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|Year : 2013 | Volume
| Issue : 2 | Page : 107-110
Protein aggregates and regional disease spread in ALS is reminiscent of prion-like pathogenesis
Department of Neurology, University of Miami Miller School of Medicine, Clinical Research Building, 1120 NW 14 Street, Suite 1317, Miami, FL 33136, USA
|Date of Submission||27-Feb-2013|
|Date of Decision||08-Mar-2013|
|Date of Acceptance||05-Mar-2013|
|Date of Web Publication||29-Apr-2013|
Department of Neurology, Clinical Research Building, 1120 NW 14 Street, Suite 1317, Miami, FL 33136
Source of Support: None, Conflict of Interest: None
Amyotrophic lateral sclerosis (ALS) typically commences in a discrete location in a limb or bulbar territory muscles and then spreads to the adjacent anatomical regions. This pattern is consistent with a contiguous spread of the disease process in motor neuron network resulting in progressive motor weakness. The etiology of ALS onset and the mechanism of the regional ALS spread remain elusive. Over the past 5 years, identification of mutations in two RNA binding proteins, trans active response (TAR) DNA-binding protein (TDP-43) and fused in sarcoma (FUS), in patients with familial ALS has led to a major shift in our understanding of the ALS disease mechanism. In addition to their role in RNA metabolism, TDP-43 and FUS form protein aggregates in the affected neurons. More recent findings demonstrating that both TDP-43 and FUS contain glutamine/asparagine (Q/N) residue-rich prion-like domains have spurred intense research interest. This brief review discusses the prion-related domains in TDP-43 and FUS and their implication in protein aggregate formation and disease spread in ALS.
Keywords: ALS, aggregates, FUS, prion, Q/N domain, TDP-43
|How to cite this article:|
Verma A. Protein aggregates and regional disease spread in ALS is reminiscent of prion-like pathogenesis. Neurol India 2013;61:107-10
| » Introduction|| |
First described in clinical detail by Charcot  in 1869, amyotrophic lateral sclerosis (ALS or motor neuron disease, MND) is one of the earliest known neurodegenerative diseases. Following Charcot's initial description, Gowers  in 1886 commented on variable anatomic sites of disease-onset and independent and variable abnormalities of the upper motor neuron and lower motor neuron in ALS, thus broadening the clinical spectrum of the disease. Gowers particularly emphasized focal onset and contiguous spread of the disease process in his description, which is still a classic: …from the part first affected the disease spreads to other parts of the (same) limb. Before it has attained a considerable degree in one limb, it usually shows itself in the corresponding limb on the other side (homologous part)…
This original and astute clinical observation by Gowers has been repeatedly witnessed by practicing neurologists well over a century. The classical focal and asymmetric onset of ALS in spinal or bulbar region and then its spread to contiguous group of motor neurons is now so well known that an experienced physician can often predict the 'logical' spread in a particular case.  Recent detailed autopsy studies of ALS patients have confirmed that loss of motor neurons is most pronounced at the site of onset and diminishes in a gradient fashion with further distance from that site.  The pathophysiologic mechanism underlying the focal onset and regional spread of ALS is unclear. Over the past two decades, several aberrant phenomena including excessive oxidative stress, excitotoxicity, mitochondrial dysfunction, inflammation, and altered axonal transport have been implicated in ALS pathogenesis. However, it is not easily discernable how any of these generalized processes could by itself explain the focal initiation or the progressive spread of the disease through the motor neuronal pool.
Approximately 10% of ALS cases are familial (fALS), mostly autosomal dominant, and the rest are sporadic (sALS). For almost 15 years, the only gene clearly associated with fALS was the Cu-Zn superoxide dismutase 1 (SOD1) gene, which accounts for 20% of fALS cases. The identification of SOD1 mutations  in 1993 ushered in the molecular era of ALS research, and significant insight into ALS pathogenesis has been gained through identification of pathways directly affected by the toxicity of mutant SOD1. Two main discoveries in mutant SOD1-mediated ALS in rodent models are the demonstration that the SOD1 protein aggregates produce a toxic gain of function that causes neuronal loss and the disease can spread in a noncell autonomous fashion in the nervous system. ,
A major shift in our understanding of ALS pathogenesis occurred in 2006 with the identification of a 43-kDa transactive response (TAR) DNA-binding protein (TDP-43) as a key pathological substrate of cellular inclusions in sALS and non-SOD1 fALS, and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U).  It was soon followed in 2008 by the successful discovery of dominant mutations in the TDP-43 gene as a primary cause of ALS,  thus providing the proof of principle that aberrant TDP-43 can trigger neuronal degeneration and cause ALS. The identification of TDP-43 mutations was soon followed by the discovery of mutations in another RNA/DNA-binding protein, fused in sarcoma (FUS) in 2009, as a primary cause of fALS.  Together, TDP-43 and FUS gene mutations account for approximately 10% of the fALS cases. Both in TDP-43- and FUS-linked ALS, the age and site of disease-onset and clinical progression are variable, as in sporadic ALS, and both show characteristic pathological features of the sALS. Incomplete penetrance has been documented for several of TDP-43 and FUS mutations, accounting for some sALS cases.
Both TDP-43 and FUS are predominantly nuclear proteins involved in diverse aspects of RNA metabolism;  however, in ALS disease tissue both are observed as aggregates in the cytosol of affected neurons. ,,, This finding suggests that aberrant protein aggregation and its cytosolic accumulation may play a key role in ALS pathogenesis, akin to the central role of protein misfolding and aggregations observed in other neurodegenerative diseases.  Interestingly, both TDP-43 and FUS contain 'prion-related' glutamine/asparagine (Q/N) rich domains and in the case of TDP-43, almost all the ALS-associated mutations occur in this region. 
In living organisms, proteins are regularly synthesized and degraded each day. During protein synthesis, nascent polypeptide chain exits from ribosomal tunnel and passes through a highly organized and precisely monitored protein quality control systems (see review , ). The newly formed protein is folded in its three dimensional structure, steered by molecular chaperons, and transported to appropriate subcellular site(s) where it executes its physiological functions. In prokaryote and eukaryote cells, an equally efficient and cooperative protein clearance system known as ubiquitin-proteasome system (UPS) and lysosomal autophagy also exists to clear the abnormally misfolded proteins and protein aggregates in order to maintain cellular proteostasis and normal health (reviewed elsewhere  ). For clearance through UPS, misfolded peptide is first targeted and tagged to ubiquitin, a process known as ubiquitination. Ubiquitination requires an isopeptide bond between the targeted protein and ubiquitin; the covalent isopeptide bond is generated between the glycine and lysine aminoacids of the protein moieties. In this context, it is interesting that most pathogenic ALS-associated TDP-43 and FUS mutations occur in the glycine-rich regions of these proteins. Also interestingly, rare mutations in ubiquilin2  and SQSTM1 (sequestone1/p62),  which are involved in the clearance of misfolded and aggregated proteins, are shown to cause TDP-43 positive cellular aggregates and ALS in rare fALS cases. , Excess of misfolded proteins and uncleared aggregates are inherently toxic to cell, a phenomenon well demonstrated in in-vitro cell models.  Why do certain proteins, such as TDP-43, FUS, amyloid-β, tau, etc., aggregate, how these aggregates cause cell toxicity and whether such aggregate-related toxicity is transferrable from cell to cell is an area of intense current research in neurodegenerative diseases.
Currently, prion protein is the only known example of a protein capable of propagating a self-replicating conformation that can spread like infectious particles (transmissible spongiform encephalopathies) across cells and individuals. However, additional proteins are now recognized that can exhibit 'prion-like' behavior under certain circumstances. Such proteins with more than one conformation are known to exist in yeast, invertebrate and mammalian cells.  In these cases, unlike in human prion diseases, the adoption of an alternate protein conformation and template-based spreading of this altered conformation by additional conversion of normal forms, appear not to be deleterious and cause disease, but instead regulates the physiological function of the aggregated protein.
'Prion-like' behavior of proteins is best characterized in yeast Sup35, Ure2, and Rnq1 proteins.  For example, the Sup35 protein is normally required for stop-codon recognition and translational termination; however, under certain stressful conditions it can form a self-propagating fibrillar-b-sheet conformation transmissible to offsprings. Such self-propagating fibrillar-b-sheet conformation seems to be dependent on the N-terminal region, which is characteristically rich in Q/N residues. Because this Q/N rich region is required for prion-like propagation, it is referred to as the 'prion domain'. Induction of the yeast Sup35 prion state leads to loss of Sup35 function and thereby widespread read through of stop codons, allowing the rapid emergence of novel phenotypes, a molecular adaptive strategy for yeast survival under stressful conditions. 
Other examples of prion-like behavior and Q/N rich proteins include CREB (an RNA binding protein) protein in Aplysia  and Pumilio protein in Drosophila.  Q/N rich CREB and Pumilio proteins regulate synaptic activity  and postsynaptic translational suppression,  respectively. Finally, the mammalian proteome contains several Q/N rich prion domains that may similarly use self-aggregation to modulate their activity. A well-studied example is the RNA binding protein TIA1, which is a key component of stress granules, cytoplasmic RNA-protein complexes formed under conditions of cellular stress, which mediates mRNA translational suppression.  The prion-related Q/N domain of TIA1 is essential for self-aggregation and stress granule formation. Thus, one consistent theme for proteins containing prion-related Q/N rich domains from yeast to mammals is stimulus-induced (environmental stress-induced) conformational change leading to self-aggregation, which then alters protein function to orchestrate an adaptive response. Several algorithms based on the amino acid sequence have been used to predict proteins that bind to DNA/RNA and also contain prion-related domains in both yeast and human proteomes. Using one recent algorithm, Markov Model algorithm, trained on known yeast prion-domain containing proteins, FUS and TDP-43, were predicted as the 15 th and 69 th most likely to contain prion-related domains out of nearly 30,000 proteins in the human proteome.  Although the existence of the prion-related domains in TDP-43 and FUS appears convincing, investigations into their role in the normal and pathological functions of these proteins warrants further research.
Neuronal cytoplasmic inclusions of TDP-43 and FUS are observed in cases of sporadic and familial ALS. For TDP-43, the C-terminal region is highly prone to aggregation, both as purified protein in vitro or when expressed as a fragment in yeast or cultured mammalian cells. ,, This strong tendency of the Q/N rich C-terminus of TDP-43 to self-associate and form aggregate is likewise consistent with the behavior of other prion-related Q/N domain containing proteins. Curiously, TDP-43 pathology (nuclear clearing and cytoplasmic aggregates) is observed not only in ALS, but also in affected brain regions in FTLD-U, Alzheimer disease, Parkinson disease and chronic traumatic encephalopathy, and in inclusion body myopathies.  This is quite consistent with the possibility that TDP-43 aggregation is part of a general response to cellular stress, and is likely mediated by the prion-related domain.
Although wild-type TDP-43 and FUS readily aggregate in vitro, this generally does not occur in normal cells (without pathogenic mutation); the intracellular array of protein folding chaperones probably inhibits this phenomenon. In this context, it is known that the "yeast prion" domains can switch their conformation between two states: an intrinsic unfolded state and an aggregated state that can impose its conformation on its unfolded counterpart.  If such a phenomenon can occur with wild-type TDP-43 and FUS, then whether it indeed occurs in sALS and what tips the initiation and accumulation of aggregates remain important unresolved questions.
Recently, attention has been focused on the concept that misfolded proteins involved in neurodegenerations (such as amyloid-β, tau, and synuclein) could be propagated from cell to cell in a prion-like fashion. , Although experimental evidence for this hypothesis is not robust yet, it is attractive as a potential explanation for the clinically observed spread of neurodegenerative disease through particular neuronal network. Given that prion-related Q/N domains are capable of developing altered conformers, which then can recruit aggregation of the native proteins, the TDP-43 and FUS provide a potential molecular substrate to study the spread of de novo and seeded aggregates in recently created TDP-43 and FUS animal models.
| » Conclusion|| |
The identification of mutations in DNA/RNA binding proteins TDP-43 and FUS as causative of ALS, and the demonstration of TDP-43 as a major constituent of cytoplasmic inclusions in patients with sALS and non-SOD1 fALS, has been a major step toward understanding the pathobiology of the disease. Both these proteins appear to be aggregation-prone, and their ALS-linked mutants appear to exhibit greater degree of aggregation-propensity. TDP-43 and FUS also harbor prion-like domains, raising a tantalizing possibility that focal accumulation of cellular aggregates, and their progressive spread into the motor neuron pool, could be the underlying mechanism of the temporo-spatial spread of the disease. It will be interesting to see if seeded aggregates of these proteins are found to spread into the neuronal network of the nervous system. The emergence of protein aggregates as pathognomonic feature in ALS opens unparalleled opportunities toward therapeutic targets and drug discovery strategies based on the pathways of cellular protein aggregate formation and disease spread in ALS.
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