Targeting low levels of MIF expression as a potential therapeutic strategy for ALS.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.

CD4 T-cell aging exacerbates neuroinflammation in a late-onset mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1G93A and late-onset SOD1G37R mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1G37R mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1G37R mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.

Intracellular spatially-targeted chemical chaperones increase native state stability of mutant SOD1 barrel.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment.

Blocking an epitope of misfolded SOD1 ameliorates disease phenotype in a model of amyotrophic lateral sclerosis.

The current strategies to mitigate the toxicity of misfolded superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis via blocking SOD1 expression in the CNS are indiscriminative for misfolded and intact proteins, and as such, entail a risk of depriving CNS cells of their essential antioxidant potential. As an alternative approach to neutralize misfolded and spare unaffected SOD1 species, we developed scFv-SE21 antibody that blocks the ß6/ß7 loop epitope exposed exclusively in misfolded SOD1. The ß6/ß7 loop epitope has previously been proposed to initiate amyloid-like aggregation of misfolded SOD1 and mediate its prion-like activity. The adeno-associated virus-mediated expression of scFv-SE21 in the CNS of hSOD1G37R mice rescued spinal motor neurons, reduced the accumulation of misfolded SOD1, decreased gliosis and thus delayed disease onset and extended survival by 90 days. The results provide evidence for the role of the exposed ß6/ß7 loop epitope in the mechanism of neurotoxic gain-of-function of misfolded SOD1 and open avenues for the development of mechanism-based anti-SOD1 therapeutics, whose selective targeting of misfolded SOD1 species may entail a reduced risk of collateral oxidative damage to the CNS.

Therapeutic Potential of Phytocannabinoid Cannabigerol for Multiple Sclerosis: Modulation of Microglial Activation In Vitro and In Vivo.

Multiple sclerosis (MS) is a widespread chronic neuroinflammatory and neurodegenerative disease. Microglia play a crucial role in the pathogenesis of MS via the release of cytokines and reactive oxygen species, e.g., nitric oxide. Research involving the role of phytocannabinoids in neuroinflammation is currently receiving much attention. Cannabigerol is a main phytocannabinoid, which has attracted significant pharmacological interest due to its non-psychotropic nature. In this research, we studied the effects of cannabigerol on microglial inflammation in vitro, followed by an in vivo study. Cannabigerol attenuated the microglial production of nitric oxide in BV2 microglia and primary glial cells; concomitant treatment of the cells with cannabigerol and telmisartan (a neuroprotective angiotensin receptor blocker) decreased nitric oxide production additively. Inducible nitric oxide synthase (iNOS) expression was also reduced by cannabigerol. Moreover, tumor necrosis factor-α (TNF-α), a major cytokine involved in MS, was significantly reduced by cannabigerol in both cell cultures. Next, we studied the effects of cannabigerol in vivo using a mice model of MS, experimental autoimmune encephalomyelitis (EAE). The clinical scores of EAE mice were attenuated upon cannabigerol treatment; additionally, lumbar sections of EAE mice showed enhanced neuronal loss (relative to control mice), which was restored by cannabigerol treatment. Altogether, the set of experiments presented in this work indicates that cannabigerol possesses an appealing therapeutic potential for the treatment of MS.

Targeting the Mitochondrial Protein VDAC1 as a Potential Therapeutic Strategy in ALS.

Impaired mitochondrial function has been proposed as a causative factor in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), caused by motor neuron degeneration. Mutations in superoxide dismutase (SOD1) cause ALS and SOD1 mutants were shown to interact with the voltage-dependent anion channel 1 (VDAC1), affecting its normal function. VDAC1 is a multi-functional channel located at the outer mitochondrial membrane that serves as a mitochondrial gatekeeper controlling metabolic and energetic crosstalk between mitochondria and the rest of the cell and it is a key player in mitochondria-mediated apoptosis. Previously, we showed that VDAC1 interacts with SOD1 and that the VDAC1-N-terminal-derived peptide prevented mutant SOD1 cytotoxic effects. In this study, using a peptide array, we identified the SOD1 sequence that interacts with VDAC1. Synthetic peptides generated from the identified VDAC1-binding sequences in SOD1 directly interacted with purified VDAC1. We also show that VDAC1 oligomerization increased in spinal cord mitochondria isolated from mutant SOD1G93A mice and rats. Thus, we used the novel VDAC1-specific small molecules, VBIT-4 and VBIT-12, inhibiting VDAC1 oligomerization and subsequently apoptosis and associated processes such as ROS production, and increased cytosolic Ca2+. VBIT-12 was able to rescue cell death induced by mutant SOD1 in neuronal cultures. Finally, although survival was not affected, VBIT-12 administration significantly improved muscle endurance in mutant SOD1G93A mice. Therefore, VBIT-12 may represent an attractive therapy for maintaining muscle function during the progression of ALS.

4-Phenylbutyric Acid (4-PBA) Derivatives Prevent SOD1 Amyloid Aggregation In Vitro with No Effect on Disease Progression in SOD1-ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the superoxide dismutase (SOD1) gene, causing protein misfolding and aggregation, were suggested as the pathogenic mechanisms involved in familial ALS cases. In the present study, we investigated the potential therapeutic effect of C4 and C5, two derivatives of the chemical chaperone 4-phenylbutyric acid (4-PBA). By combining in vivo and in vitro techniques, we show that, although C4 and C5 successfully inhibited amyloid aggregation of recombinant mutant SOD1 in a dose-dependent manner, they failed to suppress the accumulation of misfolded SOD1. Moreover, C4 or C5 daily injections to SOD1G93A mice following onset had no effect on either the accumulation of misfolded SOD1 or the neuroinflammatory response in the spinal cord and, consequently, failed to extend the survival of SOD1G93A mice or to improve their motor symptoms. Finally, pharmacokinetic (PK) studies demonstrated that high concentrations of C4 and C5 reached the brain and spinal cord but only for a short period of time. Thus, our findings suggest that use of such chemical chaperones for ALS drug development may need to be optimized for more effective results.

MIF homolog d-dopachrome tautomerase (D-DT/MIF-2) does not inhibit accumulation and toxicity of misfolded SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons. About 20% of familial ALS cases are caused by dominant mutations in SOD1. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is unclear why misfolded SOD1 accumulates within specific tissues. We have demonstrated that macrophage migration inhibitory factor (MIF), a multifunctional protein with cytokine/chemokine and chaperone-like activity, inhibits the accumulation and aggregation of misfolded SOD1. Although MIF homolog, D-dopachrome tautomerase (D-DT/MIF-2), shares structural and genetic similarities with MIF, its biological function is not well understood. In the current study, we investigated, for the first time, the mechanism of action of D-DT in a model of ALS. We show that D-DT inhibits mutant SOD1 amyloid aggregation in vitro, promoting the formation of amorphous aggregates. Moreover, we report that D-DT interacts with mutant SOD1, but does not inhibit misfolded mutant SOD1 accumulation and toxicity in neuronal cells. Finally, we show that D-DT is expressed mainly in liver and kidney, with extremely low expression in brain and spinal cord of adult mice. Our findings contribute to better understanding of D-DT versus MIF function in the context of ALS.

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

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.

All Roads Lead to Rome: Different Molecular Players Converge to Common Toxic Pathways in Neurodegeneration.

Multiple neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) are being suggested to have common cellular and molecular pathological mechanisms, characterized mainly by protein misfolding and aggregation. These large inclusions, most likely, represent an end stage of a molecular cascade; however, the soluble misfolded proteins, which take part in earlier steps of this cascade, are the more toxic players. These pathological proteins, which characterize each specific disease, lead to the selective vulnerability of different neurons, likely resulting from a combination of different intracellular mechanisms, including mitochondrial dysfunction, ER stress, proteasome inhibition, excitotoxicity, oxidative damage, defects in nucleocytoplasmic transport, defective axonal transport and neuroinflammation. Damage within these neurons is enhanced by damage from the nonneuronal cells, via inflammatory processes that accelerate the progression of these diseases. In this review, while acknowledging the hallmark proteins which characterize the most common NDDs; we place specific focus on the common overlapping mechanisms leading to disease pathology despite these different molecular players and discuss how this convergence may occur, with the ultimate hope that therapies effective in one disease may successfully translate to another.

Exposure of ß6/ß7-Loop in Zn/Cu Superoxide Dismutase (SOD1) Is Coupled to Metal Loss and Is Transiently Reversible During Misfolding.

Upon losing its structural integrity (misfolding), SOD1 acquires neurotoxic properties to become a pathogenic protein in ALS, a neurodegenerative disease targeting motor neurons; understanding the mechanism of misfolding may enable new treatment strategies for ALS. Here, we reported a monoclonal antibody, SE21, targeting the ß6/ß7-loop region of SOD1. The exposure of this region is coupled to metal loss and is entirely reversible during the early stages of misfolding. By using SE21 mAb, we demonstrated that, in apo-SOD1 incubated under the misfolding-promoting conditions, the reversible phase, during which SOD1 is capable of restoring its nativelike conformation in the presence of metals, is followed by an irreversible structural transition, autocatalytic in nature, which takes place prior to the onset of SOD1 aggregation and results in the formation of atypical apo-SOD1 that is unable to bind metals. The reversible phase defines a window of opportunity for pharmacological intervention using metal mimetics that stabilize SOD1 structure in its nativelike conformation to attenuate the spreading of the misfolding signal and disease progression by preventing the exposure of pathogenic SOD1 epitopes. Phenotypically similar apo-SOD1 species with impaired metal binding properties may also be produced via oxidation of Cys111, underscoring the diversity of SOD1 misfolding pathways.

Empty mesoporous silica particles significantly delay disease progression and extend survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating incurable neurological disorder characterized by motor neuron (MN) death and muscle dysfunction leading to mean survival time after diagnosis of only 2-5 years. A potential ALS treatment is to delay the loss of MNs and disease progression by the delivery of trophic factors. Previously, we demonstrated that implanted mesoporous silica nanoparticles (MSPs) loaded with trophic factor peptide mimetics support survival and induce differentiation of co-implanted embryonic stem cell (ESC)-derived MNs. Here, we investigate whether MSP loaded with peptide mimetics of ciliary neurotrophic factor (Cintrofin), glial-derived neurotrophic factor (Gliafin), and vascular endothelial growth factor (Vefin1) injected into the cervical spinal cord of mutant SOD1 mice affect disease progression and extend survival. We also transplanted boundary cap neural crest stem cells (bNCSCs) which have been shown previously to have a positive effect on MN survival in vitro and in vivo. We show that mimetic-loaded MSPs and bNCSCs significantly delay disease progression and increase survival of mutant SOD1 mice, and also that empty particles significantly improve the condition of ALS mice. Our results suggest that intraspinal delivery of MSPs is a potential therapeutic approach for the treatment of ALS.

A VDAC1-Derived N-Terminal Peptide Inhibits Mutant SOD1-VDAC1 Interactions and Toxicity in the SOD1 Model of ALS.

Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.

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.

Potential roles of gut microbiome and metabolites in modulating ALS in mice.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder, in which the clinical manifestations may be influenced by genetic and unknown environmental factors. Here we show that ALS-prone Sod1 transgenic (Sod1-Tg) mice have a pre-symptomatic, vivarium-dependent dysbiosis and altered metabolite configuration, coupled with an exacerbated disease under germ-free conditions or after treatment with broad-spectrum antibiotics. We correlate eleven distinct commensal bacteria at our vivarium with the severity of ALS in mice, and by their individual supplementation into antibiotic-treated Sod1-Tg mice we demonstrate that Akkermansia muciniphila (AM) ameliorates whereas Ruminococcus torques and Parabacteroides distasonis exacerbate the symptoms of ALS. Furthermore, Sod1-Tg mice that are administered AM are found to accumulate AM-associated nicotinamide in the central nervous system, and systemic supplementation of nicotinamide improves motor symptoms and gene expression patterns in the spinal cord of Sod1-Tg mice. In humans, we identify distinct microbiome and metabolite configurations-including reduced levels of nicotinamide systemically and in the cerebrospinal fluid-in a small preliminary study that compares patients with ALS with household controls. We suggest that environmentally driven microbiome-brain interactions may modulate ALS in mice, and we call for similar investigations in the human form of the disease.

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit.

The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1WT (holo-SOD1WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H2O2 through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.

Macrophage migration inhibitory factor: A multifaceted cytokine implicated in multiple neurological diseases.

Macrophage migration inhibitory factor (MIF) is a conserved cytokine found as a homotrimer protein. It is found in a wide spectrum of cell types in the body including neuronal and non-neuronal cells. MIF is implicated in several biological processes; chemo-attraction, cytokine activity, and receptor binding, among other functions. More recently, a chaperone-like activity has been added to its repertoire. In this review, we focus on the implication of MIF in the central nervous system and peripheries, its role in neurological disorders, and the mechanisms by which MIF is regulated. Numerous studies have associated MIF with various disease settings. MIF plays an important role in advocating tumorigenic processes, Alzheimer's disease, and is also upregulated in autism-spectrum disorders and spinal cord injury where it contributes to the severity of the injured area. The protective effect of MIF has been reported in amyotrophic lateral sclerosis by its reduction of aggregated misfolded SOD1, subsequently reducing the severity of this disease. Interestingly, a protective as well as pathological role for MIF has been implicated in stroke and cerebral ischemia, as well as depression. Thus, the role of MIF in neurological disorders appears to be diverse with both beneficial and adversary effects. Furthermore, its modulation is rather complex and it is regulated by different proteins, either on a molecular or protein level. This complexity might be dependent on the pathophysiological context and/or cellular microenvironment. Hence, further clarification of its diverse roles in neurological pathologies is warranted to provide new mechanistic insights which may lead in the future to the development of therapeutic strategies based on MIF, to fight some of these neurological disorders.

Misfolded SOD1 Accumulation and Mitochondrial Association Contribute to the Selective Vulnerability of Motor Neurons in Familial ALS: Correlation to Human Disease.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, with a 10% genetic linkage, of which 20% of these cases may be attributed to mutations in superoxide dismutase (SOD1). Specific mutations in SOD1 have been associated with disease duration, which can be highly variable ranging from a life expectancy of 3 to beyond 10 years. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles disrupting their function or forms insoluble toxic aggregates. To understand whether these biophysical properties of the mutant protein may influence disease onset and duration, we generated 19 point mutations in the SOD1 gene, based on available clinical data of disease onset and progression from patients. By overexpressing these mutants in motor-neuron-like NSC-34 cells, we demonstrate a variability in misfolding capacity between the different mutants with a correlation between the degree of protein misfolding and mutation severity. We also show a clear variation of the different SOD1 mutants to associate with mitochondrial-enriched fractions with a correlation between mutation severity and this association. In summary, these findings reveal a correlation between the accumulation of misfolded SOD1 species and their mitochondrial association with disease duration but not with disease onset, and they have implications for the potential therapeutic role of suppressing the accumulation of misfolded SOD1.

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.