Aberrant Vesicular Trafficking in Huntington’s Disease
Neil Aronin, M.D.
University of Massachusetts Medical School, Worcester, MA
David Mahoney Neuroimaging Program
June 2005, for 2 years
Aberrant Vesicular Trafficking in Huntington's Disease
Huntington's disease (HD) is caused by a mutation in the gene huntingtin, which results in production of a protein, huntingtin (htt), with a polyglutamine expansion near its amino-terminus. How the mutation causes neuronal loss in the brain is not established. It is believed that mutant htt causes a gain of abnormal function in neurons that leads to neuronal dysfunction and cell death. Progress in understanding pathogenesis in HD is hindered because the role of normal htt in neurons is still unclear. It is known that htt localizes to vesicles and interacts with proteins involved in vesicle trafficking. Aggregates of mutant htt interfere with vesicle movement in axons and with endocytosis of transferrin. Demonstration of a definitive role of normal huntingtin in vesicle trafficking and of interference of mutant huntingtin in these processes remains to be accomplished.Our goal in this proposal is to use high speed, state of the art, biological imaging to elucidate subcellular mechanisms of normal huntingtin in vesicle movement and disruption of vesicle movement by mutant huntingtin. Our hypotheses are that normal huntingtin is required for the internalization of clathrin-coated vesicles and transport of recycling vesicles and that mutant huntingtin interferes with vesicle budding from the plasma membrane and from recycling endosomes.Aim 1 examines how normal huntingtin contributes to endocytosis and subsequent vesicle distribution in neurons, neuron-like cells, and embryonic stem cells with "knockout" for htt. We will use mRNA silencing techniques to nearly eliminate normal huntingtin in cells, to compare transferring uptake by total internal reflection fluorescence (TIRF) and clathrin movement and distribution by high speed, 3-D deconvolution epifluorescence microscopy.Aim 2 examines how mutant huntingtin might disrupt endocytosis and vesicle movement. Mutant huntingtin will be delivered to cells by lentivirus-containing mutant huntingtin, and we will use embryonic stem cells expressing mutant huntingtin. We will study transferrin uptake by TIRF and movement of clathrin vesicles by 3-D deconvolution epifluorescence.Our collaborators in the biological imaging core are expert in these applications and in deconvolution to provide details in time and space. The PI and Co-PI, Dr. DiFiglia, impart more than 25 years of study of Huntington's disease at cellular and subcellular levels. The collaboration presents a convergence of biological imaging and molecular strategies, in which the sum is greater than the individual parts. This proposal has clinical implications for Huntington's disease; the underlying molecular mechanisms involved in these effects need to be investigated to identify therapeutic targets to treat Huntington's disease.
Huntington's disease is caused by a mutation in a gene, huntingtin. The protein product of huntingtin has a polyglutamine expansion near its amino-terminus. The mutant huntingtin protein leads to neuronal dysfunction and loss of brain cells; patients with Huntington's disease develop dementia and severe movement abnormalities and eventually become bed-ridden. How the mutation causes the problem is not established. It is known that huntingtin localizes to vesicles and interacts with proteins involved in vesicle trafficking; aggregates of mutant huntingtin interfere with vesicle movement in axons and with endocytosis of transferrin. We have two connected hypotheses. We hypothesize that normal huntingtin is required for the internalization of clathrin-coated vesicles and transport of recycling vesicles. We further hypothesize that mutant huntingtin interferes with vesicle budding from the plasma membrane and from recycling endosomes.
Our goal is to use high speed, state of the art, biological imaging to elucidate subcellular mechanisms of normal huntingtin in vesicle movement and disruption of vesicle movement by mutant huntingtin. Accomplishing this goal is expected to provide a foundation for identifying therapeutic targets to treat Huntington's disease.
Our overall approach is to use the powerful capabilities of a novel, custom-built microscope system that combines high speed TIRF (total internal reflection fluorescence) illumination and sub-second, 3-D epifluorescence deconvolution microscopy in the same imaging apparatus. This system will enable us to study the role of normal and mutant huntingtin in the movement of single vesicles that are near the plasma membrane (100 nm) and that move within the three dimensionally rendered cell cytoplasm. The microscopy provides real-time imaging in a movie, so that actual movement can be measured.In our first set of experiments, we will examine contributions of normal huntingtin to vesicle movement after reducing huntingtin by RNA interference (RNAi). We will study movement of vesicle populations. Fluorescent-labeled transferrin (Alexa 568) bound to its receptor will be analyzed in TIRF illumination and high speed, sub-second 3-D epifluorescence deconvolution microscopy. Changes in vesicle movement will be measured in cells, in which the normal huntingtin has been reduced by RNAi, to be measured by Western blot analysis in each experiment. We use cholesterol-conjugated siRNA to facilitate siRNA delivery to primary murine cortical neurons and clonal striatal cells. The goal is to identify the role of huntingtin in movement of recycling endosomes transporting transferring receptor, the vesicle movement to be measured in real time.In the next set of experiments, we will examine effects of mutant huntingtin on vesicle trafficking. We will use primary murine cortical neurons transduced by mutant huntingtin or wild type huntingtin containing lentivirus. Neurons so treated will express mutant or wild type huntingtin protein. The effect of mutant huntingtin or wild type huntingtin on single vesicle movement will be detected by TIRF illumination and deconvolution microscopy, to measure the movement of membrane patches that internalize Alexa-568 labeled transferrin. This approach allows us to compare the time course of transport of transferring receptor to and from recycling endosomes in neurons that contain mutant huntingtin or wild type huntingtin. The high resolution and the real time capabilities provide the methods for us to test our hypothesis that mutant huntingtin interferes with vesicle movement in neurons.
Lay Results: Huntington’s disease is an inherited brain disease that occurs mostly in adults and less often in children. Patients with Huntington’s disease develop dementia and abnormal movements; they require long-term nursing care. The cause of Huntington’s disease is a mutation in gene called huntingtin. The mutant gene makes a mutant protein, called mutant huntingtin.Previous studies point to involvement of the mutant huntingtin in interfering with normal trafficking (movement) of chemicals in cells. Interruption of normal trafficking will harm cells and can lead to premature cell death. We tested the idea that mutant huntingtin disrupts cellular trafficking by examining movement of packets of information (vesicles) in cells from patients with Huntington’s disease. We used a high resolution microscope in live cells to visualize the vesicles and measure their movement. The advantage of this approach is that movement is very rapid and is beyond the detection of standard microscopy. We found that mutant huntingtin impairs trafficking of a specific set of vesicles. The results have several implications. First, normal huntingtin regulates vesicle movement back to the outer cell membrane, where it can be refilled with chemical information to recycle back into the cell. Second, mutant huntingtin impedes the movement of the recycling vesicle. The affected vesicle less effectively returns to the outer membrane of the cell and cannot readily pick up new chemical information to enter the cell. We predict disruption of vesicle recycling by mutant huntingtin weakens brain cells and contributes to premature neuronal death in Huntington’s disease.
Scientific Results: Polyglutamine expansion in huntingtin causes Huntington’s disease, a progressive neurodegenerative disease with no effective cures. How the mutation causes the disease is not clear, but might involve aberrant vesicular trafficking. We combined traditional biochemical and cell biological methods with the advanced live cell imaging to explore if mutant huntingtin impairs vesicular trafficking particularly in the endocytic pathway, which is essential for cells to take up nutrients and to tune up the response to environmental stimuli. Both biochemical and cell biological studies and live cell imaging revealed that the mutation in huntingtin impairs receptor recycling from endosomes back to the plasma membrane. Detailed studies demonstrate that the mutation in huntingtin has no effect on internalization, the first step of the endocytic pathway. However, live cell imaging revealed that fewer small vesicles were generated to carry recycled transferrin receptors in cells expressing mutant huntingtin; instead more large vesicles and long tubules were formed in these cells than in cells expressing normal huntingtin. Such a switch in the vesicle size points out that the mutation in huntingtin causes a deficit in generating small vesicles from endosomes. Live cell imaging also revealed that the transport speed of vesicles along cytoskeletons is faster in cells expressing mutant huntingtin. This might be a compensatory effect for the deficit in producing small vesicles from endosomes in the presence of mutant huntingtin.
DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, Martin E, Vonsattel J-P, Carraway R, Reeves SA, Boyce FM, Carraway R, and Aronin N: Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14:1075-1081, 1995.
Aronin N, Chase K, Young C, Sapp E, Schwarcz C, Matta N, Kornreich R, Sheth A, Landwehrmeyer B, Bird E, Vonsattel J-P, Smith T, Carraway R, Boyce FM, Beal MF, Young AB, Penney JB, and DiFiglia M: CAG expansion affects the expression of mutant huntingtin in the Huntington’s disease brain. Neuron 15:1193-1201, 1995.
DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, VonSattel and Aronin N: Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277: 1990-1993, 1997.
Velier J, Kim M, Schwarz C, Kim TW, Sapp E, Chase K, Aronin N and DiFiglia M: Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways. Exper Neurol 152:34-40, 1998.
Kim M, Sapp E,Laforet GA, Aronin N and DiFiglia M: Forskolin and dopamine D1 receptor activation increase huntingtin’s association with endosomes in immortalized neuronal cells of striatal origin. Neuroscience 89:1159-1167, 1999.
Kegel KB, Kim M, Sapp E, McIntyre C, Castano JG, Aronin N and DiFiglia M: Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. J Neurosci 20:7268-7278, 2000
Laforet GA, Sapp E, Chase K, McIntyre C, Boyce FM, Campbell M, Cadigan BA, Warzecki L, Tagle DA, Reddy H, Cepeda C, Calvert CR, Jokel ES, Klapstein GJ, Ariano MA, Levine MS, DiFiglia M and Aronin N: Mutant huntingtin dependent cellular changes in the cortex predict behavior and electrophysiological abnormalities. J Neurosci 21:9112-9123, 2001
.Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, Kegel KB, Qin Z-H, Aronin N and DiFiglia M: Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington’s disease brains, associate with membranes, and undergo calpain-dependent proteolysis. Proc Natl Acad Sci USA 98:12784-12789, 2001.
Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, and Zamore PD: Unexpected asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199-208, 2003.
Qin ZH, Wang Y, Sapp E, Cuiffo B, Wanker E, Hayden MR, Kegel KB, Aronin N, and DiFiglia M: Huntingtin bodies sequester vesicle-associated proteins by a polyproline-dependent interaction. J Neurosci 24:269-281, 2004.