Elavl3 is essential for the maintenance of Purkinje neuron axons

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3−/− mice. Elavl3−/− mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3−/− Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3−/− Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3−/− mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3−/− mice will provide new insight regarding axonal degenerative processes.


Supplementary materials and methods
Immunohistochemistry. Mice were deeply anesthetized and tissues were fixed by perfusion with 4% PFA buffered with 0.1 M phosphate buffer. Brains were removed and postfixed in 4% PFA at 4 °C overnight. After that, cerebella were cryoprotected with 10%, 20%, and 30% sucrose solutions, embedded in OCT compound (Sakura Finetek, Tokyo, Japan), and quickly frozen. The cerebella were cut into 20-or 40-µm-thick sections using a cryostat (HM525, Thermo Fisher Scientific). Sections (20 µm thick) were placed on MAS-coated slides and immunostained, and 40-µm-thick sections were immunostained by a free-floating protocol. The 20-µm-thick sections placed on slides were incubated for 60 min at room temperature (RT) with blocking solution containing 5% bovine serum albumin (BSA) in PBS. Thereafter, the sections were incubated overnight at 4 °C with a mixture of primary antibodies. The antibodies were then rinsed three times with PBS and incubated with the appropriate secondary antibodies conjugated with Alexa TM 488, 546 or 647 (1:400; Molecular Probes, OR, USA) for 2 hr at RT. When necessary, sections were autoclaved in 10 mM citrate buffer (pH 6.0) at 105 °C for 10 min for antigen retrieval before the blocking step. For 40-µm-thick sections, a free-floating protocol was used. In this case, according to the above protocol for 20-µm-thick sections, 0.1% TritonX-100 was added to each blocking and antibody solutions, and all procedures were performed at RT. Electron microscopy. Mice were fixed with 2% paraformaldehyde and 2% glutaraldehyde buffered with 0.1 M sodium cacodylate buffer. The brains were removed, and the cerebella were postfixed with 2% OsO 4 with 0.1 M sodium cacodylate buffer, block-stained in 1% uranyl acetate, dehydrated with a graded series of alcohol, and embedded in Epon 812 (TAAB, Reading, UK). Ultrathin sections were cut with a UCT ultramicrotome (Leica Microsystems), stained with uranyl acetate and lead citrate and observed with an electron microscope (H-7100 and HT7700; Hitachi).

Production of recombinant adenoviral vector.
An adenoviral vector expressing Mito-Venus was constructed as below. Venus with a mitochondria-targeted signal was a gift from Dr. Hiroyuki Miyoshi (RIKEN BRC; taken from lentiviral vector CSII-CMV-Venus-mito-IRES2-Bsd). The DNA fragment of mito-Venus was subcloned into pCAGGS vectors 1 , which allowed the expression of mito-Venus under control of the CAG promoter. An adenoviral vector was generated using pAD/PL-DEST via the Gateway System (Thermo Fisher Scientific).

Production of recombinant lentiviral vector.
A lentiviral vector expressing mito-KikGR was constructed as below. Photoconvertible fluorescent protein KikGR was purchased by MBL (Nagoya, Japan), and the mitochondria-targeted signal COX VIII was conjugated by PCR. The DNA fragment of mito-KikGR was subcloned into CS-CDF-CG-PRE vectors (kindly gifted by Dr. Hiroyuki Miyoshi, RIKEN BRC, Ibaraki, Japan) instead of the GFP region. For Purkinje cell-specific gene expression, the CMV promoter region was exchanged with the Purkinje cell-specific L7 promoter. Lentiviral vectors were generated according to the RIKEN BRC lentiviral vector preparation protocol.
Production of recombinant ssAAV9 vectors. The coding regions of KIF3A (NM_008443.4) and KIF3C (NM_008445.2) were cloned from a cDNA library derived from mouse brain extract. Each obtained clone was inserted into the expression plasmid pAAV/TREp-GFP-P2A in-frame. Recombinant ssAAV9 vectors were produced by transfection of HEK293T cells with pAAV2/9 (kindly provided by Dr. J. Wilson), a helper plasmid (Stratagene, La Jolla, CA, USA) and pAAV/TREp-GFP-P2A-KIF3A, pAAV/TREp-GFP-P2A-KIF3C, or pAAV/TREp-GFP expression plasmids, as previously described 2 . To activate the TRE promoter specifically in Purkinje cells, ssAAV9 vectors expressing tTA under control of the L7 promoter were similarly produced using the pAAV/L7p-tTA expression plasmid. The tTA-expressing vectors were mixed with one of the vectors carrying the TRE promoter before injection.
Protein extraction and western blotting. Protein extraction from cultured HeLa cells was performed using NP40-based buffer containing 1% NP40, 50 mM Tris (pH 8.0), 150 mM NaCl, and protease inhibitor cocktail (Complete Mini; Roche Diagnostics). This buffer was added to each culture well. Cells were incubated for 15 min on ice, followed by centrifugation at 15,000 rpm for 15 min. The supernatants were collected as protein samples. Protein samples were diluted with an equal volume of 2 × Laemmli sample buffer (Bio Rad, Hercules, CA, USA) supplemented with 5% 2-mercaptoethanol and then incubated for 5 min at 95 °C. Samples were separated using SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% non-fat skim milk for 60 min at RT and then incubated overnight at 4 °C with the mixture of primary antibodies. The antibodies were then rinsed three times with TBS containing 0.1% Tween 20 and incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; Millipore) for 1 hr at RT. Thereafter, signals were detected using Chemiluminescence HRP Substrate (Takara Bio, Shiga, Japan) with a Luminograph I (ATTO, Tokyo, Japan).