Publications by authors named "Daryl A Bosco"

41 Publications

Mouse closed head traumatic brain injury replicates the histological tau pathology pattern of human disease: characterization of a novel model and systematic review of the literature.

Acta Neuropathol Commun 2021 06 29;9(1):118. Epub 2021 Jun 29.

Department of Neurology, Medical School, University of Massachusetts, 55 Lake Ave, Worcester, USA.

Traumatic brain injury (TBI) constitutes one of the strongest environmental risk factors for several progressive neurodegenerative disorders of cognitive impairment and dementia that are characterized by the pathological accumulation of hyperphosphorylated tau (p-Tau). It has been questioned whether mouse closed-head TBI models can replicate human TBI-associated tauopathy. We conducted longitudinal histopathological characterization of a mouse closed head TBI model, with a focus on pathological features reported in human TBI-associated tauopathy. Male C57BL/6 J mice were subjected to once daily TBI for 5 consecutive days using a weight drop paradigm. Histological analyses (AT8, TDP-43, pTDP-43, NeuN, GFAP, Iba-1, MBP, SMI-312, Prussian blue, IgG, βAPP, alpha-synuclein) were conducted at 1 week, 4 weeks, and 24 weeks after rTBI and compared to sham operated controls. We conducted a systematic review of the literature for mouse models of closed-head injury focusing on studies referencing tau protein assessment. At 1-week post rTBI, p-Tau accumulation was restricted to the corpus callosum and perivascular spaces adjacent to the superior longitudinal fissure. Progressive p-Tau accumulation was observed in the superficial layers of the cerebral cortex, as well as in mammillary bodies and cortical perivascular, subpial, and periventricular locations at 4 to 24 weeks after rTBI. Associated cortical histopathologies included microvascular injury, neuroaxonal rarefaction, astroglial and microglial activation, and cytoplasmatic localization of TDP-43 and pTDP-43. In our systematic review, less than 1% of mouse studies (25/3756) reported p-Tau using immunostaining, of which only 3 (0.08%) reported perivascular p-Tau, which is considered a defining feature of chronic traumatic encephalopathy. Commonly reported associated pathologies included neuronal loss (23%), axonal loss (43%), microglial activation and astrogliosis (50%, each), and beta amyloid deposition (29%). Our novel model, supported by systematic review of the literature, indicates progressive tau pathology after closed head murine TBI, highlighting the suitability of mouse models to replicate pertinent human histopathology.
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http://dx.doi.org/10.1186/s40478-021-01220-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243463PMC
June 2021

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605;

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
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http://dx.doi.org/10.1073/pnas.2024605118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201830PMC
June 2021

Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

Nat Neurosci 2021 May 31. Epub 2021 May 31.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.
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http://dx.doi.org/10.1038/s41593-021-00859-9DOI Listing
May 2021

Is the primate-specific protein pLG72 affecting SOD1 functionality and superoxide formation?

Free Radic Res 2020 Jun 21;54(6):419-430. Epub 2020 Jul 21.

Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.

pLG72 is a primate-specific protein of enigmatic function that was proposed to modulate mitochondria fragmentation and the activity of the peroxisomal enzyme D-amino acid oxidase (DAAO). DAAO is deputed to degradation of the NMDA receptor co-agonist D-serine in human brain and the R199W substitution in DAAO was identified in a familial case of amyotrophic lateral sclerosis (ALS). A recent work reported that U87 glioblastoma cells ectopically expressing pLG72 showed a lower proliferation, produced superoxide radicals, induced SOD1 aggregation and decreased its activity. Because of the role of SOD1 in eliminating ROS species and its relevance in ALS we evaluated the link between pLG72 and SOD1 using both wild-type pLG72 and its R30K variant related to schizophrenia susceptibility. studies on recombinant proteins excluded the establishment of a stable complex and that pLG72 could affect SOD1 activity and stability. At cellular level, ectopic expression of pLG72 in glioblastoma U87 cells did not affect cell viability and ROS/superoxide production: only caspase activity (a marker of apoptosis) was slightly increased in cells expressing the R30K pLG72 variant. SOD1 and pLG72 did not colocalize in transfected U87 glioblastoma cells: pLG72 largely localised to mitochondria and SOD1 was largely cytosolic. Moreover, the ectopic expression of pLG72 appeared not to alter the expression of SOD1 and its aggregation. Altogether, the combination of biochemical and cellular studies allow to exclude that pLG72 modulates SOD1 function and aggregation, thus that it could play a role in ALS susceptibility.
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http://dx.doi.org/10.1080/10715762.2020.1791335DOI Listing
June 2020

Phenotypic Suppression of ALS/FTD-Associated Neurodegeneration Highlights Mechanisms of Dysfunction.

J Neurosci 2019 10;39(42):8217-8224

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912,

A fundamental question regarding the etiology of amyotrophic lateral sclerosis (ALS) is whether the various gene mutations associated with the disease converge on a single molecular pathway or act through multiple pathways to trigger neurodegeneration. Notably, several of the genes and cellular processes implicated in ALS have also been linked to frontotemporal dementia (FTD), suggesting these two diseases share common origins with varied clinical presentations. Scientists are rapidly identifying ALS/FTD suppressors that act on conserved pathways from invertebrates to vertebrates to alleviate degeneration. The elucidation of such genetic modifiers provides insight into the molecular pathways underlying this rapidly progressing neurodegenerative disease, while also revealing new targets for therapeutic development.
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http://dx.doi.org/10.1523/JNEUROSCI.1159-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794934PMC
October 2019

The RNA-binding protein FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for mRNA processing.

J Biol Chem 2019 06 15;294(26):10194-10210. Epub 2019 May 15.

From the Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 () in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.
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http://dx.doi.org/10.1074/jbc.RA118.005933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664169PMC
June 2019

Quantitative proteomics identifies proteins that resist translational repression and become dysregulated in ALS-FUS.

Hum Mol Genet 2019 07;28(13):2143-2160

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.

Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins influence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA- and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-relatedprocesses in cells expressing mutant FUS. Even in the absence of stress, COPBI localization was altered in primary and human stem cell-derived neurons expressing ALS-linked FUS variants. Our results suggest that Golgi to ER retrograde transport may be important under conditions of stress and is perturbed upon the expression of disease-linked proteins such as FUS.
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http://dx.doi.org/10.1093/hmg/ddz048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586143PMC
July 2019

Translation dysregulation in neurodegenerative disorders.

Authors:
Daryl A Bosco

Proc Natl Acad Sci U S A 2018 12 30;115(51):12842-12844. Epub 2018 Nov 30.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605

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http://dx.doi.org/10.1073/pnas.1818493115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304993PMC
December 2018

Endoplasmic reticulum stress leads to accumulation of wild-type SOD1 aggregates associated with sporadic amyotrophic lateral sclerosis.

Proc Natl Acad Sci U S A 2018 08 23;115(32):8209-8214. Epub 2018 Jul 23.

Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile 8380453;

Abnormal modifications to mutant superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (fALS). Misfolding of wild-type SOD1 (SOD1) is also observed in postmortem tissue of a subset of sporadic ALS (sALS) cases, but cellular and molecular mechanisms generating abnormal SOD1 species are unknown. We analyzed aberrant human SOD1 species over the lifetime of transgenic mice and found the accumulation of disulfide-cross-linked high-molecular-weight SOD1 aggregates during aging. Subcellular fractionation of spinal cord tissue and protein overexpression in NSC-34 motoneuron-like cells revealed that endoplasmic reticulum (ER) localization favors oxidation and disulfide-dependent aggregation of SOD1 We established a pharmacological paradigm of chronic ER stress in vivo, which recapitulated SOD1aggregation in young transgenic mice. These species were soluble in nondenaturing detergents and did not react with a SOD1 conformation-specific antibody. Interestingly, SOD1 aggregation under ER stress correlated with astrocyte activation in the spinal cord of transgenic mice. Finally, the disulfide-cross-linked SOD1 species were also found augmented in spinal cord tissue of sALS patients, correlating with the presence of ER stress markers. Overall, this study suggests that ER stress increases the susceptibility of SOD1 to aggregate during aging, operating as a possible risk factor for developing ALS.
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http://dx.doi.org/10.1073/pnas.1801109115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094144PMC
August 2018

ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation.

Sci Rep 2017 03 8;7(1):115. Epub 2017 Mar 8.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.

Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.
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http://dx.doi.org/10.1038/s41598-017-00091-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428330PMC
March 2017

Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice.

Neuron 2015 Dec;88(5):902-909

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.
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http://dx.doi.org/10.1016/j.neuron.2015.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828340PMC
December 2015

Structural basis for mutation-induced destabilization of profilin 1 in ALS.

Proc Natl Acad Sci U S A 2015 Jun 8;112(26):7984-9. Epub 2015 Jun 8.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605;

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.
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http://dx.doi.org/10.1073/pnas.1424108112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491777PMC
June 2015

Autophagy meets fused in sarcoma-positive stress granules.

Neurobiol Aging 2014 Dec 28;35(12):2832-2835. Epub 2014 Sep 28.

Neurounion Biomedical Foundation, CENPAR, Santiago, Chile; Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago, Chile; Program of Cellular and Molecular Biology, Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago, Chile. Electronic address:

Mutations in fused in sarcoma and/or translocated in liposarcoma (FUS, TLS or FUS) are linked to familial cases of amyotrophic lateral sclerosis (ALS). Mutant FUS selectively accumulates into discrete cytosolic structures known as stress granules under various stress conditions. In addition, mutant FUS expression can alter the dynamics and morphology of stress granules. Although the link between mutant FUS and stress granules is well established, the mechanisms modulating stress granule formation and disassembly in the context of ALS are poorly understood. In this issue of Neurobiology of Aging, Ryu et al. uncover the impact of autophagy on the potential toxicity of mutant FUS-positive stress granules. The authors provide evidence indicating that enhanced autophagy activity reduces the number of stress granules, which in the case of cells containing mutant FUS-positive stress granules, is neuroprotective. Overall, this study identifies an intersection between the proteostasis network and alterations in RNA metabolism in ALS through the dynamic assembly and disassembly of stress granules.
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http://dx.doi.org/10.1016/j.neurobiolaging.2014.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324442PMC
December 2014

Functions of FUS/TLS from DNA repair to stress response: implications for ALS.

ASN Neuro 2014 Jun 1;6(4). Epub 2014 Jun 1.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is a multifunctional DNA-/RNA-binding protein that is involved in a variety of cellular functions including transcription, protein translation, RNA splicing, and transport. FUS was initially identified as a fusion oncoprotein, and thus, the early literature focused on the role of FUS in cancer. With the recent discoveries revealing the role of FUS in neurodegenerative diseases, namely amyotrophic lateral sclerosis and frontotemporal lobar degeneration, there has been a renewed interest in elucidating the normal functions of FUS. It is not clear which, if any, endogenous functions of FUS are involved in disease pathogenesis. Here, we review what is currently known regarding the normal functions of FUS with an emphasis on DNA damage repair, RNA processing, and cellular stress response. Further, we discuss how ALS-causing mutations can potentially alter the role of FUS in these pathways, thereby contributing to disease pathogenesis.
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http://dx.doi.org/10.1177/1759091414544472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189536PMC
June 2014

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

J Biol Chem 2014 Oct 27;289(41):28527-38. Epub 2014 Aug 27.

From the Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp(92) and Asp(96) as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS.
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http://dx.doi.org/10.1074/jbc.M114.581801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192504PMC
October 2014

Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.

Proteomics 2014 May 17;14(10):1152-7. Epub 2014 Apr 17.

Department of Chemistry and Chemical Biology, Barnett Institute, Northeastern University, Boston, MA, USA; Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA; Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA; Department of Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA.

Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S-thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S-thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen-dependent artifactual S-thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S-thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50-fold reduction in S-thiolation occurred indicating that the majority of S-thiolation observed aerobically was artifact. Tissue-specific artifacts were explored by comparing brain- and blood-derived protein, with remarkably more artifacts observed in brain-derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S-thiolation of highly reactive, surface-exposed, cysteine residues can result.
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http://dx.doi.org/10.1002/pmic.201300450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507715PMC
May 2014

An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis.

Front Cell Neurosci 2013 Dec 16;7:253. Epub 2013 Dec 16.

Department of Neurology, University of Massachusetts Medical Center Worcester, MA, USA.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s) of sporadic ALS, which accounts for ~90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1) protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a "toxic conformation" that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.
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http://dx.doi.org/10.3389/fncel.2013.00253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3863749PMC
December 2013

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics.

Mol Neurodegener 2013 Aug 31;8:30. Epub 2013 Aug 31.

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.

Background: Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.

Results: We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.

Conclusions: Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.
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http://dx.doi.org/10.1186/1750-1326-8-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766239PMC
August 2013

Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage.

Biochemistry 2013 Sep 26;52(36):6137-44. Epub 2013 Aug 26.

Departments of Biochemistry and Chemistry and Rosenstiel Basic Medical Sciences Research Center, Brandeis University , Waltham, Massachusetts 02454, United States.

Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.
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http://dx.doi.org/10.1021/bi4006122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859700PMC
September 2013

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.

PLoS One 2013 12;8(6):e65235. Epub 2013 Jun 12.

Depart of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois, United States of America.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065235PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680447PMC
January 2014

FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress.

J Cell Physiol 2013 Nov;228(11):2222-31

Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS-associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non-disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re-distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress.
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http://dx.doi.org/10.1002/jcp.24395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000275PMC
November 2013

Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1.

PLoS One 2013 17;8(4):e61210. Epub 2013 Apr 17.

MassBiologics, University of Massachusetts Medical School, Boston, Massachusetts, United States of America.

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061210PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629177PMC
November 2013

Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis.

Nature 2012 Aug;488(7412):499-503

Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.
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http://dx.doi.org/10.1038/nature11280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575525PMC
August 2012

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Proc Natl Acad Sci U S A 2012 Mar 13;109(13):5074-9. Epub 2012 Mar 13.

Weinberg Unit for ALS Research, Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.
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http://dx.doi.org/10.1073/pnas.1115402109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324021PMC
March 2012

Anti-superoxide dismutase antibodies are associated with survival in patients with sporadic amyotrophic lateral sclerosis.

Amyotroph Lateral Scler 2011 Nov;12(6):430-8

Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts, USA.

Our objective was to test the hypothesis that aberrantly modified forms of superoxide dismutase (SOD1) influence the disease course for sporadic amyotrophic lateral sclerosis (SALS). We probed for anti-SOD1 antibodies (IgM and IgG) against both the normal and aberrantly oxidized-SOD1 (SODox) antigens in sera from patients with SALS, subjects diagnosed with other neurological disorders and healthy individuals, and correlated the levels of these antibodies to disease duration and/or severity. Anti-SOD1 antibodies were detected in all cohorts; however, a subset of ∼5-10% of SALS cases exhibited elevated levels of anti-SOD1 antibodies. Those SALS cases with relatively high levels of IgM antibodies against SODox exhibit a longer survival of 6.4 years, compared to subjects lacking these antibodies. By contrast, SALS subjects expressing higher levels of IgG antibodies reactive for the normal WT-SOD1 antigen exhibit a shorter survival of 4.1 years. Anti-SOD1 antibody levels did not correlate with disease severity in either the Alzheimer's or Parkinson's disease cohorts. In conclusion, the association of longer survival with elevated levels of anti-SODox antibodies suggests that these antibodies may be protective. By extension, these data implicate aberrantly modified forms of WT-SOD1 (e.g. oxidized SOD1) in SALS pathogenesis. In contrast, an immune response against the normal WT-SOD1 appears to be disadvantageous in SALS, possibly because the anti-oxidizing activity of normal WT-SOD1 is beneficial to SALS individuals.
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http://dx.doi.org/10.3109/17482968.2011.585163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446817PMC
November 2011

Proteostasis and movement disorders: Parkinson's disease and amyotrophic lateral sclerosis.

Cold Spring Harb Perspect Biol 2011 Oct 1;3(10):a007500. Epub 2011 Oct 1.

Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA.

Parkinson's disease (PD) is a movement disorder that afflicts over one million in the U.S.; amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is less prevalent but also has a high incidence. The two disorders sometimes present together, making a comparative study of interest. Both ALS and PD are neurodegenerative diseases, and are characterized by the presence of intraneuronal inclusions; however, different classes of neurons are affected and the primary protein in the inclusions differs between the diseases, and in some cases is different in distinct forms of the same disease. These observations might suggest that the more general approach of proteostasis pathway alteration would be a powerful one in treating these disorders. Examining results from human genetics and studies in model organisms, as well as from biochemical and biophysical characterization of the proteins involved in both diseases, we find that most instances of PD can be considered as arising from the misfolding, and self-association to a toxic species, of the small neuronal protein α-synuclein, and that proteostasis strategies are likely to be of value for this disorder. For ALS, the situation is much more complex and less clear-cut; the available data are most consistent with a view that ALS may actually be a family of disorders, presenting similarly but arising from distinct and nonoverlapping causes, including mislocalization of some properly folded proteins and derangement of RNA quality control pathways. Applying proteostasis approaches to this disease may require rethinking or broadening the concept of what proteostasis means.
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http://dx.doi.org/10.1101/cshperspect.a007500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179340PMC
October 2011

A yeast model of FUS/TLS-dependent cytotoxicity.

PLoS Biol 2011 Apr 26;9(4):e1001052. Epub 2011 Apr 26.

Department of Biochemistry and Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, United States of America.

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.
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http://dx.doi.org/10.1371/journal.pbio.1001052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082520PMC
April 2011

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS.

Nat Neurosci 2010 Nov 17;13(11):1396-403. Epub 2010 Oct 17.

Department of Neurology, University of Massachusetts Medical Center, Worcester, Massachusetts, USA.

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.
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http://dx.doi.org/10.1038/nn.2660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967729PMC
November 2010

Genetic determinants of amyotrophic lateral sclerosis as therapeutic targets.

CNS Neurol Disord Drug Targets 2010 Dec;9(6):779-90

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Amyotrophic lateral sclerosis (ALS) is an incurable disease resulting from the deterioration of motor neurons. The onset of disease typically occurs in the fifth decade of life and progresses rapidly; death occurs for 75% of patients within 5 years. The only drug that is available to treat ALS is riluzole, which extends survival by just 2-3 months. Thus, new therapeutic directions are being sought to prolong the lifespan of ALS patients. Since the discovery of SOD1 as a genetic determinant of ALS in 1993, SOD1-models of ALS have been extensively employed for the development of ALS therapeutics. Novel genetic targets are now under investigation following the recent discoveries linking TDP-43, FUS/TLS, angiogenin, KIFAP3 and UNC13A to ALS. In this review, we present several of the genetic contributors to both sporadic and familial forms of ALS and discuss their potential as therapeutic targets for this devastating disease.
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http://dx.doi.org/10.2174/187152710793237494DOI Listing
December 2010

Dissecting the microscopic steps of the cyclophilin A enzymatic cycle on the biological HIV-1 capsid substrate by NMR.

J Mol Biol 2010 Nov 12;403(5):723-38. Epub 2010 Aug 12.

Department of Biochemistry and Howard Hughes Medical Institute, MS 009, Brandeis University, Waltham, MA 02454, USA.

Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA(N)) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
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http://dx.doi.org/10.1016/j.jmb.2010.08.001DOI Listing
November 2010
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