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Androgen receptor with elongated polyglutamine tract forms aggregates that alter axonal trafficking and mitochondrial distribution in motor neuronal processes¹

FEDERICA PICCIONI^*, PAOLO PINTON, SILVIA SIMEONI^*, PAOLA POZZI^*, UMBERTO FASCIO, GUGLIELMO VISMARA^*, LUCIANO MARTINI^*, ROSARIO RIZZUTO and ANGELO POLETTI^*2

^* Institute of Endocrinology, Centre of Excellence for the Study and Treatment of Neurodegenerative Diseases, University of Milan, Milano, Italy;
Interdisciplinary Center for the Study of Inflammation and Telethon Center for Cell Imaging, Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy; and
Department of Biology, section of Zoology and Interdipartimental Center for Advanced Microscopy, University of Milan, Italy

²Correspondence: Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano Italy. E-mail: Angelo.Poletti{at}unimi.it

SPECIFIC AIMS

The CAG/polyglutamine (polyGln) -related diseases include nine different members that together form the most common class of inherited neurodegenerative disorders; neurodegeneration is linked to the same type of mutation found in unrelated genes, consisting of an abnormal expansion of a polyGln tract normally present in wild-type proteins. The androgen receptor (AR), one of these proteins, is responsible for spinal bulbar muscular atrophy (SBMA), an X-linked recessive disease characterized by motor neuronal death. Androgen-dependent ‘neuropil’ aggregates are formed by SBMA AR, but their role is still debated. In this study, we analyzed whether neuropil inclusion may alter distribution of mitochondria and the axonal transport mediated by kinesin in cell processes of motor neurons.

PRINCIPAL FINDINGS

1. Alteration of mitochondrial distribution and neurite swelling in cells bearing aggregates of the androgen receptor with an elongated polyglutamine tract
Analysis performed on living cells showed that in immortalized motor neurons transfected with a chimera of wild-type AR with GFP (GFP-AR.Q0 or GFP-AR.Q22), the AR protein is normally concentrated into the nuclei, whereas the mitochondria (evidenced by a blue FP containing a mitochondrial localization signal: mtBFP) are well distributed in the cell cytoplasm. Cells transfected with mutated AR containing an elongated polyGln tract (GFP-AR.Q48) show aggregates localized in the cytoplasm and cell processes. Figure 1 shows the appearance of representative NSC34 cells cotransfected with control GFP-AR.Q0 (Fig. 1A, B ) or GFP-AR.Q22 (Fig. 1C, D ) and mtBFP after testosterone treatment. AR proteins (Q0 or Q22) are confined in the nuclei, whereas mitochondria are distributed in the cell cytoplasms and homogeneously along cell processes. In NSC34 expressing GFP-AR.Q48 and mtBFP, accumulation of mitochondria in the cell processes is detectable in close association with neuropil aggregates. Figure 1E, F shows that in NSC34/GFP-AR.Q48/mtBFP, mitochondria are associated with two small aggregates; the neurite is unexpectedly large with a constant diameter (Fig. 1F ). Figure G, H shows representative NSC34/GFP-AR.Q48/mtBFP with an aberrant neurite morphology due to neuropil aggregates. In this case, the fiber has a smaller diameter (Fig. 1H ) than the one shown in Fig. 1F ; the accumulation of insoluble proteinaceous material (green) results in three abnormal enlargements of the neurite caliber, accompanied by an increased number of mitochondria (in blue) in the swelled portions. Figure 1I, J shows a phenotype of NSC34/GFP-AR.Q48/mtBFP cells similar to that depicted in Fig. 1H , but apparently induced by the intersection of two processes arising from different cells (higher magnification in Fig. 1K ).

View larger version (99K): [in this window] [in a new window]   Figure 1. Swelling of processes containing neuropil aggregates. NSC34 were cotransfected with GFP-AR.Q(n)s proteins and a mtBFP treated with testosterone. A) NSC34 expressing GFP-AR.Q0 (green) and mtBFP. B) The merge of panel A with corresponding contrast phase microscopy analysis to visualize cell morphology. C) NSC34 expressing GFP-AR.Q22 (green) and mtBFP. D) The merge of panel C with the corresponding contrast phase microscopy analysis to visualize cell morphology. E, G, I) NSC34 expressing GFP-AR.Q48 and bearing neuropil aggregates (green) and mtBFP (blue). F, H, J) Merge of panels E, G, I with contrast phase microscopy analysis to visualize cell morphology. E, F) NSC34/GFP-AR.Q48/mtBFP bearing jams in the cell processes: mitochondria are entrapped by two small aggregates. The cell neurite appears unexpectedly large and constant in diameter, with a substantially normal phenotype. G, H) NSC34/GFP-AR.Q48/mtBFP: mitochondria and small AR aggregates: the accumulation of insoluble material results in abnormal and localized enlargements of the neurite accompanied by an increase number of mitochondria in the swelled neurite. I, J) A NSC34/GFP-AR.Q48/mtBFP shows a neurite swelling similar to that depicted in panel G and H, but this is apparently induced by the intersection of two processes arising from different cells. Higher magnification of these two intersecting neurites is reported in panel K. Scale bars = 10 µm.

2. Motor protein kinesin is abnormally distributed in motor neuronal cells containing neuropil aggregates of androgen receptor with the elongated polyglutamine tract
To evaluate the potential neurotoxicity of GFP-AR.Q48 neuropil aggregates in immortalized motor neurons, we investigated whether neuropil aggregates may alter axonal transport in immortalized motor neurons transfected with the SBMA AR. Since mitochondria are rapidly transported on microtubules through the motor protein kinesin, we analyzed the distribution of kinesin in cells bearing neuropil aggregates. Figure 2 shows confocal microscopy images on kinesin immunoreactivity in NSC34/GFP-AR.Q(n)s. In control NSC34/GFP-AR.Q0 (Fig. 2A , green) or NSC34/GFP-AR.Q22 (Fig. 2C , green), kinesin (red) is detectable in the cytoplasm, with homogeneous distribution along the cell processes (Fig. 2B, D : light-transmitted microscopy). The kinesin distribution in cells bearing neuropil aggregates is depicted in Fig. E, which shows two contiguous cells (see Fig. 2H ). Figure 2F shows that both NSC34 cells are GFP-AR.Q48 positive in the two nuclei, but only the upper cell contains neuropil aggregates. Figure 2G shows that kinesin is normally distributed in the lower cell devoid of neuropil aggregates; the presence of inclusions in the processes (upper cell) heavily alters kinesin distribution in the neurites, similar to the case of the mitochondrial study. At higher magnification (Fig. 2I : double fluorescence; Fig. 2J : light transmission), the two parallel neurites are evident with aggregate location within the upper neurite (merging in Fig. 2K ).

View larger version (78K): [in this window] [in a new window]   Figure 2. Kinesin distribution in NSC34 cells containing neuropil aggregates. NSC34 were transiently transfected with GFP-AR.Q(n)s and treated with testosterone. The cells were processed using an anti-kinesin monoclonal antibody (MAB1614) and visualized with a TRICT secondary antibody. A) Distribution of kinesin (red) in control GFP-AR.Q0 (green) transfected NSC34 cells. B) Light-transmitted microscopy of cells in panel A. C) Distribution of kinesin (red) in control GFP-AR.Q22 (green) transfected NSC34 cells. D) Light-transmitted microscopy of cells in panel C. Images were obtained with a confocal fluorescence microscope. E–H) Analysis of GFP-AR.Q48 transfected cells. E) Kinesin immunoreactivity in two contiguous cells expressing GFP-AR.Q48. F) Fluorescence analysis to detect GFP-AR.Q48, both NSC34 cells were positive for AR, but only one contained neuropil aggregates. G) Merging analysis of panels E, F. H) Light transmission analysis of the same cells. I, J) Higher magnification of the neurites depicted in panels G, H representing the region of the neurites containing the AR aggregates and kinesin accumulation. K) Merging analysis of panels I and J. Little distortion of the axonal caliber was observed also in this case (J). Scale bars = 10 µm.

CONCLUSIONS AND SIGNIFICANCE

The mutated AR with the polyGln tract elongation, responsible for SBMA, seems to confer a toxic gain-of-function to the mutant protein. Several reports have shown that this gain-of-function may be linked to formation of intracellular aggregates. We have now analyzed neuropil aggregates formation in immortalized motor neurons and shown that when they are present, the intracellular distribution of the motor protein kinesin is impaired and consequently the distribution of mitochondria (actively transported by kinesin) is altered in the same processes. This suggests that all the kinesin-mediated fast axonal transport is blocked by the inclusion and leads us to hypothesize that the transport of other important components vesiculated by other motor proteins are also impaired. Alterations of axonal functions, in SBMA, as in other polyGln-related disorders, may represent one of the multifactorial events able to induce cell death, since neuropil aggregates may also deprive neuronal processes of factors/components important for axonal/dendritic functions, becoming toxic by acting as one of the multistep processes activated during neurodegeneration.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1035fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.01-1035fje

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