Maria Nagy

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotides γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.

Synergistic Cooperation between Two ClpB Isoforms in Aggregate Reactivation

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

RNA-Seq Profiling of Spinal Cord Motor Neurons from a Presymptomatic SOD1 ALS Mouse

Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrigs Disease) are poorly understood, but genetically inherited forms, comprising ∼10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5–6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.

Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS

Significance We describe a method that allows, for the first time to our knowledge, the preparation of viable acute spinal cord slices from adult mice, enabling patch-clamp recording from fluorescent motor neurons. From electrophysiological parameters, four subtypes of motor neurons were identified. Two fast firing subtypes innervated fast twitch muscle, whereas the two slow firing subtypes innervated slow twitch muscle. In superoxide dismutase 1-linked amyotrophic lateral sclerosis mice, the same four firing types were observed before the onset of symptoms, but the fastest firing type was lost after symptoms developed. Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) affects motor neurons (MNs) in the brain and spinal cord. Understanding the pathophysiology of this condition seems crucial for therapeutic design, yet few electrophysiological studies in actively degenerating animal models have been reported. Here, we report a novel preparation of acute slices from adult mouse spinal cord, allowing visualized whole cell patch-clamp recordings of fluorescent lumbar MN cell bodies from ChAT-eGFP or superoxide dismutase 1-yellow fluorescent protein (SOD1YFP) transgenic animals up to 6 mo of age. We examined 11 intrinsic electrophysiologic properties of adult ChAT-eGFP mouse MNs and classified them into four subtypes based on these parameters. The subtypes could be principally correlated with instantaneous (initial) and steady-state firing rates. We used retrograde tracing using fluorescent dye injected into fast or slow twitch lower extremity muscle with slice recordings from the fluorescent-labeled lumbar MN cell bodies to establish that fast and slow firing MNs are connected with fast and slow twitch muscle, respectively. In a G85R SOD1YFP transgenic mouse model of ALS, which becomes paralyzed by 5–6 mo, where MN cell bodies are fluorescent, enabling the same type of recording from spinal cord tissue slices, we observed that all four MN subtypes were present at 2 mo of age. At 4 mo, by which time substantial neuronal SOD1YFP aggregation and cell loss has occurred and symptoms have developed, one of the fast firing subtypes that innvervates fast twitch muscle was lost. These results begin to describe an order of the pathophysiologic events in ALS.

Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110

Significance Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) is a progressive paralyzing condition affecting ∼1:1,000 adults, associated with death of motor neurons, for which there is no effective treatment. ALS is inherited in 10% of cases, whereas the remainder are “sporadic,” yet all behave very similarly. In mice, the condition has been modeled by transgenesis with mutant versions of superoxide dismutase 1 (SOD1) (∼2% of human cases). Mutant SOD1s misfold and form aggregates inside the cytosol of motor neurons in the spinal cord. Recently, a disaggregating machinery has been described consisting of three chaperones, one of which, Hsp110, plays a rate limiting role. Here, we have transgenically overexpressed Hsp110 in motor neurons of a mutant SOD1 strain and observed extended survival. Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoter-driven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ∼5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery.

Absence of lipofuscin in motor neurons of SOD1-linked ALS mice

Significance This is the first report, to our knowledge, of the absence of lipofuscin, “aging pigment,” in a setting of motor neuron neurodegenerative disease, mutant SOD1-linked ALS. Although the initial hypothesis was that this must be due to a block in the autophagy/lysosome pathway, instead studies of autophagy markers and of ALS mice treated with chloroquine suggest the opposite, that there is hyperactivity of the autophagy/lysosome pathway, at least in part mediated through MTORC1. Lipofuscin, or aging pigment, is accreted as red autofluorescence in the lysosomes of motor neuron cell bodies in the ventral horn of WT mice by 3 mo of age. Strikingly, in two presymptomatic ALS mouse strains transgenic for mutant human Cu/Zn superoxide dismutase (SOD1), G85R SOD1YFP and G93A SOD1, little or no lipofuscin was detected in motor neuron cell bodies. Two markers of autophagy, sequestosome 1 (SQSTM1/p62) and microtubule-associated protein 1 light chain 3 (LC3), were examined in the motor neuron cell bodies of G85R SOD1YFP mice and found to be reduced relative to WT SOD1YFP transgenic mice. To elucidate whether the autophagy/lysosome pathway was either impaired or hyperactive in motor neurons, chloroquine was administered to 3-mo-old G85R SOD1YFP mice to block lysosomal hydrolysis. After 2 wk, lipofuscin was now observed in motor neurons, and SQSTM1 and LC3 levels approached those of WT SOD1YFP mice, suggesting that the autophagy/lysosome pathway is hyperactive in motor neurons of SOD1-linked ALS mice. This seems to be mediated at least in part through the mammalian target of rapamycin complex 1 (MTORC1) pathway, because levels of Ser757-phosphorylated Unc-51-like kinase 1 (ULK1), an MTORC1 target, were greatly reduced in the G85R SOD1YFP motor neurons, correspondent to an activated state of ULK1 that initiates autophagy.

Flexible connection of the N-terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency.

ClpB reactivates aggregated proteins in cooperation with DnaK/J. The ClpB monomer contains two nucleotide‐binding domains (D1, D2), a coiled‐coil domain, and an N‐terminal domain attached to D1 with a 17‐residue‐long unstructured linker containing a Gly‐Gly motif. The ClpB‐mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB, but the events preceding the translocation are poorly understood. The N‐terminal domains form a ring surrounding the entrance to the channel and contribute to the aggregate binding. It was suggested that the N‐terminal domains mobility that is maintained by the unstructured linker might control the efficiency of aggregate reactivation. We produced seven variants of ClpB with modified sequence of the N‐terminal linker. To increase the linkers conformational flexibility, we inserted up to four Gly next to the GG motif. To decrease the linkers flexibility, we deleted the GG motif and converted it into GP and PP. We found that none of the linker modifications inhibited the basal ClpB ATPase activity or its capability to form oligomers. However, the modified linker ClpB variants showed lower reactivation rates for aggregated glucose‐6‐phosphate dehydrogenase and firefly luciferase and a lower aggregate‐binding efficiency than wt ClpB. We conclude that the linker does not merely connect the N‐terminal domain, but it supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. Moreover, our results suggest that selective pressure on the linker sequence may be crucial for maintaining the optimal efficiency of aggregate reactivation by ClpB. Proteins 2012;

Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2

In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~106 copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt’s lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~106 copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.