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.

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.

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

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 SOD1(G85R) 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 SOD1(G85R)-MIF(-/-) mice than in their SOD1(G85R)-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 SOD1(G85R) 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.

Loss of mouse Stmn2 function causes motor neuropathy.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.

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.

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.