**CELLULAR AND MOLECULAR MECHANISMS OF MOTOR NEURON DEATH IN AMYOTROPHIC LATERAL SCLEROSIS**

**Topic Editor Ricardo Tapia**

### *FRONTIERS COPYRIGHT STATEMENT*

© Copyright 2007-2015 Frontiers Media SA. All rights reserved.

All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.

Cover image provided by Ibbl sarl, Lausanne CH

**ISSN** 1664-8714 **ISBN** 978-2-88919-376-9 **DOI** 10.3389/978-2-88919-376-9

## *ABOUT FRONTIERS*

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

## *FRONTIERS JOURNAL SERIES*

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing.

All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

## *DEDICATION TO QUALITY*

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view.

By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

## *WHAT ARE FRONTIERS RESEARCH TOPICS?*

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area!

Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
## **CELLULAR AND MOLECULAR MECHANISMS OF MOTOR NEURON DEATH IN AMYOTROPHIC LATERAL SCLEROSIS**

Topic Editor: **Ricardo Tapia,** Universidad Nacional Autónoma de México, Mexico

Amyotrophic lateral sclerosis (ALS), which was described since 1869 by Jean Martin Charcot, is a devastating neurodegenerative disease characterized by the selective and progressive loss of upper and lower motor neurons of the cerebral cortex, brainstem and the spinal cord. The cognitive process is not affected and is not merely the result of aging because may occur at young ages. The only known cause of the disease is associated with genetic mutations, mainly in the gene encoding superoxide dismutase 1 (familial ALS), whereas there is no known cause of the sporadic form of ALS (SALS), which comprises >90% of cases. Both ALS types develop similar histopathological and clinical characteristics, and there is no treatment or prevention of the disease. Because effective treatments for ALS, as for other neurodegenerative diseases, can only result from the knowledge of their cellular and molecular pathophysiological mechanisms, research on such mechanisms is essential. Although progress in neurochemical, physiological and clinical investigations in the last decades has identified several mechanisms that seem to be involved in the cell death process, such as glutamate-mediated excitotoxicity, alterations of inhibitory circuits, inflammatory events, axonal transport deficits, oxidative stress, mitochondrial dysfunction and energy failure, the understanding of the origin and temporal progress of the disease is still incomplete and insufficient.

Clearly, there is a need of further experimental models and approaches to discern the importance of such mechanisms and to discover the factors that determine the selective death of motor neurons characteristic of ALS, in contrast to other neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Whereas studies in vitro in cell cultures, tissue slices or organotypic preparations can give useful information regarding cellular and molecular mechanisms, the experiments in living animal models obviously reflect more closely the situation in the human disease, provided that the symptoms and their development during time mimics as close as possible those of the human disease. It is necessary to correlate the experimental findings in vitro with those in vivo, as well as those obtained in genetic models with those in non-genetic models, aiming at designing and testing therapeutic strategies based on the results obtained.

# Table of Contents

*05 Cellular and Molecular Mechanisms of Motor Neuron Death in Amyotrophic Lateral Sclerosis: A Perspective*

Ricardo Tapia

*08 Investigating Cell Death Mechanisms in Amyotrophic Lateral Sclerosis Using Transcriptomics*

Paul R. Heath, Janine Kirby and Pamela J. Shaw

*18 Differential Autophagy Power in the Spinal Cord and Muscle of Transgenic ALS Mice*

Valeria Crippa, Alessandra Boncoraglio, Mariarita Galbiati, Tanya Aggarwal, Paola Rusmini, Elisa Giorgetti, Riccardo Cristofani, Serena Carra, Maria Pennuto and Angelo Poletti

*30 Early Gene Expression Changes in Spinal Cord From SOD1G93A Amyotrophic Lateral Sclerosis Animal Model*

Gabriela P. de Oliveira, Chrystian J. Alves and Gerson Chadi

*47 Mitochondrial DNMT3A and DNA Methylation in Skeletal Muscle and CNS of Transgenic Mouse Models of ALS*

Margaret Wong, Barry Gertz, Barry A. Chestnut and Lee J. Martin

Emiliano Trias, Pablo Díaz-Amarilla, Silvia Olivera-Bravo, Eugenia Isasi, Derek A. Drechsel, Nathan Lopez, C. Samuel Bradford, Kyle E. Ireton, Joseph S. Beckman and Luis Hector Barbeito

*86 Beta-2 Microglobulin is Important for Disease Progression in a Murine Model for Amyotrophic Lateral Sclerosis*

Kim A. Staats, Susann Schonefeldt, Marieke Van Rillaer, Annelies Van Hoecke, Philip Van Damme, Wim Robberecht, Adrian Liston and Ludo Van Den Bosch

*91 Neuroimmunity Dynamics and the Development of Therapeutic Strategies for Amyotrophic Lateral Sclerosis*

Melissa Bowerman, Thierry Vincent, Frédérique Scamps, Florence E. Perrin, William Camu and Cédric Raoul

*101 Redox Environment is an Intracellular Factor to Operate Distinct Pathways for Aggregation of Cu,Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis* Yoshiaki Furukawa

Mariko Ogawa and Yoshiaki Furukawa

Luis B. Tovar-y-Romo, Uri Nimrod Ramírez-Jarquín, Rafael Lazo-Gómez and Ricardo Tapia

	- Jerònia Lladó, Laia Tolosa and Gabriel Olmos

Florent Schmitt, Ghulam Hussain, Luc Dupuis, Jean-Philippe Loeffler and Alexandre Henriques

*182 Blood-CNS Barrier Impairment in ALS Patients Versus an Animal Model* Svitlana Garbuzova-Davis and Paul R. Sanberg

## Cellular and molecular mechanisms of motor neuron death in amyotrophic lateral sclerosis: a perspective

## *Ricardo Tapia\**

*División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico \*Correspondence<EMAIL_ADDRESS>
### *Edited and reviewed by:*

*Egidio D'Angelo, University of Pavia, Italy*

**Keywords: motor neuron degeneration, Amyotrophic lateral sclerosis (ALS), spinal cord, muscle, skeletal, genetic expression, trophic factors, neuroinflammation**

Amyotrophic lateral sclerosis (ALS), which was described since 1869 by Jean Martin Charcot, is a devastating neurodegenerative disease characterized by the selective and progressive loss of upper and lower motor neurons of the cerebral cortex, brainstem and the spinal cord. The result of this loss is a progressive and irreversible paralysis leading to a complete incapacity of movements and finally to respiratory failure, but the cognitive functions are not affected and is not merely the result of aging because may occur at young adult ages. There is still no treatment, prevention or reliable biomarkers of ALS and it is clear that this could only be accomplished, as for other neurodegenerative diseases, through the knowledge of the cellular and molecular pathophysiological mechanisms involved. Research on such mechanisms is, therefore, essential. Although progress in neurochemical, physiological, genetic and clinical investigations in the last decades has identified several cellular processes and mechanisms that seem to be involved in the neuronal death, such as glutamate receptors-mediated excitotoxicity, disruption of spinal inhibitory circuits, inflammatory events, axonal transport deficits, oxidative stress, mitochondrial dysfunction, energy failure, intracellular Ca2<sup>+</sup> dishomeostasis, protein aggregation and misfolding, changes in gene expression, astrocytes alterations, and non-cell autonomous toxic factors, the understanding of the origin and temporal progress of the disease is still incomplete and insufficient. Clearly, there is a need of further experimental models and approaches to discern the importance of such mechanisms and to discover the factors that determine the selective death of motor neurons characteristic of ALS, in contrast to other neurodegenerative diseases such as Parkinson's and Alzheimer's disease in which other neuronal types located in other CNS regions are predominantly affected.

The only known cause of the disease is associated with genetic mutations, mainly in the gene encoding superoxide dismutase 1 (SOD1) which is the most frequent case of familial ALS (FALS), representing about 20% of cases of FALS, albeit over one hundred mutations have been described in FALS. In contrast, there is no known cause of the sporadic form of ALS (SALS), that comprises *>*90% of all cases, although several other mutations have recently been identified also in SALS. Both ALS types show similar histopathological and clinical characteristics, and in spite of numerous investigations using tissue from ALS patients and relevant advances in the design of several experimental models of motor neuron degeneration, it has not been possible to establish a clear cause-effect relationship regarding the loss of motor neurons or the motor alterations characteristic of the disease, such as fasciculations, spasticity, and progressive paralysis.

Studies *in vitro* in cell cultures, tissue slices or organotypic preparations have given useful information regarding the cellular and molecular mechanisms of motor neuron death. However, except for transgenic rodents expressing mutant forms of human SOD1 (mSOD1), which are the most extensively used model *in vivo* in spite of the low frequency of FALS as compared to SALS, experiments in living animals are scarce. These models should reflect more closely the situation in the human disease, provided that the symptoms and their development during time mimics as close as possible those of the human disease. It is therefore necessary to correlate the experimental findings *in vitro* with those *in vivo*, as well as those obtained in genetic models with those in non-genetic models, in order to design and test therapeutic strategies based on the results obtained. The aim of the original research articles and reviews of this Research Topic is to contribute to the progress in this important field.

Genetic alterations, including epigenetic and gene expression changes in both neurons and glial cells, mainly astrocytes, as well as studies *ex vivo* of nervous and muscular tissue from ALS patients and from mutant SOD1 transgenic mice, constitute the majority of the articles of this Research Topic. Such changes have been studied in whole tissue, microdissected isolated cells and subcellular fractions, mainly mitochondria, using several molecular biology techniques, not only in the CNS but also in muscle of mSOD mice, and have shown that there is a large variety of alterations in gene expression. These results are the subject of the articles by Heath et al. (2013); Crippa et al. (2013); de Oliveira et al. (2013), and Wong et al. (2013), which amply cover the literature on this subject and also provide new data, such as the relation of some of these genetic alterations with autophagy, epigenetic changes of mitochondrial DNA in the spinal cord and in muscle, and comparative analysis of early and late gene expression changes.

The role of astrocytes and microglia from mSOD mice in noncell autonomous processes, including the release of toxic factors, is studied by Rojas et al. (2014) and Trias et al. (2013), with a useful *ex vivo* approach. Related studies by Staats et al. (2013) and Bowerman et al. (2013) show the involvement of microglia and neuroimmunological events in the progress of ALS and suggest possible therapeutic strategies. The molecular modifications of mSOD1 protein, and how such alterations, like oxidation, aggregation or misfolding, result in its toxicity, are reviewed by Furukawa (2013); Rotunno and Bosco (2013), and Ogawa and Furukawa (2014). Again in mSOD1 mice but with a different approach, Mòdol et al. (2014) show a possible role of the potassium chloride cotransporter 2 in spasticity, including results on the role of spinal inhibitory interneurons.

A partial protective effect of the NMDA receptor antagonist gacyclidine on the motor deficits in mSOD1 mice is shown by Gerber et al. (2013), and the role of trophic factors in the modulation of motor neurons activity as well as their protective effect in several models of ALS, is reviewed by Tovar-y-Romo et al. (2014) and Lladó et al. (2013). It is emphasized in particular the potent protective action of vascular endothelial growth factor (VEGF). Other potential protective agents, reviewed by Lazo-Gómez et al. (2013), are activators or inhibitors of histone deacetylases, enzymes that regulate gene expression and may be involved in motor neuron degeneration.

A general panorama of the role of lipids in ALS is presented by Schmitt et al. (2014), with emphasis on energy and signaling mechanisms. In a more systemic approach, Garbuzova-Davis and Sanberg (2014) review the vascular changes that may alter blood brain barrier and thus be involved in the pathogenesis of ALS, as compared to the transgenic rodent models.

The large amount of information contained in this Research Topic, in both the comprehensive reviews and the original research articles, reflect the considerable advances made but at the same time the necessity for new experimental models and approaches for gaining clearer responses to several still unresolved questions, such as the following: why the clinical manifestations of motor neuron death in both FALS and SALS appear in young adulthood but are not age-related, particularly the former, in spite of the fact that genetic alterations are present since the beginning of individual life? Why the motor neuron degeneration progresses rapidly after the initial symptoms appear? What is the cause of SALS? It is now clear that the loss of motor neurons in ALS is multifactorial, but many of the deleterious factors seem to occur also in other neurodegenerative diseases like Parkinson's and Alzheimer' disease; then, maybe the crucial question to be answered is: what determines the predominant, if not totally selective characteristic death of motor neurons in ALS, sparing other neuronal types?

In spite of these unsolved questions, it is clear that the available information permits the design and testing of diverse therapeutic strategies that have already shown valuable results in delaying or preventing neuronal death in experimental models, for example the administration of VEGF. However, the translation of possibly effective treatments from experimental procedures to ALS patients represents different problems, such as the routes, doses and timing of administration and the potential undesirable collateral effects. Because we have no indication or biomarker of the susceptibility to ALS and of when will it manifest, it is extremely difficult to prevent or treat the disease. When symptoms have already started and the diagnosis is made it is probably too late to impede the progression of neuronal degeneration. A verifiable resolution of the above questions seems a requisite for efficient and reliable treatments or prevention of this dreadful disease.

### **ACKNOWLEDGMENTS**

This work was supported by Consejo Nacional de Ciencia y Tecnología (project 128229) and Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (UNAM) (IN201013).

### **REFERENCES**

Trias, E., Díaz-Amarilla, P., Olivera-Bravo, S., Isasi, E., Drechsel, D. A., Lopez, N., et al. (2013). Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS. *Front. Cell. Neurosci.* 7:274. doi: 10.3389/fncel.2013.00274

Wong, M., Gertz, B., Chestnut, B. A., and Martin, L. J. (2013). Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS. *Front. Cell. Neurosci*. 7:279. doi: 10.3389/fncel.2013.00279

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 23 May 2014; accepted: 29 July 2014; published online: 14 August 2014. Citation: Tapia R (2014) Cellular and molecular mechanisms of motor neuron death in amyotrophic lateral sclerosis: a perspective. Front. Cell. Neurosci. 8:241. doi: 10.3389/ fncel.2014.00241*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Tapia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Investigating cell death mechanisms in amyotrophic lateral sclerosis using transcriptomics

## *Paul R. Heath\*, Janine Kirby and Pamela J. Shaw*

*Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK*

### *Edited by:*

*Ricardo Tapia, Universidad Nacional Autónoma de México, Mexico*

### *Reviewed by:*

*Brian K. Kaspar, The Research Institute at Nationwide Children's Hospital, USA Roger Pamphlett, The University of Sydney, Australia*

*\*Correspondence:*

*Paul R. Heath, Sheffield Institute for Translational Neuroscience, University of Sheffield, 385a Glossop Road, Sheffield S10 2HQ, UK e-mail<EMAIL_ADDRESS>
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of upper and lower motor neurons from the motor cortex, brainstem and spinal cord although evidence is suggesting that there is further involvement of other cell types in the surrounding tissue. Transcriptomic analysis by gene expression profiling using microarray technology has enabled the determination of patterns of cell death in the degenerating tissues. This work has examined gene expression at the level of the tissue and individual cell types in both sporadic and familial forms of the disease. In addition, further studies have examined the differential vulnerability of neuronal cells in different regions of the central nervous system. Model systems have also provided further information to help unravel the mechanisms that lead to death of the motor neurons in disease and also provided novel insights. In this review we shall describe the methods that have been used in these investigations and describe how they have contributed to our knowledge of the cell death mechanisms in ALS.

**Keywords: transcriptomics, cell death, amyotrophic lateral sclerosis, microarray**

### **INTRODUCTION**

Amyotrophic lateral sclerosis (ALS) is described as a degenerative disease involving loss of the upper and lower motor neurons from motor cortex, brain stem and spinal cord leading to muscle denervation, wasting and death. There is now increasing evidence that other cell types are involved and the precise pattern of degeneration is unclear (Ince et al., 2011). Multiple mutually compatible mechanisms have been implicated in the pathogenesis of the disease including oxidative stress, excitotoxicity, ubiquitin and proteasome dysfunction, inflammatory activation, RNA processing dysregulation, mitochondrial dysfunction, cytoskeletal abnormalities and activation of apoptosis (Ferraiuolo et al., 2011). However, there remain many unanswered questions as to the origins of the disease and the precise pathogenetic mechanism. The use of microarray technology to explore gene expression at a particular time point and in particular cells is enabling us to unpick some of the specific changes undergone by cells or tissues involved in the disease and also to determine what processes act to protect cells from degeneration. The technology for different array types has been described and reviewed previously and we will not at this time extend this (Clark et al., 2007; Kirby et al., 2007). Whilst there are inconsistencies between the findings of transcriptomic studies and the precise biological mechanisms involved in a disease situation there is no doubt that properly designed studies which generate functionally verifiable results can be invaluable in assisting in the description of the disease process (Bolstad et al., 2004; Rosenfeld, 2010; Henriques and Gonzalez De Aguilar, 2011). For the purposes of this review we concentrated our attention upon articles that were found from a PubMed search using the search terms microarray and ALS.

### **RNA QUALITY**

A recurring dilemma in studies of gene expression in post mortem tissue has been the residual debate concerning the quality of RNA derived from such tissue and whether it is appropriate for these studies. However, it is now accepted that gene expression studies in post mortem tissue are possible as long as appropriate quality control measures are followed (Harrison et al., 1995; Trabzuni et al., 2011). For most model studies standardized procedures can be set in place in order to reduce potential factors that might cause RNA degradation but even in an established and well organized human brain banking situation there are variables relating to post mortem delay, agonal state and cause of death all of which can affect RNA quality (Tomita et al., 2004; Popova et al., 2008; Durrenberger et al., 2009; Trabzuni et al., 2011). As indicated a number of studies have examined the effect of multiple potential RNA degrading features but the overall consensus is that providing adequate care is taken and good experimental design is incorporated in order to eliminate gross differences between the material being used then a reliable study can be carried out. In order to monitor these potential confounding factors it is important to control for any RNase introduction by carrying out all experimental work in a clean and tidy environment using RNase free materials. In addition it is essential that quality control steps should be included to measure both quantity and quality of the RNA being extracted and used (Copois et al., 2007; Wilkes et al., 2010; Trabzuni et al., 2011). Spectrophotometric methods can be used to measure nucleic acid quantity by examining absorbance at 260 nm. This can give an indication of RNA quality and the 260/280 ratio provides a measure of protein contamination whilst the 260/230 ratio provides a measure of organic solvent carryover. However, these metrics do not give a clear idea of RNA quality. For this, electrophoretic methods are required to examine the RNA directly. In the past a denaturing agarose gel (including formamide in the gel matrix) allowed the RNA to be visualized following staining with ethidium bromide. However, these methods required a large input of total RNA and have now been largely superseded by capillary based methodology such as the Agilent Bioanalyser and/or the TapeStation. In effect, both these devices drive the RNA sample through a capillary and over a laser which monitors absorbance at 260 nm over time hence providing a profile of the absorbance which can be compared to the profile of a molecular weight ladder run at the same time. If total RNA is the test sample then one would expect a small series of peaks at low molecular weight followed by the two ribosomal RNA peaks (the smaller peaks might be removed if a column based separation method is used). Software integral to the machine is then able to calculate an RNA integrity number (RIN) which is scaled from 0 to 10 and depends upon the ratio of the 28S to 18S rRNA peaks and the amount of degraded material that is present. It should be expected that for material derived from tissue culture experiments that a high RIN value is routinely achieved. However, post mortem human material is often quite degraded and RIN values of around 4 might be measured. It has been demonstrated in microarray, RNASeq and qRT-PCR that these values do not preclude a valid experiment (Preece et al., 2003). However, it is important to maintain equivalence in the experimental material and endeavor to compare samples of similar quality as well as support findings through appropriate validation methodologies.

### **HUMAN STUDIES IN WHOLE TISSUE**

Microarray studies on whole tissue generate an overview of the gene expression profile of the whole tissue at the time of the RNA collection. There have been a number of microarray studies which were designed to examine some aspect of gene expression changes in ALS by examining a particular tissue. A very early study by Malaspina et al. (2001), used gridded cDNA membrane arrays to examine the expression of 18,400 genes in the anterior horn of lumbar spinal cord from four ALS cases and four controls (with a further four patients and controls used as verification subjects). Fourteen transcripts were identified as being significantly differentially expressed, and these were shown to belong to processes relevant to ALS including; oxidative stress and neuro-protection, motor neuron function, lipid metabolism, neuroinflammation and anti-apoptotic factors. This was an important demonstration of the potential of this sort of study and whilst it depended upon a predefined group of cDNAs that were present upon the array, the results gave an overview of gene expression in the anterior horn of the lumbar spinal cord. It also confirmed that post mortem tissue could be used in such a study. Ishigaki et al. (2002) took a different approach. Using lumbar spinal cord from 8 sporadic ALS cases and controls they initially used one case and control for molecular indexing which identified 576 cDNA fragments in the sALS material that could be compared to the control to select identifiers for further analysis. In this way spotted microarrays were prepared for 84 cDNAs deemed to be differentially expressed between the sALS and control individuals that were further examined on a wider group of patients and controls. Ultimately six differentially expressed genes were identified; four corresponded to previously known genes, 30-kDa TATA-binding protein-associated factor (*TAFII30*), macrophage-inhibiting factor-related protein-8 (*MRP8*), metallothionein-3 (*MT-3*) and ubiquitin-like protein 5 (*UBL5*) whilst the remaining two were uncharacterized expressed sequence tags. This was an interesting approach but the limited numbers of genes identified made it difficult to identify processes related to cell death.

An early study by Dangond et al. (2004), used Affymetrix FL arrays to compare the expression profile of grey matter from the lumbar spinal cord of seven ALS cases and four controls. A robust analytical process identified 93 genes which discriminated between the two groups. There was some attempt to differentiate between sporadic and familial disease, however, the numbers in the familial group made this analysis less robust. The 93 significant genes were subdivided into groupings defined by known biological functions giving some 10 sub-groups including; transcription and RNA binding, excitotoxicity, cell survival and growth, neuronal function, inflammation, cell receptors and signaling and stress response. Wang et al. (2006), examined the motor cortex in five ALS cases compared to three controls using the newer Affymetrix U133A Gene Chips containing 14,500 genes for microarray analysis. They identified 275 significantly differentially expressed genes, of which 265 were downregulated and they were able to verify the changes in 22 of these genes by quantitative real time polymerase chain reaction (QRT-PCR). Interestingly different groups of genes seemed to be important in sALS motor cortex compared to the spinal cord, but there were similarities in that stress response and excitoxicity were featured in both groups, as were features of mitochondrial metabolism. Unfortunately, the different cell populations of the two regions might be having a profound impact upon the data. A further study of motor cortex (Lederer et al., 2007), used 11 sALS cases and 9 controls in an Agilent slide array based study. Fifty-seven genes were identified as differentially expressed following the filtering and pathway based analysis; 40 down and 17 upregulated. Such a small group of genes were readily verifiable by alternative methods and the list was compared to previous studies including that of Dangond et al. (2004), and 14 genes showed a similar response. Overall a very thorough analysis produced an interesting interacting pathway diagram examining the interplay of differentially expressed genes in the motor cortex of sALS individuals. It is important to note that without describing individual genes it is possible to discern similar patterns in the lists of genes differentially expressed in the whole tissue experiments described here. Recurrent themes are mitochondrial metabolism, excitotoxicity, calcium homeostasis, protein turnover and neuronal maintenance and signaling.

Using a different approach Offen et al. (2009) examined the gene expression profile in spinal cord of sALS patients and compared some of the changes to the Glycine 93 to Alanine (G93A) SOD1 mouse model of fALS. In the study of four sALS cases compared to four matched controls using the Clontech membrane arrays, 60 gene changes were identified in the 1176 probe sets available to be examined. On this occasion the genes identified were grouped into biological processes such as; apoptosis, protein turnover, cell cycle functions, extracellular carrier/transport functions, cytoskeletal and intracellular proteins and proteins relating to transcription and translation. Six genes were further investigated in the G93A *SOD1* mouse model of fALS to determine if the changes seen post mortem were recapitulated during the lifespan of the mouse. These genes were; cathepsins *B* and *D*, apolipoprotein *E*, epidermal growth factor receptor (*EFGR)*, ferritin and lysosomal trafficking regulator (*LYST)*. These transcripts did seem to increase with disease progression in the mouse model suggesting some overlap between the human situation and the mouse model which does add some credence to the use of the model for human ALS.

Two recent studies have taken a quite different approach. Bernardini et al. (2013), concentrated upon mitochondrial related genes and Figueroa-Romero et al. (2012), tried to relate gene expression changes to methylation events in the genome. The Figueroa-Romero study was focussed on relating genome methylation events to gene expression changes found in sALS spinal cord. 12 sALS cases and 11 age and sex matched controls were used to determine the methylated genetic regions using Illumina Methylation 27 Bead Chips, whilst gene expression analysis was carried out using the Affymetrix Human U133 Plus 2 GeneChips. In the methylation chip assay 3574 regions associated with genes were found to be differentially regulated. In the gene expression study some 1182 genes were found to be differentially expressed in the sALS spinal cord. When comparing the two datasets 51 genes that were hypomethylated were upregulated and 61 genes that were hypermethylated were downregulated. These concordant "epigenes" could be functionally grouped into categories such as; immune response, defence response, neuron adhesion and plasma membrane. Overall this work suggested an epigenetic, or environmental, influence upon the pathogenesis of sALS which may be relevant to the process of cell survival. In the Bernardini study they concentrated upon skeletal muscle which was a new, but potentially relevant, tissue to examine. They hypothesized that muscle degeneration might be functionally important in the process of neurodegeneration and that differentially expressed genes in this tissue could be key in the pathogenic process. They examined quadriceps or biceps muscles in seven sALS cases and seven age and sex-matched controls. A focussed Affymetrix GeneChip array was used and using a fold change cutoff of 1 and *p*-value cutoff of 0.05, 96 differentially expressed genes were identified. Unlike in previous studies on this occasion more genes (80), were seen in the upregulated group. In total 13 genes related to muscle structure were distinguished and when ontology relationships were investigated using the DAVID software (Huang da et al., 2009) platform, other genes were found to be grouped in areas such as the actin cytoskeleton and glycogenesis, with mitochondrial function also identified. Further studies would be needed to determine the relevance of this work particularly with regard to further subtyping of the ALS cases. A study by Shtilbans et al. (2011) also looked at gene expression differences in muscle biopsies from ALS cases and controls. The study was hampered in that mixed groups of familial and sporadic cases were examined and different muscle groups were included in mixed proportions in each comparative group. The differences they found were in genes related to muscle structure, the cytoskeleton and cellular metabolism.

Two studies have used peripheral blood to look for gene expression differences with the underlying hypothesis that changes found in this tissue might reflect changes associated with the disease as has been seen in other neurodegenerative diseases. Saris et al. (2009) used whole blood and a designed analysis tool to uncover disease specific differences whereas Mougeot et al. (2011) isolated lymphocytes and examined these directly. In the Saris study, the weighted co-expression analysis found that genes in pathways related to apoptosis, the mitochondrion, stress response, calcium binding ubiquitination and vesicle transport were differentially expressed between the ALS patients and controls. In contrast, Mougeot used an ontology relationship tool called SAFE and found that a set of genes previously grouped into an ALS related KEGG pathway were differentially expressed in the lymphocytes. Genes in this pathway related to motor neuron degeneration in ALS (KEGG:05014), and included SOD1, caspases 1,3,9, neurofilament light, medium and heavy (*NFL, NFM* and *NFH)*, and N-methyl-D-Aspartate receptors *GRIN 1, 2A, 2B, 2C* and *2D*. In addition, an ontology analysis based upon individual genes identified changes in pathways related to DNA metabolism, RNA splicing, mitochondrial function, oxidation, endoplasmic reticulum,ubiquitin proteasome system, post-transcriptional modification and neurological function. The overall conclusion to be drawn from both studies in blood is that although it is a peripheral tissue, it does show gene expression changes and pathways previously implicated by the use of central nervous system (CNS) material, including apoptosis, mitochondria, RNA splicing and, surprisingly, neurological function.

The studies of whole tissue have reached different conclusions, perhaps as a result of using tissue from different stages of disease and location, but there are common themes involved in the processes that are found to be differentially expressed in the ALS tissue which have been associated to motor neuronal cell death. In particular these are apoptosis, oxidative stress, protein processing, mitochondrial function and the cytoskeleton.

## **SINGLE CELL ANALYSIS OF HUMAN MOTOR NEURONS**

The previous studies are valuable but they make it difficult to determine the precise input of each cell. In order to obviate this, several methods have been developed to examine the gene expression profile of individual cells isolated from tissue. Two recent reviews have discussed the available methods for cell specific transcriptome analysis (Kannanayakal and Eberwine, 2005; Okaty et al., 2011), and we shall not discuss these further. In terms of ALS, most studies seem to have adopted a laser capture microdissection approach. In 2005, Jiang et al. carried out a microarray study of laser captured motor neurons from the lumbar region of 14 sporadic ALS cases and 13 controls (Jiang et al., 2005). They used a PALM microdissector and applied the labeled RNA to BD Atlas glass microarrays. They found 52 up and 144 down regulated genes from the 4845 interrogated. Owing to the fact that they compared the isolated spinal motor neurons data to ventral horn spinal cord tissue data they were able to distinguish genes whose differential expression was restricted to the motor neurons and which was not detected when the whole tissue was examined. It was found that genes associated with cell death and cell signaling were upregulated in the disease related motor neurons whereas those associated with the cytoskeleton and transcription were downregulated. This study provided information concerning gene expression changes that were specific to the spinal cord motor neurons and demonstrated the value of this approach over whole tissue studies. Our group has carried out a number of published studies examining the gene expression profile of motor neurons isolated by laser capture microdissection in several models. In her study in 2010, Cox et al. (2010) examined the differences between three individuals who had charged multivesicular body protein 2B (*CHMP2B)* mutations leading to an ALS phenotype and 7 matched normal controls. Following gene expression analysis using Affymetrix Human U133 Plus 2 GeneChips, 890 genes were downregulated and 55 upregulated at a fold change cutoff of 2 and *p*-value 0.05. These were categorized using DAVID software into ontological groupings and groups associated with axon guidance, regulation of actin cytoskeleton and soluble NSF attachment protein receptor (SNARE) interactions in vesicular transport, mammalian target of rapamycin (*mTOR*) signaling and regulation of autophagy, mitogen activated kinase (*MAPK*) signaling, calcium signaling, and cell cycle and apoptosis were found to be the most enriched. Further functional studies were able to confirm a dysfunction in the autophagy pathway in an *in vitro* model. A further study (Kirby et al., 2011), examined gene expression differences in cervical spinal motor neurons between three fALS cases carrying SOD1 mutations and seven normal controls using the Affymetrix Human U133 Plus 2 arrays. In total, 524 probe sets were found to be increased and 646 decreased. These were characterized using the DAVID software package and the major enriched categories were transcription, signaling and metabolism. Importantly further investigations demonstrated the relevance of the cell survival pathway involving *PTEN/AKT* in the motor neurons with anti-apoptotic genes being downregulated in the surviving motor neurons indicating an attempt by these cells to mount a pro-survival response. Comparison of these studies indicates that the different genetic variants have distinct gene expression changes which ultimately lead to motor neuron death.

In contrast to the case control scenario, Brockington et al. (2013), examined features that distinguish the motor neurons from the oculomotor nucleus and the lumbar spinal cord in normal individuals to determine those features that enable the oculomotor motor neurons to be selectively resistant to the cell death undergone by spinal motor neurons in ALS. Tissue from four neurologically normal individuals was collected and laser capture microdissection used to isolate motor neurons from the oculomotor nucleus and lumbar spinal cord. The labeled RNA from these was applied to the Affymetrix Human U133 Plus 2 GeneChip. 1521 gene expression differences were identified as being differentially expressed in the oculomotor neurons. Gene ontology analysis determined that genes involved in synaptic transmission, ubiquitin mediated protein degradation and mitochondrial oxidative phosphorylation were upregulated in oculomotor motor neurons; these pathways had shown decreased expression in the ALS spinal cord and motor cortex in previous studies. This work was supplemented by carrying out comparison studies with other gene expression data derived from an online database and the analysis showed that the differences observed in human oculomotor neurons were also found in two other species confirming that the oculomotor motor neurons had a particular profile of synaptic neurotransmitter receptors, particularly gamma aminobutyric acid (*GABA)* and glutamate which made them less vulnerable to excitotoxic cell death. Electrophysiological studies complemented and supported this conclusion.

Again some common features can be derived that seem to associate the differential expression of genes related to the mechanisms of cell death. These include; cell signaling, autophagy, phosphatase and tensin homologue/protein kinase B (*PTEN/AKT)* cell signaling pathway, ubiquitin and mitochondrial function, cytoskeleton, transcription and apoptosis.

## **ANIMAL MODELS USING WHOLE TISSUE**

Whilst human tissue can only be accessed at end stage, the use of animal models allows progression of the disease to be monitored. An early study by Yoshihara et al. (2002) examined the gene expression differences between tissue homogenates of lumbar spinal cord of G93A SOD1 versus non-transgenic littermates at three ages; 7, 14 and 17 weeks corresponding to presymptomatic, onset and end stage of disease. Using mouse Atlas arrays from Clontech they found an upregulation of inflammatory related genes associated with activated microglia and astrocytes. This was induced by 11 weeks of age and continued to advance up to the 17 week time point.

Fukada et al. (2007) examined the gene expression profile of whole spinal cord from two time points, presymptomatic (98 days) and post symptomatic (154/176 days), in a different mutant SOD1 transgenic model carrying the L126delTT mutation. They used the AceGeneMouse Oligo Chip. At the presymptomatic stage 11 genes were upregulated and two downregulated at a fold change of greater than two whilst at the postsymptomatic stage 54 were upregulated and four downregulated. With such small numbers of differences ontological relationships were hard to carry out and since whole spinal cord was examined the precise relationship of the gene changes to the disease process is difficult to disentangle.

It is thought that physical exercise might have a beneficial effect upon disease progression in ALS and two studies investigated this using microarrays and mouse models (Ferraiuolo et al., 2009; Hashimoto et al., 2009). Hashimoto et al. (2009) examined the response to exercise in normal mice by subjecting the mice to two exercise regimes; a single burst of 30 min exercise or 2 weeks of 30 min exercise per day on a treadmill. Whole spinal cord RNA was examined using the OpArray Mouse V4 array from Operon. Only a small number of changes in gene expression were identified; after the single burst of exercise 3 genes were upregulated and 29 downregulated whilst following the extended exercise regime 1 gene was found to be increased and 13 decreased. As seen in some previous studies the numbers are rather small for pathway analysis and since whole tissue has been studied it is difficult to dissect out precise effects. Ferraiuolo et al. (2009) examined the gene expression changes in gastrocnemius muscle as well as motor neurons isolated from lumbar spinal cord of three female mice subjected to a voluntary exercise regime of 21 days on a running wheel compared to three sedentary mice. Data were also collected concerning the exercise behavior of the mice. In the muscle tissue, 194 genes were upregulated and 176 downregulated using the same parameters. These genes were functionally found to be associated with vascularization and myogenic processes involved with extracellular matrix reorganization. The profiles exhibited by the gastrocnemius muscle are representative of the mixed cell population of the whole tissue and indicate some overall changes in the muscle as a response to the exercise regime. Overall, 203 genes were upregulated and 241 down at a fold change of at least two and *p*-value of 0.05 or less in the motor neurons. Functionally these genes were categorized into signaling, cytoskeleton and transcription regulation. The response of the motor neurons to exercise was similar to that of hippocampal neurons to repetitive stimulation, akin to a long term potentiation. There is a body of evidence that links exercise to the processes of cell death observed in ALS. This work goes some way to highlight similarities between the physiological response of MNs and skeletal muscle to exercise and the pathophysiology of ALS.

Two recent studies from the same group (Chen et al., 2010; Hu et al., 2013), have used Mouse Exon 1 arrays from Affymetrix to examine gene expression changes and alternative splicing in the G93A SOD1 mouse model. In the first study they examined animals at onset versus litter mate controls and in the second study 30 day transgenic mice were compared to litter mate controls of the same age and 120 day transgenic mice. They examined both differential expression and alternative splicing events. There were only 202 differentially expressed genes with a *p*-value cutoff of 0.05 found when comparing the 30 day old transgenic to nontransgenic mice but only one of these was at a fold change of *>* 2. There were in total 2869 differentially expressed genes events found when comparing the 120–30 day transgenic mice with 263 up and 71 down when a fold change of two and *p*-value of *<* 0.05 was imposed. Gene ontology analysis of the differentially expressed genes revealed that pathways involving cytokine receptor, cell adhesion, haematopoesis, and cell signaling included the most genes. In the onset group 322 transcripts were differentially expressed with 309 upregulated in the transgenic group when of a fold change of two and *p <* 0.05 was applied. Similar pathways were identified in this study to the previous one. In both studies a splicing index analysis was carried out in order to determine the levels of alternatively spliced transcripts. Comparing the 120 day transgenic to 30 day control 563 transcripts were alternatively spliced, of which 537 were considered as exon inclusion events, whilst only 85 alternative splicing events were identified in the 30 transgenic versus 30 day control comparison and on this occasion 61 were classified as exon inclusion. When the splicing data was examined by pathway analysis it was interesting that similar pathways were highlighted as found in the differentially expressed genes analysis. The data was interpreted to demonstrate that as the disease progressed then an increased number of splicing changes were seen. To some extent this is borne out by the onset study where 333 probe sets showed an altered splicing index indicating possible alternative splicing. However, the comparison of 120 day transgenic mice versus controls is missing which would allow a better comparison relative to normal ageing effects. Whilst some qRTPCR validation was completed the studies represent early work in using the exon arrays to examine the changes invoked by the presence of the G93A *SOD1* mutation on alternative splicing events.

The studies discussed thus far have used mouse models but Hedlund et al. (2010) used the G93A SOD1 rat model for a study of selective motor neuron vulnerability. They quantified the extent of motor neuron loss in different motor neuron nuclei; oculomotor and trochlear (CN3/4), facial (CN7), trigeminal (CN5), hypoglossal (CN12) and cervical spinal cord in the transgenic model. This directed the laser capture studies where motor neurons were collected from the CN3/4, CN12 and cervical spinal cord of normal rats and applied the isolated RNA to whole genome rat microarrays (Rat 230 2 Affymetrix). The microarray analysis showed that the more vulnerable motor neurons of the cervical spinal cord and hypoglossal nerve were more similar than either group compared to the less vulnerable CN3/4 motor neurons. There were interesting differences in Hox gene expression between the motor neuronal groups which were concomitant to the relative positions of the neurons in the anterior to posterior axis. There were also differences in RNA processing between the groups which also seemed to relate to the known association of RNA processing genes with ALS (Baumer et al., 2010). This report highlighted the usefulness of alternative animal models for the study of ALS and took an interesting approach to uncovering some of the underlying differences between selectively vulnerable and resistant motor neurons.

Dupuis and Loeffler (2009) examined neuromuscular changes in transgenic models of ALS. This led from the observation that the first event in the disease process seen in the transgenic mouse model of ALS is the destruction of the neuromuscular junction. Hence, studies concentrating upon the motor neurons may be identifying effects rather than cause of disease. However, it must be borne in mind that the SOD1 mouse model represents an overexpression model of disease and that SOD1 mutations correspond to only 2% of all ALS.

Finally in this section, Kumimoto et al. (2013) have undertaken to study ALS in a Drosophila model. They used a GAL4-UAS promoter to drive expression of wild type Drosophila SOD1 or the human SOD1 mutation glycine 85 to arginine (G85R) in motor neurons and glia. They carried out microarray analysis upon whole flies 5 days old (young), and 45 days (old), using the Drosophila 2 GeneChips from Affymetrix. They used several analyses to examine gene expression changes at the different ages and in the different cell types as well as a meta-analysis to uncover general changes. The mutant *SOD1* G85R in motor neurons alone caused 58 gene expression changes (33 up) at 5 days and 102 transcripts (73 up) at 45 days whereas in glia alone these numbers were 65 (33 up) and 105 (57 up) respectively. When the transgene was expressed in both cell types simultaneously the numbers were; 70 (34 up), and 83 (38 up). The overexpression of G85R SOD1 in the cells of the Drosophila caused changes in gene expression in oxidative stress, mitochondria, lipid metabolism and neurodevelopmental and signaling genes. Since the microarray analysis was carried out upon whole flies it is again difficult to determine the precise cellular effect of the overexpression strategy but there were commonalities in the pathways implicated with previous microarray studies and the impression is that the study offers a new model approach to understanding ALS. Since these studies were somewhat varied in the animal models, the tissue being investigated and the number of genetic changes identified it is difficult to describe common themes in the gene expression changes being seen and their relationship to cell death. However, cell signaling, mitochondrial dysfunction and oxidative stress continue to be implicated in the pathogenesis of ALS in animal models.

## **SINGLE CELL ANALYSIS OF NON-HUMAN MODEL MOTOR NEURONS**

Perrin et al. (2005) combined laser capture dissection with microarray to examine gene expression in the spinal cord motor neurons of the G93A *SOD1* mice. Lumbar spinal motor neurons from male mice at presymptomatic (60 days), onset (90 days) and end stage of disease (120 days) were analyzed using the Mouse 430 2 GeneChip from Affymetrix. At 60 days only 27 genes were differentially expressed with this number increasing to 150 at 90 days and more than 400 by 120 days. At all the time points more genes were found to be upregulated in the G93A SOD1 mice; 17, 95 and 389 respectively. Thirteen of these transcripts were found to be differentially expressed at all stages. A similar study from our group (Ferraiuolo et al., 2007), looked at the effect of both the G93A SOD1 and the wild type SOD1 compared to nontransgenic littermate controls on a homogeneous background of C57bl6. Few changes were seen in motor neurons isolated from the WTSOD1 mice but 252 genes were differentially expressed at 60 days (234 upregulated), 51 at 90 days (32 up) and 167 at 120 days (81 up), in the G93A SOD1 mice. These changes at the presymptomatic, symptomatic and end stage of the disease process in the mice indicate that at an early time point the motor neurons try to mount a metabolic response to the burgeoning disease process by upregulating several pathways. However, by the endstage, this process having failed, the cells appear to try to re-enter the cell cycle which is a doomed response. There are notable differences between the two studies in terms of the overall changes in gene expression profile, although both groups do see changes in the intermediate filament protein vimentin, it is likely that the differences might relate to the actual genetic background of the mice being used, the transgene copy number and also the analytical processes used in examining the data. A study by Saxena et al. (2009) took a different approach to examine the matter of selective vulnerability of motor neurons. They injected RITC-dextran into the gastrocnemius or soleus muscles and following retrograde transport they were able to identify and selectively isolate the motor neurons associated with the vulnerable phenotype which project to the fast fibre gastrocnemius muscle and those that are relatively resistant and project to the soleus muscle which has fatigue resistant or slow fibres. The RNA isolated from the selectively microdissected motor neurons was applied to the Mouse 430 2 arrays from Affymetrix. The longitudinal study showed that the vulnerable motor neurons are selectively prone to endoplasmic reticulum stress and a progressive failure to mount a proper unfolded protein clearance response.

This group of studies are more focussed and underline the importance of the single cell analysis at different stages of disease progression.

## **SINGLE CELL ANALYSIS OF NON-HUMAN NON-MOTOR NEURON CELL TYPES**

Using the G93A Mutant SOD1 mouse model Ferraiuolo et al. (2011) attempted to examine the relationship between motor neurons and astrocytes in this model of disease (Ferraiuolo et al., 2011). Astrocytes were collected from lumbar spinal cord of three male 60 day old mice and three non transgenic litter mate controls, and the labeled RNA applied to Mouse 430 2 Gene Chips from Affymetrix. 583 transcripts were found to be upregulated 526 downregulated in the G93A SOD1 astrocytes. Ontological analysis showed that multiple transcripts associated with carbohydrate metabolism were differentially expressed and an overall decline in the activity of the lactate shuttle was described in the mutant astrocytes indicating an inability to support the metabolic requirements of the motor neurons. The altered metabolism was further investigated and it was shown that non-transgenic motor neurons grown on G93A SOD1 astrocytes demonstrated an increase in the ratio between pro- nerve growth factor and mature nerve growth factor (NGF) which was associated with an over-expression of the p75 receptor for NGF. Hence it was shown that the G93A SOD1astrocytes provided reduced metabolic support to the motor neurons as a result of the downregulation of the lactate shuttle and the activation of the p75 receptor and these factors contributed to the toxicity of the mutant astrocytes to the co-cultured motor neurons.

### **CELL CULTURE MODEL STUDIES**

Two studies have examined the gene expression profile of the NSC 34 cells carrying different human SOD1 mutations. The first study (Kirby et al., 2002), utilized a designed membrane microarray from Clontech with cDNAs for just 588 genes. The experiment was carried out on cells carrying G93A, G85R or isoleucine 113 to threonine (I113T) SOD1 mutations at basal conditions and cells which had undergone stress exerted by serum withdrawal. At the basal condition differences were seen in the numbers of differentially expressed genes for the different SOD1 mutation but a combined analysis showed only 29 genes to be differentially expressed as a result of the SOD1 mutation with seven being downregulated. Very few genes were found to be differentially expressed following the induction of a stress imposed by serum withdrawal which might be a result of the analytical stringency being applied. In a subsequent study (Kirby et al., 2005), a comparison was made between NSC34 cells expressing G93A mutant SOD1 and those expressing wild type human SOD1. On this occasion the mouse U74Av2A GeneChip from Affymetrix was used. At a fold change of two with a *p*-value of 0.05, 268 transcripts were differentially regulated in the presence of mutant SOD1, of which 197 were downregulated. Following thorough ontological analysis it was found that the presence of the mutant SOD1 caused a significant down-regulation of the cells capacity to deal with oxidative stress. In particular the group of genes with an antioxidant response element in their promoter, termed programmed cell life genes, which would be activated by the transcription factor nuclear factor (erythroid-derived) like 2 *(Nrf2)* were downregulated indicating a failure of the cells to mount a proper therapeutic antioxidant stress response. Subsequent work by Mead et al. (2013) has demonstrated that induction of the *Nrf2* response by S(+)-Apomorphine improves the motor function of the G93A *SOD1* mice underlining the importance of increasing the expression of the programmed cell life genes in a therapeutic strategy to treat ALS.

Vargas et al. (2008) isolated primary astrocytes from both G93A SOD1 mutant and normal littermate control embryonic rats and examined the profile of gene expression using Rat genome 230 2 arrays from Affymetrix. A total of 81 transcripts were differentially expressed by the mutant SOD1 bearing astrocytes with 55 being upregulated. The genes were characterized according to their biological function and were separated into groups; extracellular matrix and cell migration, signaling and receptor activity, transcription, cell proliferation, response to stress, oxidoreductase activity, catalytic activity and unknown. It is interesting to note that despite the fact that different cell types are being investigated there are some similarities in the effect of the *SOD1* mutation in astrocytes and motor neurons from mice and rats.

In the study by Boutahar et al. (2011) they derived primary cortical motor neurons from transgenic mice either with or lacking the G93A *SOD1* mutation. The cells were derived from E14 embryos and maintained in culture for a brief period until the presence of the transgene was confirmed. Some cultures were subjected to stress with hydrogen peroxide or NMDA treatment. Microarrays using Mouse 430 2 GeneChips from Affymetrix were carried out and the results analyzed to determine fold changes of two or more. In the untreated cells 260 transcripts were differentially regulated with 70 upregulated. Following hydrogen peroxide treatment the number was 163 with 59 upregulated and following NMDA treatment 181 with 62 upregulated. Few genes were shared between the three treatments. Gene ontological analysis showed an increase in transcripts associated with the proteasome, some associated with autophagy and cytoskeletal organization and axonal transport. There was some downregulation in transcripts associated with ion transport. It was noted that the presence of the transgene alone increased cell death but this process was mitigated when the additional stresses, hydrogen peroxide or NMDA, were introduced. Hence they identified a small group of genes which were upregulated in the untreated G93A cells but downregulated following the introduction of an additional stress. These genes were; Cathepsin H,(*CSTH*) and Autophagy related 4 homolog D (*ATG4D*) which are involved in protein degradation, tubulin beta-4B chain (*TUBB2C*) and Rho guanine nucleotide exchange factor 11 (*ARHGEF11*) which are cytoskeletal, cell division cycle 25 homolog (*CDC25*) which is involved in cell cycle regulation, solute carrier 7 (*SLC7A12*), DEAD box 43 (*DDX43*) and leucine rich recognition motif 4 (*LRRTM4*) which are involved in the transmembrane domain and transcripts *ACN9, SSBP9* and *TRIM36* involved in transcription regulation.

The studies in cellular culture models all seem to underline the importance of oxidative stress in the pathogenesis of ALS and the difficulty that cells containing mutant SOD1 have in dealing with this metabolic insult.

## **SYSTEMS BIOLOGY**

Some studies have taken a more broad brush approach to the analysis of microarray data and attempted to incorporate data from a number of sources. Kudo et al. (2010) developed an integrative approach to examine post mortem human material, mouse models and microarray methods. They used the mouse models of ALS, G93A SOD1 and frontotemporal dementia (FTD), P30L Tau and isolated both motor neurons and glia for microarray analysis. Using Agilent whole mouse genome microarrays 251 transcripts were identified as being differentially expressed in at least one of the four comparisons. Of these 186 corresponded to known genes and ontological analysis revealed that the most enriched biological processes were associated with protein modification/phosphorylation, signaling, regulation of muscle contraction, stress response, the immune system and cell communication. The data were confirmed both in further murine tissue by semi-quantitative PCR and in human tissue from sALS cases with tissue microarrays. Hence they used complementary methodology to identify common mechanisms in the SOD1 and TAU mouse models of motor neuron degeneration and human sALS. The genes *CNGA3, CRB1* and *OTUB2* were common to all the models and *CNGA3, CRB1, OTUB2, SLK, DDX58, RSPO2* and *MMP14* were related to sALS. In addition, 13 transcripts found differentially regulated in the SOD1 motor neurons were also found to be altered in their expression in the blood of G93A SOD1 mice.

Baciu et al. (2012) adopted a reanalysis approach where they performed an in depth analysis of a microarray experiment carried out using purified peripheral blood lymphocytes from 11 ALS patients and 11 age matched controls. The RNA had been interrogated using Agilent 4 × 44k human arrays and the authors used a modified BaFL pipeline and at the same time a severe TM4 statistical analysis to remove potential confounding probes sets and "purify" the dataset. The methodology was validated using a previous dataset carried out using similar microarrays and a coronary heart disease study. By carrying out the two different analytical methods the authors attempted to account for errors that might be inherent to either alone. Ultimately, a series of seven genes was identified as being upregulated in the sALS patients, were common to both methods and were verifiable by qRTPCR. These genes were; *B2M, ACTG1, DYNLT1, SKIV2L2, C12orf35, TARDBP* and *ILKAP* (which cross-hybridises with TARDBP).

A recent meta-analysis from Saris et al. (2013) looked to examine the datasets from a number of different microarray studies of both murine and human material. They included seven human studies; three of lumbar cord, two cervical cord and two motor cortex. In addition there were nine studies of transgenic mouse model material; seven of G93A SOD1, one L126delTT SOD1 and one study of the progressive motor neuropathy and wobbler mice. As the authors conclude, despite the variety of input material and different array platforms used, there was a level of consistency in the altered gene expression output. Hence they found altered gene expression in the areas of protein turnover, immune response and apoptosis in both human and murine studies. The mouse studies also showed consistent differences in genes involved in the lysosome, metal ion binding and mitochondrial function. This study is an interesting new approach to attempt to summate all the data that has been produced thus far. There are likely to be further assays of this type which will take advantage of the large datasets in order to generate informative data with greater statistical power owing to the larger numbers of samples being included.

### **CONCLUDING REMARKS**

This short review has attempted to summarize the depth of study that has been carried out using transcriptomic technology to examine the gene expression changes that can be identified in comparisons of ALS tissue and controls. With the variety of studies that have been done it is quite difficult to accurately summarize the information but some overall differences can be seen. In terms of mechanisms that have been uncovered in these studies that are influential in the processes that underlie cell death in ALS it can be seen that certain pathways are repeatedly enriched as a consequence of the disease process. These include; oxidative stress, mitochondrial function, apoptosis, cytoskeleton, neuroinflammation and protein processing. It is interesting to note that when peripheral tissues have been used similar processes have been identified as distinguishing the material from individuals with disease. Whilst many of the studies delineated differences between disease and control at the level of the transcriptome an important development has been developing functional studies to demonstrate the validity of the identified changes and then subsequent studies to investigate mechanisms to ameliorate them.

It is likely that the use of the microarray technology is likely to become more of an adjunct to support a body of work as seen in the study from Egawa et al. (2012). In this case the transcriptomic element of the work was used to characterize human induced pluripotent stem cells derived from dermal fibroblasts of normal individuals and several TDP43 mutants. In the past we have seen the transcriptomic study as being the lead in the experimental approach but on this occasion the development of the induced stem cells was the primary goal and the microarray analysis was simply a tool to assist in the characterization of derived cells and define potential routes for drug intervention. It is likely that with the maturity and confidence that several years of transcriptomic research have generated this type of approach will become more widespread.

A further development in the examination of gene expression differences between tissues is going to be the use of next generation sequencing to examination gene expression directly rather than as an indirect measure based upon manufactured probes. The next generation sequencing methodology provides a more detailed examination of the transcriptome which allows for a more in depth examination of novel transcripts, splice junctions and non-coding RNAs in a single step rather than having to carry out multiple different array experiments. As with the early microarray work there is not yet a defined analytical pathway for interpreting the data from RNA Seq experiments but this is progressing rapidly. There is evidence that the two tools are being used in a combinatorial approach (Kogenaru et al., 2012).

Transcriptomics has been used successfully to investigate the mechanisms underlying the processes of cell death and has provided insights into the pathways that may be dysregulated. As the tools are becoming more established it is likely that more studies will use them to assist in the functional studies needed to develop new treatments for ALS.

### **AUTHOR CONTRIBUTIONS**

Paul R. Heath devised and produced the manuscript, Janine Kirby and Pamela J. Shaw contributed editing and revising of the manuscript.

## **ACKNOWLEDGMENTS**

Paul R. Heath is supported by funding from the BBSRC. Pamela J. Shaw and Janine Kirby are supported by funding from the European Union: Seventh Framework Programme (FP7/2007-2013) under the Euro-MOTOR project (No: 259867, http://www.euromotorproject.eu/) and Pamela J. Shaw and Janine Kirby by the EU Joint Programme–Neurodegenerative Disease Research (JPND), Sampling and biomarker OPtimization and Harmonization In ALS and other motor neuron diseases (SOPHIA). This is an EU Joint Programme— Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organizations under the aegis of JPND—www.jpnd.eu: France, Agence Nationale de la Recherche (ANR); Germany, Bundesministerium für Bildung und Forschung (BMBF); Ireland, Health Research Board (HRB); Italy, Ministero della Salute; The Netherlands, The Netherlands Organization for Health Research and Development (ZonMw); Poland, Narodowe Centrum Badan i Rozwoju; Portugal, Fundação a ´ Ciência e a Tecnologia; Spain, Ministerio de Ciencia e Innovación; Switzerland, Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (SNF); Turkey, Tübitak; United Kingdom, Medical Research Council (MRC). Pamela J. Shaw is supported as an NIHR Senior Investigator.

## **REFERENCES**

otrophic lateral sclerosis mouse model. *Brain Res.* 1340, 52–69. doi: 10.1016/j. brainres.2010.03.075

linked mutant SOD1. *J. Neurosci. Res.* 86, 3515–3525. doi: 10.1002/jnr. 21797

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 July 2013; paper pending published: 21 October 2013; accepted: 28 November 2013; published online: 17 December 2013*.

*Citation: Heath PR, Kirby J and Shaw PJ (2013) Investigating cell death mechanisms in amyotrophic lateral sclerosis using transcriptomics. Front. Cell. Neurosci. 7:259. doi: 10.3389/fncel.2013.00259*

*This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2013 Heath, Kirby and Shaw. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

## Differential autophagy power in the spinal cord and muscle of transgenic ALS mice

### *Valeria Crippa1,2, Alessandra Boncoraglio1,2,3, Mariarita Galbiati 1,2, Tanya Aggarwal 4, Paola Rusmini 1,2, Elisa Giorgetti 1,2, Riccardo Cristofani 1,2, Serena Carra3,5, Maria Pennuto4,6 and Angelo Poletti 1,2\**

*<sup>1</sup> Sezione di Biomedicina e Endocrinologia, Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano, Italy*

*<sup>2</sup> Centro InterUniversitario sulle Malattie Neurodegenerative, Università degli Studi di Firenze, Milano, Genova e Roma Tor Vergata, Italy*

### *Edited by:*

*Ricardo Tapia, Universidad Nacional Autónoma de México, Mexico*

### *Reviewed by:*

*Jie Zhang, University of Texas Health Science Center at San Antonio, USA Lee J. Martin, Johns Hopkins University School of Medicine, USA*

### *\*Correspondence:*

*Angelo Poletti, Sezione di Biomedicina e Endocrinologia, Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy e-mail<EMAIL_ADDRESS>
Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and to a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analyzed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 mice at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences were also considered. No changes of HSPB8, BAG3, and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analyzed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cord. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded proteins accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.

### **Keywords: amyotrophic lateral sclerosis, motoneurons, autophagy, HSPB8, BAG3, BAG1, protein quality control**

### **INTRODUCTION**

Autophagy is a fundamental intracellular degradative pathway activated to respond to the accumulation of aberrantly folded (misfolded) proteins (Mizushima and Komatsu, 2011). Autophagy is required for the cellular protein quality control (PQC) system, which also includes molecular chaperones and the ubiquitin-proteasome degradative system (UPS) (Carra et al., 2012). These systems work together protecting cells particularly sensitive to misfolded protein toxicity, such as motoneurons (Rusmini et al., 2010; Bendotti et al., 2012; Carra et al., 2013). Motoneurons are major targets of toxicity in diseases linked to mutant proteins prone to misfold, such as in amyotrophic lateral sclerosis (ALS) (Pasinelli and Brown, 2006; Rusmini et al., 2010; Sau et al., 2011; Strong and Yang, 2011; Carra et al., 2012, 2013; Robberecht and Philips, 2013). Most ALS cases appear in sporadic (sALS) forms; only about 10–15% have familial (fALS) history, and are clinically indistinguishable from sALS. fALSs have been associated with mutations in different genes, such as the superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), optineurin (Robberecht and Philips, 2013), or the C9ORF72 gene (Dejesus-Hernandez et al., 2011; Renton et al., 2011; Ash et al., 2013; Lashley et al., 2013; Mori et al., 2013). Notably, also the wild type (wt) forms of the mutated fALS proteins may show aberrant behavior in sALS (e.g., oxydized wtSOD1, cleaved C-terminus of wtTDP-43, etc.) (Neumann et al., 2006; Daoud et al., 2009; Bosco and Landers, 2010; Bosco et al., 2010), suggesting the existence of a common pathological mechanism. An explanation for this is that these proteins (either the modified wt or the mutant forms) have the propensity to misfold and aggregate forming insoluble inclusions that are a key neuropathological hallmark of ALS.

In recent years, emerging evidences support the idea that also non-neuronal cells (e.g., surrounding astrocytes or Schwann cells, chemotactically attracted microglial cells, and target muscle cells) might contribute to disease onset and progression (Boillée et al., 2006), by making motoneuronal cells more sensitive to protein toxicity. For example, selective expression of mutant SOD1 (mutSOD1) in skeletal muscle induced atrophy associated with the loss of motoneurons in the anterior horn of the spinal cord (Dobrowolny et al., 2008a; Corti et al., 2009; Wong and Martin, 2010). However, mutSOD1 largely accumulates in spinal cord of transgenic mice expressing human G93A-SOD1 (Tg G93A-SOD1) (Cheroni et al., 2005, 2009; Basso et al., 2006, 2009; Bendotti et al., 2012), but not in skeletal muscle of the same mice at any stage of disease (Galbiati et al., 2012; Wei et al., 2012); thus, muscle cells better cope with misfolded mut-SOD1 species than motoneuronal cells. This may be due to an higher muscular degradative capabilities compared to motoneurons (Onesto et al., 2011). In fact, in both cells, mutSOD1 clearance involves both UPS and autophagy, but both the proteasome and autophagy activities are higher in muscle cells than in motoneuronal cells.

The interplay between UPS and autophagy is finely regulated through a mechanism that involves the co-chaperones BAG1 and BAG3, which act as a switch between the two pathways (Luders et al., 2000; Carra et al., 2008a; Gamerdinger et al., 2009; Arndt et al., 2010; Zhang and Qian, 2011). BAG1 is expressed at relatively higher levels in young tissues, paralleled by higher UPS activity, while BAG3 is expressed at higher levels in aged tissues, characterized by higher autophagic activity (Gamerdinger et al., 2009), and the BAG3:BAG1 ratio determines the fate of client proteins to be degraded through either UPS or autophagy. Indeed, BAG1 routes substrates to UPS by interacting with a complex formed by the chaperone HSC70 and the ubiquitinating enzyme CHIP. UPS overwhelming or impairment, due to misfolded proteins, results in upregulation of BAG3 and its partner, the chaperone HSPB8 (a small heat shock protein involved in misfolded proteins recognition) (Wang et al., 2008; Du et al., 2009; Crippa et al., 2010b; Gentilella and Khalili, 2011; Carra et al., 2013). The increased BAG3:BAG1 ratio favors a stoichiometric HSC70/CHIP complex association to BAG3/HSPB8 complex, allowing the p62 mediated autophagic removal of CHIP-ubiquitinated substrates (Crippa et al., 2010b; Gamerdinger et al., 2011; Zhang and Qian, 2011; Carra et al., 2012, 2013). Interestingly, we found that mut-SOD1 blocks the autophagic flux in motoneurons, but not in muscle cells (Onesto et al., 2011). This autophagic flux blockage can be counteracted by HSPB8, which participates together with BAG3/HSC70/CHIP in the autophagic clearance of several different misfolded proteins (polyQ containing proteins, mutSOD1, a truncated form of TDP-43) (Carra et al., 2005, 2008a,b, 2013; Crippa et al., 2010b). In addition, HSPB8 induces translation attenuation via phosphorylation of eIF2alpha (Carra et al., 2009), thereby decreasing the amount of aggregation-prone proteins to levels manageable by PQC.

In this study, we analyzed whether spinal cord, which comprises the two main cell targets of SOD1 toxicity, namely motoneuronal and astroglial cells, and skeletal muscle, differentially activate the HSPB8 mediated PQC system in response to mutSOD1 in tg G93A-SOD1 mice. The results demonstrate that the autophagic response of muscle tissue to the mutSOD1 expression is much higher than that found in the spinal cord of the same mice, suggesting that if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded protein accumulation.

## **MATERIALS AND METHODS**

### **ANIMALS**

All the procedures involving animals and their care were carried out following the institutional guidelines and in accordance with national (D.L. no. 116, G.U. suppl. 40, Feb. 18, 1992), and international laws and policies (EEC Council Directives 86/609, OJ L 358, 1 DEC.12, 1987), and were approved by the Italian Institute of Technology Animal Care Committee. All the animals were kept under controlled temperature and humidity conditions with standardized dark/light cycles of 12 h each. Food (standard pellets) and water were supplied *ad libitum*. BL6JL Tg(SOD1)2Gur/J (Stock number 002297, Charles River, Wilmington, MA, USA) male mice or BL6JL-Tg(SOD1∗G93A)2Gur/J (Stock number 002726, Charles River) male mice were crossed with wt female mice purchased (Stock number 100012, Charles River) or obtained in house by crossing C57Bl/6J female and SJL male mice. All the experiments were performed in mice coming from the F1 generation of the cross described above. Non-transgenic littermates were used as controls (NTg). Mice were genotyped by PCR on tail DNA as previously described (Gurney et al., 1994), using REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, MO, USA). To evaluate disease stages, starting from the 8th week of age and twice a week, mice were tested for deficit by rotarod, and hanging wire by the same operator as previously described (Palazzolo et al., 2009). Body weight loss was also monitored. Disease onset was set as the time at which the mouse permanently starts to lose body weight. Four mice per group were anesthetized with isoflurane and sacrificed at 8 or 16 weeks of age, corresponding to presymptomatic (PS) or symptomatic (S) stage of disease. Quadriceps muscles and spinal cord were rapidly collected after the sacrifice, snap frozen in liquid nitrogen, and conserved at −80◦C until RNA and protein extraction.

### **RNA EXTRACTION**

Total RNA from frozen spinal cords or muscles was extracted using the standard TRI Reagent protocol based on the method developed by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). RNA was subsequently extracted in accordance to manufacturer's protocol (Sigma-Aldrich). The precipitated RNA was dissolved in RNase-free water. Total RNA (1µg) was treated for 15 min at room temperature with 1 U of DNaseI (Sigma-Aldrich). Samples were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions, in a 20µL volume. Primers for selected genes were designed via the Primer Express software (Life Technologies Corporation) and purchased from MWG Biotech (Ebersberg, Germany). Primer sequences were as follows: mouse HSPB8: 5 - ATA CGT GGA AGT TTC AGG CA -3 (forward), 5 - TCC TTT GAC CTA ACG CAA CC -3 (reverse); mouse BAG3: 5 - ATG GAC CTG AGC GAT CTC A -3 (forward), 5 - CAC GGG GAT GGG GAT GTA -3 (reverse); mouse BAG1: 5 - GAA ACA CCG TTG TCA GCA CT -3 (forward), 5 - GCT CCA CTG TGT CAC ACT C -3 (reverse); mouse MAP-LC3b: 5 - CGT CCT GGA CAA GAC CA -3 (forward), 5 - CCA TTC ACC AGG AGG AA - 3 (reverse); mouse p62: 5 - AGG GAA CAC AGC AAG CT -3 (forward), 5 - GCC AAA GTG TCC ATG TTT CA -3 (reverse); mouse GAPDH: 5 -CCA GAA CAT CAT CCC TGC AT-3 (forward), 5 -CAG TGA GCT TCC CGT TCA-3 (reverse). The evaluated efficiency of each set of primers was close to 100% for both target and reference gene. Real-time PCR was performed using the CFX 96 Real Time System (Bio-Rad Laboratories, Hercules, CA, USA), in a 10µL total volume, using the iTaq SYBR Green Supermix (BioRad), and with 500 nmol primers. PCR cycling conditions were as follows: 94◦C for 10 min, 40 cycles at 94◦C for 15 s, and 60◦C for 1 min. Melting curve analysis was performed at the end of each PCR assay to control specificity. Data was expressed as Ct values and used for the relative quantification of targets with the *--*Ct calculation to give N-fold changes in gene expression. Values were normalized to those of GAPDH. To exclude potential bias due to averaging data transformed through the equation 2−*--*Ct, all statistics were performed with *-*Ct values. Each experiment was carried out with four independent samples, and each sample was run in duplicate wells.

The BAG3:BAG1 relative ratio was obtained considering the group of 8 weeks-old NTg female mice as control for the basal physiological condition of the PQC system. In particular, for each sample a single BAG3:BAG1 ratio was determined [measuring the (BAG3 2−*--*Ct/BAG1 2−*--*Ct)sample] and the mean BAG3:BAG1 ratio of control was subtracted [(BAG3 2−*--*Ct/BAG1 2−*--*Ct)control]. Finally, the resulting values were normalized by the mean BAG3:BAG1 ratio of the control 8 weeks-old NTg female mice.

### **WESTERN BLOTTING AND FILTER RETARDATION ASSAY (WB AND FRA)**

Total proteins from frozen spinal cord or muscles were extracted in 1% SDS using the standard TRI Reagent protocol, in accordance with the manufacturer's protocol (Sigma-Aldrich). Protein concentration was determined with the bicinchoninic acid method (BCA assay, EuroClone, Pero, Milan, Italy). WB analysis was performed on 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis loading 15µg of total proteins. Samples were then electrotransferred to Nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo (Bio-Rad). The membranes were treated with a blocking solution containing 5% non-fat dried milk powder (EuroClone) in TBS-T for 1 h and then incubated with the primary antibodies: (1) home-made rabbit polyclonal anti-HSPB8 (kindly provided by Dr Jacques Landry, Quèbec, Canada, dilution 1:1000); (2) rabbit-polyclonal anti-BAG3 antibody (Abcam, Cambridge, UK; dilution 1:1000); (3) rabbit-polyclonal anti-BAG1 (Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000); (4) rabbit polyclonal anti-LC3 (Sigma; dilution 1:1000); (5) rabbit polyclonal anti-SQSTM1/p62 (Abcam; dilution 1:1000); (6) rabbit-polyclonal anti-GAPDH (Santa Cruz Biotech; dilution 1:1000); (7) rabbit-polyclonal anti-SOD1 (SOD-100; StressMarq, Victoria, BC, Canada; dilution 1:1000). Immunoreactivity was detected using goat antirabbit (sc-2004, Santa Cruz Biotech, dilution 1:5000) secondary peroxidase-conjugated antibody. Immunoreactivity was then visualized using the enhanced chemiluminescence detection kit reagent (ECL prime Western Blotting Substrate, GE Healthcare, Maidstone, UK). The same membranes were subsequently processed with different antibodies to detect the levels of different proteins in the same samples loaded on the gel, after stripping for 10 min at room temperature (StripABlot, EuroClone). Each experiment was carried out twice, with four independent samples.

FRA was performed using a slot-blot apparatus (Bio-Rad). 3µg of total proteins were loaded on a 0.2µm cellulose acetate membrane (Whatman, GE Healthcare), pre-incubated with 20% methanol, washed with water and then incubated with 1% SDS solution. After slight vacuum filtration, slot-blot membranes were probed as described for WB.

A ChemiDoc XRS System (Bio-Rad) was used for the image acquisition of WB and FRA.

### **STATISTICAL ANALYSIS**

Statistical analysis was performed through two-tailed Student *t-test* for comparisons between 8 and 16 weeks mice of the same group (NTg/Tg wtSOD1/Tg G93A-SOD1) and two-way analysis of variance (ANOVA) for group comparisons, using the PRISM software (GraphPad, San Diego, CA, USA). Specific group pair(s) statistical difference was determined by the Bonferroni *post-hoc* test. Data were expressed as mean ± SD of four independent samples.

## **RESULTS**

Our study has been performed on 8 (presymptomatic stage, PS) and 16 weeks-old (symptomatic stage, S) Tg G93A-SOD1 mice, compared to age-matched non-transgenic (NTg) and transgenic human wild-type SOD1 (Tg wtSOD1) mice. In addition, since gender differences have been reported to affect the age of onset and disease progression both in ALS patients (Kurtzke, 1982; Giagheddu et al., 1983; Rudnicki, 1999; Manjaly et al., 2010; McCombe and Henderson, 2010; Lee et al., 2013a) and rodent models of ALS (Veldink et al., 2003; Suzuki et al., 2007), with male gender being more susceptible, we analyzed males and females separately. Based on our data on the protective role of HSPB8 in ALS, which is mediated by autophagy, we analyzed the expression of this small chaperone in the spinal cord and muscle of Tg G93A-SOD1 mice and control mice. We also analyzed the expression of the two co-chaperones BAG3 and BAG1, which may mediate the PQC activity by selecting the proper degradative system to be activated in cells in response to proteotoxicity. Finally, we analyzed two widely used autophagic markers, LC3 and p62. Using homogenates of spinal cord and skeletal muscle derived from Tg G93A-SOD1 mice and age-matched NTg or Tg wtSOD1 mice, we first compared the levels of wtSOD1 and mutSOD1 in both the soluble and insoluble fractions in 8 (corresponding to PS stage) and 16 weeks (corresponding to S stage) male and female mice. As shown in **Figure 1**, soluble monomeric wtSOD1 and mutSOD1 were analyzed by western blot (WB; **A**: spinal cord; **B**: muscle). In the spinal cord of Tg mice the levels of wtSOD1 and mut-SOD1 proteins were similar, while in skeletal muscle mutSOD1 levels were apparently lower as compared to wtSOD1 and no gender differences were observed. Using motoneuronal and muscle ALS models as well as tg G93A-SOD1 mice, we recently demonstrated that mutSOD1 accumulated both in motoneuronal cells and in spinal cord of tg mice (already at PS, increasing at S stage), but did not accumulate in ALS muscle cells and ALS muscle tissues at any age tested (Onesto et al., 2011; Galbiati et al., 2012). These data were recently confirmed and extended by Wei and coll. (Wei et al., 2013) using a different strain of tg G93A-SOD1 mice. Thus, here we repeated these experiments to provide a general view of mutSOD1 behavior in our mice. A representative filter retardation assay (FRA; **C**: spinal cord; **D**: muscle) illustrating the corresponding insoluble fractions of mutSOD1 is reported. The FRA analysis confirmed the presence of relevant amounts of mut-SOD1 insoluble species in samples of S stage tg G93A-SOD1 mice. In muscle tissue, no mutSOD1 insoluble species were present, with the exception of samples obtained from tg G93A-SOD1 male mice at S stage (16 weeks), suggesting that muscle tissue may better cope with the misfolded fraction of mutSOD1 as compared to cells in the spinal cord.

Since we already showed that overexpression of HSPB8 prevents mutSOD1 intracellular accumulation by facilitating its clearance through autophagy (Crippa et al., 2010a,b), we analyzed HSPB8 mRNA levels in the spinal cord and skeletal muscle in response to wt or mutSOD1 expression. In spinal cord, we observed a significant increase of HSPB8 mRNA transcript levels in response to mutSOD1 expression in S stage (16 weeks) Tg G93A-SOD1 male mice as compared to age-matched NTg or Tg wtSOD1 mice (**Figure 2A**) (Crippa et al., 2010b). Interestingly, Tg G93A-SOD1 female mice did not show any significant increase of HSPB8 transcript level in response to mutSOD1expression,

**D**, FRA) of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (wtSOD1), and of suggesting the existence of gender differences in the PQC system activated by misfolded proteins in spinal cord. In male 16-weekold NTg mice, the expression level of HSPB8 tended to decrease, even if it did not become statistically significant. However, we already shown that HSPB8 expression significantly decreases when older NTg mice (age-matched with Tg G93A-SOD1 mice at end stage of disease) were analyzed (Crippa et al., 2010a,b). In contrast to what we found in spinal cord, in the skeletal muscles of S stage (16 weeks) Tg G93A-SOD1 mice we observed a dramatic increase of HSPB8 mRNA (up to 8-fold) (**Figure 2B**). Importantly, this HSPB8 mRNA upregulation occurred in both sexes. HSPB8 mRNA levels were unaffected in both sexes in NTg and Tg wtSOD1 mice at all ages considered.

The robust induction of HSPB8 expression in response to mutSOD1 in muscle might serve to initiate and/or facilitate the clearance of aberrant mutSOD1 species. This may avoid the aggregation of misfolded protein and the accumulation to levels that cannot be managed by the PQC system. Considering that mutSOD1 species are degraded by the saturable UPS, but it can be preferentially degraded by the highly efficient autophagic system in presence of high levels of HSPB8 (Crippa et al., 2010b) and that the pro-degradative activity of HSPB8 is mediated by its partner BAG3, we analyzed the expression levels of BAG3 in both spinal cord and muscle (Carra et al., 2008b; Carra, 2009). We found that in the spinal cord of both male and female mutSOD1 mice BAG3 expression remains unchanged at PS stage (8 weeks), but it was significantly increased by approximately 3-fold compared to NTg or Tg wtSOD1 mice at S stage (16 weeks, **Figure 3A**). Furthermore, the mutSOD1 induced appearance of symptoms correlated with a great response of the HSPB8-BAG3-mediated PQC system also in muscle (**Figure 3B**). Curiously, in female mice, BAG3 expression differed from that of HSPB8, since it was upregulated by mutSOD1 both in spinal cord and in muscle of tg G93A-SOD1 mice at S stage.

murine SOD1.

loading. hSOD1, transgenic human SOD1; mSOD1, endogenous

In light of our previous results showing that muscle cells possess a higher UPS activity with respect to motoneuronal cells (Onesto et al., 2011), we analyzed the expression of BAG1, which is involved in the re-routing of the misfolded proteins to proteasome-mediated substrates degradation. In spinal cord, BAG1 transcript levels were not affected by mutSOD1 expression (**Figure 3C**). On the contrary, BAG1 expression increased 2-folds in the skeletal muscle of S stage Tg G93A-SOD1 mice compared to all other groups analyzed (**Figure 3D**). This increase was similar in both sexes. These data suggest that proteasomemediated substrate degradation is highly favored in muscle cells under proteotoxic conditions (e.g., misfolded mutSOD1 overexpression). We also estimated the ratio BAG3:BAG1 in each sample (**Figures 3E,F**) and found that this is always more elevated in muscle tissue, both in control and tg G93A-SOD1 mice, suggesting a predominance of the autophagic pathway, but it reaches higher levels at S stage of disease in response to mutSOD1. In the case of spinal cord, the BAG3:BAG1 ratio increases only at S stage (16 weeks) both in male and female tg G93A-SOD1 mice, suggesting that misfolded mutSOD1 could be re-routed to the autophagic system at this stage of disease (possibly not fully removed because of autophagic flux blockage).

Next, we asked whether the upregulation of the transcript levels of the genes described above results in an increase in protein expression. As shown in **Figure 4**, in the spinal cord **(A)**, HSPB8, BAG1, and BAG3 protein levels correlated with the data of gene transcription observed in the same mice. In skeletal muscle, we found that HSPB8, BAG3, and BAG1 were all up-regulated in Tg G93A-SOD1 mice at the S stage (16 weeks) of disease compared to Ntg and Tg wtSOD1 mice (**Figure 4B**). In the case of BAG3, the protein was up-regulated also at the PS stage (8 weeks). Little variation was observed in Tg G93A-SOD1 mice at S stage, while no major sex differences were noted. Also these data were perfectly in line and corroborate the mRNA expression analysis data reported above.

Since HSPB8 overexpression has beneficial effects in cell models of fALS, it is upregulated in human tissues from patients, and its mode of action requires the partner BAG3 and a functional autophagy system, we then analyzed the expression of two widely used markers of the autophagic flux: LC3 (which is overexpressed and associated with the autophagosome when autophagy is activated) and p62 (which is also upregulated during autophagy activation and is responsible for the insertion of ubiquitinated misfolded protein species into the autophagosomes). We found a moderate increase of LC3 mRNA selectively in the spinal cord of S stage (16 weeks) Tg G93A-SOD1 female mice (**Figure 5A**). In contrast, in skeletal muscle of Tg G93A-SOD1 mice (**Figure 5B**), LC3 expression was robustly increased at S stage both in male and female animals. p62 mRNA levels remained unchanged in spinal cord (**Figure 5C**), while they were increased in muscle of S stage female mice (**Figure 5D**). Notably, p62 levels increased also in Tg G93A-SOD1 male mice at S stage, even if this induction was not statistically significant.

Protein levels of LC3 and p62 as well as the conversion of free LC3-I to autophagosome-associated lipidated form of LC3-II were analyzed by WB (**Figure 6**). In spinal cord samples no major variations of p62 protein were observed, while the levels of LC3 protein were higher in Tg G93A-SOD1 mice (**Figure 6A**). Surprisingly, no conversion of LC3-I to LC3-II was observed, suggesting that the autophagic process is not massively activated in the spinal cord at these stages of disease. In skeletal muscle, p62 protein levels robustly increased in Tg G93A-SOD1 mice at both ages and in both sexes (**Figure 6B**). Moreover, at S stage (16 weeks), in skeletal muscle samples, the lipidated form of LC3 (LC3-II), which is the only one capable to associate with the newly formed autophagosomes, was increased in Tg G93A-SOD1 mice.

**FIGURE 3 | BAG3 and BAG1 expression in spinal cord and muscle of ALS animal models.** RT-qPCRs were performed on total RNA extracted from whole spinal cord **(A,C)** or quadriceps muscles **(B,D)** of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). Data have been normalized to the amount of GAPDH mRNA, expressed relative to the levels determined in age-matched PS female NTg mice, taken as internal reference, and expressed as fold changes. Data are mean ± SD of four independent replicates. **(A)** RT-qPCR on BAG3 mRNA expression levels in whole spinal cord. ∗∗*p <* 0*.*01 vs. ageand sex-matched NTg and Tg wtSOD1 mice; ∧*p <* 0*.*05 vs. age- and sex-matched NTg mice; §§*p <* 0*.*01 vs. age- and sex-matched Tg wtSOD1 mice. **(B)** RT-qPCR on BAG3 mRNA expression levels in quadriceps

muscles. ∗∗*p <* 0*.*01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ◦*p <* 0*.*05 vs. PS (8 weeks) female Tg G93A-SOD1; ##*p <* 0*.*01 vs. PS (8 weeks) male Tg G93A-SOD1. **(C)** RT-qPCR on BAG1 mRNA expression levels in whole spinal cord. **(D)** RT-qPCR on BAG1 mRNA expression levels in quadriceps muscles. ∗∗*p <* 0*.*01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ◦*p <* 0*.*05 vs. sex-matched PS (8 weeks) Tg G93A-SOD1. **(E)** BAG3:BAG1 relative ratio of mRNA expression levels in whole spinal cord. Data represent variations of the relative levels of BAG3 and BAG1 normalized over the relative BAG3 and BAG1 levels of age-matched PS (8 weeks) female NTg mice (taken as internal reference, see Materials and Methods for details). ∗∗*p <* 0*.*01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ##*p <* 0*.*01 vs. PS (8 weeks) male Tg G93A-SOD1. **(F)** BAG3:BAG1 relative ratio of mRNA expression levels in quadriceps muscles. Data have been calculated as in **(E)**. ∗*p <* 0*.*05 and ∗∗*p <* 0*.*01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ◦*p <* 0*.*05 vs. PS (8 weeks) male Tg G93A-SOD1.

**FIGURE 4 | HSPB8-BAG1-BAG3 protein levels in spinal cord and muscle of ALS animal models.** Western Blot (WB) analysis on proteins extracted from whole spinal cord **(A)** or quadriceps muscles **(B)** of female and male non-transgenic (NTg) mice, of mice expressing the wild type human SOD1 transgene (Tg wtSOD1), and of mice expressing the G93A mutant form of human SOD1 (Tg G93A-SOD1), at 8 (corresponding to presymptomatic stage, PS, in Tg G93A-SOD1 mice) or 16 weeks (corresponding to symptomatic stage, S, in Tg G93A-SOD1 mice). GAPDH was used to normalize protein loading.

NTg mice, taken as internal reference, and expressed as fold changes. Data are mean ± SD of four independent replicates. **(A)** RT-qPCR on LC3 mRNA expression levels in whole spinal cord. ∗∗*p <* 0*.*01 vs. ageand sex-matched NTg and Tg wtSOD1 mice. **(B)** RT-qPCR on LC3 mRNA expression levels in quadriceps muscles. ∧*p <* 0*.*05 vs. age- and sex-matched NTg mice; §§*p <* 0*.*01 vs. age- and sex-matched Tg wtSOD1 mice. **(C)** RT-qPCR on p62 mRNA expression levels in whole spinal cord. **(D)** RT-qPCR on p62 mRNA expression levels in quadriceps muscles. ∗∗*p <* 0*.*01 vs. age- and sex-matched NTg and Tg wtSOD1 mice; ◦◦*p <* 0*.*01 vs. PS female (8 weeks) Tg G93A-SOD1.

Collectively, these data strongly suggest that muscle, probably because of higher HSPB8, BAG1, and BAG3 expression, not only may better cope with misfolded mutant proteins, but also has a more intense autophagic response power compared to cells in the spinal cord.

## **DISCUSSION**

In a previous study, we showed that spinal cord and muscle differently cope with mutSOD1 (Onesto et al., 2011). In fact, in Tg G93A-SOD1 mice mutSOD1 tends to accumulate in the spinal cord, while no accumulation of high molecular weight (HMW) species was found in muscles (Galbiati et al., 2012). Thus, the specific ability of spinal cord and skeletal muscle to handle misfolded protein aggregation and clearance (Cheroni et al., 2005, 2009; Basso et al., 2006, 2009; Bendotti et al., 2012; Galbiati et al., 2012; Wei et al., 2012) could be related to differences in their response to proteotoxicity, mediated by the PQC system. In this study we further investigated this aspect and found that mutSOD1 poorly accumulates in skeletal muscle, and only a mild increase of SDSinsoluble species of mutSOD1 is detectable in this tissue in Tg G93A-SOD1 male mice at the S stage (16 weeks), but not in female mice. This suggests that differences in the PQC system may also exist between the two sexes. Consequently, we then evaluated whether the expression pattern of specific factors of the PQC system shows a different expression profile in the spinal cord and muscle of a tg ALS mice model also considering possible gender differences.

To this purpose, here, we evaluated whether modifications occur at the initial stages of the PQC system, by focusing our attention on the HSPB8-BAG3 mediated pathway, involved in the autophagic process as well as on BAG1 pathway, involved in UPS mediated PQC. We then further investigated the autophagic process, since mutSOD1 induced alterations of UPS have already been analyzed by several investigators, reviewed in Bendotti et al. (2012), including us (Onesto et al., 2011). Autophagic response was analyzed using two well-known autophagic markers: LC3 and p62.

Our data clearly demonstrated that in both spinal cord and muscle HSPB8, BAG1, and BAG3 expression levels are not affected at early stage of disease (PS). The HSPB8-mediated PQC response was induced at the S stage of disease, and this activation was lower in spinal cord than muscle. Therefore, in spinal cord, the presence of misfolded mutSOD1 might not be sufficient to induce the activation of the PQC system *per se*, unless the disease is in an advanced phase, when motoneurons loss and/or alterations of muscle structures are present (Crippa et al., 2010b). Considering BAG3 and BAG1 expression, we found that their behavior was similar to that observed for HSPB8 at S stage (16 weeks). However, the BAG3 response appeared higher than that of BAG1, and BAG3 induction was also present in the spinal cord at S stage. Thus, cells in spinal cord have a lower capability than muscle cells to activate the HSPB8/BAG3- and BAG1-mediated PQC responses, and this might explain the larger accumulation of mutant misfolded proteins in this structure. It is of note that mutSOD1 expression results in the induction of both HSPB8 and BAG3 in the same structures, since HSPB8 activity is enhanced by BAG3 recruitment. Once the HSPB8-BAG3 interaction occurs, the complex recognizes the misfolded proteins and recruits the constitutively expressed chaperone HSC70 bound to the CHIP ubiquitinating enzyme (Arndt et al., 2010), allowing misfolded proteins removal by autophagy (Carra et al., 2008a,b, 2012, 2013; Arndt et al., 2010; Crippa et al., 2010b). In fact, CHIP ubiquitinates the target misfolded protein, which is then recognized by the protein p62 for its delivery to the nascent autophagosome (Arndt et al., 2010). Thus, HSPB8, BAG3, and the autophagy pathway may represent key players in the stress response elicited by mutSOD1 expression. Therefore, changes in their expression levels may correlate with the efficiency with which spinal cord and muscle cope with aggregate-prone mutSOD1.

On the other hand, the complex HSC70/CHIP can also associate to BAG1 to re-route substrates to UPS. When the proteasome component of UPS is overwhelmed or impaired by the misfolded proteins, HSPB8 and BAG3 are both highly induced. UPS inhibition is indeed a condition which occurs in motoneurons and muscle cells expressing mutSOD1 (Onesto et al., 2011), and the increase in the BAG3:BAG1 ratio allows BAG3 to more efficiently direct the HSC70/CHIP–associated substrates to autophagy for degradation (Zhang and Qian, 2011). Our data are in line with this working hypothesis, since in muscle we estimated that the BAG3:BAG1 ratio in tg G93A-SOD1 mice at S stage (16 weeks) is much higher than that observed in control mice. Conversely, in the spinal cord of tg G93A-SOD1 mice at S stage the BAG1 response is not detectable.

By following the auophagic response in muscle, our data here clearly show that at the S stage, muscle cells are still able to upregulate two essential players of the autophagic process, (a) p62, which recognizes the CHIP-ubiquitinated misfolded proteins in the HSPB8-BAG3-HSC70-CHIP complex, allowing their insertion into the autophagosome, and (b) LC3, which, after its activation to LC3, it is recognized by p62 and assists formation of autophagosomes. These steps are critical to initiate a correct autophagic flux required to clear the large excess of misfolded proteins from the cells during PQC response. This response does not efficiently take place in the spinal cord, since at S stage (16 weeks), misfolded mutSOD1 does not induce the overexpression of p62 and LC3, suggesting that the autophagic response in this structure is not as efficient as it is in muscle. In addition, in spinal cord very low basal levels of LC3-II form can be detected, suggesting that at these ages very few autophagosomes can be generated in response to mutSOD1. These data are consistent with previous reports obtained in the spinal cord of early symptomatic tg G93A-SOD1 mice, in which the LC3-I to LC3-II conversion, and thus, autophagy activation, occurs at very low levels, and it efficiently takes place only after the 17 week of age (Tian et al., 2011; Lee et al., 2013b). With disease progression, several autophagosomes appear in spinal cord motoneurons surviving at the end stage of disease (Li et al., 2008; Crippa et al., 2010b). This autophagic response can also be selectively activated in different cell types; for example LC3-I to LC3-II conversion can be visualized in cultured astrocytes in response to mutSOD1 (Kim et al., 2013).

With regards to the possible gender differences existing in the pathways here analyzed, we only found that, at S stage in tg G93A-SOD1 mice, the expression of p62 is significantly induced in skeletal muscle of female but not in that of male mice. These observations may help to explain why in skeletal muscle of tg G93A-SOD1 male mice mutSOD1 tends to accumulate in insoluble forms (see above). Interestingly, a mild increase of HSPB8 expression was present in spinal cord at S stage (16 weeks) of tg G93A-SOD1 male mice, but not in female mice. The molecular mechanism at the basis of this peculiar gender differences is unknown. Interestingly enough, there is a positive correlation between mutSOD1 accumulation and HSPB8 expression in the spinal cord of male tg G93A-SOD1 mice at S stage (16 weeks). This suggests that HSPB8 gene transcription increases in response to misfolded species accumulation. These data are in line with our previous results showing that an UPS impairment (which is know to result in a further mutSOD1 aggregation), lead to HSPB8 overexpression and stabilization (Crippa et al., 2010a,b; Bendotti et al., 2012; Carra et al., 2012, 2013). It remains to be determined why this HSPB8 overproduction fails to clear mutSOD1 insoluble species via autophagy (Crippa et al., 2010b; Rusmini et al., 2010).

By analysing the molecular and biochemical behavior of mut-SOD1, we have previously elucidated several potential mechanisms of neurotoxicity associated with accumulation of misfolded proteins in motoneuronal cells (Sau et al., 2007, 2011; Crippa et al., 2010a,b; Onesto et al., 2011). For instance, the formation of HMW mutSOD1 species and aggregation correlated with an altered intracellular mutSOD1 distribution (Sau et al., 2007; Crippa et al., 2010b). In these conditions, mutSOD1 nuclear bioavailability was reduced in motoneuronal, but not in muscle cells (Sau et al., 2007; Crippa et al., 2010a,b; Onesto et al., 2011), an aberrant biochemical behavior that may result in a decreased nuclear protection from free radical species (Sau et al., 2007). In addition, by evaluating mitochondrial superoxide induced oxidation, using MitoSOX (a mitochondrial specific fluorogenic dye oxidized by superoxide) in motoneuronal NSC34 cell models of ALS, we found that mutSOD1 expressing motoneuronal cells are more sensitive to a superoxide-induced oxidative stress when compared to wtSOD1 expressing motoneuronal cells (Onesto et al., 2011). These mitochondrial alterations were not found in muscle C2C12 cells expressing either wt or mutSOD1 (Onesto et al., 2011). Therefore, mutSOD1 altered intracellular bioavailability, due to sequestration into aggregates, might result in a less efficient protection against free radical species damages in different subcellular compartments (Sau et al., 2007; Onesto et al., 2011).

Our results are in line with a recent report showing that protein misfolding, mitochondrial dysfunction and muscle loss are not directly dependent on soluble and aggregated mutSOD1 in skeletal muscle of ALS (Wei et al., 2012). Moreover, soluble mutSOD1 does not have direct effects on mitochondrial dysfunction as determined by quantifying the release of reactive oxygen species (ROS) in skeletal muscle mitochondria (Wei et al., 2012).

Interestingly, dysfunction of skeletal muscle and degeneration of neuromuscular junctions precede disease onset and motoneurons loss in tg ALS mice (Frey et al., 2000; Fischer et al., 2004). Notably, while the reduction of mutSOD1 expression in the skeletal muscle of tg ALS mice has no effect on disease progression (Miller et al., 2006), the selective expression of mutSOD1 in skeletal muscle induces atrophy and mitochondrial abnormalities in this tissue (Dobrowolny et al., 2008a; Corti et al., 2009; Wong and Martin, 2010). Moreover, mutSOD1-induced muscle damage leads to loss of mutSOD1-negative motoneurons in the anterior horn of the spinal cord, suggesting that mutSOD1 neurotoxicity can be exerted at different levels and its restricted expression in muscle can be sufficient to induce an ALS-like disease in mice (Dobrowolny et al., 2005, 2008a,b).

The data presented here clarify that mutSOD1 activates distinct pathogenetic pathways in muscle and motoneuron cells. In addition, the aggregation-independent mutSOD1 toxicity in muscle can be monitored at transcriptional levels. In fact, in our muscle cell models of ALS we observed specific alterations in the expression of typical genes controlling muscle pathways activated by nerve injury, or muscular atrophy [e.g., MyoD, myogenin (two myogenic regulatory factors), and TGFbeta1, which are markers for muscle fiber damage or atrophy] (Galbiati et al., 2012). Thus, muscle cells are able to better manage misfolded mutSOD1 species, even if misfolding is an intrinsic property of mutSOD1, and it does not depend on the cell environment (Dobson, 2003). It remains to be clarified whether the autophagic activation induced by mutSOD1 in muscle initially protective, when excessive, may become one of the toxic events mediating the deleterious activity associated with this mutant protein (Nassif and Hetz, 2011; Zhang et al., 2011). This hypothesis has already been proved in a ALS-related motoneuron disease, the Spinal and Bulbar Muscular Atrophy (SBMA) linked to an expansion of a polyQ stretch in the androgen receptor (ARpolyQ) (Poletti, 2004; Rusmini et al., 2013). Even in this motoneuron disease the mutant ARpolyQ affects both spinal cord and muscle, but it has been shown that an enhanced autophagy exacerbated skeletal muscle atrophy (Yu et al., 2011). Indeed, the genetic inhibition of autophagy in muscle, through haploinsufficiency for Beclin-1, a component of the autophagic initiation complex, mitigates skeletal muscle atrophy and prolongs survival of a tg SBMA mouse model (Yu et al., 2011).

Therefore, the toxicity exerted by mutant misfolded proteins in muscle cells is probably not related to the classical mechanism of intracellular protein aggregation, but other mechanisms of toxicity may be involved.

### **ACKNOWLEDGMENTS**

AriSLA Foundation Italy (ALS\_HSPB8 to Angelo Poletti and Serena Carra), Telethon—Italy (GGP06063 and GGP07063 to Angelo Poletti, GGP10037 to Maria Pennuto); Italian Ministry of Labour, Health and Social Affairs (Convenzione Fondazione Mondino/UNIMI to Angelo Poletti); Regione Lombardia (to Angelo Poletti and Valeria Crippa); Universita' degli Studi di Milano (to Angelo Poletti and Valeria Crippa); Fondazione CARIPLO (2008-2307 to Angelo Poletti); Fondation Thierry Latran, France (to Angelo Poletti and Maria Pennuto), Association Française contre les Myopathies (to Angelo Poletti), Muscular Dystrophy Association (196646 to Maria Pennuto), and Marie-Curie Reintegration Grant (FP7-256448 to Serena Carra).

### **REFERENCES**

and a common pathogenic pathway in ALS. *Nat. Neurosci.* 13, 1396–1403. doi: 10.1038/nn.2660

target of SOD1G93A-mediated toxicity. *Cell Metab.* 8, 425–436. doi: 10.1016/j.cmet.2008.09.002

*Biochem. Biophys. Res. Commun.* 365, 381–385. doi: 10.1016/j.bbrc.2007. 11.001

Zhang, X., and Qian, S. B. (2011). Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes. *Mol. Biol. Cell* 22, 3277–3288. doi: 10.1091/mbc.E11-05-0388

**Conflict of Interest Statement:** Maria Pennuto and Angelo Poletti received support from Siena Biotech (Italy). The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 06 September 2013; accepted: 07 November 2013; published online: 26 November 2013.*

*Citation: Crippa V, Boncoraglio A, Galbiati M, Aggarwal T, Rusmini P, Giorgetti E, Cristofani R, Carra S, Pennuto M and Poletti A (2013) Differential autophagy power in the spinal cord and muscle of transgenic ALS mice. Front. Cell. Neurosci. 7:234. doi: 10.3389/fncel.2013.00234*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2013 Crippa, Boncoraglio, Galbiati, Aggarwal, Rusmini, Giorgetti, Cristofani, Carra, Pennuto and Poletti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Early gene expression changes in spinal cord from SOD1G93A Amyotrophic Lateral Sclerosis animal model

## *Gabriela P. de Oliveira , Chrystian J. Alves and Gerson Chadi\**

*Department of Neurology, Neuroregeneration Center, University of São Paulo School of Medicine, São Paulo, Brazil*

### *Edited by:*

*Ricardo Tapia, Universidad Nacional Autónoma de México, Mexico*

### *Reviewed by:*

*Hermona Soreq, The Hebrew University of Jerusalem, Israel Takumi Takizawa, Gunma University, Japan*

#### *\*Correspondence:*

*Gerson Chadi, Department of Neurology, University of São Paulo, Av. Dr. Arnaldo, 455, 2nd floor, room 2119, 01246-903-São Paulo, Brazil e-mail<EMAIL_ADDRESS>
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and fast progression neurodegenerative disease that leads to the loss of motor neurons. Mechanisms of selective motor neuron loss in ALS are unknown. The early events occurring in the spinal cord that may contribute to motor neuron death are not described, neither astrocytes participation in the pre-symptomatic phases of the disease. In order to identify ALS early events, we performed a microarray analysis employing a whole mouse genome platform to evaluate the gene expression pattern of lumbar spinal cords of transgenic SOD1G93A mice and their littermate controls at pre-symptomatic ages of 40 and 80 days. Differentially expressed genes were identified by means of the Bioconductor packages Agi4×44Preprocess and limma. FunNet web based tool was used for analysis of over-represented pathways. Furthermore, immunolabeled astrocytes from 40 and 80 days old mice were submitted to laser microdissection and RNA was extracted for evaluation of a selected gene by qPCR. Statistical analysis has pointed to 492 differentially expressed genes (155 up and 337 down regulated) in 40 days and 1105 (433 up and 672 down) in 80 days old ALS mice. KEGG analysis demonstrated the over-represented pathways tight junction, antigen processing and presentation, oxidative phosphorylation, endocytosis, chemokine signaling pathway, ubiquitin mediated proteolysis and glutamatergic synapse at both pre-symptomatic ages. *Ube2i* gene expression was evaluated in astrocytes from both transgenic ages, being up regulated in 40 and 80 days astrocytes enriched samples. Our data points to important early molecular events occurring in pre-symptomatic phases of ALS in mouse model. Early SUMOylation process linked to astrocytes might account to non-autonomous cell toxicity in ALS. Further studies on the signaling pathways presented here may provide new insights to better understand the events triggering motor neuron death in this devastating disorder.

**Keywords: ALS, SOD1G93A, pre-symptomatic, spinal cord, microarray, laser microdissection, astrocytes**

### **INTRODUCTION**

Amyotrophic Lateral Sclerosis (ALS) is a fast disabling neurodegenerative disease characterized by upper and lower motor neuron loss of motor cortex, brainstem, and spinal cord leading to respiratory insufficiency and death (Turner et al., 2013). The incidence of ALS ranges from 1.7 to 2.3 cases per 100,000 population per year worldwide (Beghi et al., 2006). The mechanisms underlying neurodegeneration in ALS are multifactorial, and seem to involve neurons and non-neuronal cells (Boillee et al., 2006a,b; Yamanaka et al., 2008; Wang et al., 2011a) as well as several molecular pathways (Boillee et al., 2006a; Ferraiuolo et al., 2011b; Kiernan et al., 2011; Usuki et al., 2012). Approximately 5% of ALS cases are familial, and 20% of these have been linked to mutations in Cu/Zn superoxide dismutase 1 (SOD1) (Rosen et al., 1993; Andersen and Al-Chalabi, 2011). The first symptoms define the beginning of the clinical phase of the diagnosed cases of the more prevalent sporadic forms, consisting in muscle atrophy, weakness, fasciculations, and spasticity (Brooks et al., 2000). There is a lack of pathological studies on post mortem spinal cord from ALS patients that could add information about the triggering, initial time of motor neuron death and mechanisms of the disease. In fact, Fischer et al. (2004) reported the postmortem evaluation in a patient with a short history of ALS, whose electromyography showed signs of acute and chronic denervation, coming out with an unexpected die without peripheral and central motor neuron death together with autolytic changes and a little axonal degeneration. Histological evaluations at the neuromuscular junctions and also electrophysiological analysis at the peripheral nerves in ALS patients have allowed authors to claim that motor neuron death correlates to the begging of clinical classical symptoms (Veugelers et al., 1996; Liu et al., 2013).

As the majority of familial ALS cases are linked to the mutations in SOD1 gene (Dion et al., 2009), transgenic mice expressing human mutant SOD1 (mSOD1) developing agedependent clinical and pathological features of human ALS are current largely employed in the physiopathological studies of the disorder (Turner and Talbot, 2008). Using this mouse model, we previously described early behavior and electrophysiological alterations, prior the classical neurological symptoms and the beginning of motor neuron death (Alves et al., 2011). In fact, several early events demonstrated in animal models seemed to precede the neuronal death, remarkably the activation of glial cells (microglia and astrocytes) close to motor neurons (Graber et al., 2010; Wang et al., 2011b; Gerber et al., 2012), retraction of motor neuron fibers and neuromuscular junction displacement (Fischer et al., 2004; De Winter et al., 2006; Narai et al., 2009). It is still unknown whether the most claimed pathogenic processes for ALS, for instance oxidative stress, mitochondrial and neurofilament dysfunction, excitotoxicity, inflammation, non-autonomous cell toxicity, protein misfolding and abnormal RNA processing (Rothstein et al., 1992; Bergeron et al., 1994; Boillee et al., 2006a; Lemmens et al., 2010; Bendotti et al., 2012; Richardson et al., 2013) are taking place at the pre-symptomatic period of the disease.

The central question in understanding ALS facing therapeutic target development involves a further knowledge about the toxic mechanisms that trigger motor neuron death (Boillee et al., 2006a). Until scientific technology approaches do not overstep ethical limitations of clinical studies, the mutant SOD1 expressing mouse model may offer opportunity for a detailed analysis of intra and intercellular signaling-related to motor neuron toxicity.

The profiling of gene expression using different platforms have been largely employed in the ALS model in several stages of the disease course (Olsen et al., 2001; Dangond et al., 2004; Malaspina and De Belleroche, 2004; Jiang et al., 2005; Perrin et al., 2005; Ferraiuolo et al., 2007, 2011a; Yamamoto et al., 2007; Offen et al., 2009; Brockington et al., 2010; D'arrigo et al., 2010; Guipponi et al., 2010; Saris et al., 2013b; Yu et al., 2013), including the early symptomatic phase (Olsen et al., 2001; Yoshihara et al., 2002; Ferraiuolo et al., 2007; Yu et al., 2013), however, there is a lack of information on differential gene expression taking place before classical clinical symptoms (Olsen et al., 2001; Yoshihara et al., 2002; Ferraiuolo et al., 2007; Guipponi et al., 2010). Olsen et al. (2001) inaugurated that issue by looking at patterns of gene expression from SOD1G93A spinal cord by means of a murine restricted platform of oligonucleotide microarray and by describing negligible changes in the transcript profile at the pre-symptomatic phases. Other authors that have examined gene profiling in presymptomatic phases of ALS disease employed restricted platforms of cDNA arrays, used animals with an uncommon symptom onset (Yoshihara et al., 2002; Guipponi et al., 2010) or evaluated gene profiling in specific spinal cord cells (Ferraiuolo et al., 2007, 2011a). Authors have encountered gene expressions related to inflammation, apoptosis, oxidative stress, ATP biosynthesis, myelination, axonal transport as candidates of biological processes taking place in the pre-symptomatic periods of ALS.

By means of a high-density oligonucleotide microarrays linked to specific tools capable to identify enriched pathways, the aim of this work was to identify early molecular changes in the presymptomatic stage in the spinal cord of the SOD1G93A mouse model. The data showed important alterations at early 40 days pre-symptomatic period of disease and in 80 days old presymptomatic mice.

## **MATERIALS AND METHODS**

### **SAMPLES**