffer, drop worms were placed on microscope cover glasses (HSU-0101242; Marienfeld Laboratory Glassware, Lauda-Königshofen, Germany), and covered with a coverslip. All images were obtained using Olympus FV1000 confocal laser scanning microscopy (Olympus, Tokyo, Japan).
Pharyngeal pumping rate of each line was monitored for 1 min at room temperature using Axio observer a1 inverted microscope (Carl Zeiss MicroImaging Inc., Göttingen, Germany). The pumping count was symbolized as PPM (Pumps Per Minute).
Life span analyses were performed as previously described . Eggs produced from gravid worms were synchronously grown up to the larval 4 (L4)-stage on NGM plates seeded with
All experiments were performed blind-coded and repeated at least three times. The graphs were drawn using Prism 5 software (Graphpad Software Inc., San Diego, CA, USA) and the values in the figures are represented as mean±s.e.m. All data were analyzed, compared for statistical significance by one-way ANOVA with Tukey's post-hoc test using Graphpad InStat version 3.05 software (Graphpad Software Inc., San Diego, CA, USA).
BiFC has been applied to visualization of dimerization and oligomerization of proteins in cells . This technique has also been successfully used to investigate the cell-to-cell transmission of α-synuclein in mammalian cells  and
To determine the effects of the length of polyQ on aggregate transmission, we analyzed the BiFC signals in the pharynx using confocal microscopy. The BiFC fluorescence in our system solely depends on the extent of transmission rather than the aggregation propensity, because the cell-autonomous protein aggregation would not generate BiFC fluorescence. All lines we generated produced BiFC fluorescence in both the pharyngeal muscle and neighboring neurons (Fig. 2A). This suggests that the transmission does occur between these two cell types and in a bidirectional manner. However, the lines with long polyQ tracts (Q97) exhibited stronger BiFC fluorescence than the lines with short polyQ length (Fig. 2A and B).
The transmission of both wild type and mutant polyQ proteins was increased with age. At all ages, the Q97 lines showed higher BiFC signal than the Q25 lines. While transgenic animals with Q25 exhibited largely diffuse patterns in the pharynx until day 8 (Fig. 2A), some inclusions did appear at day 13 in these animals (Fig. 2B). On the other hand, the Q97 animals developed the inclusion bodies as early as two day after the L4-stage (Fig. 2C~E), and the numbers of BiFC-positive inclusion bodies were considerably higher in the Q97 lines compared to the Q25 lines at all ages (Fig. 2C and D). These data indicate that both transgenic animals showed an age-dependent increase in aggregate transmission with polyQ expansion accelerating the rate of aggregate transmission between muscles and adjacent neurons.
In order to investigate the effects of different polyQ lengths on the degenerative phenotypes in
To analyze behavioral changes in these animals, we measured the pharyngeal pumping rates. The pumping rates of transgenic worms containing Q25, compared to the wild-type N2, did not change significantly at day 8 (Fig. 3C). However, at day 13, the reduction of pumping rates in Q25 animals had become significant (Fig. 3C). The pumping rates of the Q97 animals were significantly decreased already at day 8 and exhibited more deficits at all ages than the Q25 animals (Fig. 3C).
In life span analyses, transgenic worms with Q25 exhibited a slightly, but not significantly, decreased life span compared to the N2 worms at day 8 (Fig. 3D), whereas the life span of Q97 animals were significantly decreased than the other lines (Fig. 3D). There is no significant difference of the survival rates among three independent lines in each transgenic model, validating consistency of the data. When average life span was concerned, the transgenic lines with Q25 exhibited shortened life span compare with N2 worms, while the Q97 worms showed further reduced mean lifespan (Fig. 3E).
Previous studies have shown that aggregates generated by HTT fragments with expanded polyQ propagated between cells in both cultured neurons and animal models [16,17,18,27,28]. This occurs transsynaptically  as well as between neurons and glia . In humans, evidence suggested spreading of HTT aggregates, showing the presence of mutant HTT in neuronal grafts in the cortex . In the current study, we developed an
Here, we suggested that the rate of transmission correlated with degenerative phenotype. However, overexpression of mutant polyQ can be toxic cell-autonomously even without transmission. Therefore, the degenerative phenotypes we observed may not necessarily be due solely to the transmission of polyQ. In the current study, we cannot rule out the possibility of cell-autonomous cytotoxicity of polyQ proteins.
We chose to use the pharyngeal system to study the relationship between aggregate transmission and the associated phenotypes. The pharynx has its own autonomic nervous system, which are known as pharyngeal nervous system, and its neuronal processes are connected with adjacent pharyngeal muscles . Because pharyngeal specific motor neurons regulate movement of the muscle, degeneration of axonal processes would lead to abnormal motor symptoms and affect the lifespan. One of the strengths of our
Another strength of the model is the age-dependent manner of aggregate transmission, which would allow for the study of effects of aging, a biggest risk factor for many neurodegenerative diseases. In the previous study using
In conclusion, we have developed a novel