AAV2/9-mediated overexpression of MIF inhibits SOD1 misfolding, delays disease onset, and extends survival in mouse models of ALS

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the loss of upper and lower motor neurons. Transgenic mice that overexpress mutant SOD1 develop paralysis and accumulate misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit mutant SOD1 misfolding and binding to intracellular membranes. In addition, complete elimination of endogenous MIF accelerated disease onset and late disease progression, as well as shortened the lifespan of mutant SOD1 mice with higher amounts of misfolded SOD1 detected within the spinal cord. Based on these findings, we used adeno-associated viral (AAV) vectors to overexpress MIF in the spinal cord of mutant SOD1G93A and loxSOD1G37R mice. Our data show that MIF mRNA and protein levels were increased in the spinal cords of AAV2/9-MIF–injected mice. Furthermore, mutant SOD1G93A and loxSOD1G37R mice injected with AAV2/9-MIF demonstrated a significant delay in disease onset and prolonged survival compared with their AAV2/9-GFP–injected or noninjected littermates. Moreover, these mice accumulated reduced amounts of misfolded SOD1 in their spinal cords, with no observed effect on glial overactivation as a result of MIF up-regulation. Our findings indicate that MIF plays a significant role in SOD1 folding and misfolding mechanisms and strengthen the hypothesis that MIF acts as a chaperone for misfolded SOD1 in vivo and may have further implications regarding the therapeutic potential role of up-regulation of MIF in modulating the specific accumulation of misfolded SOD1.

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Endogenous macrophage migration inhibitory factor reduces the accumulation and toxicity of misfolded SOD1 in a mouse model of ALS

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604600113 ◽

2016 ◽

Vol 113 (36) ◽

pp. 10198-10203 ◽

Cited By ~ 21

Author(s):

Marcel F. Leyton-Jaimes ◽

Clara Benaim ◽

Salah Abu-Hamad ◽

Joy Kahn ◽

Amos Guetta ◽

...

Keyword(s):

Migration Inhibitory Factor ◽

Macrophage Migration Inhibitory Factor ◽

Disease Onset ◽

Inhibitory Factor ◽

Neuronal Cultures ◽

Macrophage Migration ◽

Mutant Sod1 ◽

Misfolded Sod1

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons in the brain and spinal cord. It has been suggested that the toxicity of mutant SOD1 results from its misfolding and accumulation on the cytoplasmic faces of intracellular organelles, including the mitochondria and endoplasmic reticulum (ER) of ALS-affected tissues. Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit the accumulation of misfolded SOD1 and its binding to intracellular membranes, but the role of endogenous MIF in modulating SOD1 misfolding in vivo remains unknown. To elucidate this role, we bred MIF-deficient mice with SOD1G85R mice, which express a dismutase-inactive mutant of SOD1 and are considered a model of familial ALS. We found that the accumulation of misfolded SOD1, its association with mitochondrial and ER membranes, and the levels of sedimentable insoluble SOD1 aggregates were significantly higher in the spinal cords of SOD1G85R-MIF−/− mice than in their SOD1G85R-MIF+/+ littermates. Moreover, increasing MIF expression in neuronal cultures inhibited the accumulation of misfolded SOD1 and rescued from mutant SOD1-induced cell death. In contrast, the complete elimination of endogenous MIF accelerated disease onset and late disease progression and shortened the lifespan of the SOD1G85R mutant mice. These findings indicate that MIF plays a significant role in the folding and misfolding of SOD1 in vivo, and they have implications for the potential therapeutic role of up-regulating MIF within the nervous system to modulate the selective accumulation of misfolded SOD1.

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Early upregulation of cytosolic phospholipase A2α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

Journal of Neuroinflammation ◽

10.1186/s12974-021-02326-5 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Yafa Fetfet Malada Edelstein ◽

Yulia Solomonov ◽

Nurit Hadad ◽

Leenor Alfahel ◽

Adrian Israelson ◽

...

Keyword(s):

Spinal Cord ◽

Amyotrophic Lateral Sclerosis ◽

Transgenic Mice ◽

Motor Neurons ◽

Dependent Manner ◽

Mutant Sod1 ◽

Over Expression ◽

Cytosolic Phospholipase ◽

Misfolded Sod1 ◽

Lateral Sclerosis

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA2α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1G93A) was detected long before the development of the disease, and inhibition of cPLA2α upregulation delayed the disease’s onset. The aim of the present study was to determine the mechanism for cPLA2α upregulation. Methods Immunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA2α and inflammatory markers were performed in the spinal cord sections of SOD1G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells. Results Misfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1G93A mice before cPLA2α upregulation. Elevated expression of both misfolded SOD1 and cPLA2α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA2α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA2α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA2α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA2α upregulation in a dose dependent manner. Conclusions Motor neurons expressing elevated cPLA2α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA2α upregulation via induction of TNFα.

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The Timing and Extent of Motor Neuron Vulnerability in ALS Correlates with Accumulation of Misfolded SOD1 Protein in the Cortex and in the Spinal Cord

Cells ◽

10.3390/cells9020502 ◽

2020 ◽

Vol 9 (2) ◽

pp. 502

Author(s):

Baris Genc ◽

Oge Gozutok ◽

Nuran Kocak ◽

P. Hande Ozdinler

Keyword(s):

Spinal Cord ◽

Drug Discovery ◽

Motor Neuron ◽

Motor Neurons ◽

Selective Vulnerability ◽

Motor Neuron Diseases ◽

Neuron Populations ◽

Misfolded Sod1 ◽

Over Time

Understanding the cellular and molecular basis of selective vulnerability has been challenging, especially for motor neuron diseases. Developing drugs that improve the health of neurons that display selective vulnerability relies on in vivo cell-based models and quantitative readout measures that translate to patient outcome. We initially developed and characterized UCHL1-eGFP mice, in which motor neurons are labeled with eGFP that is stable and long-lasting. By crossing UCHL1-eGFP to amyotrophic lateral sclerosis (ALS) disease models, we generated ALS mouse models with fluorescently labeled motor neurons. Their examination over time began to reveal the cellular basis of selective vulnerability even within the related motor neuron pools. Accumulation of misfolded SOD1 protein both in the corticospinal and spinal motor neurons over time correlated with the timing and extent of degeneration. This further proved simultaneous degeneration of both upper and lower motor neurons, and the requirement to consider both upper and lower motor neuron populations in drug discovery efforts. Demonstration of the direct correlation between misfolded SOD1 accumulation and motor neuron degeneration in both cortex and spinal cord is important for building cell-based assays in vivo. Our report sets the stage for shifting focus from mice to diseased neurons for drug discovery efforts, especially for motor neuron diseases.

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Faculty Opinions recommendation of Transfer of pathogenic and nonpathogenic cytosolic proteins between spinal cord motor neurons in vivo in chimeric mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727458673.793530611 ◽

2017 ◽

Author(s):

Judith S Eisen

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Chimeric Mice ◽

Cytosolic Proteins

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LanCL1 promotes motor neuron survival and extends the lifespan of amyotrophic lateral sclerosis mice

Cell Death and Differentiation ◽

10.1038/s41418-019-0422-6 ◽

2019 ◽

Vol 27 (4) ◽

pp. 1369-1382 ◽

Cited By ~ 4

Author(s):

Honglin Tan ◽

Mina Chen ◽

Dejiang Pang ◽

Xiaoqiang Xia ◽

Chongyangzi Du ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

Neuronal Survival ◽

Disease Onset ◽

Alternative Mechanism ◽

Specific Expression ◽

Motor Neuron Survival ◽

Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Improving neuronal survival in ALS remains a significant challenge. Previously, we identified Lanthionine synthetase C-like protein 1 (LanCL1) as a neuronal antioxidant defense gene, the genetic deletion of which causes apoptotic neurodegeneration in the brain. Here, we report in vivo data using the transgenic SOD1G93A mouse model of ALS indicating that CNS-specific expression of LanCL1 transgene extends lifespan, delays disease onset, decelerates symptomatic progression, and improves motor performance of SOD1G93A mice. Conversely, CNS-specific deletion of LanCL1 leads to neurodegenerative phenotypes, including motor neuron loss, neuroinflammation, and oxidative damage. Analysis reveals that LanCL1 is a positive regulator of AKT activity, and LanCL1 overexpression restores the impaired AKT activity in ALS model mice. These findings indicate that LanCL1 regulates neuronal survival through an alternative mechanism, and suggest a new therapeutic target in ALS.

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Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515526113 ◽

2016 ◽

Vol 113 (9) ◽

pp. 2514-2519 ◽

Cited By ~ 12

Author(s):

Drew L. Sellers ◽

Jamie M. Bergen ◽

Russell N. Johnson ◽

Heidi Back ◽

John M. Ravits ◽

...

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Unmet Need ◽

Nerve Roots ◽

Biologic Drugs ◽

Administration Routes ◽

Macromolecular Drugs ◽

The Central Nervous System ◽

Clinical Potential

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

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FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

10.21203/rs.3.rs-493867/v1 ◽

2021 ◽

Author(s):

Bin Qiu ◽

Zhaohui Zhong ◽

Shawn Righter ◽

Yuxue Xu ◽

Jun Wang ◽

...

Keyword(s):

Hippocampal Neurons ◽

Translational Regulation ◽

Disease Onset ◽

Fold Increase ◽

Knock Out ◽

Fk506 Binding Protein ◽

Protein Levels ◽

And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

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Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice

Protein Science ◽

10.1002/pro.700 ◽

2011 ◽

Vol 20 (10) ◽

pp. 1692-1696 ◽

Cited By ~ 7

Author(s):

George W. Farr ◽

Zheng Ying ◽

Wayne A. Fenton ◽

Arthur L. Horwich

Keyword(s):

Spinal Cord ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Mutant Sod1 ◽

Hydrogen Deuterium

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“Stretch-Growth” of Motor Axons in Custom Mechanobioreactors to Generate Long-Projecting Axonal and Axonal-Myocyte Constructs

10.1101/598755 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kritika S. Katiyar ◽

Laura A. Struzyna ◽

Suradip Das ◽

D. Kacy Cullen

Keyword(s):

Spinal Cord ◽

Nervous System ◽

Motor Neuron ◽

Motor Neurons ◽

Motor Axon ◽

Central Feature ◽

Motor Axons ◽

Ganglion Neurons

AbstractThe central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to an often-distant target muscle. However, to date in vitro models have not replicated this central feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of micro-tissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from the spinal cords of rats and induced to form engineered micro-spheres via forced aggregation in custom micro-wells. This three-dimensional micro-tissue yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were systematically optimized to generate parameters for robust and efficient “stretch-growth” of motor axons. We found that axons projecting from motor neuron aggregates were able to respond to axon displacement rates at least 10 times greater than that tolerated by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared to benchmark stretch-grown axons from sensory dorsal root ganglion neurons, revealing similar axon densities yet increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and then stretch-grown to create novel long-projecting axonal-myocyte constructs that better recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal cord motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue engineered “living scaffolds” for targeted axon tract reconstruction following nervous system injury or disease.Significance StatementWe have developed novel axon tracts of unprecedented lengths spanning either two discrete populations of neurons or a population of neurons and skeletal myocytes. This is the first demonstration of “stretch-grown” motor axons that recapitulate the structure of spinal motor neurons in vivo by projecting long axons from a pool of motor neurons to distant targets, and may have applications as anatomically inspired in vitro test beds to study mechanisms of axon growth, development, and neuromuscular function in anatomically accurate axo-myo constructs; as well as serve as “living scaffolds” in vivo for targeted axon tract reconstruction following nervous system trauma.

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The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

10.1101/305284 ◽

2018 ◽

Cited By ~ 2

Author(s):

Lewie Zeng ◽

Rachid El Bejjani ◽

Marc Hammarlund

Keyword(s):

Motor Neurons ◽

Neuronal Development ◽

Axon Regeneration ◽

Neuronal Response ◽

Small Gtpase ◽

C Elegans ◽

Protein Levels ◽

Mature Neurons ◽

And Function

AbstractMembers of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.

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