Absence of alsin function leads to corticospinal motor neuron vulnerability via novel disease mechanisms

Human Molecular Genetics, 2016, Vol. 25, No. 6 1074–1087 doi: 10.1093/hmg/ddv631 Advance Access Publication Date: 10 January 2016 Original Article ORIGINAL ARTICLE Absence of alsin function leads to corticospinal motor neuron vulnerability via novel disease mechanisms 1 Department of Neurology and, 2Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA, 3Robert H. Lurie Comprehensive Cancer Center and 4Cognitive Neurology and Alzheimer’s Disease Center, Northwestern University, Chicago, IL 60611, USA *To whom correspondence should be addressed. Tel: +312 503 2774; Email: Abstract Mutations in the ALS2 gene result in early-onset amyotrophic lateral sclerosis, infantile-onset ascending hereditary spastic paraplegia and juvenile primary lateral sclerosis, suggesting prominent upper motor neuron involvement. However, the importance of alsin function for corticospinal motor neuron (CSMN) health and stability remains unknown. To date, four separate alsin knockout (AlsinKO) mouse models have been generated, and despite hopes of mimicking human pathology, none displayed profound motor function defects. This, however, does not rule out the possibility of neuronal defects within CSMN, which is not easy to detect in these mice. Detailed cellular analysis of CSMN has been hampered due to their limited numbers and the complex and heterogeneous structure of the cerebral cortex. In an effort to visualize CSMN in vivo and to investigate precise aspects of neuronal abnormalities in the absence of alsin function, we generated AlsinKO-UeGFP mice, by crossing AlsinKO and UCHL1-eGFP mice, a CSMN reporter line. We find that CSMN display vacuolated apical dendrites with increased autophagy, shrinkage of soma size and axonal pathology even in the pons region. Immunocytochemistry coupled with electron microscopy reveal that alsin is important for maintaining cellular cytoarchitecture and integrity of cellular organelles. In its absence, CSMN displays selective defects both in mitochondria and Golgi apparatus. UCHL1-eGFP mice help understand the underlying cellular factors that lead to CSMN vulnerability in diseases, and our findings reveal unique importance of alsin function for CSMN health and stability. Introduction To date, numerous mutations have been identified to amyotrophic lateral sclerosis (ALS) and other related motor neuron diseases. However, mutations in the Alsin2 (ALS2) gene are particularly important because, unlike other mutations, it mainly affects young people. Mutations in this gene are detected in juvenile cases of ALS, patients with juvenile primary lateral sclerosis (PLS) (1), infantile-onset ascending hereditary spastic paraplegia (IAHSP) (2) and are the most common cause of autosomal recessive juvenile ALS (1). Alsin is a ubiquitous protein, expressed mainly in the central nervous system, and is encoded by the ALS2 gene. ALS2 is located on chromosome 2q33 contains 33 introns and 34 exons, and encodes two splice variant of alsin protein: a long form of 1657 amino acids and a short form of 396 amino acids (3). To date, 14 different mutations have been identified in the ALS2 gene in 24 ALS patients (4). In addition, a novel c.2761C > T mutation was found to † These authors contributed equally to this work. Received: November 30, 2015. Revised: November 30, 2015. Accepted: December 29, 2015 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 1074 Mukesh Gautam1, Javier H. Jara1,†, Gabriella Sekerkova2,†, Marina V. Yasvoina1, Marco Martina2 and P. Hande Özdinler1,3,4, * Human Molecular Genetics, 2016, Vol. 25, No. 6 line of AlsinKO, we reveal that apical dendrite degeneration is a common cellular pathology for CSMN that become diseased for different causes. In addition, we identify mitochondria and the Golgi apparatus of CSMN as a target of cellular dysfunction in the absence of alsin, thus revealing a novel importance of alsin function for CSMN health and stability. Results Generation of a CSMN reporter line of AlsinKO mice Because visualization and detailed cellular analysis of CSMN has been a challenge in mouse models, we used UCHL1-eGFP mice as a tool to generate reporter lines of motor neuron diseases in which CSMN express eGFP that is stable even at postnatal day (P) 800—2.5 years old—in vivo (20). AlsinKO mice, which lack alsin function and display very minor motor neuron circuitry defects by P300 have been generated and characterized (17). Here, CSMN reporter line of AlsinKO mice, AlsinKO-UeGFP were generated by two consecutive cross-breedings: AlsinHTmice were bred with UCHL1-eGFP to get AlsinHT-UeGFP, which were crossed back to AlsinHT mice to generate AlsinKO-UeGFP mice (Fig. 1A and B). All mice resulting from these cross-breedings were viable and fertile, without obvious developmental defects, and were born according to Mendelian ratios. Neurons expressing eGFP (eGFP+) in AlsinWT-UeGFP and AlsinKOUeGFP mice were located in layer V of the motor cortex, displayed pyramidal neuron morphology and a prominent apical dendrite (Fig. 1C–E). In addition, eGFP+ neurons showed co-localization with Ctip2 (subcerebral projection neuron marker (21), 95%; n = 246 neurons), but not with Satb2 (callosal projection neuron marker (22), 5%; n = 192 neurons). eGFP+ neurons retained CSMN identity in AlsinKO-UeGFP mice, even at P500 (Fig. 1F–I, arrows). CST axons, labeled with eGFP, were detected in ventral pons as previously documented in UCHL1-eGFP mice (20). CST axons display the signs of degeneration Silver staining revealed CST axon fiber degeneration in AlsinKO mice, by P300 (17). To further investigate whether AlsinKO-UeGFP mice recapitulate the CST axon pathology, we next studied the health of the CST axons at the level of pons (Fig. 2A). Because eGFP expression in CSMN allows visualization of single axon fibers and overall health of the CST tract, details of axonal pathology were revealed. The average fiber density was comparable between AlsinWT-UeGFP: (120.7 ± 9/unit area) and AlsinKO-UeGFP: (99.6 ± 2.2/ unit area) at P300 (Fig. 2D), but there was a significant reduction by P500 AlsinWT-UeGFP: (92.1 ± 9.9/unit area); AlsinKO-UeGFP: (50.1 ± 6.8/unit area, P < 0.002) (Fig. 2D). The average axon fiber cross-sectional area was reduced both at P300 (AlsinWT-UeGFP: 3.1 ± 0.4 µm2, n = 31 axons; AlsinKO-UeGFP: 1.5 ± 0.2 µm2, n = 29 axons; P < 0.03) and P500 (AlsinWT-UeGFP: 2.5 ± 0.1 µm2, n = 28 axons; AlsinKO-UeGFP: 1.4 ± 0.1 µm2, n = 30 axons; P < 0.004; Fig. 2E). GFP immunocytochemistry coupled with electron microscopic (EM) analysis further delineated the presence of many degenerating axon (...truncated)